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Journal of Pharmaceutical and Biomedical Analysis 52 (2010) 110–113

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journa l homepage: www.e lsev ier .com/ locate / jpba

ltra high-pressure liquid chromatographic assay of moxifloxacin in rabbitqueous humor after topical instillation of moxifloxacin nanoparticles

aurav K. Jaina,∗, Nilu Jaina, Shadab A. Pathana, Sohail Akhtera, Sushma Talegaonkara,rakash Chanderb, Roop K. Khara, Farhan J. Ahmada

Department of Pharmaceutics, F/O Pharmacy, Hamdard University, Hamdard Nagar, New Delhi 110062, IndiaWaters India Pvt Ltd., CS-08/09, 7th Floor, Lobe 2, Tower A, The Corenthum, Plot No. A-41, Sector 62, Noida 201301, UP, India

r t i c l e i n f o

rticle history:eceived 21 September 2009eceived in revised form 2 December 2009ccepted 3 December 2009vailable online 11 December 2009

eywords:oxifloxacin

a b s t r a c t

The present report describes a rapid and sensitive ultra high-pressure liquid chromatography (UHPLC)method with UV detection to quantify moxifloxacin in rabbit aqueous humor. After deproteinisationwith acetonitrile, gradient separation of moxifloxacin was achieved on a Waters Acquity BEH C18(50 mm × 2.1 mm, 1.7 �m) column at 50 ◦C. The mobile phase consisted of 0.1% trifluoroacetic acid inwater and acetonitrile at a flow rate of 0.4 ml/min. Detection of moxifloxacin was done at 296 nm. Methodwas found to be selective, linear (r = 0.9997), accurate (recovery, 97.30–99.99%) and precise (RSD, ≤1.72%)in the selected concentration range of 10–1000 ng/ml. Detection and quantitation limit of moxifloxacinin aqueous humor were 0.75 and 2.5 ng/ml, respectively. The aqueous humor levels of moxifloxacin after

queous humor

ltra high-pressure liquid chromatographyanoparticlescular pharmacokinetics

single topical instillation in three formulations, i.e. moxifloxacin solution (Moxi-SOL), anionic nanopar-ticles (Moxi-ANP) and cationic nanoparticles (Moxi-CNP) were investigated. A fourfold increase in therelative bioavailability was observed with the Moxi-CNP (AUC0→t, 3.14 �g h/ml) compared with Moxi-SOL (AUC0→t, 0.79 �g h/ml) and Moxi-ANP (AUC0→t, 0.72 �g h/ml) formulation. The results indicate thatthe cationic nanoparticle increases ocular bioavailability of moxifloxacin and prolong its residence time

in the eye.

. Introduction

Moxifloxacin, a fourth generation fluoroquinolone, is a broad-pectrum antibiotic used in the prevention and treatment of aariety of ocular infections [1]. Recent reports based on severaln vivo studies have shown the potency of moxifloxacin in pre-enting anterior eye infections such as bacterial conjunctivitisnd keratitis [2]. Traditionally the plasma concentration of mox-floxacin and its relation to the minimum inhibitory concentrationas been used to predict its likely efficacy against ocular infec-ions and therefore most of the bioanalytical methods currentlyvailable for the quantification of moxifloxacin are restricted to

easuring plasma or serum concentration of moxifloxacin in phar-acokinetic studies [3–16]. For extravascular infections such as

cular infections, the ability of antibiotic to kill and eradicate theathogens at the site of infection (aqueous humor) is an important

∗ Corresponding author. Tel.: +91 09811127909; fax: +91 11 26059663.E-mail addresses: drgkjain@gmail.com (G.K. Jain), jneelu78@yahoo.com (N. Jain),

hadab.ahmad1@gmail.com (S.A. Pathan), sohailakhtermph@gmail.com (S. Akhter),talegaonkar@jamiahamdard.ac.in (S. Talegaonkar), prakash chander@waters.comP. Chander), rkkhar@jamiahamdard.ac.in (R.K. Khar), farhanja 2000@yahoo.comF.J. Ahmad).

731-7085/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.jpba.2009.12.008

© 2009 Elsevier B.V. All rights reserved.

goal and therefore determination of drug concentration in aqueoushumor is desirable and is needed. As for moxifloxacin concentra-tion in eye, the only assay described in the literature was based onHPLC with amino acid HPLC column and concerned the measure-ment of moxifloxacin using fluorescent detection [17]. However,the chromatographic run time of the method was long with reten-tion time of moxifloxacin at 16.7 min [17]. In this paper, the UHPLCmethod with UV detection for determination of moxifloxacinin rabbit aqueous humor is described. We propose the proce-dure with minimal sample pre-treatment using direct injectioninto chromatographic column. Furthermore, the rabbit aqueoushumor concentrations of moxifloxacin solution (Moxi-SOL), itsnegatively charged nanoparticles (Moxi-ANP) and its positivelycharged nanoparticles (Moxi-CNP) following topical administra-tion were evaluated.

2. Experimental

2.1. Chemicals

Gift sample of moxifloxacin was provided by Ranbaxy Labora-tories Ltd. (Gurgaon, Haryana, India). Acetonitrile of HPLC gradewas obtained from Qualigens Fine Chemicals (Mumbai, India) andwater was produced in the laboratory by a Milli-Q purification sys-

G.K. Jain et al. / Journal of Pharmaceutical and

Table 1Gradient elution programme for the analysis of moxifloxacin.

S. no. Time (min) Flow rate ml/min A (%) B (%) Curve

1 Initial 0.40 80.0 20.0 62 0.50 0.40 80.0 20.0 63 1.00 0.40 70.0 30.0 64 2.00 0.40 70.0 30.0 6

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: 0.1% TFA in water; B: acetonitrile.

em (Millipore, Billerica, MA, USA). Trifluoroacetic acid (TFA) ofhrom Lichro grade was purchased from Merck Ltd. (Worli, Mum-ai, India). All other reagents used were of analytical grade.

.2. Chromatography

UHPLC analysis was performed on a Waters Acquity UHPLC sys-em (Milford, MA, USA) equipped with a binary solvent manager,n auto sampler, column manager composed of a column oven, are-column heater and a photo diode array detector. Five microliterf final analytical solution was injected into a Waters Acquity BEH18 (50 mm × 2.1 mm, 1.7 �m) UHPLC column kept at 50 ◦C andhe chromatographic separation was performed by gradient elutionTable 1). The mobile phase consisting of a mixture of A: 0.1% TFA inater (pH 3.5) and B: acetonitrile, with the flow rate of 0.4 ml/minas employed. The analysis was performed at 296 nm wavelengthith total run time of 4 min. Data acquisition, data handling and

nstrument control were performed by Empower Software v1.0.

.3. Sample preparation

A 50 �l aliquot of rabbit aqueous humor was pipetted into a.0 ml Eppendorf tube and 100 �l of acetonitrile was added. Theamples were vortex mixed for 2 min followed by filtration through.22 �m nylon filter and 5 �l of filtrate was directly injected into theHPLC system. All the rabbit aqueous humor samples were storedt −20 ◦C and were allowed to thaw at room temperature prior toample preparation.

.4. Calibration

A stock solution of moxifloxacin (1.0 �g/ml) was prepared byissolving an appropriate amount of moxifloxacin in acetonitrile.orking standard solutions of moxifloxacin were prepared daily by

ilution of stock solution with acetonitrile. To prepare the aqueousumor calibration standards, aliquots of 50 �l of aqueous humorere placed in each Eppendorf tube and spiked with increasing

oncentrations of working standard solutions to give moxifloxacinoncentrations of 10, 20, 100, 200, 400, 600, 800 and 1000 ng/ml.alibration standards were processed according to sample prepa-ation procedure and were analyzed by UHPLC method.

.5. Validation and stability

The UHPLC method was validated in terms of linearity,pecificity, sensitivity, precision, accuracy, system suitability andobustness [18]. The stability of moxifloxacin in aqueous humor atand 20 ◦C and after freeze–thaw cycles was also determined.

.6. Preparation of moxifloxacin nanoparticles

For the preparation of cationic nanoparticles (Moxi-CNP) theouble emulsification solvent diffusion method was used [19].riefly, chitosan (0.1%, w/v) was dissolved in 50 ml of acetic aciduffer, pH 4.4, which also contained polyvinyl alcohol (1%, w/v). Pri-ary w/o emulsion formed by dropwise addition and subsequent

Biomedical Analysis 52 (2010) 110–113 111

stirring of 1 ml of aqueous solution of moxifloxacin (1.0%, w/v) into10 ml of dichloromethane containing poly (lactide-co-glycolide)(1.0%, w/v) was poured into the chitosan aqueous solution. Theemulsion was stirred at 1500 rpm till complete evaporation ofdichloromethane. The nanosuspension so formed is passed throughthree cycles of homogenization at 10,000 bar pressure. Afterparticle formation the entire dispersed system was centrifuged(10,000 rpm, 15 min) and the sediment was resuspended in Milli-Qwater. This process was repeated and the resultant dispersion wassubjected to freeze–drying. Anionic Moxi-ANP particles (with nochitosan) were also prepared for comparison. The mean hydrody-namic diameter and zeta potential of Moxi-ANP and Moxi-CNP, asmeasured using Malvern Zetasizer Nano-ZS90, were found to be110.5 nm (−23.5 mV) and 120.7 nm (+32.5 mV), respectively.

2.7. Ocular pharmacokinetic study

Three groups, each having seven New Zealand Albino rabbits(2.25 ± 0.25 kg), were used for the ocular study. The protocol wasapproved by Institutional Animal Ethics Committee, Jamia Ham-dard (approval no. 469) and their guidelines were followed. Fortopical instillation, weighed amount of lyophilized Moxi-ANP andMoxi-CNP was dispersed in isotonic buffer (pH 7.2) to form 0.5%moxifloxacin suspension. Moxi-SOL (0.5%) was also prepared inthe same vehicle. Each group received single topical instillationof Moxi-SOL, Moxi-ANP and Moxi-CNP. The formulations wereinstilled in both the eyes and approximately 50 �l of aqueoushumor was collected before instillation of formulations and posttreatment at 0.5, 1, 2, 4, 6 and 12 h. Aqueous humor was collectedfrom one rabbit (both eyes) at each time point. All aqueous humorsamples were collected in pre-labeled eppendorf tubes, sealed andstored at −20 ◦C until UHPLC analysis. The aqueous humor sampleswere prepared as above mentioned.

3. Results and discussion

3.1. Method development

Moxifloxacin exists in solution as cationic, anionic, zwitterionicand neutral forms owing to the presence of two protonation sites,carboxyl and secondary amino piperazinyl group [7]. These neutraland ionic forms of moxifloxacin have significant difference in theirapparent hydrophobicity and thus tend to migrate through columnwith different velocities resulting in poor peak shape, tailing anddecreased sensitivity during HPLC method development [20]. TheHPLC methods described previously for plasma pharmacokineticsof moxifloxacin were based on either pre-column derivatization oron-column focusing or fluorescence detection in order to achievecolumn efficiency, selectivity and sensitivity. The present studyattempts to improve the analyte retention and chromatographicselectivity, using smaller Bridged Ethyl Hybrid column packedwith small sized (1.7 �m) particles employing ultra high pressures.The small sized particles reduce plate height and consequentlyallow the number of theoretical plates to be increased. They alsofavour faster linear velocities. The use of smaller particles there-fore allows reduction of analysis time and improved peak shape.Protein precipitation with acetonitrile and direct injection intochromatographic column enables high recovery of moxifloxacin.Gradient elution with varying compositions of TFA and acetonitrilewas tried to improve the chromatographic separations. TFA acts as

acidic modifier and provides counter ions which affect the moxi-floxacin solvation. The ionic retention of protonated moxifloxacinwith oppositely charged species results in formation of stable ionpairs which improves retention of moxifloxacin. The theoreticalplates, USP tailing factor and retention time obtained for mox-

112 G.K. Jain et al. / Journal of Pharmaceutical and Biomedical Analysis 52 (2010) 110–113

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Table 2Precision and accuracy of UHPLC method.

Nominal amount(ng/ml)

Amount found(ng/ml)

Precision Accuracy (%)

SD SE RSD (%)

Intra-day10 9.73 0.09 0.05 0.92 97.30100 98.57 1.08 0.62 1.09 98.571000 998.29 0.68 0.39 0.07 99.83

Inter-day

ig. 1. UHPLC chromatograms obtained from (a) blank aqueous humor, (b) aqueousumor spiked with moxifloxacin (400 ng/ml) and (c) a rabbit’s aqueous humor, 1 hfter topical instillation of Moxi-CNP; moxifloxacin Rt = 2.10 min.

floxacin in aqueous humor matrix showed improvement whenolumn was kept at a temperature of 50 ◦C.

.2. Method validation

.2.1. LinearityThe linearity of the detector response for the moxifloxacin

as evaluated by injecting a total of eight calibration (work-ng) standard solutions (10–1000 ng/ml) covering the workingange of the assay. The calibration curves were constructed bylotting peak area of moxifloxacin against corresponding concen-rations. The correlation coefficient for the calibration regressionine was 0.9997 whereas the equation of calibration curve was= (903.29 ± 3.42)x + (7200.7 ± 17.2) where, x is the concentrationf moxifloxacin in aqueous humor, and y is the peak area of moxi-oxacin. Standard errors for slope and intercept were 1.4 and 7.0%,espectively.

.2.2. SelectivitySelectivity was demonstrated by the ability to assess unequiv-

cally the analyte in the presence of endogenous matrixonstituents. UHPLC chromatograms of blank aqueous humorFig. 1a), aqueous humor spiked with moxifloxacin (Fig. 1b) and

10 9.83 0.10 0.06 1.02 98.30100 98.02 1.69 0.98 1.72 98.021000 999.86 1.66 0.96 0.17 99.99

a rabbit’s aqueous humor after topical instillation of Moxi-CNP(Fig. 1c) were compared to show the selectivity of the proposedprocedure. The retention time of moxifloxacin was 2.10 min and nointerference was observed either by matrix or by formulation ingre-dient, near the retention time, demonstrating method’s selectivity(Fig. 1).

3.2.3. Precision and accuracyPrecision and accuracy were performed by triplicate analysis

of aqueous humor samples spiked with moxifloxacin at concen-trations of 10, 100 and 1000 ng/ml followed by their comparisonwith the calibration curves prepared on the same day and on threedifferent days. Precision was expressed as the percentage relativestandard deviation of measured concentrations for each calibra-tion level, whereas accuracy was expressed as percent recovery[amount found/nominal amount × 100] of drug added to the blankaqueous humor. Table 2 summarizes the results of intra and inter-day precision and accuracy of the moxifloxacin assay.

3.2.4. Detection limits (DL) and quantitation limits (QL)DL and QL were experimentally estimated by analysis of aque-

ous humor samples spiked with serially diluted moxifloxacinstandard until the signal-to-noise ratio reached 3 and 10, respec-tively. DL and QL were found to be 0.75 ng/ml and 2.5 ng/ml,respectively. The present method has a 13-times higher sensitivitythan that reported in previous assay for moxifloxacin determina-tion in humor samples [17].

3.2.5. System suitabilitySystem suitability was determined by six replicate injections at

a concentration of 200 ng/ml. The results passed all the commonUSP acceptance criteria (Table 3).

3.2.6. RobustnessThe low values of % RSD (≤1.74) and SE (<1) obtained after intro-

ducing small deliberate changes in the developed UHPLC methodindicated the robustness of the method.

3.3. Stability studies

Moxifloxacin was found to be stable in aqueous humor at 20 ◦Cfor at least 24 h and at 4 ◦C for 2 days with average recovery of 95.7and 97.6%, respectively. The freeze–thaw data indicated that threecycles can be tolerated without losses greater than 10%. Determi-nation of the stock solutions stability in mobile phase revealed nosignificant losses for at least 5 days at 20 ◦C.

3.4. Ocular pharmacokinetic study

The UHPLC method was successfully used to quantify mox-ifloxacin in aqueous humor samples collected following topical

G.K. Jain et al. / Journal of Pharmaceutical and

Table 3System suitability for moxifloxacin in aqueous humor.

Injectiona Rt (min) Peak area USP tailing

1 2.10 187,859 1.12 2.10 191,356 1.13 2.10 186,782 1.14 2.10 189,580 1.15 2.10 187,466 1.16 2.10 187,598 1.1

Mean 2.10 188,440 1.1% RSD 0.0 0.9 0.0

a Replicate injections of 200 ng/ml moxifloxacin spiked aqueous humor.

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ig. 2. Aqueous humor concentration–time profile of moxifloxacin after topicalnstillation of Moxi-SOL, Moxi-ANP and Moxi-CNP to rabbit eyes.

nstillation of Moxi-SOL, Moxi-ANP and Moxi-CNP to rabbit eye. Theesulting moxifloxacin concentrations measured in aqueous humorollected at 0.5, 1, 2, 4, 6 and 12 h are shown in Fig. 2. In the groupreated with the Moxi-SOL and Moxi-ANP the aqueous humor lev-ls of drug were undetectable after 4 h, attributed to their rapidrecorneal loss. In contrast, drug was detected in aqueous humoror at least 12 h (214 ± 19.09 ng) following Moxi-CNP. A fourfoldncrease in the relative bioavailability was observed with the Moxi-NP (AUC0→t, 3.14 �g h/ml) compared with Moxi-SOL (AUC0→t,.79 �g h/ml) and Moxi-ANP (AUC0→t, 0.72 �g h/ml) formulation.he results indicate that the positively charged Moxi-CNP interactsith negatively charged cornea and conjunctiva prolonging ocular

esidence time and thus maintaining prolonged transcorneal drugoncentration gradient. It could be attributed to the presence ofhitosan which has ability to enhance the paracellular drugs trans-ort by opening intracellular tight junctions of cornea. The resultsemonstrate that positively charged nanoparticles increases thecular bioavailability of moxifloxacin compared to moxifloxacinolution or negatively charged nanoparticles.

. Conclusion

A novel UV-UHPLC method having high reproducibility and sen-itivity for the determination of moxifloxacin in rabbit aqueousumor was developed in this study. The advantages of our methodre the short analysis time (4 min), high sensitivity (QL: 2.5 ng/ml)

nd a simple sample extraction. Our laboratory is actually involvedn development of nano-formulations for ocular delivery of anti-nfectives. The established method provides a reliable bioanalytical

ethodology for moxifloxacin pharmacokinetics in rabbit aqueousumor.

[

Biomedical Analysis 52 (2010) 110–113 113

Acknowledgements

The author would like to acknowledge ACQUITY program teamat Waters India Pvt Ltd., particularly Mr. Prakash Chander, Mr. Tijen-der Sharma and Mr. D.P. Joshi for the scientific support. We wouldlike to thank Mr. Ravi Shankar Prasad, Ph.D. Candidate, College ofPharmacy and Nutrition, University of Saskatchewan, Canada, forhis suggestions during pharmacokinetic studies.

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[6] H.A. Nguyen, J. Grellet, B.B. Ba, C. Quentin, M.C. Saux, Simultaneous deter-mination of levofloxacin, gatifloxacin and moxifloxacin in serum by liquidchromatography with column switching, J. Chromatogr. B 810 (2004) 77–83.

[7] A.L. Djurdjevic, M.J. Stankov, P. Djurdjevic, Optimization and validation of thedirect HPLC method for the determination of moxifloxacin in plasma, J. Chro-matogr. B 844 (2006) 104–111.

[8] S. Schulte, T. Ackermann, N. Bertram, T. Sauerbruch, W.D. Paar, Determina-tion of the newer quinolones levofloxacin and moxifloxacin in plasma byhigh-performance liquid chromatography with fluorescence detection, J. Chro-matogr. Sci. 44 (2006) 205–208.

[9] A.K.H. Kumar, G. Ramachandran, Simple and rapid liquid chromatographymethod for determination of moxifloxacin in plasma, J. Chromatogr. B 877(2009) 1205–1208.

10] J.D. Smet, K. Boussery, K. Colpaert, P.D. Sutter, P.D. Paepe, J. Decruyenaere,J. Bocxlaer, Pharmacokinetics of fluoroquinolones in critical care patients: abio-analytical HPLC method for the simultaneous quantification of ofloxacin,ciprofloxacin and moxifloxacin in human plasma, J. Chromatogr. B 877 (2009)961–967.

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12] T. Lemoine, D. Breilh, D. Ducint, J. Dubrez, J. Jougon, J.F. Velly, M.C. Saux,Determination of moxifloxacin (BAY 12-8039) in plasma and lung tissue byhigh-performance liquid chromatography with ultraviolet detection using afully automated extraction method with a new polymeric cartridge, J. Chro-matogr. B 742 (2000) 247–254.

13] N. Srinivas, L. Narasu, B.P. Shankar, R. Mullangi, Development and validationof a HPLC method for simultaneous quantitation of gatifloxacin, sparfloxacinand moxifloxacin using levofloxacin as internal standard in human plasma:application to a clinical pharmacokinetic study, Biomed. Chromatogr. 22 (2008)1288–1295.

14] L. Baietto, A. D’Avolio, F.G. De Rosa, S. Garazzino, S. Patanella, M. Siccardi, M.Sciandra, G. Di Perri, Simultaneous quantification of linezolid, rifampicin, lev-ofloxacin, and moxifloxacin in human plasma using high-performance liquidchromatography with UV, Ther. Drug Monit. 31 (2009) 104–109.

15] N. Erk, Voltammetric behaviour and determination of moxifloxacin in phar-maceutical products and human plasma, Anal. Bioanal. Chem. 378 (2004)1351–1356.

16] J.G. Moller, H. Stass, R. Heinig, G. Blaschke, Capillary electrophoresis with laser-induced fluorescence: a routine method to determine moxifloxacin in humanbody fluids in very small sample volumes, J. Chromatogr. B 716 (1998) 325–334.

17] K.P. Chan, K.O. Chu, W.W. Lai, K.W. Choy, C.C. Wang, D.S. Lam, C.P. Pang,Determination of ofloxacin and moxifloxacin and their penetration in humanaqueous and vitreous humor by using high-performance liquid chromatogra-phy fluorescence detection, Anal. Biochem. 353 (2006) 30–36.

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Colloids and Surfaces B: Biointerfaces 82 (2011) 397–403

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

journa l homepage: www.e lsev ier .com/ locate /co lsur fb

icroscopic and spectroscopic evaluation of novel PLGA–chitosan Nanoplexes asn ocular delivery system

aurav K. Jaina,∗, Shadab A. Pathana, Sohail Akhtera, Nirmal Jayabalanb, Sushma Talegaonkara,oop K. Khara, Farhan J. Ahmada

Department of Pharmaceutics, F/O Pharmacy, Hamdard University, Hamdard Nagar, New Delhi 110062, IndiaDepartment of Ocular Pharmacology and Pharmacy, Dr Rajender Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Science, New Delhi 110029, India

r t i c l e i n f o

rticle history:eceived 2 June 2010eceived in revised form 15 August 2010ccepted 13 September 2010vailable online 18 September 2010

eywords:

a b s t r a c t

The interaction of PLGA–chitosan Nanoplexes with ocular mucosa was investigated ex vivo and in vivoto assess their potential as ocular delivery system. Fluorescent Rhodamine Nanoplexes (Rd-Nanoplexes)were prepared by ionotropic gelation method. The size and morphology of Nanoplexes was investigatedby TEM, SEM and PCS. The corneal retention, uptake and penetration of Nanoplexes were analyzed byspectrofluorimetry and confocal microscopy. Corneas from Rd-Nanoplexes-treated rabbits were eval-uated for the in vivo uptake and ocular tolerance. The Nanoplexes prepared were round with a mean

onfocal microscopyhitosananoplexescular drug deliveryLGA

diameter of 115.6 ± 17 nm and the encapsulation efficiency of Rd was 59.4 ± 2.5%. Data from ex vivo andin vivo studies showed that the amounts of Rd in the cornea were significantly higher for Nanoplexesthan for a control Rd solution, these amounts being fairly constant for up to 24 h. Confocal microscopyof the corneas revealed paracellular and transcellular uptake of the Nanoplexes. The uptake mechanismpostulated was adsorptive-mediated endocytosis and opening of the tight junctions between epithelial

icroonstr

cells. No alteration was mtogether, these data dem

. Introduction

Topical instillation represents the most convenient route of ocu-ar drug delivery. However, this route is impeded by poor ocularioavailability (<5%), mainly attributed to low corneal permeabil-

ty of drugs, tear turn over, and drug elimination via conjunctivand sclera [1]. Efforts to enhance ocular bioavailability from topicalnstillation have been accomplished either using prodrug design,ermeation enhancing formulations, longer residence formula-ions or nano-sized formulations [2–4]. Nano-sized formulationsave been evaluated as ocular drug delivery systems to enhancehe absorption of therapeutic drugs, improve bioavailability, reduceystemic side effects, and sustain intraocular drug levels [5–17].he use of polymeric nanoparticles is an attractive strategy tonhance the ocular bioavailability of topically administered drugsecause they offer unique features while preserving the ease ofelivery in liquid form [9–17]. Polymeric nanoparticles have been

tilized to improve the corneal and conjunctival penetration ofherapeutic drugs and peptides, sustain drug levels and reduceystemic side effects [9–17]. PLGA, a copolymer of poly (d,l-lactide-o-glycolide), is an ideal biodegradable polymer for nanoparticle

∗ Corresponding author. Tel.: +91 09 811127909; fax: +91 11 26059663.E-mail address: drgkjain@gmail.com (G.K. Jain).

927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2010.09.010

scopically observed after ocular surface exposure to Nanoplexes. Takenate that Nanoplexes are potentially useful as ocular drug carriers.

© 2010 Elsevier B.V. All rights reserved.

formulation due to its biocompatibility, safety, regulatory approvaland wide use [12–17]. PLGA nanoparticles are well tolerated inanimal models and their potentialities in ophthalmology are welldocumented [12–17]. In fact, several experiments have shownthat PLGA nanoparticle-entrapped drugs have improved ocularbioavailability [15–17]. However, the short residence time of thesenanosystems represents a limitation in their therapeutic use [18].On the other hand, the potential of chitosan for topical oculardrug delivery is particularly promising due to its unique proper-ties such as mucoadhesion, tolerability, biodegradability and abilityto enhance the paracellular transport of drugs [19–22]. Chitosannanoparticles and nanocapsules have shown interesting propertiesand promising results as drug carriers across the ocular mucosa.Although chitosan nanoparticles reside on the ocular surface fora prolonged period, they lack proper control over drug releaseowing to rapid drug diffusion. Some previous reports demonstratedimproved performance with chitosan-coated nanocapsules [23].

However, to achieve therapeutic concentrations in eye thedosage form must reside in the cul-de-sac for prolonged period andthe entrapped drug moiety should be released from the nanopar-

ticles at an appropriate rate. Consequently, the purpose of ourwork is to design nanoparticulate system consisting of complexesof release-controlling anionic polymer – PLGA and mucoadhe-sive cationic polymer – chitosan. Our hypothesis was that thisPLGA–chitosan nanocomplex (Nanoplex), which presumably could

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98 G.K. Jain et al. / Colloids and Surfa

ombine the properties of PLGA and chitosan, would be easilypplied as eye-drops, well tolerated by the ocular mucosa, and ableo overcome the ocular mucosal barrier.

In the present study, we used microscopic and spectro-copic techniques to evaluate Rhodamine-loaded NanoplexesRd-Nanoplexes) that can be used topically for ocular deliv-ry. Nanoplexes were characterized by transmission electronicroscopy (TEM), scanning electron microscopy (SEM) and pho-

on correlation spectroscopy (PCS). Retention of Nanoplexes in theornea was quantified by spectrofluorimetric determination of Rd.orneal retention, uptake and penetration-enhancing propertiesere qualitatively evaluated ex vivo and in vivo by confocal laser

canning microscopy (CLSM). Ex vivo and the in vivo tolerance ofhese Nanoplexes was also evaluated by means of microscopy.

. Materials and methods

.1. Materials

PLGA (Resomer® 503 H; d,l-lactide:glycolide ratio of 50:50;3 kDa) was purchased from Boehringer Ingelheim (Ingelheim,ermany). Chitosan (with a deacetylation degree >80%) was

eceived as a gift sample from India Sea Foods (Kerala, India). Rd wasbtained from Sigma–Aldrich (St. Louis, MO, USA). Water was pro-uced in the laboratory by a Milli-Q purification system (Millipore,illerica, MA, USA). All other chemicals and reagents used weref analytical grade and were purchased from Merck Ltd. (Mumbai,ndia).

.2. Rd-Nanoplex preparation

Rd-Nanoplexes were prepared by a slight modification of theonotropic gelation technique [24,25]. Briefly, 20 mg of PLGA poly-

er was dissolved in 2 mL of dichloromethane. 10 mL of a 2 mg/mLolution of Rd was added to the polymer/solvent mixture. Sub-equently, 10 mL of 0.5 M acetate buffer, pH 4.4, consisting of aixture of 0.25% (w/v) chitosan and 1% (w/v) PVA, was prepared

eparately. The Rd/PLGA mixture was added dropwise to the chi-osan/PVA solution under continuous stirring at 1800 rpm using

agnetic stirrer (REMI, India). The stirring was continued for 5 ho allow for dichloromethane evaporation. Colloidal suspensionhus formed was passed through a high pressure homogenizerEmulsiFlex-C5, Avestin Inc., Canada). The suspension was pro-essed for 3 cycles at 10,000 psi to obtain Rd-Nanoplexes of sizeuitable for ocular delivery. Rd-Nanoplexes were collected by aentrifugation, washed 3 times with Milli-Q water, and finallyesuspended in 4 mL of Milli-Q water and dried in a lyophilizerHeto DRYWINNER, Germany).

.3. Rd-Nanoplex characterization

.3.1. Rd-Nanoplex encapsulation efficiency and release profileFluorescence spectroscopy was used to evaluate the actual

mount of Rd encapsulated in the Nanoplexes and its release pro-le. Calibration curve at known concentrations of the dye wasrepared that allowed for quantification of the percent loading ofhe dye and the release profile of Rd from the Rd-Nanoplexes. Toetermine the encapsulation efficiency, a known quantity of Rd-anoplex was dissolved in 1 mL of dimethyl sulfoxide. The sampleas centrifuged at 10,000 rpm for 5 min and the supernatant was

nalyzed. Release was characterized by suspending a known quan-

ity of Rd-Nanoplex in phosphate buffered saline (PBS) enclosed indialysis bag (MW cut off 12 kDa, Sigma–Aldrich, USA). The dialysisag was immersed in a beaker containing PBS at 37 ◦C under mildgitation. Samples were collected from beaker at designated timeoints and analyzed. Both the analysis were done in triplicate.

Biointerfaces 82 (2011) 397–403

2.3.2. Rd-Nanoplex size and morphologyThe mean particle size and zeta potential of the Rd-Nanoplexes

was determined by PCS using a Zetasizer Nano ZS (Malvern Instru-ments, Malvern, UK). For size determination, sample suspensionwas diluted to the appropriate concentration with Milli-Q water(pH 5.8) and analysis was performed at 25 ◦C with a detection angleof 90◦. The analysis was done in triplicate. Zeta potential mea-surements were performed at 25 ◦C in a disposable capillary cell.Samples were measured in triplicate with 20 sub-runs howeverthe equipment was set by default at 100 sub-runs. The morpholog-ical examination of the Rd-Nanoplexes was performed by TEM andSEM. Rd-Nanoplexes treated on copper grids (Polysciences, War-rington, PA) with 1% uranyl acetate for negative staining, followedby sample drying, were analyzed by TEM (Philips CM 10, Holland)at an accelerating voltage of 100 kV. Data acquisition was done onthe AMT Image Capture Engine. For SEM analysis, Rd-Nanoplexeswere fixed to aluminum sample stubs with double-sided carbonadhesive tape and sputter-coated with conductive gold–palladium.They were viewed with an EVO LS 10 scanning electron microscope(Zeiss, Carl Zeiss Inc., Germany) operating at an accelerating voltageof 13.52 kV under high vacuum.

2.4. Ex vivo evaluation of Rd-Nanoplexes

Ex vivo studies were performed using a self-designed OcuFlowapparatus (designed to differentiate between non-mucoadhesiveand mucoadhesive formulations) on corneas isolated from goateyes obtained from freshly slaughtered animals at a local abattoir.5 mL of bicarbonate buffered Ringer’s solution, preadjusted to pH7.4, was placed in both compartments thermostated at 37 ◦C andthe system was maintained for 15 min to stabilize the corneal tis-sue. The buffer of the donor compartment was then substitutedwith 1 mL aqueous suspension of Rd-Nanoplexes (Rd equivalent to2 �g/mL) or the control Rd solution. The contact of the free or encap-sulated Rd with the corneas was maintained for 1, 4, 8, 12 and 24 h.Ocular retention of formulation was quantitatively evaluated byspectrofluorimetric analysis of Rd. Before analysis, the goat corneaswere removed from the perfusion cell, transferred to the test tubeswhere they were digested using tissue homogenizer. The Rd wasextracted using butanol as an extraction solvent 3 times, followedby centrifugation at 15,000 rpm for 15 min and fluorescence wasthen measured. The studies were performed in triplicate. In orderto evaluate the retention, uptake and penetration capacity of Rd-Nanoplexes into the corneal epithelium, corneal specimens fromex vivo perfusion experiments were directly mounted, epithelialside up, on a glass slide and examined without further tissue pro-cessing by CLSM (Olympus FluoView FV 1000, Hamburg, Germany).Samples were excited with green helium neon 543 nm laser beam.Images were taken employing a 20× oil objective, assembled in anintegral image processor and displayed on a digital video moni-tor. To confirm the penetration of Rd-Nanoplexes, stacks of serial4.4 �m optical sections were captured along the Z-axis.

To evaluate the irritation potential, fresh goat corneas wereincubated with Nanoplexes suspension. At stipulated incubationtime, the cornea was removed, washed with PBS, and immediatelyfixed with 8% (w/w) formalin solution. The tissue was dehydratedwith an alcohol gradient, put in melted paraffin and solidified inblock form. Cross-sections were cut, stained with haematoxylineand eosine, and microscopically observed for modifications.

2.5. In vivo evaluation of Rd-Nanoplexes

Five groups, each having three New Zealand Albino rabbits(2.25 ± 0.25 kg), were used for the in vivo study. The protocolwas approved by the Institutional Animal Ethics Committee, JamiaHamdard (approval no. 469), and the Association for Research in

G.K. Jain et al. / Colloids and Surfaces B: Biointerfaces 82 (2011) 397–403 399

) Tran

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formation was kept at pH 4.4. Lower pH values were not tried sinceit is known that PLGA undergoes degradation at low pH values,while at higher pH values Nanoplexes were not formed due to pHdependent solubility of chitosan. Particle size analysis of the opti-

Fig. 1. Representative electron micrographs of Rd-Nanoplexes. (A

ision and Ophthalmology (ARVO) guidelines for usage of ani-als for ophthalmic use were followed. A weighed amount of

yophilized Rd-Nanoplexes was dispersed in PBS and administeredo the cul-de-sac of conscious rabbits in order to quantify their inivo interaction with the cornea. Aqueous solution of Rd as controlormulation was also administered. The animals were maintainedn an upright position using restraining boxes. They were then sac-ificed at 1, 2, 4, 8, and 24 h after the instillation of Rd-Nanoplexessing an overdose of sodium pentobarbital given through intra-enous route. The eyes were enucleated and corneal specimens,reshly excised, were observed for Rd-Nanoplexes retention, pen-tration and irritation potential as described in Section 2.4.

.6. Statistical analysis

Data were expressed as mean ± standard deviation (SD). Thetatistical significance of the differences between Nanoplexes andontrols at each time point was analyzed by paired t-test (Sig-aStat program; Jandel Scientific, Version 1.0). Differences were

onsidered to be significant when p < 0.05.

. Results and discussion

.1. Rd-Nanoplexes preparation and characterization

Our aim was to evaluate PLGA–chitosan Nanoplexes as ocularelivery systems by fluorescence spectroscopy and CLSM. In ourttempt to evaluate the interaction of PLGA–chitosan Nanoplexesith the ocular surface, the first step was the development of Rd-

oaded PLGA–chitosan Nanoplexes. Influence of type of organichase solvent, PLGA concentration in the organic phase, chitosannd PVA concentration in the aqueous phase and the aqueous phaseH on Rd-Nanoplexes size and encapsulation efficiency was eval-ated and optimized. Nanoplexes formed with dichloromethaneDCM) as solvent had lower particle size compared to that formedith ethyl acetate (EA). This was attributed to low thermodynamic

uality of DCM for PLGA and subsequently low viscosity of PLGA-CM solution. Further, low encapsulation efficiency of Rd wasbserved with EA, which favours Rd partitioning from inner aque-us phase to the organic phase, owing to higher water solubility ofA (8.7 wt%) compared to DCM (1.32 wt%).

The size of the Nanoplex system depends upon the net shear

tress available for droplet breakdown. Increasing, either the PLGAoncentration above 1% in the organic phase or chitosan concen-ration above 0.25% in the aqueous phase resulted in increasedanoplex size. Increased polymer concentration led to increasediscous forces which resist droplet break down. At high PLGA con-

smission electron micrograph; (B) scanning electron micrograph.

centrations or at relatively low PVA concentrations, the amountof PVA is insufficient to stabilize the emulsion droplets and thusresulted in bimodal distributions. Nanoplexes with minimum par-ticle size were obtained when PVA was used at a concentrationof 1%. Above 1% PVA concentration, the viscosity of the aqueousphase was increased and the net shear stress available for dropletbreakdown was reduced. The aqueous phase pH during Nanoplex

Fig. 2. Rhodamine (Rd) amount transported across the cornea after the instillationof Rd-Nanoplexes (�) and Rd-solution (�) during (A) ex vivo studies (n = 3); (B) invivo studies (n = 3).

400 G.K. Jain et al. / Colloids and Surfaces B: Biointerfaces 82 (2011) 397–403

F ake ofe cells (r the a

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ig. 3. Confocal images of goat corneal epithelium (ex vivo studies) showing uptpithelium at depths of 20 and 60 �m are provided. Nanoplexes are seen within theeferences to color in this figure legend, the reader is referred to the web version of

ized formulation revealed an average diameter of 115.6 ± 17 nmith a zeta potential of +32.5 mV. Representative TEM (Fig. 1A)

nd SEM (Fig. 1B) micrographs illustrated that Nanoplexes had aharacteristic round shape and were monodisperse. Encapsulationfficiency of Rd was 59.4% (w/w). The release of Rd from the Rd-anoplexes was sustained over a period of 48 h, and thus it is andequate fluorescent marker.

.2. Corneal retention of Nanoplexes – quantitative evaluation

In order to estimate the corneal retention of the Nanoplexes,e evaluated the Rd content in cornea at different time pointsost-incubation. Fig. 2 shows the concentration of Rd in the corneaollowing instillation of the Rd-Nanoplexes and Rd solution as con-rol. These results showed that the behaviour of the Rd-Nanoplexesas remarkably different from that of Rd solution. Rd-Nanoplexesrovided greater concentrations of Rd than Rd solution. The dif-erences in Rd concentrations for the Rd-Nanoplexes and the Rdolution were statistically significant at all time points (p < 0.05).

he results (Fig. 2) also indicated that following instillation of thed-Nanoplexes, the concentration of Rd in cornea remained fairlyonstant for up to 24 h. The reason for this could be due to theechanism of interaction of PLGA and chitosan with the corneal

pithelium. The proposed mechanism of interaction of PLGA with

Rd-Nanoplexes after 1 and 24 h of incubation. Colour overlay images of cornealyellow circles) and in between the cells (yellow squares). (For interpretation of the

rticle.)

the cornea is adsorptive-mediated endocytosis [26,27] and that forchitosan is electrostatic interaction and mucoadhesion [28–30], allbeing susceptible to saturation. Further, in the non-physiologicalex vivo study there was no reconstruction of the mucin layer andit is possible to assume a saturation of interaction sites for Rd-Nanoplexes. In contrast, the levels associated with the Rd solutiondecreased gradually with time. In other words, the Nanoplexes hadbetter retention and more persistent interaction with the ocularsurface compared to solution. The prolonged ocular retention ofthe Nanoplexes compared to solution is in good agreement witha previous work that showed prolonged corneal retention of col-loidal particles [5]. The results obtained ex vivo (Fig. 2A) were quitesimilar to those obtained in vivo (Fig. 2B). Further, the difference inRd content observed ex vivo and in vivo might be due to interplay ofa number of factors including difference in dose applied, tear turnover, blinking latency and pressure applied by the eyelids.

3.3. Corneal uptake and penetration of Nanoplexes – qualitativeevaluation

In order to elucidate the disposition of Nanoplexes in the cornea,we examined cross-sections of the cornea by CLSM. The confocalimages of different cross-sections of the goat cornea (ex vivo study)exposed to the Rd-Nanoplexes are shown in Fig. 3. Qualitative

G.K. Jain et al. / Colloids and Surfaces B: Biointerfaces 82 (2011) 397–403 401

F take oe cells (r the a

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ig. 4. Confocal images of rabbit corneal epithelium (in vivo studies) showing uppithelium at depths of 20 and 60 �m are provided. Nanoplexes are seen within theeferences to color in this figure legend, the reader is referred to the web version of

ssessment of confocal images revealed particle-like round fluo-escent spots located in between and inside the epithelial cells. Inur study, these fluorescent spots are thought to be Rd-Nanoplexes,hereas blurred areas and diffused fluorescence are thought to beue to free Rd released into the medium and subsequent intracellu-

ar uptake of the dye. Experiments in which Rd solution was appliedonfirmed this hypothesis (data not shown). The presence of rounduorescent spots in between as well as inside the cells (Fig. 3)uggested the uptake of Nanoplexes via paracellular (movementetween the cells through ‘leaky’ tight junctions) and transcellularmovement across plasma membrane of the cells) routes. Our find-ngs suggested that the behaviour of Nanoplexes is different fromhat reported previously for chitosan nanoparticles taken up viahe paracellular routes [21], and also from poly(alkylcyanoacrylate)31] and PECL nanoparticles [32] taken up via transcellular route.

Nevertheless, the trans- para-cellular transport of Nanoplexesould be better explained by the intrinsic properties of both PLGAnd chitosan nanoparticles. Previous studies have demonstratedhat PLGA nanoparticles uptake occurs through transcellular mech-nism, while chitosan nanoparticles are preferably transportedy paracellular route. The mechanism postulated was adsorptive-

ediated endocytosis mitigating intracellular localization of PLGA

anoparticles and the ability of chitosan to open the tight junctionsetween epithelial cells to allow paracellular transport. The Rd-anoplexes investigated in this work had a positive zeta-potential

hat could favour the attachment of the nanosystems onto the

f Rd-Nanoplexes after 1 and 24 h of instillation. Colour overlay images of cornealyellow circles) and in between the cells (yellow squares). (For interpretation of the

rticle.)

ocular surface. The positively charged Nanoplexes interact withnegatively charged sites on the cornea and tight junctions, andresult in loosening or opening of the tight junctions owing to alter-ation in the relative concentrations of specific ion species in thepore volume. Thus, the intercellular and intracellular penetrationrather than simple mucoadhesion could explain the prolonged res-idence time (up to 24 h) of the particles on the cornea.

Fig. 3 also shows confocal images observed at 20 and 60 �mdepths of the corneal epithelium. It is not surprising to see that theNanoplexes were capable of gaining access to the deeper corneallayers. The Z-series micrographs confirmed that the Nanoplexeswere deep inside and not simply adsorbed onto the outer cornealsurface (results not shown). The evidence of the paracellular andtranscellular uptake was also confirmed by confocal images of therabbit cornea (in vivo studies) following in vivo administration ofthe Nanoplexes (Fig. 4). Based on the results observed, the proposedpathway for uptake of Nanoplexes is shown in Fig. 5.

3.4. Ocular tolerance of Nanoplexes

Tolerance to Nanoplexes was studied because they crossed

the plasma membrane. Histopathology study on goat cornea (exvivo studies, Fig. 6A) and rabbit cornea (in vivo studies, Fig. 6B)confirmed the presence of normal ocular surface structures, withcells maintaining normal morphology, in both control and treatedeyes. The internalization, as observed for Nanoplexes, could not

402 G.K. Jain et al. / Colloids and Surfaces B: Biointerfaces 82 (2011) 397–403

Fig. 5. Schematic representation of proposed pathway for corneal uptake and retention of Nanoplexes.

Fig. 6. Light microscopy of control and Nanoplexes treated sections of (A) goat corneal tissue (ex vivo study); (B) rabbit corneal tissue (in vivo study). Nanoplex-treated tissuesections showed no alterations in morphological details compared to control eyes.

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G.K. Jain et al. / Colloids and Surfa

e attributed to a disruption of cellular membranes because theanoplexes did not contribute to changes in the epidermal layer.urthermore, the rabbits in control and all other experimentalroups showed no signs of discomfort during the 24 h assay. Takingnto account the adequate ocular tolerance previously reported foroth chitosan and PLGA nanoparticles, our results showed that thessayed Nanoplexes were well tolerated and were non-irritating tohe ocular surface.

. Conclusion

PLGA–chitosan Nanoplexes are able to interact and remain asso-iated with the ocular mucosa for extended period of time, thuseing promising carriers for enhancing and controlling the releasef drugs to the ocular surface. Confocal microscopy offered insightnto the uptake and fate of Nanoplexes, and serve as a baseline forhe design of Nanoplexes intended for improved, targeted ocularelivery.

cknowledgements

The authors are thankful to Ms. Charu Tanwar, Imaging special-st, Advanced Instrumentation Research Facility, Jawaharlal Nehruniversity, New Delhi, India for interpretation of the confocal

mages. Ms. Neha Malik is acknowledged for her inputs with respecto checking of the manuscript.

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lable at ScienceDirect

Polymer Degradation and Stability 95 (2010) 2360e2366

Contents lists avai

Polymer Degradation and Stability

journal homepage: www.elsevier .com/locate/polydegstab

Mechanistic study of hydrolytic erosion and drug release behaviour of PLGAnanoparticles: Influence of chitosan

Gaurav K. Jain a,b,*, Shadab A. Pathan a,b, Sohail Akhter a,b, Niyaz Ahmad b, Nilu Jain a,Sushma Talegaonkar a, Roop K. Khar a,b, Farhan J. Ahmad a,b

aDepartment of Pharmaceutics, F/o Pharmacy, Hamdard University, Hamdard Nagar, New Delhi 110062, IndiabNanomedicine Lab., Pharmacy Research Block, Hamdard University, New Delhi 110062, India

a r t i c l e i n f o

Article history:Received 2 June 2010Received in revised form9 August 2010Accepted 18 August 2010Available online 16 September 2010

Keywords:ErosionChitosanNanoparticlesDrug releasePoly(D,L-lactide-co-glycolide)

* Corresponding author. Department of PharmaceuUniversity, Hamdard Nagar, New Delhi 110062, Indiaþ91 11 26059663.

E-mail address: drgkjain@gmail.com (G.K. Jain).

0141-3910/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.polymdegradstab.2010.08.015

a b s t r a c t

The hydrolytic erosion of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (PLGA-NPs) was investi-gated in vitro. The changes in physical properties of the nanoparticles with time were evaluated by ultrahigh-pressure liquid chromatographic (UHPLC) analysis, particle size analysis and scanning electronmicroscopy (SEM). Mass reduction data demonstrated a triphasic erosion pattern for PLGA-NPs withnearly no mass loss (3.0%) up to a week, followed by a rapid mass loss (weeks 1e3, 61.4%), and furtherfollowed by slow mass loss (weeks 3e5, 19.8%). SEM revealed microcavitation on the surface of nano-particles, which tended to increase with the erosion time and eventually particle fragmentation wasevident at 5 weeks. A significant increase in particle size was observed at 4 weeks which can beattributed to particle aggregation, however, at about 5 weeks, the particle size decreased significantlyowing to particle fragmentation. The hydrolytic erosion of PLGA-NPs was found to be specifically protoncatalyzed. The release profile of the model drug, moxifloxacin, from PLGA-NPs was closely related tonanoparticle erosion except for the initial burst release which was based on diffusion. The presence ofchitosan in the PLGA-NPs accelerated the rate of erosion of the nanoparticles and reduced the burstrelease of the drug. An understanding of the erosion mechanism and alteration in erosion by chitosancould give desirable and more uniform drug release kinetics from PLGA-NPs.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Biodegradable polyesters composed of poly(D,L-lactide-co-gly-colide) (PLGA) have been successfully employed to design drugdelivery systems such as implants [1], scaffolds [2,3], microparti-cles [4] and nanoparticles [5,6]. PLGA-based nanoparticles havebeen extensively used over the past many years since they canoffer a number of advantages for drug delivery purposes includingbiocompatibility, biodegradability, entrapment of a variety oftherapeutic moieties, and their protection from degradation[7e10]. PLGA-based nanoparticulate formulations, designed to beadministered orally, topically, subcutaneously, or transmucosallyhave the advantage of supplying a continuous amount of drug overa long period of time (from days to months). These polymericdevices generally release drugs by diffusion and/or by erosion ofthe polymer, or by a combination of both [11e24]. It is generally

tics, F/o Pharmacy, Hamdard. Tel.: þ91 9811127909; fax:

All rights reserved.

accepted that PLGA undergoes hydrolytic degradation via bulkerosion, leading to the formation of water and carbon dioxidethrough the intermediate lactic acid and glycolic acid monomers(Fig. 1). Since these polymers are designed to degrade, theirerosion behaviours, both in vitro and in vivo, have always beena subject of study of the biomaterials. In most situations, an in vitrostudy is always used, first to screen the candidate materials, fol-lowed by their in vivo studies. Understanding of the in vitrohydrolytic erosion mechanisms will help to design better deliverysystems with the desirable and uniform drug release kinetics.Although there are a few reports on the study of in vitro degra-dation of PLGA including scanning electron microscopy (SEM) [17],X-ray diffraction (XRD) [18], differential scanning calorimetry(DSC) [19], gel permeation chromatography (GPC) [19], and liquidchromatography of degradation products [20], much less is pub-lished on the behaviour of PLGA-NPs. In the present work,hydrolytic erosion of the PLGA-NPs was investigated underconditions selected to mimic physiological milieu. The relation-ships between mass loss, particle size, size distribution, surfaceand cross-section morphologies and erosion time were investi-gated to elucidate PLGA-NPs erosion mechanism. The influence of

Fig. 1. Hydrolytic degradation of PLGA.

G.K. Jain et al. / Polymer Degradation and Stability 95 (2010) 2360e2366 2361

pH of the degrading medium on mass loss measurements was alsocarried out. Further, the influence of addition of chitosan on theerosion of PLGA-NPs and on the release of model drug moxi-floxacin from PLGA-NPs was also investigated.

2. Material and methods

2.1. Materials

Moxifloxacin was received as a gift sample from RanbaxyLaboratories Limited (Gurgaon, India). PLGA (Resomer� 503 H;D,L-lactide:glycolide ratio of 50:50; 33,000 g/mol) was purchasedfrom Boehringer Ingelheim (Ingelheim, Germany). Chitosan (witha deacetylation degree >80%) was received as a gift sample fromIndia Sea Foods (India). LCeMS grade acetonitrile, CHROMASOLV�

and analytical grade ammonium acetate were purchased fromSigma Aldrich (St. Louis, MO, USA). Water was produced in thelaboratory by a Milli-Q purification system (Millipore, Billerica, MA,USA). All other chemicals and reagents used were of analyticalgrade and were purchased from Merck Ltd. (Mumbai, India).

2.2. Nanoparticle preparation

Nanoparticles were prepared using the emulsification solventevaporationmethod [25]. Briefly,100.0mg of PLGAwas dissolved in10 mL of dichloromethane and this solution was added dropwisewith continuous stirring into 50.0 mL of 1.0% (w/v) PVA aqueoussolution. The resultant emulsion was agitated for 6 h at roomtemperature to completely remove dichloromethane. Colloidalsuspension thus formed was passed through high-pressurehomogenizer EmulsiFlex-C5 (Avestin Inc., Ontario, Canada). Thesuspension was processed for 3 cycles at 10,000 psi to obtainnanoparticles of desired size (150.7 nm). PLGA-NPs were collectedby centrifugation at 15,000 rpm for 15 min, washed three timeswith distilled water and freeze-dried. For the preparation ofPLGAechitosan nanoparticles (PLGAeCS-NPs), a solution consistingof chitosan (0.05%e0.25% w/v, in 0.5 M acetate buffer, pH 4.4) andPVA (1.0% w/v, in distilled water) was used as the dispersing phase.The drug loaded PLGA-NPs and/or PLGAeCS-NPs were preparedusing the same technique as mentioned above, except that moxi-floxacin (1.0% w/v) was added to the aqueous phase.

2.3. Ageing of the nanoparticles

Ten milligrams of nanoparticles, enclosed in a dialysis bag(cellulose membrane, MW cut off 12000 kDa, Sigma) containing2.0 mL of phosphate buffered saline (PBS, pH 7.4, internal media),were immersed in 20.0 mL of the same medium (incubationmedium) at 37 �C under mild stirring for various lengths of time. Atthe end of each incubation period, the internal medium wasremoved, and nanoparticles’ mass, size and morphology weredetermined. The incubation mediumwas completely replaced withfresh PBS at stipulated time intervals to minimize pH changes.PLGA-NPs were incubated in PBS with pH adjusted to 3.0, 5.0, 7.4,and 9.0 with either phosphoric acid or sodium hydroxide solutionto determine the influence of pH on erosion while PLGAeCS-NPswere used to determine the influence of chitosan on erosion.

2.4. Nanoparticle erosion studies

2.4.1. Nanoparticle massFor the determination of mass loss from the nanoparticles, the

internal media were removed at predetermined time intervals andnanoparticles were separated by centrifugation (28,000 rpm for1 h) after washing two times with deionisedwater. The supernatantwas discarded and the solid residue was reconstituted with anappropriate volume of acetone and 5.0 mL of this solution wasinjected into the UHPLC system to detect the amount of PLGAremaining.

UHPLC analysis of PLGA was performed on a Waters AcquityUHPLC system (Milford, MA, USA) equipped with a binary solventmanager, an auto sampler, a column manager composed ofa column oven, a pre-column heater and a photo diode arraydetector. Five microlitres of the final analytical solution was injec-ted into Acquity UPLC BEH C8 (100 mm � 2.1 mm, 1.7 mm) columnmaintained at 30 �C. The mobile phase consisting of acetonitrile:1.0 mM ammonium acetate (90:10 v/v) with a flow rate of0.25 mL/minwas employed. The analysis was performed at 220 nmwavelength with a total run time of 2.0 min. The method was foundto be linear (r ¼ 0.9915), accurate (recovery, 99.89e100.16%), andprecise (CV, �15.3%). Quantification limits were found to be0.2 mg/mL. Data acquisition, data handling and instrument controlwere performed by Empower Software v1.0. The erosion rate ofnanoparticles was calculated by subtracting the amount of PLGAremaining from the initial amount of PLGA-NPs taken.

2.4.2. Nanoparticle morphologyFor the determination of the morphology of the nanoparticles,

the samples were removed from the incubation medium at pre-determined time intervals and lyophilized. The samples weremounted on aluminium stubs using double-sided carbon adhesivetape and sputter-coated with conductive goldepalladium. Theywere viewed with an EVO LS 10 scanning electron microscope(Zeiss, Carl Zeiss Inc., Germany) operating at an accelerating voltageof 13.52 kV under high vacuum. The particles were examined forsurface characteristics like shape, size, pores, pits and presence ofaggregation.

2.4.3. Nanoparticle sizeThe nanoparticle size was determined by dynamic light scat-

tering (DLS; Zetasizer Nano ZS, Malvern Instruments, Malvern, UK)at 25 �C at an angle of 90�, taking an average of three measure-ments. The polydispersity index (PI) indicating thewidth of the sizedistribution was also determined.

2.5. Drug release studies

A 10 mg quantity of moxifloxacin-loaded PLGA-NPs andPLGAeCS-NPswere enclosedwithin dialysis bags containing 2.0mLof PBS, pH 7.4, and immersed in 20.0 mL of the same medium ina beaker under mild agitation. At stipulated time points, sampleswere withdrawn from the incubation medium and analyzed usingUHPLC method reported previously by our group [26]. Themeasurements were done in triplicate, and the average and stan-dard deviations were calculated. An identical volume of fresh PBS

Fig. 2. Mass loss of hydrolytically eroded PLGA-NPs plotted as a function of erosiontime.

Fig. 4. Particle size and size distribution of PLGA-NPs as a function of erosion time.

G.K. Jain et al. / Polymer Degradation and Stability 95 (2010) 2360e23662362

was added back into the beaker after each removal. The percentdrug released was determined using a standard curve.

3. Results

3.1. Nanoparticle erosion studies

3.1.1. Nanoparticle massThe nanoparticles were allowed to age at 37 �C in PBS, pH 7.4,

and mass reduction of PLGA-NPs was monitored using UHPLCmethod. As shown in Fig. 2, the PLGA-NPs exhibited a triphasicerosion pattern (initial lag phase, followed by rapid erosion andslow erosion). During the first week (lag phase), only 3.0%mass losswas observed followed by 61.4% mass loss in the weeks 2 and 3(rapid erosion phase). Further during weeks 4 and 5 (slow erosionphase) 19.8% mass loss was observed with a total mass lossreaching 84.2% after 5 weeks.

3.1.2. Nanoparticle morphologySEM micrographs revealed that the PLGA-NPs prepared by

emulsification solvent evaporation method initially exhibited

Fig. 3. SEM micrographs of PLGA-NPs at: (a) 0 day; (b) 1 week; (c)

a spherical geometry with smooth surface (Fig. 3a). No aggregationwas visible between the particles and they did not show anyinternal or external porosity before exposure to the incubationmedium (Fig. 3a). As erosion proceeded at pH 7.4, the formation ofmicrocavities on the surface of nanoparticle was observed at day 5and this increased with time (Fig. 3bed). By 3 weeks, the effect ofincubation medium on the changes in inner morphology of thePLGA-NPs became significant (Fig. 3e). Aggregation of particles wasnoticeable only at 4 week time point. As expected, the nanoparticleporosity increased with erosion time and eventually particle frag-mentation was evident at 5 weeks. The fusion of fragmentedparticles appeared to occur and a large mass rather than particleswas visible after about 6 weeks (micrograph not shown).

3.1.3. Nanoparticle sizeFig. 4 plots the mean particle size of the PLGA-NPs as a function

of erosion time. At the start of the erosion, PLGA-NPs had a meansize of 150.7 � 20.8 nm and a PI of 0.06. Until 3 weeks, only a slightincrease in particle size was observed. At about 4 weeks theincrease in particle size (345.0 � 18.1 nm) became significant. Also,a dramatic increase in PI (0.8) was observed indicating particleaggregation. In contrast, a significant decrease in particle size

2 weeks; (d) 3 weeks; (e) 4 weeks; and (e) 5 weeks of erosion.

Fig. 5. Mass loss of PLGA-NPs at different pH plotted as a function of erosion time(main graph). Insert: Plot of log k as a function of pH.

G.K. Jain et al. / Polymer Degradation and Stability 95 (2010) 2360e2366 2363

(120.4� 50.2 nm) was observed after about 5 weeks and on furtherageing, the Zetasizer was unable to estimate the particle size.

3.2. Influence of pH

Fig. 5 shows the influence of pH of the incubation medium onerosion behaviour of the PLGA-NPs. As seen in Fig. 5, the pH affectsthe rate of erosion of the PLGA-NPs and with decrease in pH of themedium, the nanoparticle erosion rate increased. At acidic condi-tions of pH 3, the PLGA-NPs were completely degraded within 28days. At 28 day time point, in the incubation medium of pH 5, 7.4and 9, the erosion was 84%, 70% and 59.7%, respectively. The logkobs e pH profile (Fig. 5 insert) showed that the hydrolysis wasspecific proton catalyzed at pH values below 9, since the slope inthis part of the curve approached �1.

3.3. Influence of chitosan

As seen in Fig. 6, PLGAeCS-NPs had faster erosion rates atphysiological pH. The rate of mass loss from the nanoparticles alsodepended on the nanoparticle composition, increasing when thechitosan content of the nanoparticles increased. PLGAeCS-NPs

Fig. 6. Influence of chitosan on mass loss of PLGA-NPs plotted as a function of erosiontime.

formulated with 0.05% chitosan underwent complete erosionwithin 14 days, whereas, for PLGAeCS-NPs prepared with 0.25%chitosan, complete erosion was observed within 3 days. The tri-phasic erosion profile often observed with the PLGA-NPs wassmoothed somewhat to a monophasic profile. Further, the overallerosion rate was accelerated.

3.4. Drug release studies

The cumulative percent release of moxifloxacin from PLGA-NPsand PLGAeCS-NPs with 0.05, 0.10, and 0.25% chitosan is plotted inFig. 7. The in vitro drug release from the PLGA-NPs showed a hugeinitial burst of around 44.0% after one day of incubation in PBS(Fig. 7, main graph). It was believed that the huge initial burst wasdue to rapid dissolution of the moxifloxacin loosely adsorbed onthe surface of the nanoparticles. Thereafter, the moxifloxacinrelease pattern related closely to the erosion profile of the polymerindicating that drug release is through polymer erosion (Fig. 7,insert). The presence of chitosan in nanoparticles significantlyreduced the initial drug burst owing to a high drug loading and lowadsorption of the drug on nanoparticle surface. As expected, a fasterdrug release was observed from PLGAeCS-NPs compared to PLGA-NPs. Further, based on erosion of PLGAeCS-NPs, with an increase inchitosan content of the nanoparticles the drug release increased.

4. Discussion

The main objective of the present research was to study themechanism of in vitro erosion of PLGA-based nanoparticles underconditions mimicking the physiological milieu. A UHPLC methodwas used for the quantitative analysis of PLGA remaining after invitro erosion of PLGA-NPs. Although PLGA mass determinationusing gravimetry is a simple technique but was not used in thepresent study since it requires completely dried samples. Drying ofPLGA at high temperature might have resulted in its degradationwhile drying at low temperature might have resulted in unneces-sary contact of PLGA-NPs with incubation medium, thereby intro-ducing error in measurements. Furthermore, the developed UHPLC

Fig. 7. Fractional cumulative release of moxifloxacin from PLGA-NPs and PLGAeCS-NPsin PBS (pH 7.0) at 37 �C. Main graph showing initial burst release from PLGA-NPs andPLGAeCS-NPs; insert showing slow release of moxifloxacin from PLGA-NPs.

G.K. Jain et al. / Polymer Degradation and Stability 95 (2010) 2360e23662364

method for the determination of PLGA was rapid and precise, anddrying was not required for analysis.

Apart from nanoparticles mass reduction, morphology and sizeof the incubated nanoparticles were also analyzed to study theerosion mechanism. The mass loss of PLGA-NPs as a function oferosion time followed a triphasic pattern with nearly no mass lossduring week 1 (lag phase) followed by rapid mass loss duringweeks 2 and 3 and slow mass loss during weeks 4 and 5 (Fig. 2).Although, PLGA-NPs are insoluble in water, they are hydrolyticallyunstable and erode via hydrolysis of ester bonds leading to theproduction of water-soluble oligomers and their correspondingmonomeric units. It can be anticipated that initially in contact withwater, random chain scission would prevail resulting in a decreasein themolecular weight of PLGA. Slow penetration of water into thePLGA-NPs and poor diffusivity of the oligomers out of the nano-particles matrix were responsible for the lag phase observedinitially. The absence of signs of microcavitation upon day 5 verifiedthat mass loss has not occurred during the initial phase. Poordiffusivity of the oligomers and their strong water affinity allowedinflux of water molecules and build-up of osmotic pressure withinthe nanoparticles matrix. Interestingly, during weeks 2 and 3 underthese storage conditions, a dramatic decrease in the PLGA contentwas observed, however, the mean diameter of the nanoparticleswas increased slightly (Fig. 4). This could be explained via anunderstanding of the erosion process of PLGA. The term ‘erosion’ isused to describe mass loss. Depending on the relative rates of waterdiffusion into the polymeric nanoparticles matrix and erosion ofthe polymer, two erosion processes can be distinguished. Whenpolymer erosion is faster than diffusion of water into the matrix,erosion becomes a surface phenomenon. In the opposite case,

Fig. 8. Schematic representation of the mechanis

where diffusion of water into the matrix is faster than polymererosion, erosion becomes bulk phenomenon such that the nano-particle tends to maintain its original size for longer while erodingfrom within. PLGA is known to undergo bulk erosion as waterdiffusion into thematrix is faster than polymer erosion. The built upof osmotic pressure within the nanoparticles matrix, initiated crackpropagation and the formation of microcavities. As ageing went on,these microcavities grew in number and size (Fig. 3bed). Eventu-ally, when the inner cavities became connected to the outer poresand thewater-soluble polymers were small enough, diffusion drovethe fragments to the incubation medium and only then the nano-particles demonstrated a rapid mass loss, i.e., they eroded. A slightincrease in the particle size is attributed to higher swelling of theparticles due to the formation of carboxylic and hydroxyl groups asreported previously [27]. The presence of PVA seemed to preventnanoparticles aggregation until 3 weeks. The possible involvementof PVA in preventing nanoparticle aggregation has been suggestedbefore [27]. However, the particle size and the PI increased rapidly,thereby indicating particle aggregation attributed to a largenumber of hydrophilic end groups. Further, as hydrolytic attackprogressed from the edge towards the glassy core, retardation inerosion has been observed. With further erosion, the matrix was nolonger mechanically stable and the nanoparticles were brittle andfragmented easily. Particle fragmentation was responsible fora significant decrease in the particle size as determined for 5 weektime point. These small-sized particles under went rapid fusion anda large mass rather than particles was visible after about 6 weeks(micrograph not shown). A schematic representation of themechanism of in vitro erosion of PLGA-NPs as studied is depicted inFig. 8.

m of in vitro hydrolytic erosion of PLGA-NPs.

G.K. Jain et al. / Polymer Degradation and Stability 95 (2010) 2360e2366 2365

4.1. Influence of pH

It was speculated that the acidic condition of media catalyzesthe hydrolysis of polymer linkages, which was ascribed to the fastererosion of PLGA-NPs. In other words, non-enzymatic hydrolysis ofPLGA-NPs was specific proton catalyzed and was facilitated byacidic condition of media.

4.2. Influence of chitosan

PLGAeCS-NPs exhibited erosion profiles significantly differentfrom that of PLGA-NPs. The mass loss profiles showed that chitosaninfluenced the rate of hydrolysis of nanoparticles. Chitosan isa hydrophilic polymer which dissolves in water at pH below 5.5.Both these properties of chitosan are assumed to be responsible forfaster nanoparticle erosion following monophasic profile observedfor PLGAeCS-NPs. The presence of hydrophilic chitosan in thenanoparticles allows fast influx of water molecules and build-up ofosmotic pressure within the nanoparticle matrix resulting ina reduction of the lag phase. Further, formation of acidic degrada-tion products decreased the pH of nanoparticle matrix resulting inchitosan dissolution and enhanced nanoparticle erosion. Thepresence of amino groups in chitosan further facilitated the erosionrate of PLGA-NPs. The accelerated in vitro and in vivo erosion ofPLGA in the presence of amino compound, thioridazine, has beenreported by Maulding et al. [28]. Further, the overall erosion ratewas accelerated.

4.3. Drug release studies

Upon immersion of PLGA-NPs into PBS, the drug which wasloosely bound to the surface of the nanoparticles came into contactwith aqueous media and was released into the surroundingmedium resulting in a burst effect (Fig. 7, main graph). However,the time taken for the complete release of moxifloxacin into thesurroundings was dependent upon the rate of hydrolytic erosion ofthe nanoparticles (Fig. 7, insert). After an initial burst release, thelag phase observed was due to a slow influx of water into thenanoparticle matrix. Even though chain scissionwas taking place inthe polymer matrix, no significant loss of material or change in thesurface morphology was observed (Figs. 2 and 3). With furtherimmersion in PBS, the polymeric matrix swelled with further wateruptake. Slowly, cracks, small pores or microcavities were formedwithin the polymeric matrix due to hydrolytic erosion (Fig. 3bed)and the fast release of moxifloxacin was observed in conjunctionwith the onset of mass loss. With further increase in incubationtime, the drug had to migrate from the core to the surface of thepolymeric matrix and was slowly released into the PBS. On theother hand, PLGAeCS-NPs (0.25% chitosan) with the fastest erosionrate had the fastest drug release profile. Advanced erosion of thepolymer matrix owing to the presence of chitosan had createda microporous matrix, which provided additional pathways fordiffusion of drugs trapped in the polymer matrix resulting ina faster and more uniform drug release.

5. Conclusions

The mechanism of in vitro hydrolytic erosion of PLGA-basednanoparticles was investigated. The PLGA-NPs exhibited a triphasicerosion patternwith nearly no erosion during week 1, rapid erosionduring weeks 2 and 3, slow erosion during weeks 4 and 5, followedby loss of integrity of nanoparticles. Except for the initial burstrelease, drug release from the PLGA-NPs was based on polymererosion. The erosion rate of PLGA-NPs was accelerated and theinitial drug burst was significantly reduced by the presence of

chitosan in the nanoparticles. Further, the hydrolytic erosion wasfound to be specifically proton catalyzed.

Acknowledgement

We thank ACQUITY program team at Waters India Pvt. Ltd.,particularly Mr. Prakash Chander, Mr. Tijender Sharma and Mr. D.P.Joshi for their scientific support. Ms. Neha Malik is acknowledgedfor her inputs with respect to checking of the manuscript.

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ACCEPTED UNCORRECTED MANUSCRIPT

Ocular pharmacokinetics of ganciclovir nanoformulations by

ultra pressure liquid chromatographic (UPLC) method

http://mc.manuscriptcentral.com/bmc

Biomedical Chromatography

ACCEPTED MANUSCRIPT

Accepted Date: March 18, 2012Revised Date: March 3, 2012Received Date: November 4, 2011

This is a PDF file of an unedited manuscript that has been accepted for publication.

This manuscript will undergo copyediting, typesetting, and review of the resulting proof before it publish in the final form

ACCEPTED UNCORRECTED MANUSCRIPTOcular Pharmacokinetics of Ganciclovir loaded to nano-systems using UPLC for its determination in rabbits

Sohail Akhtera, c, Mohammed Anwara, c, Prakash Chanderb , Mohammad Zaki Ahmade,

Mohammad A. Siddiquia, Samim Ahmadd, Zeenat Iqbala, Sushma Talegaonkara, Roop K Khara,

Farhan J Ahmada, c*

a Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, Hamdard Nagar, New

Delhi 110062, India

b Waters India Pvt Ltd, CS-08/09, 7th Floor, Lobe 2, Tower A, The Corenthum, Plot No. A-41,

Sector 62, Noida 201301, U.P., India

cNanomedicine Research Lab, Hamdard University, Hamdard Nagar, New Delhi 110062, India

dDepartment of chemistry, Faculty of science, Hamdard University, Hamdard Nagar, New Delhi

110062, India

eDreamz College of Pharmacy, Khilra-Meramesit, Mandi-175036, India

Running heading: Ocular pharmacokinetics of GCV nanoformulations by UPLC

*Corresponding author: Tel: +91-9810720387

E-mail address: farhanja_2000@yahoo.com

Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, Hamdard Nagar, New

Delhi 110062, India.

ACCEPTED UNCORRECTED MANUSCRIPT

2

Abstract

The present report describes a rapid and sensitive ultra pressure liquid chromatography (UPLC)

method to compare ocular pharmacokinetics of ganciclovir (GCV) nanoformulations after single

topical instillation to rabbit eye. Separation of GCV was achieved on a Waters Acquity BEH C18

(50 mm x 2.1 mm, 1.7 µm) column. The mobile phase consisted of 0.1% trifluoroacetic acid

(TFA) in water (pH 3.5) and acetonitrile (95:5, v/v) at a flow rate of 0.45 mL/min. GCV analysis

was performed at a wavelength of 254 nm with total run time of 3 min. Method was found to be

selective, linear (r2 = 0.999), accurate (recovery, 97.0–100.2%) and precise (CV,≤ 3.1%) in the

concentration range of 0.1–1.0 µg/mL. Limit of detection and quantitation of GCV in aqueous

humor were 3.0 and 10.0 ng/mL, respectively. GCV nanocomplexes (AUC0→t, 3797.7±21.8

ng.hr/mL) and GCV niosomal dispersions (AUC0→t, 3428.1±29.4 ng.h/mL) provided

approximately 6 fold increase in the relative ocular bioavailability compared with GCV solution

(AUC0→t, 559.6±14.2 ng.h/mL) and nearly 2.5 fold higher than the GCV nanoparticles (AUC0→t,

1441.9±20.0 ng.h/mL). The results indicate that the nanocomplexes and niosomal dispersions

increases ocular bioavailability of GCV and prolong its residence time in the eye.

Key words: Aqueous humor; Ganciclovir; Nanoformulations; Ocular pharmacokinetics; Ultra

pressure liquid chromatography.

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1. Introduction

Ganciclovir (GCV) is a synthetic acyclic nucleoside analog of 2’-deoxyguanosine, which exhibits

antiviral activity against herpes simplex virus and cytomegalovirus at relatively low inhibitory

concentrations [1, 2]. Therefore, GCV plays an important role in the treatment of ocular viral

infections. Conventional treatment involves oral administration of GCV at a dose of 3.0 g/day.

Such a high dose results in dose-related toxicity including bone marrow suppression and

neutropenia [2]. Compared to systemic therapy, intravitreal injection of GCV provides higher

intraocular drug concentrations but repeated intravitreal injections are poorly tolerated. Topical

ocular delivery of GCV is valuable, but is limited due to poor ocular availability owing to its

hydrophilic character and rapid elimination. Development of GCV nanoformulations for the

treatment of ocular infections is worthwhile since they are expected to prolong the pre-ocular

retention and increase the ocular bioavailability. With growing interest in GCV

nanoformulations, development of a simple and rapid analytical method for studying the ocular

pharmacokinetics of GCV through these novel formulations is of obvious significance. The

quantitation of GCV in biological samples poses an important challenge because these drugs are

structurally similar to endogenous substances. Hence, this complicates their analysis and requires

the use of a highly selective analytical method. Some analytical methods have been proposed for

the analysis of GCV in plasma through high-performance liquid chromatography (HPLC) with

UV detection [3-8], fluorescence detection [9-12] and tandem mass spectrometry [13]. However,

so far no analytical method allows the measurement of GCV in ocular fluids. Here, we describe a

simple but efficient method for the determination of GCV in rabbit aqueous humor using ultra

high-pressure liquid chromatography (UPLC) with UV detection. With small amounts of aqueous

humor, this method reaches the level of selectivity and reproducibility required for

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4

pharmacokinetic studies of GCV in aqueous humor. Furthermore, the method was utilized to

compare the aqueous humor pharmacokinetics of GCV solution and GCV nanoformulations

following its topical administration to rabbit eye.

2. Experimental

2.1. Chemicals

Gift sample of GCV was provided by Ranbaxy Laboratories Ltd. (Gurgaon, Haryana, India).

Poly (lactide-co-glycolide) (PLGA, RES 503H, lactic acid/glycolic acid, 50:50) was purchased

from Boheringer Ingheleim (Germany). Chitosan (CS, deacetylation degree >80%) was received

as a gift sample from India Sea Foods (India). Span 60 and cholesterol were purchased from S.D.

Fine-chem Ltd. (Mumbai, India). Acetonitrile and TFA of HPLC grade were obtained from

Qualigens Fine Chemicals (Mumbai, India). Water was produced in the laboratory by a Milli-Q

purification system (Millipore, Billerica, M.A., U.S.A.). All other reagents used were of

analytical grade.

2.2. Chromatography

UPLC analysis was performed on a Waters Acquity UPLC system (Milford, MA, USA) equipped

with a binary solvent manager, an autosampler, column manager composed of a column oven, a

precolumn heater and a photo diode array detector. Five microliters of the final analytical

solution was injected into a Waters Acquity BEH C18 (50 mm x 2.1 mm, 1.7 µm) UPLC column

kept at 50°C. The mobile phase consisting of a mixture of 0.1% TFA in water (adjusted to pH 3.5

using 5.0% dilute ammonia) and acetonitrile (95:5, v/v) with the flow rate of 0.45 mL/min was

employed. The analysis was performed at a wavelength of 254 nm with total run time of 3 min.

Data acquisition, data handling and instrument control were performed by Empower Software

v1.0.

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2.3. Sample preparation

A 50 µL aliquot of rabbit aqueous humor (blank, standard, control, or animal sample) was

pipetted into a 1.0 mL eppendorf tube, followed by the addition of 100 µL of acetonitrile. The

samples were vortexed for 5 min followed by 5 min of centrifugation at 10,000 rpm. The samples

were filtered through a 0.22 µm nylon filter and 5 µL of the filtrate was directly injected into the

UPLC system. All the rabbit aqueous humor samples were stored at -20°C and allowed to thaw at

room temperature prior to sample preparation.

2.4. Calibration

Standard solutions of GCV were prepared with the mobile phase at a concentration of 100

µg/mL. Working solutions of GCV were prepared daily from the standard solution by diluting the

appropriate aliquot with the mobile phase. To prepare the aqueous humor calibration standards,

aliquots of 50 µL of aqueous humor were placed in each eppendorf tube and spiked with

increasing concentrations of working standard solutions to give GCV concentrations of 0.01, 0.1,

0.2, 0.4, 0.6, 0.8 and 1.0 µg/mL. Calibration standards were processed according to the sample

preparation procedure and analyzed by the UPLC method. The quality control (QC) samples used

in analytical method validation were prepared in the same way as the standard calibration

samples.

2.5. Assay validation

Linearity were investigated by the assay in parallel of triplicate rabbit’s aqueous humor samples

spiked with GCV to concentrations of 0.01,0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 µg/mL. The GCV mean

peak area was plotted against GCV concentration and regression analysis was performed.

Precision was determined by repeat assay (n =6) of aqueous humor samples spiked with 0.1, 0.4

and 0.8 µg/mL GCV and expressed as the percentage coefficient of variation (CV) of peak area.

Accuracy expressed as % recovery was calculated as mean back calculated concentration/

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theoretical concentration ×100. The limit of detection (LOD) and limit of quantification (LOQ) in

aqueous humor were determined based on the signal-to-noise ratios using analytical responses of

three and ten times the background noise, respectively. The extraction efficiency was determined

in triplicate at 0.4 µg/mL for GCV in aqueous humor. The peak areas obtained after extraction

were compared with peak areas resulting from standard solutions at the same concentration.

Recovery studies were carried out by applying the method to GCV nanoformulations to which

known amount of GCV corresponding to 100% of the GCV label claim had been added. The

analysis was done in triplicate. The stability of GCV in rabbit aqueous humor was investigated.

Spiked aqueous humor samples were stored at 20°C, 4°C and −20°C and were analyzed by

UPLC after 24 h, 5 days and 30 days, respectively. For freeze-thaw stability, three aliquots of

sample were stored at –20˚C for 24 h; they were then left to completely thaw at room

temperature. The cycle was repeated three times and the samples were then analyzed by UPLC.

The stability studies were done in triplicate.

2.6. Preparation of GCV nanoformulations

2.6.1. Preparation of chitosan nanoparticles (GCV-NPs)

GCV-NPs were prepared according to the ionotropic gelation method [14]. Briefly, chitosan

(0.2% w/v) was dissolved in acetic acid solution (pH adjusted to 5.5). For the preparation of

drug-loaded nanoparticles, GCV aqueous solutions were added to the chitosan solution. To 2.5

mL of the chitosan solution, 0.8 mL of the aqueous tripolyphosphate (TPP) solution (0.2% w/v)

was added, leading to the formation of GCV-NPs as a result of the interaction between the

negatively-charged phosphate groups of TPP and the positively-charged amino groups of

chitosan.

2.6.2. Preparation of PLGA-chitosan nanocomplexes (GCV-NCs)

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GCV-NCs were prepared using a water–oil–water (w/o/w) emulsion solvent evaporation method

[15]. One hundred microliter of phosphate buffer saline (pH 7.4) containing GCV (0.4 %) was

emulsified in 2 mL dichloromethane containing 100 mg PLGA by sonication. The resulting

primary emulsion was added to 10 mL of a mixed solution consisting of CS (0.25% w/v, in 0.5 M

acetate buffer, pH 4.4) and polyvinyl alcohol (PVA, 1.0% w/v in distilled water) with continuous

stirring to form a double emulsion. The emulsion was stirred at 1500 rpm till the complete

evaporation of dichloromethane. The nanosuspension so formed was passed through 3 cycles of

homogenization at a pressure of 10 000 bars. GCV-NCs were collected by centrifugation at 15

000 rpm for 10 min at 4ºC, washed three times with distilled water, and freeze-dried.

2.6.3. Preparation of chitosan-coated GCV niosomal dispersion (GCV-NDs)

GCV-NDs were prepared by the reverse-phase evaporation technique [16]. Briefly, GCV-loaded

niosomes were prepared by dissolving Span 60 and cholesterol (3:2%, w/w) in diethyl ether and

adding 2 mL of phosphate buffer saline (pH 7.4) containing GCV to it. The mixture was

sonicated for 5 min and a thick emulsion was obtained which was vortexed to remove any

residual ether. To this emulsion 3 mL of phosphate buffer saline was added in order to hydrate

the vesicles. The vesicles thus formed were incubated with 0.2% w/w chitosan solution for 2 hrs.

2.7. Characterization of GCV nanoformulations

2.7.1. Transmission electron microscopy (TEM)

Surface morphology of GCV nanoformulations were studied with the help of TEM. Formulations

treated on copper grids (Polysciences, Warrington, PA) with 1% uranyl acetate for negative

staining, followed by sample drying were analyzed by TEM (Philips CM 10, Holland) at an

accelerating voltage of 100kV.

2.7.2. Particle size distribution

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The mean particle size of GCV nanoformulations were determined by photon correlation

spectroscopy using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). For size

determination, suspension sample was diluted to the appropriate concentration with Milli-Q water

and analysis was performed at 25°C with an angle detection of 90°.

2.8. Ocular pharmacokinetic study

Four groups, each having seven New Zealand Albino rabbits (2.25±0.25 kg), were used for the

ocular study. The protocol was approved by the Institutional Animal Ethics Committee, Jamia

Hamdard, and its guidelines were followed. Each group received, in both the eyes, a single

topical instillation (50 µL) of GCV-solution, GCV-NPs, GCV-NCs and GCV-NDs dose

equivalent to 0.3% w/v of GCV. Eyes were anesthetized using topical application of 4%

xylocaine- MPF sterile solution (AstraZeneca LP) and 50 µL of the aqueous humor was collected

using 30 gauze needle before instillation of formulations and post treatment at 0.5, 1, 2, 4, 6, 8

and 12 hr. All aqueous humor samples were collected in pre-labeled eppendorf tubes, sealed and

stored at -20ºC until UPLC analysis. The aqueous humor samples were prepared as mentioned

above. Pharmacokinetic parameters (PK) were calculated by noncompartmental analysis also

called as model independent analysis using WinNonLin version 4.0 (Pharsight Corp., Mountain

View, CA).

3. Results

3.1. Chromatography

Using the chromatographic conditions described, rapid elution of GCV from aqueous humor was

achieved at 0.928 min (Fig.1). UPLC chromatograms of blank aqueous humor (Fig. 1A), aqueous

humor spiked with GCV (Fig. 1B), and a rabbit’s aqueous humor after topical instillation of GCV

nanoformulations (Fig. 2) were compared to show the selectivity of the proposed procedure. No

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interference was observed either by matrix or by formulation ingredients near the retention time,

demonstrating the method’s selectivity.

3.2. Method Validation

Results of the validation are summarized in Table 1. The method was found to be selective,

precise, accurate and robust. The method enabled the LOQ for GCV to be 10 ng/mL employing

only 50 µL aqueous humor, which was more sensitive than the results from the previous studies.

3.3. Stability Studies

The results of the stability studies showed that GCV was stable in aqueous humor at 20°C for 24

hrs at 4°C for 5 days and at −20°C for 30 days. The percentage recovery of GCV was 95.8±2.2%,

96.7±2.9 and 98.4±3.1 at 20°C for 24 hrs, at 4°C for 5 days and at −20°C for 30 days,

respectively. The freeze-thaw data indicated that three cycles can be tolerated without losses

greater than 10% w/v (figure. 3).

3.4. GCV nanoformulation characterization

The TEM and the particle size distribution of the GCV nanoformulations are shown in Figures 4

and 5, respectively. All of the three formulations, GCV-NDs, GCV-NPs and GCV-NCs were

evenly round in shape (Fig. 3) with mean particle size in the range of 180-200 nm (Fig. 5).

Values of polydispersity index (PI), which is a measure of uniformity of size within the

formulation, were also calculated. The GCV-NDs exhibited a narrow size distribution (PI, 0.181)

compared to GCV-NCs (PI, 0.65) and GCV-NPs (PI, 1.15).

3.5. Ocular pharmacokinetic study

The resulting GCV concentrations measured in aqueous humor collected at 0.5, 1, 2, 4, 8 and 12

h are shown in Figure 6 whereas pharmacokinetic parameters are detailed in Table 2. In the group

treated with the GCV solution, low ocular bioavailability (AUC 0-t, 559.6±14.2 ng.h/mL) was

observed and the aqueous humor levels of the drug were undetectable after 2 hrs, attributed to

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rapid pre-corneal loss. Topical instillation of GCV-NCs and GCV-NDs provided similar ocular

bioavailability of 3797.7±21.8 and 3428.1±29.4 ng.h/mL, respectively. The bioavailability was

approximately 6 fold greater than the GCV solution and nearly 2.5 fold higher than the GCV-

NPs. The increased ocular bioavailability was due to enhanced ocular retention and sustained

release of entrapped drug. Enhanced ocular retention was due to presence of mucoadhesive

chitosan in both GCV-NCs and GCV-NDs formulations. Sustained release of entrapped drug

might be due to presence of hydrophobic PLGA in GCV-NC and hydrophobic surfactant in

GCV-ND formulation. Further, GCV-NPs made up of hydrophilic chitosan, has AUC 0-t

(1441.9±20.0 ng.h/mL) less than that of GCV-NCs and GCV-NDs, possibly due to absence of

hydrophobic component.

4. Discussion

Nucleoside analogues such as GCV exist as both weak acids and weak bases [17]. These ionic

forms of GCV have significant differences in their apparent hydrophobicity, and thus tend to

migrate through the column with different velocities resulting in a poor peak shape, tailing and

decreased sensitivity during HPLC method development. Various HPLC methods have been

described previously for analyzing GCV pharmacokinetics in plasma. However, the LOQ for UV

detection were above 50 ng/mL, which may not be sensitive enough for ocular pharmacokinetic

studies [3-8]. In order to achieve sensitivity, studies with fluorescent or mass detections have

been reported. We attempted to overcome the above limitations using smaller Bridged Ethyl

Hybrid column packed with small sized (1.7 µm) particles, and employing ultra-high pressures.

The small-sized particles reduce plate height and, consequently allow the number of theoretical

plates to be increased [18]. They also favour faster linear velocities and allow the reduction of

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analysis time, and improve peak shape. The UPLC method runs only 3 min which allowed the

analysis of a large number of samples in a short period of time.

Nucleoside analogues are polar compounds, so the addition of ion pairing agents or organic

modifiers in the mobile phase could increase their retention in reversed-phase chromatography.

Moreover, as they are both weak acids and weak bases, it is feasible to separate nucleoside

analogues as ion-pairs with a strong base as the counter-ion. TFA is commonly used as an ion

pairing agent and pH modifier in the mobile phase. To obtain the best chromatographic

separation and sensitivity in a short time, different ratios of aqueous TFA and acetonitrile were

systematically investigated. The best separation was achieved with a mixture of 0.1% w/v

aqueous TFA and acetonitrile (95:5%, v/v) as the mobile phase. Protein precipitation with

acetonitrile and direct injection into the chromatographic column enables high recovery of GCV.

The application of the developed method was demonstrated by comparing aqueous humor

bioavailability of GCV following topical instillation of GCV solution, GCV-NCs, GCV-NPs and

GCV-NDs to rabbit eye. As determined by UPLC method, all the three GCV nanoformulations

provided significantly higher ocular bioavailability as compared to GCV solution. This could be

attributed to increased pre-corneal retention of the GCV nanoformulations owing to presence of

mucoadhesive chitosan and increased corneal penetration of nano-sized particles. All the

nanoformulations which are being compared in the study have similar sizes and have positively

charged surfaces, but differ in their release mechanisms. The results indicate that not only the size

and mucoadhesion, but also the proper drug release is required for ocular bioavailability

enhancement. In conclusion, the assay procedure presented in this report provides a simple, rapid

and sensitive procedure for the determination of GCV in aqueous humor. The achieved

pharmacokinetic results may be useful for formulation development of GCV, which could be

effective in the treatment of ocular infections after single topical instillation.

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Conclusion

In conclusion, the assay procedure presented in this report provides a new, simple, rapid and

sensitive procedure for the determination of GCV in aqueous humor. The achieved

pharmacokinetic results may be useful for formulation development of GCV, which could be

effective in the treatment of ocular infections after single topical instillation.

5. Acknowledgement

The author would like to acknowledge Department of Biotechnology, Government of India for

providing financial support to carry out the work. ACQUITY program team at Waters India Pvt.

Ltd., particularly Mr. Tijender Sharma and Mr. D.P. Joshi are acknowledged for their scientific

support. Ms. Neha Malik, Jamia Hamdard, is greatly appreciated for her inputs with respect to

checking the manuscript for English and language style.

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Table 1 Method validation data for GCV in aqueous humor

Parameter Value

Linearity (n=6)

Range (µg/mL)

Correlation coefficient (r2)

Slope (SD) Intercept (SD)

0.01 – 1.0 0.999 9.873 (0.042) 1832.48 (13.35)

Sensitivity

LOD (ng/mL) LOQ (ng/mL)

3.0 10.0

Reproducibility (n=6)

Intraday GCV spiked (µg/mL)

GCV found(µg/mL)

SD Precision a Accuracy b

0.01 (LOQ) 0.011 0.002 18.2 110.0

0.1 0.097 0.003 3.1 97.0

0.4 0.393 0.007 1.8 98.2

0.8 0.802 0.004 0.5 100.2

Interday

0.01 (LOQ) 0.009 0.001 11.1 90.0

0.1 0.099 0.002 2.0 99.0

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a Precision as CV (%) = 100 × Standard deviation/ Mean concentration found b Accuracy (as %

recovery) = 100 × Mean concentration found/Concentration spiked

Table 2 Pharmacokinetic parameters of GCV after topical instillation of GCV solution and GCV

nanoformulations to rabbit eye

0.4 0.390 0.010 2.6 97.5

0.8 0.795 0.007 0.9 99.4

Extraction efficiency (n=3)

GCV spiked (µg/mL)

GCV found(µg/mL)

SD Mean recovery (%)

0.4 0.39 0.01 97.5

Recovery (SD; n=3)

GCV-NPs GCV-NCs GCV-NDs

99.20 (2.2)% 99.18 (1.5)% 98.87 (1.9)%

Parameter Formulations

GCV-sol GCV-NPs GCV-NCs GCV-NDs

tmax (h) 0.9 1.0 1.4 1.0

Cmax (ng/mL) 325±5.1 589±8.9 449±6.5 523±8.2

Ke (1/h) 1.50±0.11 0.67±0.08 0.08±0.01 0.13±0.02

AUC 0-t ( ng.h/mL ) 559.6±14.2 1441.9±20.0 3797.7±21.8 3428.1±29.4

AUC 0-∞ ( ng.h/mL ) 720.5±19.6 1738.7±22.80 5622.1±39.5 4160.7±46.9

AUMC0-t ( ng.h/mL ) 692.0±26.2 2763.9±46.1 19402.5±40.4 15543.7±35.8

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Figure Legends

Fig. 1 UHPLC chromatograms obtained from (A) blank aqueous humor, and (B) aqueous humor

spiked with GCV (0.2 µg/mL).

Fig. 2 UHPLC chromatograms of rabbit’s aqueous humor obtained 30 min after topical

instillation of GCV nanoformulations (A) GCV-NPs, (B) GCV-NCs, and (C) GCV-NDs.

Fig. 3 Stability profile in terms of recovery for GCV in aqueous humor at different temperature in

24hrs.

Fig. 4 Transmission electron microphotographs of GCV nanoformulations (A) GCV-NPs, (B)

GCV-NCs, and (C) GCV-NDs.

Fig. 5 Particle size distribution of GCV nanoformulations (A) GCV-NPs, (B) GCV-NCs, and (C)

GCV-NDs.

Fig. 6 Aqueous humor concentration–time profile of GCV after topical instillation of GCV-

solution, GCV-NPs, GCV-NCs and GCV-NDs to rabbit eyes.

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ACCEPTED UNCORRECTED MANUSCRIPTTable 1 Method validation data for GCV in aqueous humor

a Precision as CV (%) = 100 × Standard deviation/ Mean concentration found

b Accuracy (as % recovery) = 100 × Mean concentration found/Concentration spiked

Parameter Value

Linearity (n=6)

Range (µg/mL)

Correlation coefficient (r2)

Slope (SD) Intercept (SD)

0.01 – 1.0 0.999 9.873 (0.042) 1832.48 (13.35)

Sensitivity

LOD (ng/mL) LOQ (ng/mL)

3.0 10.0

Reproducibility (n=6)

Intraday GCV spiked (µg/mL)

GCV found(µg/mL)

SD Precision a Accuracy b

0.01 (LOQ) 0.011 0.002 18.2 110.0

0.1 0.097 0.003 3.1 97.0

0.4 0.393 0.007 1.8 98.2

0.8 0.802 0.004 0.5 100.2

Interday

0.01 (LOQ) 0.009 0.001 11.1 90.0

0.1 0.099 0.002 2.0 99.0

0.4 0.390 0.010 2.6 97.5

0.8 0.795 0.007 0.9 99.4

Extraction efficiency (n=3)

GCV spiked (µg/mL)

GCV found(µg/mL)

SD Mean recovery(%)

0.4 0.39 0.01 97.5

Recovery (SD; n=3)

GCV-NPs GCV-NCs GCV-NDs

99.20 (2.2)% 99.18 (1.5)% 98.87 (1.9)%

ACCEPTED UNCORRECTED MANUSCRIPTTable 2 Pharmacokinetic parameters of GCV after topical instillation of GCV solution and GCV

nanoformulations to rabbit eye

Parameter Formulations

GCV-sol GCV-NPs GCV-NCs GCV-NDs

tmax (h) 0.9 1.0 1.4 1.0

Cmax (ng/mL) 325±5.1 589±8.9 449±6.5 523±8.2

Ke (1/h) 1.50±0.11 0.67±0.08 0.08±0.01 0.13±0.02

AUC 0-t ( ng.h/mL ) 559.6±14.2 1441.9±20.0 3797.7±21.8 3428.1±29.4

AUC 0-∞ ( ng.h/mL ) 720.5±19.6 1738.7±22.80 5622.1±39.5 4160.7±46.9

AUMC0-t ( ng.h/mL ) 692.0±26.2 2763.9±46.1 19402.5±40.4 15543.7±35.8

1. Introduction

2. Theranostic nanomedicines or

multifunctional nanoparticles

3. Possible improvement in

characteristics of

multifunctional nanoparticles

4. Advancement related to

patents

5. Conclusion

6. Expert opinion

Review

Advancement in multifunctionalnanoparticles for the effectivetreatment of cancerMahfoozur Rahman, Mohammad Zaki Ahmad, Imran Kazmi, Sohail Akhter,Muhammad Afzal, Gaurav Gupta, Farhan Jalees Ahmed† & Firoz Anwar†Dreamz College of Pharmacy, Himachal Pradesh, India

Introduction: Nanotechnology has gained wider importance for the treat-

ment of various diseases, including cancer. Multifunctional or theranostic

agents are emerging as promising therapeutic paradigms, which provide

attractive vehicles for both image and therapeutic agents. Nanosystems are

capable of diagnosis, specific targeted drug therapy and monitoring thera-

peutic response. Due to their well-developed surface nature, nanomolecules

are easy to anchor with multifunctional groups.

Areas covered: The present review aims to give an extensive account on the

progress of multifunctional nanoparticles throughout the blooming research

with regards to their clinical application in cancer. This paper discusses gra-

phene, a newly developed multifunctional vehicle in nanotechnology. Fur-

thermore, it focuses on the development of tumor cells, the advantages of

novel multifunctional nanoparticles over traditional methods and the use of

nanoparticles in cancer therapy. In addition, patents issued by the US office

are also included.

Expert opinion: Despite numerous advantages, multifunctional nanoparticles

are still at an infancy stage. Many great achievements have been attained in

this field to date, but many challenges still remain. A problem that limits

the use of multifunctional nanoparticles is toxicity. If this toxicity can be over-

come then the advancement in nanocomposite material science will be well

on the way to a prospective treatment of cancer.

Keywords: dendrimer, gold nanoparticles, graphene, multifunctional nanoparticles, patents,

quantum dots, silica nanoparticles, tumor cell

Expert Opin. Drug Deliv. (2012) 9(4):367-381

1. Introduction

Cancer is a serious global health threat and in developed countries it is the secondleading cause of cell death [1]. Cancer is a multistep process which involves numer-ous changes such as cells signaling and apoptosis to name a few [2,3]. The conver-sion of proto-oncogene to oncogene is responsible for development of abnormalimmatured group of cells, which is responsible for tumor formation. Develop-ment of tumor and its increase in size changes the normal function of adjacenthealthy cells. This results in initiation of apoptosis in healthy cells. The maximumsize of most of the tumors is 2 mm2. After achieving maximum size these cellsmove to other parts of body initiating the process of metastasis, which makes can-cer incurable [4]. An illustration of tumor development from single cell to maxi-mum size tumor is depicted in Figure 1. Cancer is a very complex disease due toits molecular heterogeneity (multiphenotype) and adaptive resistance found invarious tumor cells, and this makes it more challengeable for its treatment. Con-ventional treatment approaches like surgery, radiation, biological therapies(immunotherapy) and chemotherapy; these have poor specificity, non-recognition

10.1517/17425247.2012.668522 © 2012 Informa UK, Ltd. ISSN 1742-5247 367All rights reserved: reproduction in whole or in part not permitted

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of tumor markers, along with dose-related toxicity, targetedaction, poor bioavailability, neurotoxicity and risk of dam-age to vital organs [5]. Hence, it is the need of the hour todevelop new and innovative technologies that will help inprevention of adaptive resistance, identify tumor markercells and micrometastases. Combination of nanotechnologywith oncology is a new field of interdisciplinary research,comprising biology, chemistry, engineering and medicine(designing of materials at nanoscale levels to create productsthat exhibit novel properties), which have profound impacton disease prevention, diagnosis and their treatment [6].Multifunctional nanoparticles are the novel technologicalinnovations developed recently to fight against cancer. Thisis the most effective approach to recognize the molecularheterogeneity and adaptive resistance found in cancer cells.It reduces the problems associated with conventional therapywith respect to diagnosis, imaging and real-time controlleddrug release, followed by reduction in toxicity and makingtreatment duration shorter [7,8].

2. Theranostic nanomedicines ormultifunctional nanoparticles

It is an integrated nanotherapeutic system. The term theranosticnanotechnology means combination of individual technique

to form a single nanoplatform by mounting therapeutic func-tion on them. This is a combination of diagnostic test with tar-geted therapy at controlled rate, which co-delivers therapeuticagents and imaging functions (Figures 2 and 3). For successfuldevelopment of theranostic nanomedicines (TNM), we musthave deep knowledge regarding the materials science and nano-composite materials like particle surface chemistry, non-covalent binding, electrovalent strategies, biospecific interac-tion, hydrophobic adsorption and safety. These combinationsprovide controlled and improved reproducibility of TNM [5].Amorphous or semi-crystalline colloidal system which has10 -- 200 nm of particle size is best suited for TNM. It possesbetter optical, magnetic and thermodynamic properties [9],and is efficient in targeting high drug loading, high specificityon tumor cell. Further, it improves bioavailability, imagingcell sensitivity, reduces multidrug administration, high spatialresolution, tomographic capability, easy and early onset ofdetection and delivers selective therapeutic agents [9]. The func-tion of TNM depends on different subunits as anticancer drugor tumor-targeting moieties [10]. These nano-based TNMs havefour special properties that distinguish them from other cancertreatment options: i) the TNM can themselves have therapeutic(drug delivery, gene delivery, drug targeting and photothermalproperty), diagnostic and imaging properties (Figures 2 and 3).It further achieves synergistic effects by blocking differentreceptors; ii) TNM can be attached to multivalent different tar-geting ligand, which results in high affinity and specificity fordifferent markers; iii) TNM can be made to carry multipledrug molecules that simultaneously enable combinatorial can-cer therapy and iv) TNM can bypass traditional drug resistance,molecular heterogeneity and adoptive resistance mechanisms.TNM can achieve increased intracellular concentration byusing both passive and active targeting strategies. This resultsin enhanced anticancer effects, reduction in systemic toxicityand minimum toxicity to normal cells [10,11]. Several TNMshave been employed as the carriers of diagnostic agents anddrugs. Lukianova-Hleb et al. studied the optical generationand detection of plasmonic nanobubbles (PNBs) around goldnanoparticles (GNPs) in individual living cells, and evaluatedthe multifunctional properties of PNB [12]. Recently, muchwork has been reported and discussed about characteristicsand biomedical applications of magnetic nanoparticles, andhas concluded that it can simultaneously act as diagnosticmolecular imaging agents and with therapeuticproperties for different types of drug carriers [13]. Shim et al.reported that coated small-interfering RNA-encapsulatingpolyplexes attached to small GNPs via acid-cleavablelinkages for the development of combined (theranostic)stimuli-responsive multimodal optical imaging and stimuli-enhanced gene silencing [14]. Several nanoparticles such asiron oxide, quantum dots (QD), silica nanoparticles,carbon nanotubes (CNTs), gold, dendrimer and graphenehave been investigated as multifunctional nanoparticles. Thisreview introduces multifunctional agents, in which a linkagebetween nanoplatforms and functional entities has been

Article highlights.

. Cancer is a serious and complex disease across theworld. Multiphenotype and adaptive resistance found invarious tumor cells makes it more challengeable forcancer treatment.

. In oncology research, theranostic nanomedicine (TNM)has garnered increasing attention in the treatment ofcancer and has emerged as promisingtherapeutic paradigm.

. TNM is called an all-in-one system. It means mountingof different therapeutic function on single nanosystems,resulting in co-delivery of therapeutic agents andimaging function. Several TNMs have been discussedsuch as iron oxide nanoparticle (IONP), quantum dots(QD), silica nanoparticle, carbon nanotube (CNT), goldnanoparticle (GNP), dendrimer and graphene. All thesemultifunctional nanoparticles have integration of cancerresearch, therapeutic diagnosis and imaging.

. Stability and pharmacokinetic profile of TNM can beimproved by using several polymer, dye, ligand, PHlabile group, photosensitive, thermosensetive groups.

. So far, many TNMs have been approved by the FDAsuch as AMI-121, AMI-277, Feridex� and Combidex�.

. Recently, patents have been issued by the US office inthis nanotechnological arena on TNM, due to theireffectiveness found at different stages of clinical trials.

. One of the major limitations associated with TNM istoxicity. Advancement in nanocomposite materialsscience may overcome toxicity. In the future, TNM holdsa great potential to be used as a therapy for cancer.

This box summarizes key points contained in the article.

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developed. It has been discussed in text and also summarizedin Table 1.

2.1 Iron oxide nanoparticleIron oxide nanoparticles (IONPs)-based theranostic agentsare a magnetite or hematite nanocrystals. They are used ascontrasting agents for magnetic resonance imaging (MRI)and targeted drug delivery to the tumor cells [15,16]. Two typesof iron oxides nanoparticles have been reported, namely

supermagnetic iron oxide and ultra-small superparamagneticiron oxide (SPIO). Among these, SPIO has superior actiondue to its biocompatible and biodegradable properties ofiron, which can be recycled for iron metabolism via biochem-ical pathway. Many articles are available, where SPIOs havebeen used in combination with molecules and monoclonalantibodies to detect variety of cancers. They have also beenused with peptides to target transferring and pancreaticreceptor and augmented to image the cancer cell [17-22].

Promotors

Virus

Normal cellTumors

Mutation

Mutated cell

Figure 1. Tumor development.

Therapeutic agents

Core

Imaging agent

Drugs A

Targeting moiety

Drugs B

Figure 2. TNM have specific targeting agent, imaging agent, diagnostic agent and chemotherapeutic agent (drugs), all are in

one platform system.

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IONP synthesized by co-precipitation of Fe(II) and Fe(III)precursors and with advancement of high temperature decom-position method are utilized to synthesize them [23,24]. Co-precipitation techniques improve the colloidal suspendabilityof the particles. Generally, hydrophilic polymers such asPVP, polyaniline and dextran, are used during particle forma-tion, which results in prevention of particle aggrega-tion [23,25,26]. Several polymeric IONP as MRI contrastagents are available in market, like Feridex� particles(AMAG Pharmaceuticals, Inc., Cambridge, MA, US) fordetection of liver and spleen lesions, and Combidex� is usedat clinical trial III stage, where lymph node image isrequired [27,28]. In high temperature decomposition method,

pyrolysis synthesis results in organic solvent by the tempera-ture treatment, particle growth is carried in regular controlledfashion and particles have better crystallinity and higher magne-tism than traditional method. Drug molecules can be easilycoupled by appropriate coating on IONP [29]. Zhang and col-laborators investigated this coupling effect on methotrexate(MTX). Similarly, multiple coupling of paclitaxel (PTX), her-ceptin antibody molecules on IONP already have beenreported [30-33]. Jain et al. reported loading of doxorubicin(DOX) and PTX onto IONP by using oleic acid. Recently,Yu et al. reported that loaded DOX by using antibiofoulingpolymer-coated IONP showed better clinical pharmacokineticand therapeutic effect than only DOX. Hyeon group [36]

reported that many drug molecules which are smaller in sizecan be loaded on to hollow nanostructure by process of physicalabsorption such as loading of DOX on to hollow spindle shapeof b-FeOOH nanoparticles which are made from FeCl3 hydro-lysis. Hollow IONP are made by controlled oxidation andetching of Fe particles. This combination targets the ErbB2/Neu-positive breast cancer cell and delivery of cisplatin at acontrolled rate is obtained [34-37]. Presently, genes are deliveredthrough nanoparticles-based delivery system which can antago-nize abnormal gene regulation through target cellmembranes [38].

2.2 Quantum dotsThey are nanocrystals which are made of semiconductor mate-rials. They have unique light-emitting properties that can beoptimized by tuning their size and composition. They areclassified into two parts: i) first generation of QD consist of

Targetting and drug delivery

Theranostic nanoparticle

Gene delivery

Tumor cell

Diagnostic and imaging agent Photothermal therapy

Figure 3. Various applications of multifunctional nanoparticles in therapy.

Table 1. Examples of different multifunctional

nanoparticles [27,28,119].

Multifunctional

nanoparticles

Target organs FDA status

AMI-121 (SPIOs) Gastrointestinal lumen ApprovedAMI-277 (SPIOs) Blood node, lymph

nodeApproved

SHU 555A (SPIOs) Spleen/liver ApprovedFeridex� Spleen/liver ApprovedCombidex� Lymph node Stage IIIDOX-TCL-SPION(DOX thermallycross-linked SPIOs)

Breast cancer andimaging

Approved

DOX: Doxorubicin; FDA: Food and Drug Administration;

SPIO: Superparamagnetic iron oxide.

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Cdse, CdTe and Pbs, changes of their size gave rise to nanome-tarials that lie in visible spectrum. Major limitations associ-ated with these dots are their ineffective/limited tissuepenetration. This was overcome by use of CdTe/CdSe,Cd3P2, InAs/ZnSe and InAs/InP/ZnSe and also by enhan-cing the quantum photoluminiscent properties. ZnS coatsenhance the efficiency of QD nanoparticles. QDs are pre-pared by heating organometallic precursors in high boilingpoint organic solvent. Surfactants such trioctylphosphine(TOP) and trioctylphosphine oxide (TOPO) were used tocontrol the particle growth (Figure 4) [39-43]. Alkyl groupspresent on QDs make them water insoluble, and thiogroup is attached to overcome this problem which formsdisulfide linkage with core shell (ZnS) of QDs. Major lim-itation of such nanoparticles is fragile disulfide linkage. Tostrengthen this linkage and to improve the longevity of QD,DiHydroLipoic Acid (DHLA) oligomeric phosphines,cysteine-rich peptides and multidentate polymers wereadded [39,44-46]. First-generation QD-based drug deliveryhas innate toxicity due to release of Cd and lead. Thistoxicity leads to the development of second generation ofQDs (InAs/InP/ZnSe), which are free from Cd and arepotentially better carriers [39-41]. They were made watersoluble by the addition of poly(ethylene glycol) (PEG)-10and 12-pentacosadiynoic acid (PEG-PCDA) (Figure 4).Furthermore, QD nanostructure were stabilized by appli-cation of UV-irradiation, which cross-linked the coatingshell and reduced the leakage of core materials fromQDs. Recently, Bagalkot et al. investigated QD--aptamer(Apt)--DOX conjugated [42], which has been used forco-delivery of therapy and therapeutic agents (Figure 5).

DOX loading on QD surface by using A10 RNA resultsin controlled release of DOX which initiates therapeuticfunction and recovery of QD fluorescence (Figure 5).Yuan et al. reported that loading of MTX onto QD surfaceby the mechanism of physical adsorption induced photolu-minescence quenching [42,43]. QDs are used as gene deliveryvehicles, by mounting polymers such as lipofectamine andpoly(maleic-anhydride-alt-1-decene), which result in moreefficient delivery and reduce toxicity [44,45]. Matrix metallo-peptidase 9 (MMP-9) is the main part of the blood--brainbarrier and lies in the brain microvascular endothelialcell. Poly(diallyledimethyl ammonium chloride) (PDDAC)on QDs, which forms a complex with MMP-9-siRNA,can modulate the activity of MMP-9. This results incollagen I, IV and V expression and a decrease in endothe-lial permeability. QDs have unique properties like photo-stability, broad absorption spectra, narrow size, stableemission spectra and mounting of several agents on them.For these unique properties, they have great potential inphotodynamic therapy as a photosensitizer. The mechanisminvolved is activation by light and transfer of unpaired elec-tron state energy to near oxygen molecule. This generatesreactive oxygen intermediate, which causes abnormal celldamage [46-48].

2.3 Silica nanoparticles-based theranostic agentsIt is the most common biosafe material, used as surgicalimplant. Silica nanoparticles do not possess the potential forimaging, but they can be made theranostic by the applicationof broad range of imaging group and therapeutic functions onthem. Hence, different moieties can be delivered through thismultifunctional carrier. Generally, two techniques, hydrolysisand condensation are used for preparation of silica nanopar-ticles. Theranostic properties can be developed by mountingof aminopropylmethoxysilane (MPS) on tetraethylorthosili-cate (TEOS). MPS acts as a co-precursor which brings amineor thiol group to the particle surface. During particle forma-tion, organic dyes and GD-DTPA can be incorporated intosilica particles matrix for developing the magneticproperties [49-51]. Sathe et al. and Koole et al. developedIONPs, Au NPs and QDs incorporated into single silicananoparticles, resulting in dual core shell--shell nanoparticlesin which better magnetic and optical properties wereobserved [52,53]. Roy et al. and Kim et al. reported the incorpo-ration of hydrophobic photosensitizing anticancer drug like2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide (HPPH)into silica matrices which efficiently kills cancer cell. More-over, photon absorbing dye such as 9,10-bis [4-(4-amino-styryl) styryl]anthracene (BDSA) can be incorporated toupconvert the near-infrared (NIR) light that can transfer theintraparticle energy to HPPH which activates the PDT func-tion [54,55]. Some new changes have been developed in prepa-ration, by using chemical and physical techniques to obtainmesoporous structure. In chemical process, at the particlestage development, an N-alkyltrialkoxysilane or other surfac-tants were mixed with precursors and then were incorporatedinto matrices; subsequently, surfactants were removed bypost-synthesis solvent extraction or calcinations method tomake a toxic-free preparation. In physical process, physicalinteraction plays important role through which many thera-peutic molecules can be loaded. Mesoporous structure con-sists of accurate pore size, more than 100 channels areavailable that result in large surface area (900 m2/g) and thisinhibits premature drug release and makes them excellent res-ervoir for many therapeutic moiety in cancer drug deliverysystem. PTX when loaded on mesoporous structure and fur-ther capped with Au NPs along with QDs and IONPsobserved better drug release profile [56-59]. Luminescentporous silica nanoparticles (LPSiNPs) preparation uses physi-cal process which consists of single crystal silicon wafer poroussilicon film, filtered through a 0.22 µm membrane filter.These luminescent porous nanoparticles can be used withmany therapeutic drug molecules like DOX with a uniquefeature of self in vivo destruction and renal clearance withina short duration of time (Figure 6), rendering less chance oftheir entrapment into normal organ [60].

2.4 Carbon nanotube-based theranostic agentsCNTs are graphite-like structure and inert in nature. Itspotential application in Raman and photocoustic imaging

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have also been under investigation as a drug carrier (Figure4) [61,62]. Extreme oxidative conditions were developed to yielda defect on CNTs surface that can be used for mountingdifferent therapeutic moieties on them. Single wall nanotubes(SWNTs) when refluxed with 2.5 M HNO3 for 2 -- 36 hwith the interval of 30 min form water-soluble SWNTs, dueto the presence of carboxyl groups on nanotubes surface. Thisis the site where several molecules can be conjugated by cova-lent bonding, recently one group called azomethineylide andits derivatives were incorporated onto surface by 1,3-dipolarcycloaddition mechanism [63-66]. Similarly, many moieties likePEGlyated phospholipids can anchor onto CNT surface, andthey have shown better efficacy, better entrapment in spleenand liver without any degradation and toxicity [67].

CNTs have unique physical and surface properties, high load-ing drug molecule capacity and can also anchor the IONPs,AuNP on CNT surface but its non-biodegradability and impu-rity remain problems of concern. CNTs can be taken up by cellsthrough endocytosis, passive diffusion or other mechanism thatmay depend on the nature of surface coatings. Prato et al. cou-pled MTX onto CNT by 1,3-dipolar cycloaddition, reportsare available where multiple amines were used to load and deliverthe DNA plasmid [70]. PTX when coupled on branched PEGy-lated CNTs, improved pharmacokinetics, stability and bettertumor suppression. Moon et al. [71] investigated the use ofCNTs as a photothermal therapy, where PEGylated SWNTand NIR irradiation, caused extirpate of tumor cells and noreoccurrence after 6 months was observed [68-71].

Quantum dots

Amphiphilic polymer coating

Therapeutic agent

Affinity ligand

Drug B

Drug A

PEG

Targeting moiety

Imaging agent

Multifunctional nanoparticle

Carbon nanotube

Figure 4. New synthetic methods have been developed to design multifunctional nanoparticles like carbon nanotube and

quantum dots, QD nanoparticles, conjugated to a ligand by coating a polymeric layer, on which can encapsulate both

therapeutic and imaging agents in a single nanoplatform system.

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2.5 Gold nanoparticles-based theranostic agentsGNPs have recently emerged as an attractive candidate fordelivery of various functional groups into their targetssites [72,73]. The functional groups can be small drug moleculesor large biomolecules, like proteins, DNA or RNA (videpost), etc. GNPs are inert, easy to prepare, non-toxic and effi-cient release of therapeutic agents from them gives a betteralternate for effective therapy [74]. The unique propertiesmake GNP to be investigated in various image-related areas,such as computed tomography (CT), photocoustic andsurface-enhanced Raman spectroscopy. GNPs are availablein various forms like spheres, cubes, rods, cages andwires [75,76]. Size and morphology of GNPs influence theproperties of the products, as for example, 10 nm sphericalAu NPs have some characteristic surface plasmonic absorptionat around 520 nm. If particle size is increased from 48.3 to99.4 nm GNPs, then there is a bathochromic shift of theparticle, which shows absorption at the 533 and 575 nm,

respectively. Further change in morphology of Au NPs torod-like shape shifts the absorption to NIR region(650 -- 900 nm). These properties make GNPs applicable asa photoacoustic image or mediators in photothermal ther-apy [75,77]. Multithiolated group by layer deposition methodmakes them more stable with high drug loading capacity forantibodies and other large molecules. Better therapeuticeffects of PTX can be observed when drug is covalently cou-pled to 4-mercaptophenol using GNPs technique. Similarly,protein-based preparation have been used for loading ontoAu NPs and TNF anchoring onto PEGylated Au NPs, andthese all have better therapeutic efficacy and less toxicitywhen used as a single moiety [78-81]. In addition to Au-thiol chemistry on Au NPs, chitosan-based preparations areused when a reducing agent and coating materials aremounted to make Au NPs which are positively charged andhighly efficient in nature. DOX conjugated onto thehydrophobic shell by covalently hydrazone linkage, resulted

Receptors

Nucleus

Lysosomes

*OFF*

Drug release

ON*

Anticancer drugs

QD-Apt(Dox): *OFF*Dox

QD-Apt″ ON:

Imagingagent

Dox″ON″

Targeting tumor cell

Figure 5. Building and working mechanism of QD--Apt--DOX nanosystems. Two steps are involved: first, CdSe/ZnS QDs are

surface conjugated with the A10 PSMA aptamer. The intercalation of DOX within the A10 PSMA aptamer on the surface of

QDs. This forms QD--Apt--DOX nanosystem, which quenches fluorescence from both QD and DOX (‘OFF’ state). Second, specific

uptake of QD--Apt--DOX nanosystems into target cancer cell through endocytosis. Ultimately, release of DOX from the

QD--Apt--DOX nanosystems induces the recovery of fluorescence from both QD and DOX (‘ON’ state), thereby sensing the

intracellular delivery of DOX and synchronous fluorescent localization, resulting in killing of cancer cells.

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in selective target and efficient drug delivery in controlled wayand better image formation made new way to control thegroup of tumor [82,83]. GNPs have been mounted with alky-lated quaternary ammonium compounds on plasmid DNA.Au NPs is a good candidate for photothermal therapy;because of their unique surface plasmon resonance, stabilityand biosafety, they are easily modified. Its accumulation intumor cell results in conversion of light into heat, which killscancerous cells. One of the demerits associated with Au NPs isthe high cost of production, which diminishes its clinicalapplication [84,85].

2.6 DendrimerThese are the newer variety of nanocarriers which are stable,non-immunogenic and less than 5 nm in diameter. The applica-tion of therapeutic and diagnostic purposes in treatment of can-cer including advances in the delivery of anti-neoplastic andcontrast agent, neutron capture therapy, photodynamic therapyand recently in photothermal therapy is a matter of great impor-tance. Due to their better elucidation and unique derivativeproperties Dendrimer have been synthesized with biocompatibleretention, and better pharmacokinetic and targeted deliveryproperties are under research [86-88]. In dendrimer, drug can beincorporated with the help of polymers, either by non-covalent or covalent conjugation to form macromolecular pro-ducts. Camptothecin was conjugated with poly(glycerol-succinicacid) to obtain its dendrimer, multiple drugs can be anchored toeach dendrimer molecule [89]. Gurdag and Khandare, reportedconjugation of MTX with polyamidoamine (PAMAM) by therelease of carboxylic and amine group, which were beneficialin resistant CCRF-CEM human acute lymphoblastoid

leukemia. PTX when conjugated by PEG or G4-PAMAM,results in homogeneous distribution intracellularly. This homo-geneous distribution reduces the IC50 more than 10-fold whencompared with free drug, further it increases plasma circulationtime and also enhances tumor uptake due to enhanced perme-ability and retention (EPR) effect. Liposomal DOX has beencomplexed with G4-PAMAM for potential delivery in the breastcancer and showed highest efficacy against it [90-92]. Recently,Lee et al. reported conjugation of DOX through PEGylationwith one side substituted by 2,2-bis(hydroxymethyl)propionicacid dendrimer, which conjugates the DOX by hydrazone link-age. This results in gradual release of increased DOX and shortelimination half-life [92]. Target delivery of dendrimer, achievedby the conjugation of folic acid, peptides, monoclonal antibod-ies, which are highly capable of delivering multifunctional agentsat targets are under investigation. Acetylated PAMAM den-drimers were conjugated to folic acid (for tumor targeting),drug MTX and a fluorescent label for its imaging [93,94]. The tox-icity of 5-fluorouracil (5-FU) can be minimized by acetylation ofthis drug if conjugated to dendrimer. Starpharma (Prahran, Aus-tralia) formulated drug dendrimer-based microbicides (Viva-Gel), which are easy to administer and have improved thesafety level of patients [95]. The unique architecture of den-drimers is the potential candidate for multivalent attachmentof imaging probes and target moieties; therefore, it can be usedas a diagnostic tool for cancer imaging and drug delivery stickto cancer cell.

2.7 GrapheneGraphene is a two-dimensional honeycomb monoatomicthick building block of a carbon allotrope (CNT, fullerene,

Si(OH)4

Emission650 – 900 nm

Excitation300 – 650 nm

Biopolymer Non toxic clearable product

Multifunctional nanoparticle

Si-SiO2

Drugs

In vivoSi(OH)4

Si(OH)4

Si(OH)4

Figure 6. Illustrated structure and in vivo degradation of the silica nanoparticles.

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diamond) with a bond length of 0.142 nm. It was first pre-pared by peeling a single layer of grapheme using stickytape and a pencil in 2004. At the global level, it hasemerged as an exotic material of the 21st century, due to itsexceptional high electron mobility at room temperature(250,000 cm2/vs), exceptional thermal conductivity(5000 W/m/K) and optical and mechanical properties [96-98].On behalf of its unique properties, electronic, optoelectronicdevices, chemical sensors, nanocomposites and energy storageinstruments can be designed easily. Hence, it can be used inthe biological systems for detection of DNA, metal ion, pro-tein, pathogen, design of cell/bacterial nanodevice, drug deliv-ery carrier and drug targeting in cancer. Recently, researchershave developed an electrochemical aptasensor for label-free selective and sensitive detection of cancer cells whichcan be used for the treatment of cancers [99-101]. For earlydetection of carcinoma cells, perylene tetracarboxylic acid(PTCA) functionalized grapheme modified electrodes havebeen developed [102]. Over the past several years, scientistshave used various methods for producing graphene, such asmicromechanical exfoliation and epitaxial growth or chemicalvapor deposition (CVD) epitaxial growth. The sticky tape andpencil method is like micromechanical exfoliation or peel-off method, which can be used successfully to produce pureand single-layered graphene sheet with a honeycomb lat-tice [103,104]. The major disadvantage associated with this isof small yield. Recently, graphene synthesized by chemicalprocess is a new tool with advantages of scalable, high-volumeproduction and ease of chemical modification [105].

Biologically compatible and biodegradable natural poly-mers, such as lignin and cellulose derivatives, have beenemployed to formulate stable graphene nanosheets, whichcan be used as loading platform for biomarkers and othermultifunctional anticancer agents (Figure 7), such asadhesive-protein functionalized graphene nanosheet with theelectrostatic assembly of various metallic NPs, for example,Au, Pt, Ag and Pd to name a few. Functionalized graphenenanosheets are very useful in identification of tumor cell atthe early stage and eradication of cancer cell [106,107].

3. Possible improvement in characteristics ofmultifunctional nanoparticles

Stability and biocompatibility of TNM can be improved ifvarious groups such as PEG, modified acrylic acid polymer,phospholipids micelles are attached to nanoparticles, thiscan improve in maintaining the drug level in blood. Aptamer(oligonucleotides), carbohydrates, folic acid and peptides maybe attached to have specific target site [108]. Intracellular pen-etration peptides can be transferred through transactivatingtranscriptional activator (TAT) ligand, using positivelycharged moieties and cationic lipid polymers. This attach-ment improves pharmacokinetic and biodistribution proper-ties of drugs. Different groups may be attached to TNMsuch as QDs, magnetic nanoparticles for better

imaging [109-111]. For making the best stimulus, sensitivedrug release property must be altered by attachment of pHlabile, photosensitive, thermosensitive, magnetic sensitiveand redox sensitive groups. These all, provide better controlbioavailability and reduce the toxicity [112]. Silica-based TNM applicable for bioimaging, biosensing and releas-ing therapeutic drug is a good carrier of metals, drugs andfluorescent dye; further, it can be modified and differentligand or biomolecules can be attached [113,114]. Moreover,recently the conjugation of silica with dye fluorescein isothio-cynate is used for imaging of human bone marrow stemcell [115,116]. Now core satellite composed of rhodamine dye,silica core and multiple satellites, made up of magnetic nano-particles, when combined to human B1 antibodies, can beused as marker of neuroblastoma, lung carcinoma and Wilm’stumor if associated with polysialic acid. Mesoporous silicateTNMs can conjugate with different ligands and chemothera-peutic drug molecules, which are beneficial for diagnosis andtreatment of various types of cancers [117,118].

4. Advancement related to patents

There is extensive support for the applications of multifunc-tional nanoparticles in biological system for the diagnosticand therapeutic uses. However, the use of multifunctionalnanoparticles in cancer therapy has recently been reportedby many nanotechnologists [119].

4.1 Patents on multifunctional nanoparticlesPossible cancer therapy and diagnostic techniques which arethrough multifunctional nanoparticles such as early detectionof tumor and imaging of cancer cell along with therapeuticdelivery of drug to the targeted tissues or cancer cell havealready been or are being patented.

The US patent number 3177868 describes the biconju-gated biocompatible QD, which targets the specific targetedmoiety and it claimed that QDs emit electromagneticradiation of UV region after soft X-ray treatment [120]. TheUS patent number 67485 describes the composition of semi-conductor nanoparticles with polypeptides templates, whichalso includes cadmium selenide, cadmium telluride, zincselenide, zinc sulfide and zinc tellurides. These particleshave the properties to show multiple color luminescentsystem. This can be eminent approach for early tumor diag-nosis and therapy. Moreover, iron oxide (Fe2O3/Fe3O4)nanoparticles have supermagnetic properties, which canserve as better contrast enhancement agents for MRI [121].The US patent number 216239 describes the compositionand evaluation of nanoparticles, which are made up of mag-netic materials for targeting, diagnostics and therapeuticpurposes and can be used for early detection of tumor celland further for their treatment [122].

The US patent number 255403 describes the compositionand production of fluorescent nanoparticles, which can actas markers, indicators and light sources [123]. It is used

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for identification and location of the tumor cell andprovides therapeutic action too. The US patents number6,689,338 describes biconjugation of nanoparticles as radio-pharmaceuticals, it is a combination of radio immunotherapyand radioimmunodetection agents and is used for diagnosisand treatment of cancer [124].The US patent number 6165440 (entitled radiation and

nanoparticles for enhancement of drug delivery in solidtumor) describes conjugation of nanoparticles to many bodieslike antibodies, ligands as targeting molecules can enhance thetargeting action to desired tissue for specific action [125]. TheUS patent number 6187823 describes conjugation of CNTsto molecules like carboxyl, amino group can enhance particu-lar action [126]. The US patent number 6884478 informs thatQD nanoparticles which have different diameters but samecomposition can emit a different wavelength for therapeuticaction [127]. The US patent number 6843919 describes thearomatic structures like graphene for their MRI and NIRagents [128]. The US patent number 6001054 describesGNPs for enhancing the targeting action and better therapeu-tic effects of these nanoparticles [129]. The US patent number6, 165440, describes interaction of metallic nanoparticles withelectromagnetic pulse and ultrasonic radiation, which resultsin enhanced drug delivery in solid tumor. Ultrasonic waves-induced cavitations, resulting from perforation of cancer cellmembranes, ultimately provide enhancement of drug deliveryfrom blood to cancer cells [125].

5. Conclusion

The application of nanotechnology in the field of cancer biol-ogy has experienced exponential growth in the past few years.In this article, the authors have discussed some nanoplatformsthat are currently under investigation to build multifunctionalnanoparticles. All these nanoparticles have been previouslystudied for imaging; because of their unique optical or mag-netic properties. Every nanoparticle has its own advantagesand disadvantages. This review discussed the anchor role oftarget moieties onto nanoparticles to achieve multifunctionalnanoparticles, in which integration of imaging and therapeu-tic function can be augmented where therapeutic moleculecan be delivered to disease area and can use its imaging func-tion to improve diagnosis and therapeutic response. It istoo premature to say when one system with comprehensivefeature will be available for human use.

6. Expert opinion

Cancer is a complex disease across the world. Tumor hetero-geneity and adaptive resistance of malignant cells to drugs isa major challenge for treatment. Conventional treatmentapproaches used to remove cancer cells lies with severallimitations. However, the growing of interdisciplinaryscience at the nanoscale gives an option for nanotechnology.Nanotechnology uses development in surface chemistry

Aptamer

Nucleic acid

Avidin-biotinNP

Protein

Cell

Peptides

Figure 7. Graphene due to its unique properties can be functionalized with different moieties like peptides, proteins,

aptamer, Avidin-Biotin, multifunctional nanoparticles and cells via the physical adsorption or chemical conjugation. This

biosystem can be used to build up multifunctional biological platforms for medical applications.

M. Rahman et al.

376 Expert Opin. Drug Deliv. (2012) 9(4)

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of nanoparticles anchored with different functionalmoieties onto nanoparticles giving rise to multifunctionalnanoparticles, which can be integrated in cancer research,imaging, diagnosis and therapeutics. Despite of allthese advantages, multifunctional nanoparticles are still atan infancy stage. Many great achievements have beenattained in this field but still many challenges remain.Currently, most of the multifunctional nanoparticleshave been develop and have been approved by the Foodand Drug Administration (FDA) and many are under

clinical developmental stage. A problem that limits the useof multifunctional nanoparticles is toxicity. If this toxicitycan be overcome then the advancement in nanocompositematerials science will be a prospective way to treat the threatof cancer.

Declaration of interest

The authors state no conflict of interest and have received nopayment in preparation of this manuscript.

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AffiliationMahfoozur Rahman1,4,

Mohammad Zaki Ahmad1, Imran Kazmi2,

Sohail Akhter3, Muhammad Afzal2,

Gaurav Gupta2, Farhan Jalees Ahmed†3 &

Firoz Anwar2

†Author for correspondence1Dreamz College of Pharmacy,

Himachal Pradesh, India2Siddhartha Institute of Pharmacy,

Dehradun, Uttarakhand, India3Faculty of Pharmacy,

Department of Pharmaceutics, Jamia Hamdard,

Hamdard Nagar, New Delhi, 110 062, India4Assistant Professor,

Dreamz College of Pharmacy,

Himachal Pradesh, 175036, India

Tel: +91 9625218477;

E-mail: mahfoozkaifi@gmail.com

Advancement in multifunctional nanoparticles for the effective treatment of cancer

Expert Opin. Drug Deliv. (2012) 9(4) 381

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1834 Current Pharmaceutical Design, 2011, 17, 1834-1850

1381-6128/11 $58.00+.00 © 2011 Bentham Science Publishers

Cancer Targeted Metallic Nanoparticle: Targeting Overview, Recent Advancement and Toxicity Concern

Sohail Akhter1, Mohammad Zaki Ahmad2,* Anjali Singh3, Iqbal Ahmad3, Mahfoozur Rahman2, Mohammad Anwar3, Gaurav Kumar Jain3, Farhan Jalees Ahmad1,3 and Roop Krishen Khar1,3

1Nanoformulation Research Lab., Jamia Hamdard (Hamdard University), New Delhi-62, India, 2Dreamz College of Pharmacy, Khilra-Meramesit, Sundernagar, Himachal Pradesh-36, India, 3Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi-62, India

Abstract: The targeted delivery of theranostic agents to the cancer cells is one of the major challenges and an active field of research in the development of cancer chemotherapeutic approaches. Theranostic metallic nanoparticles (TMNPs) have garnered increasing atte ntion in recent years as a novel tool for theranostic application such as imaging, diagnosis, and therapeutic delivery of active agen ts to tumour specific cells. This paper attempts to unveil the multidimensional theranostic aspects of multifunctional metallic nanoparticles (MNPs) including passive and active targeting (HER2, Folate, Angiogenesis etc.) as well as the RES escaping approach. Special attentio n is given to the theranostic application of MNPs in oncology. Patents issued by the US office in this nanotechnological arena are also in cluded em-phasising the importance of MNPs in current cancer treatment/imaging research scenario. Keeping in mind the blooming research i n

Keywords: Metallic nanoparticles (MNPs), Active and passive targeting, Patents, Theranostic application, Quantum dots, Toxicity assess-ment.

INTRODUCTION Conventionally, the first-line treatment of solid tumours is their surgical removal followed by a regimen of chemotherapy and/ or radiation treatment. Unfortunately, these strategies often fail and the patient may discontinue the treatment before the complete eradication of tumour due to several severe side effects [1-2]. Pres-ently, a variety of anticancer agents are available belonging to dif-ferent categories like plant origin, hormones, enzymes, biologicals, semi-synthetic and synthetic molecules. However, they have certain therapeutic and toxicological limitations [3]. Intracellular transport of therapeutically active substances, particularly in cancerous cell, is a potentially challenging task for the drug delivery systems. Dis-tribution of drugs within the cellular and tissue structure essentially depends upon its physiochemical properties like pKa, hydrophilic-ity, polarity and electrostatic charge. However, all these criteria do not necessarily fit in the characteristics of tumour cell because drug can be distributed towards healthy tissue rather than the target [4]. In addition, most of the anticancer drugs are not soluble in water or physiological solution, thus necessitating the use of pharmaceutical solvents for the administration of drugs causing life threatening effects [1, 5]. Consequently, conventional chemotherapy for sys-temic delivery of chemotherapeutic agents often fails due to inade-quate delivery of the drug to target cell / tissue and has a tremen-dous impact on reducing the quality and expectancy of life. Other disadvantages that are associated with current conventional antican-cer therapy and imaging technology are - inefficient cellular uptake, uptake by the immune system (RES), mononuclear phagocyte sys-tem and accumulation in non-targeted organs [6]. Therefore, the urgent need in the treatment of tumour therapy is the designing of a delivery system that can selectively deliver the anti-cancer agents to the target tissue with highly localised bioavailability, thereby achieving therapeutic efficacy while minimising side effects. Novel drug delivery systems, such as nano-sized carriers have shown to improve the treatment of cancer with reduced dosing frequency and lesser side effects [7-12]. Advancement in nanotechnology-based

*Address correspondence to this author at the Dreamz College of Pharmacy Khilra-Meramesit Sundernagar Mandi (Himachal Pradesh). India-175036; Tel/Fax: +911905248600; Cell: +919805815080; E-mail: zaki.manipal@gmail.com

delivery approaches and imaging techniques have a growing inter-est in nanomedicine based research [2, 13]. For example, nanoparti-cle formulations, such as DoxilTM and AbraxaneTM, have demon-strated clinical significance by increasing drug efficacy and dimin-ishing toxicity [14-15]. Need for nanotechnology based drugs and drug delivery devices will grow by more than 17% annually reach-ing an approximately $53 billion market in 2011 and an estimated $18 billion in 2014 [7]. Moreover, the US National Science Foun-dation predicts that half of the pharmaceutical industry product line will comprise of central nanotechnological design features by 2015. At least 12 nanotechnology based medicines are already approved and progressively more are seen entering the active developmental stages [8]. Hence, a steady succession of new nanomedicine, imag-ing, and diagnostic agents are anticipated to seek (and possibly gain) regulatory approvals and subsequent access to human use. In recent years, theranostic metallic nanoparticles (TMNPs) have shown potential application in field of magnetic resonance imaging (MRI) and colloidal mediators for cancer magnetic hyperthermia [16]. Nanotechnology based imaging and therapy has been investi-gated independently and their understanding has now evolved to a point enabling the birth of theranostics agent. The term ‘theranos-tics’ was coined about a decade ago and was first used to describe diagnostic tests developed to guide personalised therapies [17]. It may be defined as the combination of therapeutic and diagnostic agents on a single platform i.e. the development of theranostic nanoparticles (TNPs) that may simultaneously monitor and treat disease [18]. Here diagnostic means those agents which provide enhanced visibility of specific tissues by increasing the signal to noise ratio relative to surrounding tissues and provide a quick, high-fidelity snapshot of the living system [18]. Theranostic agent en-ables an entirely new category of clinical solution for oncological disorders, permitting early recognition of disease through the use of contrast agents combined with existing imaging modalities fol-lowed by tailored release of therapeutic agent. Advantage with the use of metallic nanoparticle based theranostic system in cancer therapeutics includes: (1) tumour targeting ligands, that bind to a particular tumour cell and are capable of sequestering anticancer drugs exclusively within tumour, thus reducing the accumulation of the drugs in healthy tissues, (2) Large surface to volume ratio, that provides opportunity for surface modification with improved cell

clinical application directed nanotechnology; toxicity concerns related with MNPs are also discussed in element.

Recent Advancement and Toxicity Concern Current Pharmaceutical Design, 2011, Vol. 17, No. 18 1835

entry, (3) Protection of the therapeutic agent from the biological milieu, (5) Improved bioavailability of the anticancer agent [19-24], (6) Additionally, MNPs can detect and attack the heterogeneous crowd of tumour cells, (7) High drug loading capacity [19, 22-24], (8) Delaying the drug resistance and (9) Increasing therapeutic in-dex through oncological site specific delivery. The multifunctional nature of MNPs is briefly represented in Fig. 1, which gives an outline for the legend and bioactive binding site.

MULTIFUNCTIONAL ACTIVITIES OF TMNPS Multifunctional TMNPs can combine specific targeting and therapeutic agent, and therefore it is designed for simultaneous imaging and targeting of cancer cells. A simple proposed diagram-matic presentation of multifunctional TMNP is given in Figure 1.TMNPs have been designed to concomitantly carry a drug, a ligand responsible for targeted delivery (for example, antibodies) and/or any contrast agent. This type of multifunctional character can be used to identify tumour cell, release specific antitumour drug and monitor the treatment efficacy in given time limit. Multifunctional nanoparticles can also help to overcome multiple drug resistance by delivering the antitumour drugs to tumour cells without launching the glycoprotein pump.

Site Specific Delivery and Biological Contemplation Conventionally, anticancer drugs are designed with relatively low molecular weight having an agreement between hydrophilic and lipophilic balance (HLB), hence allowing partition across the lipid membrane easily. Therefore, drugs rapidly get distributed throughout the body including the non-target tissue /organ and rap-idly getting metabolised by liver and/or excreted by the kidneys. Ideally, for effective drug targeting, it is essential that a drug should not eliminate quickly and the drug carrier should provide a pharma-cokinetic profile that will allow the drug to interact with its target [6, 25]. Therefore, in the designing of metallic nanoparticle, the painstaking lessons learned from the polymer based and liposomal drug delivery systems must be taken into consideration. Since, it is established now that upon intravenous injection the unprotected liposomal and polymer based drug delivery systems are rapidly cleared from the blood by reticuloendothelial system (RES) and

gets accumulated in the liver conditioning their rapid first pass me-tabolism from the systemic circulation followed by metabolic deg-radation and excretion. This is an imperative consideration while designing metallic nanoparticles intended for cancer therapeutics targeting cells located nearby the mononuclear phagocyte system [26-30]. The performance of nanoparticles inside the vascular com-partment is controlled by a complex array of factors such as shape, density, size distribution, surface characteristic, zeta potential, magnetism, reactivity and release characteristics of nanoparticles. All these factors control the flow properties of nanoparticles, bifur-cation in vascular compartment as well as modulation of circulation time and mode of entry into the cell [25, 31-36].

RES (Reticuloendothelial System) Escaping A major barrier that a drug delivery system must be able to avoid in the systemic circulation is the removal of delivery system or drug by phagocytic cells of Mononuclear Phagocytes System (MPS). Since the nanoparticles are usually taken up by the liver, spleen and other parts of the reticuloendothelial system (RES) de-pending on their surface characteristics and undergo opsonisation in the blood and clearance by RES [4, 18, 37-40], the nanoparticles, therefore should be designed to avoid these interactions and their possible clearance from the vascular compartment, particularly opsonisation process. Opsonization is the process by which a pathogen, foreign particle or particulate drug carrier is marked for ingestion and destruction by a phagocyte. It involves the binding of an opsonin i.e. antibody to the surface of foreign particle due to which phagocytes are attracted towards it. Finally, the foreign particle is engulfed and digested by lysosomes [31, 40-43]. For example, when normal colloidal gold nanoparticles were intrave-nously injected into a mouse, it was observed that 95% of gold nanoparticles were cleared from the vascular compartment within 10 minutes [44], indicating the need of suppression of opsonisation, avoiding MPS recognition and receptor mediated phagocytosis while the designing of metallic nanoparticle. A newer practical approach to avoid RES uptake of nanoparticles is the modification of nanoparticle surface. In such cases, increasing surface hydrophil-icity i.e. adding a hydrophilic polymer coat to the metallic nanopar-ticle carrier, will make them imperceptible to the RES and would

Figure 1. Structural outline of multifunctional TMNP representing possible carrier site for targeting ligand and therapeutic agent.

1836 Current Pharmaceutical Design, 2011, Vol. 17, No. 18 Akhter et al.

thus reduce opsonisation and suppress macrophage recognition. This coating is referred to as stealth. Most commonly used stealth agents for this purpose are polyethylene glycol (PEG), poloxamers, carbopols and block copolymers [45-46]. It has been established for PEG that it contains high concentration of hydrated groups which sterically inhibit the interaction with blood-born opsonin [25]. In-travenous injections of sterically stabilised nanoparticles result in prolonged circulation time and accumulation in tumour [47-54]. Another factor affecting the opsonisation process are physico-chemical properties of nanoparticles like surface characteristics (size, surface charge), bio-distribution and residence times of these particles in vivo [44, 55-61]. It is commonly seen that neutral sys-tems tend to remain longer in blood circulation but their charged counterparts are cleared by RES readily [2, 44]. Furthermore, parti-cle size of around 1-2 micron undergoes phagocytosis and higher size i.e. around 6 micron is trapped in pulmonary capillaries [25]. Therefore, it is postulated that, to avoid clearance by RES, the MNPs should be formulated of not more than 100nm in size and should have sterically stabilised neutral surface.

Passive Tumour Targeting In passive targeting, the bio-distribution of MNPs is mediated by their physiological conditions. In such approaches, we take the advantage of pathological condition of tumour cells (pH condition, temperature and specific enzymes) to allow accumulation of drug carrier at the target site [62]. Enzymes such as alkaline phosphatase and plasmins are present in higher amounts at the tumour site. When the volume of tumour becomes 2mm3, it becomes diffusion limited, which is overcome by angiogenesis (growth of new capil-lary in cell/ tissue) [63]. In tumour cells, the characteristic features of angiogenesis are its aberrant tortuosity and abnormalities in basement membrane [64]. Since, tumour cells generally lack the well-defined lymphatic system as a result of which the interstitial pressure is maximum at the centre of tumour. These incomplete vasculatures of tumours result in leaky blood vessels (capillary). This hyper permeability of tumour vasculature is a key feature in passive targeting of drug carrier [65-69]. Due to leaky vasculature and poor lymphatic drainage, the drug carriers get trapped in the tumour vasculature and release the loaded drug. This process is

known as Enhanced Permeation and Retention effect (EPR effect) [57, 69-70]. Figure 2 represents the capillary with Enhanced Per-meability and EPR effect at tumour sites. However, this EPR is associated with accumulation of nanoparticles in blood capillaries near to healthy cell and can lead to recognition by MPS [71].

Active Tumour Targeting Since passive targeting does not necessarily assure the internali-sation of nanoparticles by the targeted cell, therefore nanoparticles are modified with molecular targeting ligands for active targeting. Active targeting of TMNPs involves the interaction between pe-ripherally conjugated targeting moiety and a corresponding receptor to facilitate the targeting of a carrier to a specific malignant cell [72-74]. It is now established that tumour cells over-express certain specific surface receptors, aptamers [75-76], proteins and antibodies [7, 77-79] which can be targeted for effective delivery of anticancer agents [80], facilitating the active targeting of nanoparticles. The bioconjugation of ligands, like monoclonal antibodies, proteins or peptides to the nanocarrier surface has been exploited in many cases for the purpose of concentrating therapeutic action to specific tumour cell through nanoparticles [2, 7, 23, 27, 81-83]. These ligands (antibodies, saccharides, aptamers, hormones, lectin) bind to their specific receptor on the cellular surface and trigger the in-ternalisation process of drug delivery so that the anticancer drugs act on cellular organelles (e.g., mitochondria, microtubules, nu-cleus, etc.) by means of receptor mediated endocytosis (RME) (Fig-ure 3). Several over-expressed growth factor receptors have been used for selectively targeting the cancer cell. The description of many of these targets has been reviewed [84-85].

HER2 Targeting The Human Epidermal growth factor Receptor 2 (HER2) is a transmembrane tyrosine kinase receptor which belongs to the ErbB protein family and is expressed or over expressed in 25-35% breast carcinoma cells [86]. An essential requirement for the activation of its signal pathway is the receptor dimerisation. This receptor di-merisation is responsible for the transactivation of tyrosine kinase [87-89]. Monoclonal antibodies targeting the HER2 receptor can be used to suppress this dimerisation, hence, blocking the activation of

Figure 2. Figure showing the capillary with Enhanced Permeability at tumour sites which facilitate escape of drug loaded metallic nanoparticle from the circu-lation EPR, enhanced permeability and retention in tumour cell.

Recent Advancement and Toxicity Concern Current Pharmaceutical Design, 2011, Vol. 17, No. 18 1837

HER2 and its signalling pathway. FDA-approved monoclonal anti-body, Herceptin is a popular targeting agent for the HER2 receptor for breast and ovarian cancer and has a major positive impact on the treatment of breast cancer [90-91]. Huh et al. reported the specific delivery of Herceptin-targeted MNPs to the cells expressing the HER2 cancer marker in vivo [92]. Unfortunately, this antibody can target the normal cardiac cells undergoing repair induced by the actions of a common anticancer agent, doxorubicin. Akira et al.constructed the anti-HER2 immunoliposomes containing MNPs, which act as tumour-targeting vehicles, combining anti-HER2 anti-body therapy with hyperthermia. They reported that MNPs loaded with anti-HER2 immunoliposomes exerts HER2 mediated antipro-liferative effects on SKBr3 breast cancer in vitro resulting in strong cytotoxic effects [93]. Chen et al. investigated the gold nanoshell-based TMNPs for targeting and photothermal therapy of HER2 over-expressing and drug resistant ovarian cancer OVCAR3 cells in in-vitro. They synthesised the nanocomplexes for simultaneous fluorescence optical imaging and magnetic resonance imaging agent. They reported that when nanocomplexes were targeted to OVCAR3 cells and irradiated with near infra-red (NIR) laser, selec-tive destruction of cancer cells through photothermal ablation was observed. Thus, these nanocomplexes are highly promising for image-guided photothermal therapy of ovarian cancer, as well as other HER2-overexpressing cancers [94]. In a similar fashion, Day et al. developed NIR resonate gold-gold-sulphide nanoparticles theranostic function as a dual contrast and therapeutic agents for cancer management via multiphoton microscopy followed by higher intensity photoablation. In conjugation with anti-HER2 anti-bodies, these nanoparticles specifically bind SK-BR-3 breast carci-noma cells that express the HER2 receptor, thus enabling cell to be imaged via multiphoton microscopy and induce thermal damage to the tumour cells, producing extensive membrane blubbing, leading to cell death in a fraction of a minute [95].

Tumour Angiogenesis-Associated Targeting The term angiogenesis is widely used when referring to the process of blood vessel formation. More specifically, angiogenesis denotes the formation of new capillaries from pre-existing ones. Tumour angiogenesis essentially necessitates the same sequence of events as physiological angiogenesis, however, the latter proceeds in an uncontrolled and disproportionate manner giving rise to leaky and tortuous blood vessels that are in a constant state of inflamma-tion [96-97]. It is mainly because of upregulation of angiogenic cytokines and growth factors, notably the vascular endothelial cell growth factor (VEGF) and Angiopoietin (Ang) families, as well as integrins [98-99]. Tumour cell secretes a variety of potent pro-angiogenic factors including vascular endothelial growth factors (VEGF) A, B, C, respectively, fibroblast growth factor-2 and an-giopoetin-2, epidermal growth factor (EGF) [100-102]. These molecules, as said earlier, are considered as potential cancer thera-peutic targets. Nowadays, the targeting of angiogenesis has become an attractive area of focus in targeting of tumour cells for therapeu-tics and imaging delivery. Angiogenesis appears to be one of the most crucial steps in metastatic capabilities of tumours. Thus, anti-angiogenesis approach has emerged as an effective step in limiting oncogenesis with the transformed endothelial cells of the neovascu-lature as the main targets [100]. As reported in literature, integrin �v�3 is a heterodimer and is upregulated in most of the oncological disorders. It is hence, not surprising that these molecules are often targeted in both experimental and clinical malignancy settings [103-104]. The tumour vessels have increased permeability due to aber-rant angiogenesis, thus allowing nanoparticles to passively ex-travasate into the tumour sites through the EPR effect. Nanoparti-cle- mediated targeting of the tumour vasculature in anti-angiogenic therapy has been achieved by targeting the VEGF receptors

Figure 3. Schematic diagram of intracellular uptake of drug through receptor mediated endocytosis with MNPs.

1838 Current Pharmaceutical Design, 2011, Vol. 17, No. 18 Akhter et al.

(VEGFR), �v�3 integrins and other angiogenic factors. Integrin �v�3has been by far, the most widely used targeting moiety on nanopar-ticles due to its pleiotropic upregulation in a variety of cancer set-tings, some of which have been successfully translated into several clinical trials [105-109]. Anderson et al. demonstrated the site di-rected contrast enhancement of angiogenic vessels in vivo in rabbit corneal micropocket model. The targeted contrast agent consists of Gd-perfluorocarbon nanoparticles linked to �v�3 integrin antibody DM101. They reported that the antibody-targeted agent enhances MR signal intensity in the capillary bed in a corneal micropocket model of angiogenesis and is selectively retained within the angio-genic region via specific interaction with the �v�3 epitope [110]. Schmieder et al. demonstrated the detection of �v�3 integrin expres-sion on neovasculature induced by nascent melanoma xenografts via �v�3 -targeted paramagnetic nanoparticles. They concluded that this technique may be employed to noninvasively detect very small regions of angiogenesis associated with nascent melanoma tumours and to phenotype and stage early melanoma in a clinical setting [111]. Mukherjee et al. reported that gold nanoparticles bind spe-cifically to heparin-binding growth factors, resulting in inhibition of endothelial cell proliferation in vitro and VEGF-induced permeabil-ity and angiogenesis in vivo [112]. In a similar investigation, Arvizo et al. described the mechanism of anti-angiogenic property of gold nanoparticles, emphasising that gold nanoparticles selectively dis-rupted the function of pro-angiogenic heparin-binding growth fac-tors HB-GFs in a size-dependent manner in vitro. They also demon-strated that the inhibitory effects of gold nanoparticles are due to the change in HB-GFs conformation/configuration by the nanopar-ticles, whereas the conformation of non-HB-GFs remains unaf-fected [113]. Gurunathan et al. reported the anti-angiogenic proper-ties of silver nanoparticles and demonstrated that these agents could inhibit VEGF-induced cell proliferation, migration and formation of new blood microvessels in vivo. Furthermore, their results indicated that silver nanoparticles could target the activation of PI3K/ Akt signalling pathways, thus leading to the inhibitory effect of angio-genesis [114]. Chen et al. reported that dextran coated iron-oxide nanoparticles, which were conjugated with radiolabelied (131I) anti-VEGF monoclonal antibody, significantly increased imaging reso-lution as well as destruction of liver cancer in mice [115]. Maeng etal. developed doxorubicin-loaded, folate-receptor targeted super-paramagnetic iron oxide nanoparticles that significantly inhibited tumour growth and surprisingly, did not increase systemic cytotox-icity associated with heavy metals, most likely due to their selective localisation in tumours [116]. Zhang et al. developed the ultrasmall superparamagnetic iron oxide particles (USPIO) coated with 3-aminopropyltrimethoxysilane (APTMS) and conjugated with Arg-Gly-Asp (RGD) peptides, and investigated the in vitro and in vivobinding ability to with �v�3 integrin on endothelial cells [117]. They reported that RGD-coupled, APTMS-coated USPIO efficiently label �v�3 integrins expressed on endothelial cells. Furthermore, these molecular MR imaging probes are capable of distinguishing tumours differing in the degree of �v�3 integrin expression and in their angiogenesis profile. Thus, from above discussion, we can conclude that the advent of nanotechnology provides a massive prospective for devising increasingly novel anti-angiogenic thera-peutics that can eventually be translated from bench to bed-side.

Folate Receptor Targeting Folic acid is required for essential cell functions such as synthe-sis of nucleotides (purine and pyrimidines). Folate receptors (high affinity membrane folate-binding protein) over expression has fre-quently been observed in cancers like, ovarian, lung, brain, head, neck, renal cell and breast cancers, while generally being absent in normal tissues with the exception of placenta and choroid plexus [118-122]. Normal cells transport a reduced folate across their membranes but not the transport folate conjugates, while the malig-

nant cells transport folate conjugates through the folate receptor. Therefore, folate receptors are widely used for targeting as they are easy to conjugate, stable and retain high affinity [120-123]. A pro-posed diagrammatical model for the cellular uptake of drug conju-gates targeted to the folate receptor is illustrated in Figure 4.

RECENT ADVANCEMENTS WITH SPECIAL REFERENCE TO PATENTS There is an extensive chronological support for the application of MNPs in biological system for the diagnostic and therapeutic rationales. However, the use of metallic nanoparticle in cancer therapeutics has recently been reported by many nanotechnologists [83, 124].

Patents on Metallic Nanoparticle There are a great number of researches being carried out on possible cancer therapeutics and imaging techniques which can be conveniently amalgamated with the metallic nanoparticle/device for the early detection of tumour cells, imaging of cancer cells and targeted drug delivery to the cancer cells. Recently, considerable numbers of patents are generated for TMNPs. US Patent number 6,165440 assigned to Board of reagents, the University of Texas; system describes interaction of electromagnetic pulse and ultrasonic radiation with metallic nanoparticle for enhancement of drug deliv-ery in solid tumours [125]. Patent claimed that nanoparticles at-tached to the antibodies targeted against antigens on the surface of tumour cells. Cavitations induced by ultrasonic waves result in perforation of cancer cell membranes, and therefore, provide en-hanced delivery of therapeutic agents from blood into cancer cells. US Patent number No 6,689,338, assigned to the board of reagent for Oklahoma State University describes the diagnosis and treatment of cancer by bioconjugates of nanoparticles as radiophar-maceuticals for use in combination with radioimmunotherapy and radioimmunodection [126]. Their claim indicates the nanoparticles covalently linked to a biological vector with radioactive metal ion (metal sulphide or metal oxide). This biological vector may be monoclonal antibody or fragment of antibody. This bioconjugates has utility as an effective radiopharmaceuticals to deliver a radio-labeled nanoparticle in cancer treatment. US Patent number 67485 discloses the fabrication of semiconductor nanoparticles with a protein template [127]. The semiconductors enlisted in the claims are cadmium selenide, cadmium telluride, zinc selenide, zinc sul-phide and zinc telluride. Such class of semi-conductor particles are known as Quantum Dots. They claimed that these types of particles present the opportunity to construct multiple-colour luminescent system. Magnetic resonance imaging is an appealing non-invasive approach for early cancer diagnosis and therapy. Super-magnetic property of iron oxide have been found effective in nano sized molecules and can better serve as a contrast enhancement agent for magnetic resonance imaging. US Patent number 216239 describes the fabrication and evaluation of nanoparticles having a core com-prising of a magnetic material including a targeting, diagnostic, and therapeutic agent which can be used to detect and treat tumour cells [128]. US Patent number 255403 describes the various composi-tions, methods and devices for the production of fluorescent nanoparticles, which can function as markers, indicators and light sources [129]. They claimed that fluorescent nanoparticles can be formed from fluoropore core surrounded by a biocompatible shell and these fluorescent nanoparticles can be delivered to the tissue for identification and location of the tumour cell and to provide thera-peutic effect. US Patent number 3177868 describes the composition and methods for the treatment of cancer using a bioconjugated nanoparticle comprising a biocompatible quantum dot conjugated to a target moiety [130]. They claimed that the quantum dot, upon excitation by soft X-rays, emit electromagnetic radiation of ultra-violet region, thereby allowing the disruption of the DNA found in cancer cells. US Patent number 136580 assigned to Boise State University describes the preferential killing of cancer cells and

Recent Advancement and Toxicity Concern Current Pharmaceutical Design, 2011, Vol. 17, No. 18 1839

activated human cells using zinc oxide nanoparticle. They also describe the response of normal human cells to zinc oxide nanopar-ticle under different signalling environments and its comparison against response to the cancerous cells [131]. They claimed that zinc oxide nanoparticle exhibit a strong ability to kill cancerous T cells. US Patent No.6,767, 635 assigned to Biomedical Apherse system Gabh (Jena : DE) invention relates to MNPs, their produc-tion, and their use [132]. The object of the invention is to provide nanoparticles capable of specifically forming bonds to intracellular biomacromolecules even in the intracellular region of cells, so that separation is possible by exposure to an exterior magnetic field. This is accomplished by means of MNPs having biochemical activ-ity, consisting of a metallic core particle and an envelope layer fixed to the core particle.

Theranostic Application of MNPs Escalating technology in nanoscience has provided unparalleled opportunities for the development of theranostic agents for detect-ing and treating cancer. As discussed earlier, metallic nanoparticles having the ability to treat as well as diagnose the disease can be termed as theranostics [133]. Advance in the development of metal-lic nanoparticles has significantly impacted the development of systems both as therapeutic and diagnostic agents. MNPs are attrac-tive options for theranostic application due to their stability on which desired multiple functionalities can be fabricated. For exam-ple, light activated theranostic nanoparticles have been reported for imaging and treatment of brain tumour [134]. Recently, it was re-ported that iron nanoparticles have been used as theranostic agents with specific application as contrasting agents for magnetic reso-nance imaging (MRI) and magnetically targeted drug delivery to the tumour cells [135-141]. There are mainly two types of iron oxide nanoparticle that have been reported as imaging agents [135,141], superparamagnetic iron oxide (SPIOs) and ultra-small superparamagnetic iron oxide (USPIOs). Major advantages associ-ated with SPIO nanoparticles are the biocompatible and biodegrad-able properties of iron, which can be recycled via normal biochemi-cal pathway for iron metabolism [142]. Hepatic imaging was the

first application reported on these MNPs. In liver imaging, it seems that normal hepatocytes take up SPIOs, resulting in darkening of image. However, cancerous cells are not able to take up the SPIOs, thus resulting in bright spot in tumour cell [143-146]. There are many reports in which SPIOs have been used in combination with monoclonal antibodies to detect different oncological cases to target some cancer cell associated molecules [79, 146-149]. Recently, peptide conjugated SPIOs were successfully tried for the targeting of over expressed transferrin and pancreatic receptors and synapto-tagmin conjugated SPIOs enabled imaging of the cells undergoing apoptosis during chemotherapy [150-151]. Currently, several anti-cancer drug such as doxorubicin, methotrexate and paclitaxel have been using MNPs as a carrier [152-155]. Zhang et al, developed methotrexate bonded MNPs through which the bound methotrexate (MTX) got internalised into tumour cell along with the carrier par-ticle and accumulated into the lysosome. The acidic environment there cleaved the attachment of MTX from the MNPs surface re-sulting in its controlled and site specific delivery, which is illus-trated schematically in Figure 5. This novel approach can be further utilised for tumour targeting with theranostic applications [156]. Similarly, Liang et al, has demonstrated the ability of radionuclides containing SPIOs to specifically induce cell death in liver cells [157]. In another investigation, Ross et al. successfully confirmed the therapeutic application of SPIOs through antibody Herceptin conjugated SPIOs to target the HER2/neu receptor in early stages of breast cancer [158]. Recently, Medarova et al. demonstrated the in vivo transfection of siRNA and simultaneous imaging of its local-isation in the tumour cell by magnetic resonance imaging and NIR in-vivo optical imaging using MNPs. They reported the tracking of tumour uptake of nanoparticle and optical imaging in tumour mod-els mediated via EPR (Fig. 2) [159]. As discussed earlier, magnetic nanoparticle-based hyperthermia magnetic resonance imaging (MRI) technique has emerged as a promising therapeutic approach to cancer treatment. For hyper-thermia as well as MRI, typically paramagnetic, superparamagnetic as well as ferromagnetic nanoparticle particles have been explored extensively. Manganese was one of the earliest reported examples

Figure 4. A Schematic illustration of the folate receptor mediated endocytosis for the transport of metallic nanoparticle.

1840 Current Pharmaceutical Design, 2011, Vol. 17, No. 18 Akhter et al.

of paramagnetic contrast material for MRI (magnetic resonance imaging) because of its efficient positive contrast enhancement. The development of manganese-based nanoparticles, particularly man-ganese oxides, as MRI contrast agents is ever-increasing [160-162]. Huang et al. reported the synthesis of nanospheres, nanoplates and nanocubes of Mn3O4, which exhibited paramagnetic behaviour at room temperature on the basis of superconducting quantum inter-ference device (SQUID) measurements [163]. Yang et al. synthe-sised the monodisperse silica coated manganese oxide nanoparticle and aminated through silanisation. The amine-functionalised core shell nanoparticles enable the covalent conjugation of a fluorescent dye, Rhodamine B isothiocyanate (RBITC), and folate (FA) on their surface. After magnetic resonance imaging studies, they re-ported that Mn3O4-SiO2 (RBITC)-FA nanocomposites can specifi-cally target cancer cells overexpressing FA receptors, thus the find-ings from the study suggests that silica coated Mn3O4 core shell nanoparticles could be used as a platform for bimodal imaging (MR and fluorescence) in various biological systems [164]. Similarly, Mathew et al. developed a novel folic acid conjugated car-boxymethyl chitosan coordinated to manganese doped zinc sulphide quantum dot nanoparticle loaded with 5-flouro uracil. They demon-strated the imaging, specific targeting and cytotoxicity of drug loaded nanoparticle and they suggested that the bright and stable luminescence of quantum dots can be used to image the drug carrier in cancer cells without affecting their metabolic activity and mor-phology [165]. Manganese containing nanoscale metal organic frameworks (NMOFs) with controllable morphologies were synthe-sised by Taylor et al. They reported that the cell targeting mole-cules on the Mn NMOFs enhanced their delivery to cancer cells allowing target specific MRI in vitro [166]. Huang et al. demon-strated the synthesis of manganese oxide nanoparticles with promi-nent MRI contrast in a glioblast xenograft model and they reported that the developed nanoparticles accumulated efficiently in the tumour area to effectively signal alteration [167]. In yet another investigation, multifunctional hollow manganese oxide nanoparticle were synthesised by Bae et al, using 3,4-dihydroxy-L-phenylalanine (DOPA) as an adhesive moiety for

cancer targeted delivery of therapeutic siRNA and simultaneous diagnosis via magnetic resonance imaging (MRI). These nanoparti-cles were subsequently functionalised with a therapeutic mono-clonal antibody, Herceptin, to selectively target cancer cells. They reported that the surface functionalised hollow manganese oxide nanoparticles enabled the targeted detection of cancer cells in T1-weighted MRI as well as the efficient intracellular delivery of siRNA for cell-specific gene silencing [168]. Recently Geszke et al.demonstrated a novel approach for the synthesis of water soluble manganese doped core/shell Zn/ZnS quantum dots. They further demonstrated folate receptor-mediated delivery of folic acid-conjugated ZnS: Mn/ZnS QDs using photon confocal microscopy. They reported that due to their low cytotoxicity and fair stability, these QDs should play an important role in a variety of nanocrystal based biomedical applications in the near future [169].

Gold (Au) Nanoparticles Among MNPs mediated oncological drug delivery, gold MNPs have emerged as promising carriers. Gold and its compounds have long been used as medicinal agents throughout the history of civili-sation. Gold nonomaterials have been extensively studied for poten-tial applications in the emerging and highly interdisciplinary field of nanotechnology [170-172]. Gold nanoparticles have been used to deliver antitumour agents such as tumour necrosis factor (TNF) or paclitaxel at the site of the tumour by the enhanced permeability and retention (EPR) effect [173]. Synthesis of Au NPs has been well established in the forms of spheres, rods and tubes. Their mor-phology control is important as it greatly influences the physical properties of the products and in turn affects their role as therapeu-tic and imaging probes [174]. Two characteristics of gold nanoma-terials make them particularly suitable for therapeutic applications: 1) antibodies and other bioactive molecules can be easily conju-gated to the surface of gold nanomaterials, and 2) gold nanomateri-als have absorption efficiencies that are 4 to 5 times greater than conventional photothermal dyes and are not affected by pho-tobleaching. Besides the physical and chemical properties of gold nanoparticles, their unique optical properties make them particu-

Figure 5. Schematic representation of theranostic molecules conjugation to TMNPs as targeted tumour imaging, sensing and therapeutics.

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larly attractive tools for cancer detection and therapy [175]. Once gold nanomaterials are adequately accumulated at the target site, they are activated via the absorption of irradiation of an appropriate wavelength, and thereby cause irreversible thermal cellular destruc-tion [176-178]. This property can provide an opportunity for thera-peutic treatment in deeper tissues. They have several attractive advantages like easy tailoring with ligand for tumour specific tar-geting, biocompatibility and stability [179-180]. Metallic gold MNPs have been extensively studied for potential application in targeted tumour cell drug delivery [141, 180-185]. The first report on this application was reported by Hirish and co-worker in 2003 in hyperthermal therapy of tumour cell [176]. Later on, in 2005, Loo et al. reported the HER2 antibody conjugated gold nanoshells to target breast carcinoma [186]. Optical properties of gold nanoparticles incubated in single living cancerous and noncancerous cells have been compared for different incubation methods and studied by Sayed et al. They re-ported that scattering images and the absorption spectra recorded from anti-EGFR antibody conjugated gold nanoparticles incubated with cancerous and noncancerous cells are very different and offer potential techniques for cancer diagnostics [187]. In a similar study, Hainfeld demonstrated that intravenously administered small gold nanoparticles can deliver high levels of gold to tumours (up to 0.7% by weight) with specificity and thereby improve x-ray therapy. They reported that gold nanoparticle were apparently non-toxic to mice and were largely cleared from body via kidney, thus novel use of gold nanoparticle permitted achievement of high metal content in the tumour necessary for significantly high Z radio enhancement [171]. One of the recent advancement in establishing the MNPs in cancer therapy is a development of gold nanoshell formulation. Metal nanoshell is a composite spherical nanoparticle consisting of a dielectric core, surrounded by a metallic shell which may be pref-erably gold. Nanoshell possesses unique optical and physicochemi-cal properties for imaging and therapeutics. An example of this is AuroLaseTM, which is under the phase III of clinical trials for photo-thermal tumour ablation. On intravenous administration, these MNPs get accumulated in cancer cell which is monitored by magnetic resonance imaging along with exposure to NIR that cause the thermal ablation [177, 188]. A clinical trial using gold-silica nanoshell hyperthermia following NIR light exposure has been initiated for patients with oropharyngeal malignancies is reported by Arnfield et al. They demonstrated that photothermal therapy can treat superficial tumours [189]. In another study Diagaradjane et al.designed gold nanoshell and immobilised the thiolated poly poly-ethylene glycol on the surface of the gold nanoshells to enhance circulation. After the injection of gold nanoshells in human colorec-tal cancer (HCT 116)-bearing mice via the tail vein, localised hy-perthermia was carried out. They stated that large necrotic regions were observed in the tumour after this thermoradiotherapy. Thus, optically activated gold nanoshells can be a novel and minimally invasive method to induce mild temperature hyperthermia treatment [190]. An interesting report by Chang et al. states that after absorp-tion of NIR to IR photon, gold nanorods can be transformed to spherical nanoparticles. This interesting and unique property may affect the conjugation of biomolecules to the surface of nanorods, and can be used for the controlled release of biomolecules (gene) [191]. Gobin et al. demonstrated the NIR absorptions of nanoshells that have been used to heat nanoshells within cancer cells and within the tumours, and have been shown to be efficacious in the treatment of superficial tumours [192]. Modification in the shape of gold MNPs to nanorods seems to be advantageous as scattering wavelength changes from visible to near infra-red (NIR) region due to which, the gold nanorods can be used as contrast agents for dual molecular imaging [93]. As follows-up work by Hainfed et al, who vividly described the combination of gold nanoparticle followed by X-ray treatment reduces the size of tumour cells in mice [171]. Moreover, Haung et al. reported that cellular uptake of gold nano-rods was increased two-fold in malignant cells when gold nanopar-

ticles were conjugated with antibody to target antiepidermal growth factor receptor [193]. It is the optical properties of nanorods that form the basis for biomedical applications of nanorods entirely due to its optical properties. Gold nanoparticles strongly absorb the light in the region of 700-1200nm which can be explained by localised surface Plasmon resonance (LSPR) phenomenon [194]. Thus, they can be made in different shapes in order that they may preferen-tially absorb light in this region. By changing the shape of gold nanoparticles to nanorods, the absorption and scattering wavelength changes from the visible to the NIR region and their absorption and scattering cross sections also increase [179]. For example, Huang etal. reported the synthesis of antiepidermal growth factor receptor (anti-EGFR) antibody-conjugated gold nanorods. They emphasised that when nanorods were incubated with tumour cells, their cellular uptake increased by twice than non-malignant cells due to targeting of EGFR on the malignant cell surface. Further, along with the irradiation of NIR laser, photothermal destruction was observed in both malignant and nonmalignant cells, and interestingly, increased uptake of nanorods by overexpressing EGFR malignant cells re-duces the laser energy required to cause death in normal cells [193]. Kwano et al. suggested that gold nanorods can be coated with a thermo-sensitive shell, in which drug molecules can be dispersed, and subsequently, irradiating the gold nanorods with NIR light generating heat which will induce phase transition from gel to sol and this transition can be exploited to aid drug release [195]. Hirsch reported that average temperature of tumour cell treated with gold nanoshell and NIR (Near infra red) light increased up to 38ºC at a depth of 1inch beneath the dermal surface and irreversible pho-tothermal destruction was observed, confined to the tumour area. Similarly in another study, gold nanoshells were conjugated with HER2 antibody to target the breast carcinoma cells. In this study, it was revealed that NIR irradiation caused a rise in the temperature of the target cells between 40ºC to 50ºC, which selectively destructed the carcinomas [176]. Another advantage associated with this ther-apy was that the survival rate of mice treated with HER2 conju-gated gold nanoshells and NIR irradiation was excellent as com-pared to non-specific antibodies or NIR alone [186]. Pissuwan et al.demonstrated the modification of gold nanorods targeted macro-phage cells. In their findings, they used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as their model system and a 5mW solid state diode laser as energy source. They reported that the exposure of the cells labelled with gold nanorods resulted in 81% of cell death compared to only 0.9% in control, non labelled [196]. Niidome et al. carried out the synthesis of gold nanorods targeted delivery systems for tumours. In this investigation, a peptide substrate for urokinase-type plasminogen activator (uPA), expressed specifically on malignant cells was inserted between the PEG chain and the surface of gold nanorods. They reported that PEG-peptide- modified gold nanorods showed higher accumulation in tumour cells than the control after they were injected intravenously into tumour-bearing mice, thus, PEG-peptide-modified gold nanorods, which respond to uPA activ-ity is expected to be a powerful tool for tumour bio-imaging and photothermal tumour therapy [197]. Wang et al. fabricated the dual-mode imaging probe for targeting cancer cells based on mesoporous silica coated gold nanorods. Targeting performance of this probe was investigated by conjugation of folic acid on the outer surface of the probe as a targeting ligand. They reported that as compared to the traditional imaging probes, this new type of nanoprobe had a great potential for multiplexed imaging in living cells, which can be easily realised by using fluorescence and Raman scattering SERS signals [198].

Composite Nanoparticle Composite materials combine the properties of multiple materials into a single material system. Nowadays, assembly of several functional building blocks into one multifunctional system is a common practice in nanotechnology intended for therapeutic

1842 Current Pharmaceutical Design, 2011, Vol. 17, No. 18 Akhter et al.

practice. Functionalising nanoparticles with biocompatible poly-mers and natural or rationally designed biomolecules offers a route towards engineered responsive and multifunctional composite sys-tems for cancer therapy. Although only a few such innovations have achieved success, nanocomposite materials based on functionalised metal and semiconductor nanoparticles hold an appreciable capabil-ity to transform the way cancer is diagnosed and treated. Hu et al. demonstrated the production and in vitro cancer activity of ta-moxifen-loaded magnetite/poly(l-lactic acid) composite nanoparti-cles (TMCN against MCF-7 breast cancer cells. They reported that after the incubation of MCF-7 with TMCN, approximately 80% of these cells were killed [199]. Zhao et al. reported the synthesis of Fe3O4-chitosan magnetic composite nanoparticles. The potential of Fe3O4-chitosan composite nanoparticles was evaluated for localised hyperthermia treatment of cancers. Upon exposure to the magnetic field for around half an hour, the temperatures of physiological saline suspensions containing Fe3O4-chitosan composite nanoparti-cles were 97.5 ºC and 53.7 ºC, respectively. They concluded that these types of composite nanoparticles would be a good thermo seed for the localised hyperthermia cancer therapy [201]. Ren et al.reported the preparation of magnetic composite nanoparticles by altering the block and length of poly-D,L-lactide-co-polyethylene glycol (PLA-PEG) copolymer. The prepared Fe3O4/PLA-PEG par-ticles with magnetic properties and biodegradable properties would provide useful applications in drug targeting and for controlled drug release [201]. Das et al. reported a nanoformulation of curcumin with a tripolymeric composite for drug delivery to the cancer cells. The composite nanoparticles (CNPs) were prepared by using three biocompatible polymers viz. alginate (ALG), chitosan (CS), and pluronic by ionotropic pre-gelation followed by polycationic cross-linking. Cellular internalization of curcumin loaded composite nanoparticles was confirmed from green fluorescence inside the HeLa cells [202]. Magnetic nanoparticles embedded polylactide-co-glycolide matrixes (PLGA-MNPs) as a dual drug delivery and im-aging system, capable of encapsulating both hydrophilic and hydro-phobic drugs, was synthesised by Sing et al. For targeted delivery of drugs, targeting ligands such as Herceptin was used, and such a conjugated system demonstrated enhanced cellular uptake and an augmented synergistic effect in an in vitro system when compared with native drugs. They reported that PLGA-MNPs showed a better contrast effect than commercial contrast agents due to higher T(2) relaxivity with a blood circulation half-life of about 47 min in the rat model [203]. Anti-cancer drug doxorubicin (dox) loaded mag-netite (Fe3O4)-poly-n-(isopropylacrylamide) (PNIPAM) composite MNPs relevant to multimodal cancer therapy were synthesised and studied by Purushotham et al. They explained that the composite particles loaded with 4.15 wt. % dox exhibited excellent heating properties as well as simultaneous drug release. Their work sug-gests that these dox-loaded polymer-coated MNPs show excellent in vitro hyperthermia and drug release behaviour with the ability to release drugs in the presence of magnetic field, and also realised their potential to act as agents for combined targeting, hyperthermia and controlled drug release treatment of cancer [204]. Chao et al.tested the in vitro cytotoxicity of Gold-Magnetic nanoparticle loaded with doxorubicin (Dox-GoldMag) combined with an exter-nal magnetic field on HepG2 malignant tumour cells. They reported that the Dox-GoldMag group combined with a magnetic field has increased inhibition rate for the HepG2 cell line. These results indi-cated that GoldMag nanoparticles loaded with doxorubicin com-bined with a permanent magnetic field are more cytotoxic and could be a potential targeted drug delivery system [205].

Quantum Dots Nowadays, semiconductor nanoparticles (composed of metals from the groups II-VI or III-V of the periodic table), called as Quantum dots (Qds) have been increasingly applied as imaging and labelling probe in cancer therapeutics [143,-206-208]. Most com-monly used compounds from these groups are Indium arsenide

(InAs) Cadmium telluride (CdTe) and cadmium selenide (CdSe). Qds are becoming an important class of theranostic agents; because they possess unique optical properties like high quantum yield, resistance to chemical modification and intrinsic fluorescence emis-sion spectra due to which quanta particle become highly capable of sensing and releasing the anticancer drugs at the desired site. Addi-tionally, by regulating the size and composition of Qds, their optical properties can be adjusted. For example, the quantum dot-aptamer-doxorubicin conjugate as a targeted cancer therapeutics and sensing system by functionalising surface of fluorescent Qd with A10 RNA aptamer was demonstrated by Bagalkot et al. The Qds were capable of differential uptake and imaging prostate cancer that expresses the prostate specific membrane antigen [209]. Gao et al. demonstrated multifunctional Qd probes for simultaneous tumour targeting and imaging in living animal models [208]. They reported that the in vivo study of human prostate cancer growing in mice indicated that the Qd probes accumulated by the cancer cells by both enhanced permeability and retention at tumour sites and through antibody binding to cancer specific cell surface biomarkers. In yet another finding, Yang et al. successfully developed the NIR Qd conjugated with cell penetrating peptide and labelled oral squamous carcinoma cells with Qd conjugates for visual in vivo imaging. Reports ex-posed that, due to the strong penetration power of NIR, Qd exhibits great promise for early diagnosis and individualised treatment of oral cancers [210]. In another finding, Zhiming et al. used the poly-amidoamine dendrimer to modify quantum dots to improve their water solubility. These dendrimer modified Qd can conjugate with DNA aptamer and specifically target U251 human glioblastoma cells [211]. Recently, IGF-1R antibody (a transmembrane tyrosine kinase involved in breast cancer proliferation), AVE-1642 conju-gated Qds were developed. They were bound to the cell surface which expresses IGF-1R and AVE-1642, and further goes into the cell via receptor mediated endocytosis and finally translocated into nucleus [212]. On similar lines, a fresh report described the use of conjugated indium phosphide (core)-zinc-sulphide (shell) Quantum dots. Bioconjugation of these Qds with monoclonal antibody (anti-claudin 4 and antiprostate stem cell antigen) allowed specific in vitro receptor mediated targeting of pancreatic cell and underprivi-leged in vitro targeting in nonpancreatic tumour cells which are negative to claudin-4-receptor [213]. In vivo optical imaging studies in nude mice bearing pancreatic cancer xenografts, both, subcuta-neous and orthotopic, indicated that the Qd probes accumulate at tumour sites via the cyclic RGD-peptides on the Qd surface binding to the �V�3 integrins overexpressed in the tumour vasculature fol-lowing systemic injection. In vivo tumour detection studies showed no adverse effects even at a dose roughly 6.5 times higher than that which has been reported for in vivo imaging. In another crucial study, Young et al. reported the production of manganese (Mn)-doped quantum dots (Mnd-QDs) emitting in the near-infrared (NIR). Surface functionalisations of Mnd-QDs with lysine makes them stably dispersed in aqueous media and are able to conjugate with targeting molecules. The receptor-mediated delivery of bio-conjugated Mnd-QDs into pancreatic cancer cells was demonstrated and the cytotoxicity of Mnd-QDs on live cells was evaluated. Their finding suggested that multimodal Mnd-QDs have the probable use as probes for early pancreatic cancer imaging and detection [214]. Mathew et al. reported the synthesis of folic acid conjugated car-boxymethyl Chitosan coordinated to manganese doped zinc sul-phide quantum dot (FA-CMC-ZnS: Mn) nanoparticles. They stud-ied the in vitro imaging of cancer cells with the nanoparticles using fluorescent microscopy and suggested that bright and stable lumi-nescence of quantum dots can be used to image the drug carrier in cancer cells without affecting their metabolic activity and morphol-ogy [165].

Upconverting Nanoparticle The term upconversion is an effect by which low energy near infrared radiation is converted to higher energy (visible light) radia-

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tion by multi-photon NIR absorption and subsequent emission of shorter wave length [215]. Upconverting nanoparticle (UCNP) displays the unique property of emitting visible light following photoexcitation with NIR. UCNP are chemically stable, unlike many others quantum dots, as they do not blink [216]. This property of UCNP makes them very interesting candidates for biological imaging applications owing to their insensitivity to autolumines-cence derived from the luminescence of biological samples [217] and use of low energy NIR excitation reduces photodamage and permits deeper penetration into the tissues [218]. Rare-earth-doped NPs exhibit sharp emission peaks (sharper than QDs), long fluores-cence lifetimes, high quantum yields and excellent photostability. The synthesis and optical properties of rare-earth-doped NPs have been actively studied and their application in biomedicine is being explored [219-221]. For example, PEI/NaYF(4): Yb(3+),Er(3+) nanoparticles producing green/red emission on near-infrared (NIR) and excitation were targeted to folate receptors on human colon cancer cells and imaged with high signal-to-background ratio. It was verified that these particles could be excited after deep intra-muscular injection in rats. On NIR excitation, the particles modified with zinc phthalocyanine photosensitiser released singlet oxygen and after targeted binding to cancer cells, resulted in significant cell destruction [222]. Use of UCNP for the purpose of immune labeling and virtual zero black ground imaging of cancer was reported by Wang et al. in two different investigations [223-224]. In their ex-ploration, upconversion luminescence of visible colour was tuned, following the amino-functionalisation of the silica shell with aminopropyltriethoxysilane. The nanoparticles were linked to rabbit anti-CEA8 antibodies and then used as fluorescent labels for the imaging of live HeLa cells. In another research, Maeda et al. dem-onstrated the surface modification of UCNP of Y2O3: Er with tho-lated cyclic peptide. They reported that its motif had strong affinity for �v�3 integrin that is highly expressed in invasive tumour cells, and it is possible to selectively image tumour cells in vitro by tak-ing the advantage of NIR to visible luminescence imaging [225]. In the follow up work by Xiong et al, peptide motif was used in a high contrast upconversion protocol using multicoloured, tridoped UCNPs (Yb3+/Tm3+/Er3+) modified with amino-poly(ethylene gly-col). Its surface was loaded with a cyclopeptide [c(RGDFK)] with high affinity for �v�3 integrin receptors. They recounted that in-tense fluorescence at the tumour site was still intense after 24 hours. The good signal-to-noise ratio suggests that upconverting lumines-cence imaging of tumourous tissues is an encouraging technique in the detection of cancer [226]. Wang et al. reported the functionali-sation of UCNP with a polyethylene glycol grafted amphiphilic polymer. The PEGylated UCNPs were loaded with a commonly used chemotherapeutic molecule, doxorubicin (DOX). The loading and releasing of DOX from UCNPs was controlled by varying pH, with an increased drug dissociation rate in the acidic environment, favourable for controlled drug release. They stated that that DOX is shuttled into the cells by the UCNP and released inside the cells after endocytosis. Further, they conjugated the UCNP with folic acid and demonstrated targeted drug delivery and upconversion luminescence cell imaging with UCNP [227]. Thus in short, we can say that upconversion methods give an assurance for providing a new and more sensitive technique for deep tissue imaging and early diagnosis. Despite all these advantages, metallic nanoparticles are still at early stages of development. So far some great achievements have been attained in this area but many challenges still remain unconquered. However, its multidirectional, rapid development with improved practical potential of metallic nanoparticles high-lights their potency as novel tools for future cancer therapeutic modalities.

TOXICITY ASSESSMENT OF MNPS Although, from last 3 decades important strides have been made in the production of metallic nanoparticles intended for therapeutic application, particularly in cancer research, however, there is pro-

found lack of information about the impact of these metal based nanoparticles on human health, especially the potential of MNP induced toxicity [228]. These MNPs can potentially cause adverse effects on cells, tissues, organs or at protein level due to their atypi-cal physiochemical properties. The ADME and interaction of MNPs with biological system are largely dependent on the physicochemi-cal properties of MNPs like size, shape, electric charge on the sur-face, chemical composition, surface structure (surface reactivity, surface group, inorganic or organic coatings), solubility and aggre-gation behaviour [229-231]. Once MNPs enter the systemic circula-tion and infiltrate the small capillaries, they efficiently distribute into larger vascular organs/tissues and can possibly affect the physiology of any cell. Figure 6 illustrates, in brief, the mechanism of toxicity induction in tissues at the cellular level caused by MNPs due to direct damaging of cellular component like DNA, RNA and proteins or by oxidative stress induced/directed free radical conver-sion. Earlier, as well as in the recent times, numerous reports have been published to build the concern on the toxicity associated with the future medicine like nanocarriers. With the growing positive impact of the current utility and upcoming amelioration in clinical application on MNPs, its effect on the biological system and the environment, particularly the noxious implication is needed to be addressed. Lovric et al. demonstrated that ‘naked’ CdTe quantum induced cell death through damage to the plasma membrane, mito-chondrion and the nucleus [232]. Similarly, Sayes et al. reported that Cobalt 60Co nanoparticles disrupted the integrity of plasma membrane [233]. In addition, these MNPs may cross the blood-testis barrier and reduce spermatogonial stem cell proliferation in vitro [234-237]. The free radical generation and the associated oxi-dative stress induce cell damage which may be considered as the reason for this effect [238]. Browning et al. reported that Gold nanoparticles can passively diffuse into chorionic space of the em-bryo developing into normal zebra fish, and cause teratogenic de-formities [239]. It is established in concern with MNPs that as the size of a particle decreases, its surface area to volume ratio in-creases, resulting in increased surface activity. On this line, De et al. studied the in vivo toxicity of spherical colloidal Gold nanoparti-cles in mouse, and reported that smaller particles (10-50 nm) caused more toxicity than larger particles (100-200nm) [240]. Moreover, Chen et al. studied the oral toxicity of copper nanoparticles in cor-roboration with the above finding and reported that as the size of a particle decreases, LD50 of copper nanoparticles sharply increases (nontoxic to moderately toxic level) [241]. In an another report, Yen et al. established that spherical gold nanoparticles of size 2.8 to 38nm were more toxic and induced immunological response. [242]. Pisanic et al. showed that the intracellular delivery of moderate levels of iron oxide nanoaparticles adversely affected the cell func-tions. The observations revealed that increasing concentration of anionic MNPs resulted in dose dependent deteriorating viability and capacity of nerve cells [243]. As liver is major accumulator for circulatory MNPs, in in-vitro toxicity study on hepatocytes due to silver nanoparticles, decreased mitochondrial functions, LDG leak-age and abnormal cell morphologies were reported [244]. An an-other comparative toxicity study carried out by Hussain et al. re-vealed that on equal dose levels, the silver MNPs caused more mi-tochondrial damage in the cell than the Mn MNPs in the neuroen-docrine cell line [245]. Summarised form of toxicity associated with MNPs on biological systems and different body organs/tissues that may be affected under the influence of MNPs is given in Table 1 and diagrammatically illustrated in Figure 7 respectively. Al-though in vivo findings on MNP toxicity were also reported but literatures indicate that majority of the data on the toxicity of MNPs were the results of in vitro testing. Therefore, toxicity studies di-rected towards in vivo data and development of animal models must be carried out. Furthermore, correlation should also be established between the in vitro and in vivo data for better understanding on mechanistic potential adverse response of MNPs.

1844 Current Pharmaceutical Design, 2011, Vol. 17, No. 18 Akhter et al.

CONCLUSION MNPs with theranostic applications in oncology are presently research based glossary on cancer nanotechnology in which drug/ diagnostic probe can successfully be fabricated with organ/tissue targeting and controlled release behaviour. It will reduce the che-motherapy associated predictable toxic effects and the cost of the therapy due to dose reduction. In conclusion, the reports assure the far reaching theranostic/ therapeutic applications of MNPs in oncology, in the near future, to

establish these nanocarriers as nanomedicine for successful clinical use, following developmental/safety issues have to be resolved:

I. The fabrication of MNPs is more complicated than the con-ventional dosage form.

II. Formulation stability, control of drug release and insertion of targeting nature in MNPS is an onerous task.

III. Large scale manufacturing and scale-up. IV. With the change in physicochemical properties of nanomate-

rial due to reduced size in nanoscale, they have the potential

Figure 6. Figure illustrating the toxicity/mutilation induced by MNPs to different cellular components.

Table 1. Example of Selected Comparative In Vivo Toxicity of Metallic Nanoparticle

Nanoparticle Toxic Effect References

Cd-Se Quantum dots

Cytotoxic

Transient reduction in motor activity

[232]

Copper Neurotoxic, hepatotoxicity and damage of spleen were seen mice

Histopathological changes

Alterations in gene expression

Decreased Na+/K+ ATPase activity

Dose-dependent gill lamellae damage

[241]

Gold Oxidative DNA damage, Inflammation [242]

Iron oxide (Fe2O3)

Oxidative stress induced free radical damage to cells/organs, inflammation

Neurotoxic in mice

Biochemical and Histopathological changes

Dose-dependent diminishing viability

Reduced ability to respond to nerve growth factors

[243]

Silver Dose-dependent increment of alkaline phosphatase and cholesterol

Accumulation of nanoparticle in kidney

Dose-dependent toxicity in embryo (Teratogenicity)

Distribution in brain and heart leading to neuro and cardiac toxicity

Mitochondrial disruption

Oxidative stress induced gene expression alteration

[244]

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to induce toxicity to the DNA, proteins, cellular components, tissues and other organs. Furthermore, MNPs may cause be-havioural, physiological and metabolic alteration.

V. Challenges in toxicity assessment presenting meagre in vivofindings and poor in vitro-in vivo correlation due to lack of appropriate technique/tools to directly interrogate MNPs in complex biological system.

VI. PEGylation/hydrophilic surface coating which decrease the organ distribution of MNPs, surface charge neutralization de-veloped due to size reduction to nanoscale and stabiliza-tion/coating through the charge interaction between polymer and metal used in MNPs with biodegradable or biocompatible polymers would be the probable mean to abate the toxicity associated with MNPs. In addition, it is the necessity of time that the regulatory agencies move rapidly toward new metrics to keep pace with the changing paradigms introduced by nanotechnology to address and surpass the adverse conse-quences of nanomedicine.

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Received: April 1, 2011 Accepted: May 11, 2011

1. Introduction

2. Metallic nanoparticles for

cancer theranostic

3. Targeted delivery and

biological consideration

4. Recent developments and

applications of metallic

nanoparticles in cancer

theranostics

5. Future prospects

6. Conclusions

7. Expert opinion

Review

Metallic nanoparticles:technology overview & drugdelivery applications inoncologyMohammad Zaki Ahmad†, Sohail Akhter, Gaurav Kumar Jain,Mahfoozur Rahman, Shadab Ahmad Pathan, Farhan Jalees Ahmad &Roop Krishen Khar†Dreamz College of Pharmacy, Khilra-Meramesit, Sundernagar-175036, Mandi, HP, India

Importance of the field: The targeted delivery of therapeutic agents to

tumour cells is a challenge because most of the chemotherapeutic agents dis-

tribute to the whole body, which results in general toxicity and poor accep-

tance by patients and sometimes discontinuation of the treatment. Metallic

nanoparticles have been used for a huge number of applications in various

areas of medical treatment. Metallic nanoparticles are emerging as new car-

rier and contrast agents in cancer treatment. These metallic nanoparticles

have been used for imaging of tumour cells by means of active and passive

targeting. Recent advances have opened the way to site-specific targeting

and drug delivery by these nanoparticles.

Areas covered in this review: This review summarizes the mechanisms of

passive and active targeted drug delivery by metallic nanoparticles and their

potential use in cancer theranostics.

What the reader will gain: The reader will gain information on the develop-

ment of tumour cells, advantages of modern methods of cancer treatment

over the traditional method, targeted delivery of anticancer agents using

nanoparticles, influence of nanotechnology on the quality and expectancy of

life, and challenges, implications and future prospects of metallic nanoparticles

as probes in cancer treatment.

Take home message: The development of metallic nanoparticles is rapid

and multidirectional, and the improved practical potential of metallic

nanoparticle highlights their potency as new tools for future cancer

therapeutics modalities.

Keywords: enhanced permeability and retention, metallic nanoparticle,

multifunctional nanoparticle, nanoshell, opsonisation, theranostic, tumour cell

Expert Opin. Drug Deliv. (2010) 7(8):927-942

1. Introduction

Cancer is the second leading cause of death in both developed and developing coun-tries [1]. At present, first-line cancer therapy involves invasive processes such as cath-eters to allow chemotherapy to shrink any tumour present and surgical removal ofthe tumour followed by a regimen of chemotherapy and/or radiation therapy. Themain goal of chemotherapy and radiation therapy is to kill the cancer cells. In this,the effectiveness of the therapy is directly related to the treatment’s ability to targetand kill the tumour cells while affecting as few healthy cells as possible. This in

10.1517/17425247.2010.498473 © 2010 Informa UK Ltd ISSN 1742-5247 927All rights reserved: reproduction in whole or in part not permitted

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turn is related to the quality of life and life expectancy ofpatients. Unfortunately, this strategy often fails because ofrecurrent or metastatic disease. In some cases, the patientsmust discontinue the chemotherapy before the drug has achance to eradicate the tumour because of its intense sideeffects [2,3].The use of various pharmaceutical carriers to enhance the

in vivo efficiency of anticancer drug(s) and drug delivery pro-tocols has been well established during the last four decades ofoncology research. One of the most important results of thisresearch is the ‘development of metallic nanoparticles to fightcancer’. These metallic nanoparticles may be the key to over-coming some of the limitation of conventional diagnostic andtherapeutic approaches. The use of metallic nanoparticles fortargeting cancer has significant advantages, such as increasedstability and half-life of drug carrier in circulation, requiredbiodistribution, and passive or active targeting into therequired tumour site.

1.1 Development of tumour cellsCancer is a general term used for a group of disorders causedby an abnormal and unrestricted growth of cells. Once a smallmass of tumour is formed, it will compete with the surround-ing healthy tissue for nutrients from the blood. As the healthytissues are not able to compete with cancer cells for an ade-quate supply of nutrients, the tumour cells replicate at a ratehigher than the other surrounding healthy cells and place astrain on the supply of nutrients and elimination of metabolicwastes [4-7]. As a tumour grows, its need for nutrientsincreases, and thus the number and size of blood vesselsneeded to transport them increase proportionately. To facili-tate this process the tumour produces vascular growth factorsthat promote angiogenesis (neovascularisation) and/or other

factors that normally promote angiogenesis. The exact mech-anisms that initiate angiogenesis at a tumour site are not yetknown. Further, angiogenesis is aided by the secretion of pro-teolytic enzymes, namely metalloproteinase, which facilitatethe passage of tumour through the basement membranesand into the extracellular matrix of the local connective tissue.An illustration of tumour development from a single cell isshown in Figure 1.

2. Metallic nanoparticles for cancertheranostic

Many new drugs are being synthesised for the treatment ofcancer; however, the clinical potential of such drugs is sub-jected to certain therapeutic and toxicological limitations,such as the barrier effect of the cell membrane, drug resistancedeveloped by the cell and drug disposition behaviour [8]. In acancer cell, the transportation of a drug is also governed bythe physicochemical properties of the interstitium and mole-cule (i.e., size, configuration, charge and hydrophobicity) [9].The distribution of anticancer drugs essentially depends ontheir physiochemical properties, such as pKa, hydrophilicityand electrostatic charges; however, not all of these criteria nec-essarily fit in the domain of a tumour cell. Large amounts ofthe drug can be distributed towards a healthy organ or tissuerather than the target, and this is the main limiting factor ofconventional chemotherapy [10]. In addition, at the cellularlevel drug resistance may develop in a tumour cell on accountof alterations in the P-Glycoprotein system or distortedapoptosis regulation [11].

Another dilemma associated with chemotherapy is theinherent insolubility of most of the anticancer drugs in water,which necessitates the use of pharmaceutical solvents for theirclinical administration. The use of these solvents may havelife-threatening effects [2,12].

In conventional therapy, during intravenous injection of ananticancer drug, the drug delivery system is opsonised andrapidly cleared from the bloodstream by the reticuloendo-thelial system’s defence mechanism, irrespective of particlecomposition [12-16]. Consequently, conventional anticancertherapy by systemic delivery of chemotherapeutic agents oftenfails or is inadequately delivered to the target cell/tissue andhas a tremendous impact on reducing the quality and expec-tancy of life. Some of the disadvantages of current conven-tional anticancer therapy include: inefficient cell entry,uptake by the immune system and mononuclear phagocytesystem, accumulation in non-targeted organs and tissues,and non-selective with high toxicity against normal tissues [17].The effectiveness of a cancer therapeutic device is measured byits ability to reduce and eliminate tumours without damaginghealthy tissue.

The ultimate goal of anticancer therapy should be to pro-long the survival time and increase the quality of life of thepatient. Therefore, the greatest need for the treatment oftumour is a drug delivery system that can selectively deliver

Article highlights.

. Modern treatment of cancer has become more tailoredto the individual patient and to specific tumour types.

. Metallic nanoparticle probes are emerging as a class ofnew contrast and tracking agents for tumour imaging.

. Enabled by their super molecular structures, metallicnanoparticle are capable agents in the detection,diagnosis and treatment of tumour.

. Multifunctional metallic nanoparticles can detect theearly onset of cancer in each individual and deliversuitable therapeutic agent.

. Traditionally, the treatment and diagnosis of tumourwere considered as two separate entities in the processof patient care; the emergence of the theranosticnanoconcept has blurred the boundary betweentreatment and diagnosis.

. As the capability of multifunctional metallicnanoparticles continues to increase, the integration ofoncology research, diagnostic imaging and targeteddrug delivery will be essential for cancer therapy.

This box summarizes key points contained in the article.

Multifunctional metallic nanoparticles

928 Expert Opin. Drug Deliv. (2010) 7(8)

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anticancer agents to the target tissue with required local drugconcentrations, thereby achieving therapeutic efficacy whileminimising toxic side effects. To achieve this, a multifunc-tional therapeutic agent capable of destroying the heteroge-neous population of tumour cells needs to be developed.Recently, drug delivery systems such as nanometre-sized carriers that entrap the chemotherapeutic drugs haveimproved cancer treatment [18-23].

Metallic nanoparticles for drug delivery are solid colloidalparticles ranging in size from 10 to 1000 nm that contain atherapeutic agent that is dispersed in a polymer carrier matrix,encapsulated within a polymer shell, covalently attached oradsorbed to the particle surface, or encapsulated within astructure [24-26].

Metallic nanoparticle seeks to increase the therapeuticindex of drugs through site specificity, preventing multi-drug resistance, and delivering therapeutic agents effi-ciently [3,27]. Several nanoparticle-based systems are now beingexplored for cancer treatment. The material properties of eachnanoparticle system have been developed to enhance deliveryto tumours [28-35]. Among these, metallic nanoparticle probesare emerging as a new class of contrast and tracking agent forcancer therapies [36-45].

Metallic nanoparticles are emerging as a potential applica-tion in the field of cancer diagnosis, for example MRI andcolloidal mediators for cancer magnetic hyperthermia. Theusefulness of metallic nanoparticles as probes for cancer ther-apy is mainly derived from their potential to carry a largedose of drug, which results in high concentration of anticancerdrugs at the desired site, thus avoiding toxicity and other pains-taking side effects arising owing to high drug concentration inother parts of the body [39].

There are many advantages of metallic nanoparticle in can-cer therapeutics. For example, they are designed to containtumour targeting ligands that bind to particular cells withinthe tumour to fasten the nanoparticle within solid tumour.In this way, metallic nanoparticle drug delivery systems arecapable of sequestering anticancer drugs exclusively withinthe tumour and thereby reduce the accumulation of drugs

in healthy organs. Their large surface area-to-volume ratioprovides a surface for chemical modification, which canimprove cell entry, protect the therapeutic agent in the bio-logical milieu, and improve bioavailability of the anticanceragent [13-17,46,47]. Furthermore, multifunctional metallicnanoparticles can detect and attack the heterogeneous crowdof tumours (Figure 2).

Thus, because of their unique physical properties and capa-bility to function at the cellular and molecular level, metallicnanoparticles are being actively investigated as carriers fortargeted drug delivery in cancer therapeutics [48,49]. Oneof the key reasons that these nanoparticles have promisein the treatment of cancer is that they can be targeted totumours through antigen-dependent (specific) or antigen-independent (nonspecific) mechanisms [50,51]. Specific target-ing relies on the interaction of antigens on the surface ofnanoparticles with tumour cell receptors [52]. Metallic nano-particles are used to manipulate not only the size of drug par-ticles but also the physical characteristics, and thus the extentand target of drug delivery. In brief, the use of metallic nano-particles for cancer treatment has significant advantages, forexample, the ability of metallic nanoparticles to target specifictumour cells, accumulation of therapeutic agent in the vicinityof tumour, and reduction of drug concentration in healthycells/tissue.

This sophisticated technique in cancer therapeutics is pro-gressing very quickly, in terms of both newly discovered anti-cancer agent, and advanced ways of delivering both new andold anticancer agents. The functionality of fabricated metallicnanoparticle may be exploited to enhance the specificity ofdrug delivery towards tumour cells and reduce toxicity. Usingmetallic nanoparticles, cancer therapy could be performed atthe cellular and subcellular levels, therefore side effects couldbe reduced and therapeutic efficacy greatly increased. Thus,application of metallic nanoparticles in cancer therapeutics ispotentially the largest public health contribution of nano-science. In this review, the following are covered: targetingthe tumour cells using metallic nanoparticles, developmentsand theranostic applications of metallic nanoparticles in

Tumour cell

Cancer cell divides at accelerated rateand displaces healthy tissue

Developmentof tumour

Mutated cell

Mutation

Normal cell

Virus

Figure 1. Tumour development from a single cell.

Ahmad, Akhter, Jain, Rahman, Pathan, Ahmad & Khar

Expert Opin. Drug Deliv. (2010) 7(8) 929

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oncology disorders, and an overview of risk assessment in thearea of metallic nanoparticles in cancer therapeutics.

3. Targeted delivery and biologicalconsideration

Conventionally, anticancer drugs have been designed to have arelatively low molecular mass and an agreement between thehydrophilic and lyphophilic balance (HLB), hence allowingpartitioning across the lipid membrane very easily. Therefore,drug within the systemic circulation is rapidly distributedthroughout the body, reaching the target and non-targettissue/organ, and is also rapidly metabolised by the liver and/or rapidly excreted by the kidney. Therefore, for effective tar-geting, it is essential that a drug-targeting system should notbe cleared out quickly from the body. Ideally, a drug carriershould provide a pharmacokinetic profile that will allow thedrug to interact with its target [17,53]. During the designing ofmetallic nanoparticles, the lessons learned from the polymer-based and liposomal drug delivery system must be takeninto consideration. For example, unprotected liposomal andpolymer-based drug delivery systems are rapidly cleared fromthe blood by the reticuloendothelial system (RES) and accu-mulate in the liver, conditioning their rapid first-pass metabo-lism from the systemic circulation followed by metabolicdegradation and excretion. This consideration is very beneficialwhen designing metallic nanoparticles for cancer therapeuticslocated close to the mononuclear phagocyte system [21,53-58].The performance of nanoparticles inside the vascular com-

partment is controlled by complex factors such as their shape,density, size distribution and surface characteristics. All thesefactors control the flow properties of nanoparticles, bifurca-tion in the vascular compartment, modulation of circulationtime, and mode of entry into the cell [22,59-64].

3.1 Achieving targeted deliveryA major barrier that a drug delivery system must be able toavoid in the systemic circulation is the removal of nanopar-ticles by phagocytic cells of the mononuclear phagocytesystem (MPS). Nanoparticles will usually be taken up bythe liver, spleen and other parts of the RES depending ontheir surface characteristics and undergo opsonisation in theblood and clearance by the RES [16,65-69]. Therefore, theMPS presents a significant barrier to effective drug targeting,because it has the ability to filter out and destroy a drug deliv-ery system unless appropriate formulation approaches areused to avoid this. Therefore, the nanoparticles should bedesigned to avoid these interactions, particularly opsonisa-tion, and possible clearance of the drug delivery systemfrom the vascular compartment. Opsonisation is a processin which the surface of the foreign particles such as bacteriaand particulate drug carrier is coated with blood proteins,known as opsonins. The phagocytosis of these tagged par-ticles is enhanced because surface receptors present on phag-ocytes bind to opsonins and the foreign particle is engulfedand untimely digested by various lysozymes [58,69-72]. Forexample, when unprotected colloidal nanoparticles wereintravenously injected into mouse, it was observed that 95%of the gold nanoparticles were cleared out from the vascularcompartment within 10 min [45]. Therefore, suppression ofopsonisation and avoiding MPS recognition and receptor-mediated phagocytosis are the primary concerns whendesigning metallic nanoparticles.

A more practical approach to avoid RES uptake and clear-ance of nanoparticles is the modification of nanoparticles’surface. For example, increasing the surface hydrophilicity,that is, adding a hydrophilic polymer to the metallic nano-particle carriers, made them invisible to the RES and thusreduced opsonisation and led to suppression of macrophagerecognition. This coating is referred to as the stealth moiety(e.g., Figure 2) [73,74]. The most commonly used stealth agentis polyethylene glycol (PEG) and its block copolymer. It hasbeen proposed that PEG has a high local concentration ofhydrated groups, which sterically inhibits interaction withblood-borne opsonins [50]. Intravenous injections of stericallystabilised nanoparticles result in prolonged circulation time,and their accumulation in tumour [75-82].

Another factor affecting the opsonins’ binding are physi-cochemical properties of the nanoparticles (i.e., surfacecharacteristics such as size, surface charge) [58,83-92], whichis a major factor in the characteristic biodistribution andresidence times of these particles in vivo. Neutral systemstend to remain longer in blood circulation, whereas theircharged counterparts are cleared out by the RES [3,58]. Sim-ilarly, particles of size ~ 1 -- 2 µm undergo phagocytosis,and higher sizes of ~ 6 µm are trapped in lung capillaries [53].Therefore, to avoid clearance by the RES, metallic nanopar-ticles should be formulated to be not more than 100 nmin size and should have a sterically stabilised, preferablyneutral, surface.

Tumour targetingmotif

Stealth moiety

Therapeutics 3

Therapeutics 2

Therapeutics 1

Figure 2. Multifunctional metallic nanoparticle.

Multifunctional metallic nanoparticles

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3.2 Passive targeting of tumours using metallic

nanoparticlesIn passive targeting, the distribution of nanoparticles is medi-ated by the MPS’s physiological condition. In passive targeting,advantage is taken of the pathological condition of the tumourto allow the accumulation of drug carriers at the target site [90].For example, the pH or specific enzymes present within thetumour cells can be used to facilitate the release of drugsfrom nanoparticles. Enzymes such as alkaline phosphataseand plasmins are present at a higher level at the tumour site.

Once a tumour mass has formed, normal cells are ultimatelydisplaced. Also, solid tumour secretes factors that cause newblood vessels to grow from existing blood vessels towards thetumour, and this growth process, known as angiogenesis, is ini-tiated when the volume of the tumour becomes 2 mm3

[91].Tumour vasculature originating from the host vasculature issignificantly different and is abnormal in the diseased tissue.In tumour cells, one of the characteristic features of angiogen-esis is that it has aberrant tortuousity and abnormalities inthe basement membrane [92]. This incomplete vasculature oftumour results in leaky blood vessels (capillaries). This hyper-permeability of the tumour vasculature is a key feature inpassive targeting of drug carriers [93-96]. The disorganisedpathology of angiogenic tumour vasculature with its discontin-uous endothelium leads to hyperpermeability to circulatingnanocarrier, and a lack of effective tumour lymphatic drainage,which leads to subsequent accumulation of drug carrier. This iscalled the enhanced permeability and retention (EPR) effect(Figure 3) [81,97,98]. The EPR effect has been observed in manyanimal models and also in human solid tumour. A high levelof permeability factor such as nitric oxide and vascular endo-thelial growth factors enhance the permeability of blood vessels,thus supporting EPR [64].

Although the EPR effect is very effective for intravenousdelivery of nanovector, a major obstacle associated with EPRis accumulation of drug-loaded nanovector within bloodcapillaries away from the tumour cell [45]. This is mainly owingto the formation of an intratumour clot, which results inincreased interstitial fluid pressure (IFP) of solid tumour. Asa result of the higher interstitial pressure, poor lymphaticdrainage and continued angiogenesis, movement of particulatematerials out of the tumour blood vessels and into the extra-vascular compartment is remarkably limited [45,60]. Electronmicroscopic visualisation of blood vessels in solid tumourshowed that thickness of the blood vessel wall is poorly corre-lated to its diameter; therefore, in a tumour blood flow is cha-otic [99,100]. Surface charge and hydrophobicity of metallicnanoparticles can affect their biodistribution by interaction ofmetallic nanoparticles with plasma protein, adaptive immunesystem and extracellular matrices [101,102].

3.3 Active targeting of tumour using metallic

nanoparticlesAs passive targeting does not necessarily guarantee theinternalisation of nanoparticles by the targeted cell,

nanoparticles are modified with molecular targeting ligandsfor active targeting of tumour. It is a well-known fact thatcancer cells express specific surface receptors that can be tar-geted [103], for example, some tumour cells express surfaceenzymes that might be useful to activate a prodrug once ananoparticle has been localised on the surface of thetumour [104].

Active targeting of metallic nanoparticles involves aninteraction between peripherally conjugated targeting moi-ety and a corresponding receptor to facilitate the targetingof a carrier to a specific malignant cell [105-107]. Drug deliveryto the tumour cell can be achieved by means of moleculesthat are specific to antigens or receptors expressed on the sur-face of a tumour cell [18,108]). Ligand can be designed to havespecificity for receptors that are expressed on a tumour cellbut are minimally expressed on normal cells. The introduc-tion of targeting ligand enhances the internalisation ofmetallic nanoparticles into the tumour cell. However, caremust be taken when selecting ligands for receptors on thetumour cell, as ligand--receptor interaction can affect therate of internalisation, which in turn affects the accumula-tion of metallic nanoparticles in cancer cells. Therefore,ligands used for receptor targeting in cancer treatment musthave the function of inducing receptor-mediated endocytosis(RME) [109].

These ligands (antibodies, saccharides, aptamers, hor-mones, lectin and low-molecular-mass compounds) bind totheir specific receptor on the cellular surface and trigger theinternalisation process of drug delivery so that anticancerdrugs act on intracellular targets (e.g., mitchondria, microtu-bules, nucleus, etc.) by means of RME (Figure 4) [18,110]. Var-ious molecules are used to facilitate active targeting ofnanoparticles, such as aptamers [111-113], proteins and anti-bodies (Figure 4) [113-115]. Bioconjugation of ligands, such asmonoclonal antibodies, proteins, or peptides, with the nano-carrier’s surface has been exploited in many nanoparticles forthe purpose of concentrating therapeutic action on the specifictumour cell [3,18,45,52,116-122].

Several overexpressed growth factor receptors have beenused for selectively targeting the cancer cell, and the descriptionof many of these targets has been reviewed [123].

3.3.1 Targeting with HER2/neuMonoclonal antibodies were the first targeting agents todeliver the magnetic nanoparticle [124,125]. Herceptin, anFDA-approved monoclonal antibody, is a popular targetingagent for the HER2/neu receptor for breast and ovariancancer [126]. Huh et al. reported the specific delivery ofHerceptin-targeted magnetic nanoparticles to the cells express-ing the HER2/neu cancer marker in vivo [127]. Unfortunately,this antibody can also target normal cardiac cells undergoingrepair induced by the actions of a common anticancer agent,doxorubicin. Therefore, nanoparticles having a cytotoxiccapacity and targeted using the herceptin antibody couldresult in cardiomyopathy [128].

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3.3.2 Folate receptorFolate receptor is weakly expressed in normal tissue but isoverexpressed on the surface of tumour cells, thus making itpossible to target several tumour cells. Folic acid and itsreduced form tetrahydrofolate are cofactors of severalenzymes. Folic acid and tetrahydrofolate are used as targetingmolecules because of its low molecular mass and low

immunogenicity [129]. Thus, targeting the folic acid receptorsis interesting for drug delivery in cancer treatment.

3.3.3 Targeting multivalent carriersSpecificity of nanoparticles can be improved significantlyby the attachment of multivalent targeting ligands. Thesemultifunctional nanoparticles can combine several functional

Lysosomes Receptor mediated endocytosis

Cell penetrating peptide mediatedintrcellular delivery viamacropinocytosis or electrostatic interaction

Nucleus

Mitochondria

Cytoplasm

Drug efflux

Figure 4. Schematic of intracellular drug delivery.

Tumour tissue

Angiogenic capillaries (leaky anddisorganised)

Normal capillaries

Nanoparticle loaded with drug

Normal tissue

Nanoparticle loaded withdrug and leaks out into surroundingtumour tissue

Figure 3. Enhanced permeability and retention leads to increased permeability of the capillary at sites of tumour, which can

facilitate the escape of nanoparticles loaded with drug from the circulation.

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capabilities in a single stable unit and can be used to increasethe accumulation of nanoparticles within tumour cells [130].

Several studies with enhanced binding affinity havebeen reported where multivalent targeting ligands simulta-neously bind with multiple receptors between two surfaces(Figure 5) [131-134]. Jiang et al. reported that magnetic nanopar-ticles of size range 25 -- 50 nm are most suitable for multiva-lent targeting ligands; however, it was also observed thatnanoparticles < 25 nm lack the ability to present multipleligands to specific target cells [134].

4. Recent developments and applications ofmetallic nanoparticles in cancer theranostics

There is a long history of the use metallic nanoparticlesin biological systems for diagnostic and therapeutic pur-poses [44]. However, the use of metallic nanoparticles in can-cer therapeutics has been reported only recently. As metallicnanoparticles have the ability to treat as well as to diagnosethe disease, the ability of nanoparticles both to diagnoseand to deliver the targeted drug is an emerging concept inthe nanoplatform. These nanoparticles are called theranostic(therapeutic plus diagnostic) [135].

Theranostic nanovector represents an emerging class ofimaging and therapeutic that may provide a personalised ther-apeutic response. For example, light-activated theranosticnanoparticles have been reported for imaging and treatmentof brain tumours [37]. Therapeutic efficacy of these agentswas evaluated in comparison with untargeted particles in a dis-eased animal model. In this study it was reported that survivaltime for untargeted particle groups was 13 days, whereas ani-mals treated with targeted agents had a survival of 33 days,with 2 animals disease-free within 180 days of therapy. Theroles of theranostic agent in tumour diagnosis, monitoringtumour progression and assessment of therapeutic effect haveresulted in an enhanced role. For example, ex vivo imagingof oncology biomarkers in a preclinical study was reportedby Makino et al. for the visualisation and monitoring oftumour progression by coupling the near-infrared fluorescence(NIRF) and nanoparticles in a targeted drug delivery systemfor hepatic tumour [136]. This particle works on the fact thatthe wavelength (l) of NIRF is able to penetrate deeper intotissue. In another approach, 100% cell death was observedwhen human transformed human macrophage was incubatedwith nanocarrier for 60 min and laser illumination [135]. Sim-ilarly, in another experiment Alexa 750 NIRF dye coupledwith phospholipid micelle enabled the rapid imaging oftumour in mouse model for breast cancer [137]. Theranosticphotosensitiser ADM06 was evaluated in rats bearing breastcancer; in this study, suppression of tumour activity wasobserved after 48 h [138].

Combining diagnostic and therapeutic processes into one(theranostics) and improving their selectivity to the molecular/cellular level may offer significant benefits in oncology research.Lukianova-Hleb et al. developed plasmonic nanobubbles

(PNB) based on the nanoparticle-generated transient photo-thermal vapour nanobubbles. They evaluated the PNB in livinglung carcinoma cell. After delivery and accumulation it wasobserved that PNB are capable of fast and selective damageof specific cells and guidance of the damage through thedamage-specific signals of the PNB. Thus, PNB acted as thera-nostic agents and supported diagnosis and therapy [139]. Biodis-tribution of gold nanoparticles coated with gadolinium chelatewas studied by Alric et al., they reported that functionalisedgold nanoparticle freely circulate in the blood vessels withoutundesirable accumulation in any major organ [140]. In anotherinvestigation Lindsey et al. evaluated the controlled release ofliposomes containing a molecular load and gold nanoparticles.They demonstrated an optically guided release through disrup-tion of the liposome membrane and ejection of the liposomecontents with plasmonic nanobubbles [141].

Iron nanoparticles have been used as theranostic agents withspecific application as contrasting agents for MRI and magnet-ically targeted drug deliver to the tumour cell [142-147]. Thereare mainly two types of iron oxide nanoparticle reportedfor use as imaging agents [142,148], superparamagnetic ironoxide (SPIO) and ultra-small superparamagnetic iron oxide(USPIO). The main advantages associated with SPIO nano-particles are the biocompatible and biodegradable propertiesof iron, as it can be recycled through the normal biochemicalpathway for iron metabolism [149]. Hepatic imaging was thefirst application of these magnetic nanoparticles. It was possi-ble because normal liver cells take up SPIOs, which results indarkening of the image; however, cancerous cells are not ableto take up the SPIOs, thus resulting in a bright spot in tumourcells [150-153].

There are many reports in which SPIOs have been used incombination with monoclonal antibodies to detect a varietyof cancers [154-157]. Transferrin and pancreatic receptors areoverexpressed in some tumours [158,159]. SPIOs have been con-jugated with peptides to target these receptors and image thecancer cells. Zhao et al. reported that when SPIOs were linkedwith the peptide synaptotagmin, it enabled the imaging ofcells undergoing apoptosis after chemotherapy. The magneticnanoparticles that have been approved by the FDA are listedin Table 1 [160-163].

Christopher et al. reported the synthesis and use of mag-netic nanoparticle hydrogel (MagNaGel�; Alnis Biosciences,Emeryville, California, USA) as a powerful cancer treatmentregimen. They demonstrated that these particles had the char-acteristics of ability to load chemotherapeutic agent, tumour-associated biomolecular binding and good magnetic suscepti-bility [141]. In another study, it was reported that dextran-coatedmagnetic nanoparticles showed an increased accuracy of cancernodal staging [164,165]. These modified magnetic nanoparticleshave been used for delineation of the tumour [166].

Several anticancer drugs such as doxorubicin, methotrexateand paclitaxel have been formulated with metallicnanoparticles [167-170]. Similarly, Liang et al. demonstratedthe ability of radionuclides containing SPIOs to induce

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specifically cell death in liver cells in vitro [171]. In anotherinvestigation, Ross et al. successfully demonstrated the thera-peutic application of SPIOs when they were decorated withthe antibody Herceptin to target the Her2/neu receptor inthe early stage of breast cancer [172].Semiconductor nanoparticles, known as quantum dots,

have been increasingly applied as imaging and labellingprobes in cancer therapeutics [173-177]. Major advantages asso-ciated with quantum dots include their high quantum yield,resistance to chemical modification and intrinsic fluorescenceemission spectra owing to which quantum particles possessthe ability to sense and release anticancer drugs at the desiredsite (Figure 6) [59].Chen et al. developed a dual-function NIRF probe to

assess the tumour targeting efficacy of quantum dots. They

concluded that the successful development of a quantumdot-based nanoparticle with dual function may increase theaccuracy of quantitative targeted NIRF imaging in tumourcells [178].

Similarly, metallic gold nanoparticles have been studiedextensively for their potential application in targeted tumourcell drug delivery [177,179-184]. Gold nanoparticles have severalattractive advantages in diagnostic and therapeutic applica-tions, namely, easy decoration of gold nanoparticles withantibody for tumour-specific targeting, biocompatibility andstability [173,185].

The synergistic effect of hyperthermia and radiation therapywas studied by James et al. on a mouse head and neck squa-mous cell carcinoma model to identify the various factorsaffecting the efficacy of nanogold radiation therapy. Theyobserved that radiation energy and hyperthermia influencethe potential utility of gold nanoparticle for cancer radiationtherapy [186]. Hybrid nanoparticles (HNP) of gold and ironoxide were synthesised and evaluated by Kirui et al. After bio-functionalisation of HNP with antibody that binds toA33 antigen on cancer cells, it was observed that cellular uptakeof HNP was five times higher in A33-expressing cells than innormal cells. Thus, this new class of HNP can potentially actas an effective receptor-targeted therapeutic agent for tumour

Multivalent metallicnanoparticle

Multivalent liagnd

Cell surface

Cell

Receptor on cellsurface

Figure 5. Conceptual illustration of multivalent affinity interaction between receptor on a cell surface and targeting ligands

on a metallic nanoparticle.

Table 1. FDA-approved SPIOs.

SPIOs agents Target organs

AMI-121 Gastrointestinal lumenAMI-277 Blood node, lymph nodeAMI-25 Liver/spleenSHU 555A Spleen/liver

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treatment [187]. Park et al. proposed a fusion system composedof metal multilayer-biodegradable polymer half-shell nanopar-ticles, that is, poly(lactide-co-glycolic acid) (PLGA)--magnetic(Mn/Au) half-shell nanoparticles loaded with rhodamine as amodel drug for MRI imaging, photothermal therapy anddrug delivery [188].

The first application of gold nanoshell was reported byHirsch and co-workers in 2003 in hyperthermal therapy oftumour cells [189]. In 2005, Loo et al. reported the conjugationof gold nanoshell with hER2 antibody to target breast carci-noma cells [190]. When the shape of gold nanoparticles changesfrom nanoparticle to nananorod, their absorption and scatter-ing wavelengths also change from visible to near-infraredregion [173], owing to which gold nanorods can be used as con-trast agents for dual molecular imaging. In a follow-up work byHainfeld et al., the combination of gold nanoparticles followedby X-ray treatment reduced the size of tumours in mice [191]. Inone investigation, Haung et al. reported that cellular uptake ofgold nanorods was increased twofold in malignant cells whengold nanoparticles were conjugated with antibody to targetantiepidermal growth factor receptor [192]. This therapy plusdiagnostic holds the promise in future of monitoring the effec-tiveness of therapy, and thus tailoring anticancer therapy to theindividual needs of patients. However, after successful produc-tion there will be regulatory issues of nanoparticles for molecu-lar imaging in a clinical setting. As evidence, The FDA hasestablished a nanoparticle taskforce to handle the regulatoryhurdles associated with nanomedicine [193,194].

5. Future prospects

Over the last few years, the use of metallic nanoparticlesin cancer theranostic has become relatively commonplace.

Research activities aimed at achieving specific and targeteddelivery of anticancer agents have expanded tremendouslyin the last 10 years. As the capabilities of multifunctionalnanoparticles continue to increase, the integration of cancerresearch, imaging, diagnosis and therapeutics in the futurewill be essential for antitumour therapy.

Much less research has been performed, however, on mag-netic nanoparticles for intracellular molecular imaging.Although significant effort has been devoted to developing ametallic nanoparticle drug delivery system, metallic nanoplat-forms are still at an early stage of development and muchmore research is required to overcome the problems associatedwith the nanoparticle properties influencing in vitro andin vivo toxicity assays.

6. Conclusions

The development of metallic nanoparticles is rapid and mul-tidirectional. Metallic nanotechnology has clearly impactedthe development of new theranostics in oncology disorders.Recent advances in the field of metallic nanoparticles indeedoffer the promise of better diagnostic and therapeutic options.Metallic nanoparticles are attracting attention in cancer ther-apeutics owing to their unique prospects for targeted deliveryin imaging and drug delivery to the desired site. Drug deliverybased on metallic nanotechnology seeks to increase the thera-peutic index of drugs, both by reaching their in vivo target andby exposing the drugs to malignant cells. Metallic nanotech-nology combines nanobiotechnology with molecular imagingtechniques, which has led to the development of multifunc-tional metallic nanoparticles for cancer imaging and therapy.Metallic nanoparticles have the advantage of being able totarget multiple tumour markers and deliver multiple agents

Doxorubicin

Qd Qd-Dox

Nucleus

+ Drug release

Lysosomes

Target tumour cell

Figure 6. Illustration of Qd--Dox conjugate in targeted tumour imaging, sensing and therapy.Dox: Doxorubicin; Qd: Quantum dots.

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in addressing the challenge of cancer heterogeneity and adap-tive response. It is hoped that the new generation of multifunc-tional metallic nanoparticle will eventually make it possible toinvestigate tumours and allow the collection of vast amounts ofdata important for patient care. These multifunctional metallicnanoparticles offer a new era in the application of antitumourdrugs in the near future. As summarised, this metallic nano-platform plays an important role in the field of cancer therapy,and it can be expected that this nanotechnology will continueto grow over many decades.

7. Expert opinion

Cancer is a heterogeneous population of diverse diseases.Adaptive resistance of malignant cells to drugs is a major chal-lenge to therapy. The ultimate cure for cancer is excision of thesolid tumour, namely, surgical removal of cancer cells, but asdiscussed earlier, surgery has its own limitations, for example,the inability to distinguish between cancerous cells and normalcells in certain cases. Traditionally, diagnosis and treatmentwere considered as two separate entities in the process of cancertherapy; however, the merging of biology, chemistry and phys-ics at the nanoscale has led to the emergence of nanotechnol-ogy, and in this metallic nanoparticles have blurred theboundary between diagnosis and treatment, so that these two(diagnosis and treatment) separate clinical aspects will soonmerge into a single process, for example, theranostics.Advanced developments in the nanotechnology-based drug

delivery and imaging technique allow more specific mappingof tumour cells. The larger surface area-to-volume ratio ofnanoparticles enables them to accommodate different func-tional groups on their surface. As a result of the EPR effect,metallic nanoparticles display the ability to concentrate pref-erentially at the cancer tissue. Metallic nanoparticles have alarge impact on cancer treatment. Early diagnosis and targeteddrug delivery in cancer therapeutics is one of the priorityresearch areas in which metallic nanoparticles will play a vitalrole. Metallic nanoparticles have been gradually developed asa new modality for targeted drug delivery and diagnosis in

cancer therapeutics. Tumour treatment has become moretailored to individual requirement and to a particular cancercell/tissue. The integration of theranostic nanovector withdiagnostic and imaging capability with therapy is critical foraddressing the challenge of multi-drug resistance in cancerdiversity and adaption. As the capability of multifunctionaltheranostics continues to increase, the integration of cancerresearch, imaging, diagnosis and therapeutics in the futurewill be essential for cancer therapy. With the advances in inte-gration of oncology research, diagnostic imaging, therapeuticsand explosive developments in nanocomposite materials sci-ence, there is reason to be optimistic that we are near a majorbreakthrough in antitumour therapy.

Despite all these advantages, metallic nanoparticles are stillat an early stage of development. Some great achievementshave been attained in this field, but many challenges remain.Most of the current theranostic nanoparticle systems havebeen developed for a limited number of approved drugs. Formany new anticancer drugs with diverse physicochemical prop-erties, theranostic agents need to be tailored to increase theircompatibility with these drugs to achieve precise diagnosis,imaging and therapeutic payload.

A problem that may limit the wide use of theranostic nano-technology is the toxicity of nanoparticles. The developmentof theranostic nanoparticles requires significant advances innanocomposite materials science. Use of theranostic nanopar-ticles in the clinical setting has yet to come to fruition; there-fore, the development of this agent for clinical applicationmust be viewed as a long-term matter. However, its develop-ment is rapid and multidirectional, and the improved practicalpotential of metallic nanoparticles highlights their potencyas new tools for future cancer therapeutic modalities. Mostimportantly, metallic nanotechnology must follow precisesafety study.

Declaration of interest

The authors state no conflict of interest and have received nopayment in preparation of this manuscript.

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Affiliation†1

Sohail Akhter2 MPharm PhD,

Gaurav Kumar Jain3

Mahfoozur Rahman4 MPharm,

Shadab Ahmad Pathan4 PhD,

Farhan Jalees Ahmad2 & Roop Krishen Khar2

†Author for correspondence1Dreamz College of Pharmacy,

Khilra-Meramesit,

Sundernagar-175036,

Mandi, HP, India

Tel: +91 9805759952;

E-mail: zaki.manipal@gmail.com2Faculty of Pharmacy,

Hamdard University,

New Delhi-110062, India3Assistant Professor,

Faculty of Pharmacy,

Hamdard University,

New Delhi-110062, India4Research Scholar,

Hamdard University,

New Delhi-110062, India

Multifunctional metallic nanoparticles

942 Expert Opin. Drug Deliv. (2010) 7(8)

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Mohammad Zaki Ahmad MPharm ,

84

Introduction

Ganciclovir (a synthetic acyclic nucleoside analog of 2′-deoxyguanosine) is a potent inhibitor of human and animal herpes viruses1–3 which shows excellent antiviral activity against varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus. Ganciclovir is converted to Ganciclovir-5′-triphosphate in vivo which competitively inhibits viral DNA polymerase enzyme with respect to deoxyguanosine triphosphate4–6. Phosphorylated Ganciclovir shows greater affinity for DNA polymerase. This affinity is selective as inhibiting only the viral enzyme7. Ganciclovir is the first-line therapy for cytomegalovirus in immunocompromised patients, like organ transplant

patients and patients with acquired immunodeficiency syndrome (AIDS)3,8. It is administrated through intrave-nous, oral, and intra-ocular route.

Ganciclovir is a BCS-III/IV drug having high solubility and low permeability9. Oral Ganciclovir has been shown to be effective in preventing CMV disease, but its poor oral bioavailability limits the degree of viral suppression and may predispose to the emergence of resistance4,10–14. Intravenous administration of Ganciclovir offers thera-peutically effective plasma concentration levels, but the drawbacks associated due to long-term administration of i.v. Ganciclovir are patient inconvenience, higher cost (40% higher), and incidence of catheter-related

RESEARCH ARTICLE

Development and evaluation of nanosized niosomal dispersion for oral delivery of Ganciclovir

Sohail Akhter1, Shalini Kushwaha2, Musarrat H. Warsi1, Mohammed Anwar2, Mohammad Zaki Ahmad3, Iqbal Ahmad2, Sushma Talegaonkar1,2, Zeenat I. Khan1,2, Roop K. Khar1,2, Farhan J. Ahmad1,2

1Nanoformulation Research Lab, Faculty of Pharmacy, Hamdard University, New Delhi, India, 2Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi, India, and 3Dreamz College of Pharmacy, Khilra-Meramesit, Sundernagar, Himachal Pradesh, India

AbstractEncapsulation of Ganciclovir in lipophilic vesicular structure may be expected to enhance the oral absorption and prolong the existence of the drug in the systemic circulation. So the purpose of the present study was to improve the oral bioavailability of Ganciclovir by preparing nanosized niosomal dispersion. Niosomes were prepared from Span40, Span60, and Cholesterol in the molar ratio of 1:1, 2:1, 3:1, and 3:2 using reverse evaporation method. The developed niosomal dispersions were characterized for entrapment efficiency, size, shape, in vitro drug release, release kinetic study, and in vivo performance. Optimized formulation (NG8; Span60:Cholesterol 3:2 molar ratio) has shown a significantly high encapsulation of Ganciclovir (89 ± 2.13%) with vesicle size of 144 ± 3.47 nm (polydispersity index [PDI] = 0.08). The in vitro release study signifies sustained release profile of niosomal dispersions. Release profile of prepared formulations have shown that more than 85.2 ± 0.015% drug was released in 24 h with zero-order release kinetics. The results obtained also revealed that the types of surfactant and Cholesterol content ratio altered the entrapment efficiency, size, and drug release rate from niosomes. In vivo study on rats reveals five-time increment in bioavailability of Ganciclovir after oral administration of optimized formulation (NG8) as compared with tablet. The effective drug concentration (>0.69 µg/mL in plasma) was also maintained for at least 8 h on administration of the niosomal formulation. In conclusion, niosomes can be proposed as a potential oral delivery system for the effective delivery of Ganciclovir.

Keywords: Ganciclovir, niosomes, entrapment efficiency, in vivo study

Address for Correspondence: Farhan J. Ahmad, Department of Pharmaceutics, Nanoformulation Research Lab, Faculty of Pharmacy, Hamdard University, New Delhi, India110062. Tel: + 91-9810720387. E-mail: farhanja_2000@yahoo.com

(Received 20 September 2010; revised 17 May 2011; accepted 25 May 2011)

Drug Development and Industrial Pharmacy, 2012; 38(1): 84–92© 2012 Informa Healthcare USA, Inc.ISSN 0363-9045 print/ISSN 1520-5762 onlineDOI: 10.3109/03639045.2011.592529

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infections and sepsis15 as compared with orally adminis-tered Ganciclovir. Due to hydrophilic nature (log P = −2.0) and poor membrane permeation characteristic (BCS-III/IV), the bioavailability of Ganciclovir is very poor (5–7%) which require administration of large dose per day to reach effective plasma concentration. Furthermore, poor oral bioavailability of Ganciclovir is associated with greater intersubject variability of plasma concentrations and development of drug resistance9.

So the purpose of present study was to improve the oral bioavailability of Ganciclovir by encapsulating the drug in lipophilic non-ionic surfactant-based nanosized niosomal colloidal dispersion.

Niosomes, the non-ionic surfactant vesicles are micro-scopic lamellar structures formed on admixture of differ-ent non-ionic surfactant classes such as alkyl or dialkyl polyglycerol ethers and Cholesterol, with subsequent hydration in aqueous media16–19.

The in vivo behavior and fate of niosome is similar to the liposome like prolonging the drug release and circulation of entrapped drug and altering its organ distribution and metabolic stability17. Niosomes may overcome the problems associated with liposomes, most important is chemical instability of the phospholipids, cost, oxidation susceptibility, and variable purity.

Materials and methods

MaterialsGanciclovir was obtained from Ranbaxy laboratories (Gurgaon, India). Sorbitan monopalmitate (Span40) and Sorbitan monostearate (Span60) were obtained from S.D. Fine chemicals (Delhi, India). Cholesterol was obtained from BDH chemicals (Delhi, India) and ethanol and diethyl ether, dimethyl sulfoxide from Merck India Ltd. (Mumbai, India).

MethodsAnalysis of Ganciclovir by HPTLC in dissolution media and plasmaGanciclovir was analyzed by using HPTLC. The samples were spotted in the form of bands of width 6 mm with a Camag microlitre syringe on precoated silica gel alu-minium plate 60F-254 (20 cm × 10 cm) with 200 µm thickness (E. Merck, Germany) using a Camag Linomat V (Switzerland) sample applicator. A constant applica-tion rate of 150 nL s−1 was employed and space between two bands were 10 mm. The slit dimension was kept at 5mm × 0.45 mm and 20 mm s−1 scanning speed was employed. The mobile phase consisted of butanol:acetic acid:water (60:25:15). Linear ascending development was carried out in a twin trough glass chamber saturated with mobile phase. The optimized chamber saturation time for the mobile phase was 30 min at room temperature (25 ± 2°C) at relative humidity of 55 ± 5%. The length of chromatogram run was 80 mm. Subsequent to the devel-opment, TLC plates were dried in a current of air with the help of an air-dryer. The source of radiation utilized

was a deuterium lamp. A stock solution of Ganciclovir (100 µg mL−1) was prepared in methanol. In case of plasma, the sample (1 µL) was transferred into Eppendorf tube and extracted with dichloromethane on a vortex-mixer for 2 min and centrifuged at 2100 rpm for 10 min. The supernatant was transferred to glass microvials and the solvent was evaporated at 45°C in water bath. The plasma was reconstituted with methanol (100 µL). Different vol-umes of stock solution 1, 2, 4, 6, 8 µL were spotted on the TLC plate to obtain concentrations of 100–800 ng spot−1 of Ganciclovir. The plasma samples were also spotted in the similar fashion. The data of peak areas plotted against the corresponding concentrations were treated by least square regression analysis. Densitometric scanning was performed on Camag TLC scanner III in the absorbance mode at 254 nm. The R

f was found to be 0.36. Linear least

squares regression analysis showed there was a good lin-ear relationship (r2 > 0.997) between peak area and con-centration in the range 100–800 ng per zone. The intraday and interday precisions determined as relative standard deviation (%RSD) ranged between 0.4458–2.7873% and 0.711–1.8925%, respectively, whereas in case of plasma, it was found to be 0.4491–2.574% and 0.702–1.388%, respectively. Accuracy calculated as percent recovery was in the range of 97.2484–99.7772% (including plasma sample). The LOD, expressed as 3.3σ/(slope of the cali-bration plot), and the LOQ, expressed as 10σ/(slope of the calibration plot), were 2.511 ng/spot and 7.610 ng/spot, respectively. For plasma sample, the LOD and LOQ was found to be 2.893 ng/spot and 7.992 ng/spot, which indicates the sensitivity of the method is adequate.

Preparation of niosomal formulationNoisomes were prepared by using the commonly used reverse evaporation method20. In this method, surfactant and Cholesterol (3:2 molar ratio) were dissolved in a mixture of 5 mL of ether and chloroform (1:1 v/v), called as organic phase. An aqueous phase (10 mL) contain-ing drug (20 mg) is added to this phase and sonicated at 4–5°C for 30 min. Clear gel form appeared which was fur-ther sonicated after the addition of a small amount (2 mL) of phosphate buffered saline (PBS). The organic phase is removed at 40°C under low pressure. The resulting vis-cous niosome suspension is diluted with PBS and heated on a water bath at 60°C for 10 min. The composition of the developed niosomal dispersions are given in Table 1. For the further experiment, the developed niosomes were stored at 15 ± 0.5°C under ambient humidity.

In vitro characterization of niosomesParticle size determinationMorphology of niosomal formulation was confirmed by optical microscopy (Motic, UK) and transmission elec-tron microscopy (TEM ). TEM (Morgagni 268D SEI, USA) was set at 200 KV and of a 0.18 nm capable of point-to-point resolution. Combination of bright field imaging at increasing magnification and of diffraction modes was used to reveal the form and size of the Ganciclovir

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niosomal formulation. In order to perform the TEM obser-vations, the diluted niosomal formulation was deposited on the holey film grid and observed after drying.

Vesicle size, size distribution, and zeta potentialVesicle size was determined by photon correlation spec-troscopy that analyzes the fluctuations in light scattering due to Brownian motion of the particles using a Zetasizer (Nano-ZS, Malvern Instruments, UK). The formulation (0.1 mL) was dispersed in 50 mL of water in a volumetric flask, mixed thoroughly with vigorous shaking, and light scattering was monitored at 25°C at a 90°angle. In addi-tion, zeta potential was also determined using Zetasizer (Nano-ZS, Malvern Instruments, UK).

Entrapment efficiencyUnentrapped drug was separated by centrifugation and after complete vesicle disruption using Triton X-100, the remaining amount of the drug in niosomes was determined.

Entrapment efficiency was calculated by using the following formula:

Entrapment

efficiency (EF) Amount entrapped

Total amount=

× 100

Transition temperature analysis of niosomesTransition temperature analysis was studied by DSC (Perkien Elmer, USA). A small amount of freeze-dried non-ionic surfactant vesicles and pure semisolid surfac-tant (Span40, Span60) was sealed in a 40-µL aluminum crucible and empty aluminium crucible is taken as refer-ence. The temperature of the pans was raised from 40°C to 400°C, at a rate of 10°C/min. The heat flow calibration was performed with indium. The reproducibility of the thermograms was determined by repeating the tempera-ture cycle three times for each sample.

In vitro drug release performanceIn vitro release study was performed by USP XXII method (Dissolution apparatus II at 100 rpm and 37 ± 0.5°C filled with 900 mL of phosphate buffer (pH 7.4). 2 mL of nio-somal dispersion (containing 4 mg of Ganciclovir) was placed in activated dialysis bag dialysis membrane bag (molecular weight cut off 12,000 Da). 5 mL of samples were withdrawn at regular time intervals (0, 2, 4, 6, 8, 12,

16, 20, and 24 h) and replaced with same volume of fresh phosphate buffer (pH 7.4) to maintain the sink condition. Samples were analyzed for the drug content by HPTLC.

Release kinetic studyKinetic analysis of in vitro release data of optimized for-mulation was done according to zero-order, first-order, and Higuchi model.

Zero-order model =Q kt

First-order model Log =Q kt/2.303

Higuchi model =Q k t

where, Q = amount of drug released at time t; k = dis-solution rate constant (with unit of µg/mL/h for zero-order model, 1/h for first-order model, and µg/mL/h for Higuchi model.

Estimation of Ganciclovir in plasmaApproval to carry out in vivo study was obtained from Jamia Hamdard, Institutional Animal Ethics Committee and their guidelines were followed for the studies. The optimized niosomal dispersion, which showed the high-est drug release, was taken for in vivo studies. In in vivo study, the drug analysis was performed by developed HPTLC method. Two groups containing six rats in each were taken for the study. The animals were kept under standard laboratory conditions, temperature at 25 ± 2°C and relative humidity (55 ± 5%). The animals were housed in polypropylene cages, three per cage, with free access to standard laboratory diet (Lipton feed, Mumbai, India) and water ad libitum. The formulations (niosomal dis-persion and marketed tablet) were given orally using oral feeding sonde. The rats were anesthetized using diethyl ether and blood samples (0.5 mL) were withdrawn from the tail vein of rat at 0 (pre-dose), 0.2, 0.4, 0.6, 1, 2, 2.5, 3, 4, 5, 6, 8, 12, and 24 h in vacutainer tubes, mixed and centrifuged at 5000 rpm for 20 min. The collected plasma was extracted with dichloromethane on a vortex-mixer for 2 min and centrifuged at 2100 rpm for 10 min. The combined extract was evaporated to dryness at 45°C in a water bath. At the time of analysis, all residues were reconstituted with methanol and applied on TLC plate. Pharmacokinetic parameters (PK) were calculated by

Table 1. Composition of different developed niosomal formulations.

Formulation code Surfactant CholesterolRatio of surfactant and

Cholesterol (molar ratio)Amount of drug

(mg)NG1

Span40

29 mg 1:1

20

NG2 19 mg 2:1NG3 14 mg 3:1NG4 23 mg 3:2NG5

Span60

29 mg 1:1NG6 19 mg 2:1NG7 14 mg 3:1NG8 23 mg 3:2

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non-compartmental analysis, also called as model inde-pendent analysis, using WinNonLin version 4.0 (Pharsight Corp., Mountain View, CA). Peak plasma concentration (C

max) and time of its occurrence (t

max) were read directly

from the individual plasma concentration–time profiles. Area under concentration time curve (AUC

0→t) was cal-

culated according to linear trapezoidal method.

Pharmacokinetic and statistical analysisData of in vivo analysis was expressed as mean of six animals ± SD. All pharmacokinetic parameters (t

max,

Cmax

, AUC0→t

) were calculated individually for each subject in the group and the values were expressed as mean ± SD. The data were compared for statistical sig-nificance by the one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparisons test using GraphPad Instat software (GraphPad Software Inc., CA).

Results and discussion

Photomicroscopy and transmisson electron microscopyThe photomicrograph of optimized niosomal formulation revealed the spherical shape of vesicles NG8 (Figure 1A). The electron micrograph of optimized formulation are shown in Figure 1B. They show the clear outline and the core of the well-identified vesicles displaying the vesicular structure. Similar structural feature were reported earlier21. Vesicles were presented in dispersed

and unaggregated form with the average vesicular size of 144 ± 3.47 nm (Figure 1C).

Vesicle size, size distribution, and zeta potentialThe particle size distribution and polydispersity index [PDI] of Span40- and Span60-based niosomes are given in Table 2. Average particle size was found to be 144 ± 3.47 nm with PDI = 0.08 for optimized formulation NG8 (Figure 1C). Regarding PDI, a value of zero indicates an entirely monodisperse population and a value of 1 indicates a completely polydisperse population22. So the PDI value of 0.08 for NG8 indicates the best uniformity of size among the formulation. During experiments, it was found that niosomes prepared using Span60 were slightly larger in size (133 ± 3.07 nm–144 ± 3.47 nm) than those prepared using Span40 (129 ± 3.35 nm–121 ± 3.12 nm). The result was in accordance with the previous finding23,24. Correlation between entrapment efficiency and particle size with the nature of surfactant and its ratio with Cholesterol are presented in Figure 2. The increased size of the developed niosomes may be due to the presence of Span60 which has a longer saturated alkyl chain as com-pared with Span4025. Furthermore, the larger vesicles are formed when the hydrophilic portion of the molecule is decreased relative to the hydrophobic portion26, it may also be attributed to the fact that the increase in alkyl chain length as the series is ascended from the C12 to the C18 ester would result in an increase in the value of the critical packing parameter27. Furthermore, zeta potential of all the developed niosomal dispersions were

Figure 1. (A) Photomicrograph (400×), (B) Transmission electron microscopy (TEM) image, (C) Particle size distribution curve of Ganciclovir-loaded niosomes formulation-NG8.

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determined which varied from −9.5 ± 0.9 mV to −27.9 ± 1.9 mV for NG1-NG8 (Table 2). The obtained zeta potential revealed that the developed formulations were stable and having uniformly dispersed particles.

Entrapment efficiencyData in Table 2 shows entrapment efficiencies of various developed niosomal formulations based on different molar ratio of Span40:Cholesterol and Span60:Cholesterol. The highest entrapment efficiency (89 ± 2.13%) was found for NG8 which is Span60-based niosome having surfactant:Cholesterol ratio of 3:2. Effect of nature of surfactant and its ratio against the Cholesterol is estab-lished in Figure 2. Results showed that the incorporation of Cholesterol into niosomes significantly increased the drug entrapment efficiency up to an optimum ratio (3:2) of Span60:Cholesterol. Cholesterol alters the fluidity of chains by reducing the transition of gel to liquid phase of surfactant bilayer27–29. It also increased the micro-viscosity of niosomal membrane conferring more rigidity, resulting in a higher stability which leads to the greater drug retention25. However, after increasing the Span40:Cholesterol molar ratio, it starts reducing the entrapment efficiency as it starts disrupting the regular bilayered structure leading to lowering of drug entrap-ment. Table 2 clearly shows that the entrapment effi-ciencies for Span60-based niosomes were significantly

higher than Span40-based niosomes with the same molar ratios of Span60 to Cholesterol. This might be due to the higher phase transition temperature of Span6030, long hydrophobic alkyl chain surfactant produces high entrapment23. Hence, Span60 having longer saturated alkyl chain (C18) compared with Span40 (C16) produced niosomes with higher entrapment efficiency29. In addi-tion, the length of the alkyl chain influences the HLB value of the surfactant mixture that directly affects the drug entrapment efficiency. The HLB values for Span40 and Span60 are 6.7 and 5, respectively, the lower the HLB of the surfactant, the higher will be the drug entrapment efficiency24.

Transition temperature analysis (DSC)DSC thermograms of Ganciclovir, surfactant, and drug-loaded optimized niosome composed of Span60:Cholesterol (3:2) is illustrated in Figure 3A, 3B,and 3C. DSC thermogram of Ganciclovir and Span60 showed an endothermic peak at 250.71°C (Figure 3A) and 62.165°C (Figure 3B), respectively. DSC thermogram of Ganciclovir-loaded niosomes showed disappearance of all characteristic melting endotherms (Figure 3C). This absence of all melting endotherms suggests the encap-sulation of Ganciclovir in niosomal dispersion occurs. Similar results were found earlier as well with niosomal preparation31,32.

Table 2. Mean particle size, PDI, zeta potential (mV), and entrapment efficiency (%) of niosomal dispersion.Formulation code Mean particle size ± SD (nm) PDI Zeta potential ± SD (mV) Entrapment efficiency (%) ± SDNG1 129 ± 3.35 0.21 −9.5 ± 0.9 49 ± 3.01NG2 127 ± 3.12 0.19 −11.2 ± 0.7 43 ± 5.78NG3 109 ± 2.29 0.32 −12.6 ± 1.0 39 ± 2.43NG4 121 ± 3.12 0.11 −10.3 ± 0.4 53 ± 3.67NG5 133 ± 3.07 0.12 −19.5 ± 1.1 81 ± 2.56NG6 137 ± 2.17 0.23 −16.8 ± 0.7 76 ± 4.34NG7 142 ± 2.12 0.40 −21.2 ± 1.4 71 ± 2.01NG8 144 ± 3.47 0.08 −27.9 ± 1.5 89 ± 2.13%PDI, polydispersity index.

Figure 2. Effect of different surfactants and its ratio with Cholesterol on mean particle size and % encapsulation efficiency.

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Dispersion studyNiosomes which gave the best dispersibility was Span60:Cholesterol-based NG8 formulations. In case of the other niosomal dispersions, higher sedimentation were seen that might be due to fusion of the niosomes. The sonication of the niosomal dispersion reduced the

aggregation, making them more dispersive, thereby increasing dispersibility of the vesicular dispersion24.

In vitro drug release studiesOn the basis of entrapment efficiency, we have selected only Span60-based niosomes for the in vitro drug release

Figure 3. DSC thermogram of (A) Drug, (B) Span60, (C) Ganciclovir-loaded niosomes (NG8).

Figure 4. Percentage drug release profile of niosomal dispersion NG5, NG6, NG7, and NG8.

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studies because they entrapped maximum amount of the drug used during niosomal preparation. Furthermore, maximum dose were available only when maximum entrapment occurs. A comparative in vitro release profile of four formulations [NG5, NG6, NG7, and NG8] is shown in Figure 4. Percentage release of Ganciclovir after 24 h was found to be 89.1 ± 0.016, 90.29 ± 0.17, 95.2 ± 0.014, and 87.34 ± 0.16 for NG5, NG6, NG7, and NG8, respectively. Result shows that the increase of Cholesterol molar ratio significantly reduced the efflux of Ganciclovir, showing Cholesterol membrane stabilizing ability and space-filling action20,33. Furthermore, Cholesterol is known to increase the rigidity of niosomes and abolish the gel-to-liquid phase transition of niosomal systems result-ing in niosome formulations that are less leaky34,35, thus decreasing the drug release from niosomes. Niosomal

formulations prepared using Span60 showed a signifi-cantly slower rate of drug release compared with Span40 (data are not given). This can be explained by the fact that niosomes exhibit an alkyl chain length-dependent release. The higher the chain length, the lower the release rate28. Finally, NG8 was selected for in vivo study on the basis of entrapment efficiency and in vitro study.

Release kinetic studyRelease data of optimized niosomal formulation were analyzed according to zero-order model (Figure 5A), first-order model (Figure 5B), and Higuchi model (Figure 5C). Release pattern was found to follow zero-order kinetics as average values of correlation coefficient were 0.9864 for zero-order model, 0.945 for first-order model, and 0.883 for Higuchi model.

Figure 5. Figures reperesenting the in vitro release kinetic profile for (A) Zero-order model, (B) First-order model, (C) Higuchi model.

Figure 6. Pharmacokinetic profiles of Ganciclovir after oral administration of niosomal formulation (NG8) and marketed tablet.

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In vivo studyFigure 6 shows the pharmacokinetic profiles of Ganciclovir after oral administration of niosomal for-mulation compared with oral suspension of drug. The calculated pharmacokinetic parameters are given in Table 3. For NG8 niosomal formulation, the AUC

0→∞ of

(AUC0→∞

of 11200 ± 22.5 µg/mL/h) was nearly 5-fold higher compared with orally administered drug suspen-sion (AUC

0→∞ of 2120 ± 19.56 µg/mL/h), and the statistic

calculation provided a significant difference between both values (p < 0.05). The effective drug concentration (>0.69 µg/mL in plasma) was maintained for at least 10 h through niosomal oral administration. The signifi-cant increase of C

max values and sustaining effect of the

drug in plasma for prolonged period may be owing to enhanced absorption of the Ganciclovir with niosomal formulation. This finding may be a result of the influence of the vesicle size (250 nm) of niosomes, lipophilic nature of the niosomal formulation, improved partitioning of the lipophilic system to the mucosa, prolonged localiza-tion of the drug-loaded niosomes at the site of absorp-tion, and its component (surfactants) as a penetration enhancer36-38. The in vivo data reveal that administra-tion of Ganciclovir through niosomal dispersion had a sustained and enhanced absorption. Furthermore, Ganciclovir in niosomal form is effective at lower dose level with reduced dosing frequency. So the developed formulation can play a key role in reduction of dose and its associated side effects, which is observed with con-ventional dosage form.

Conclusion

The prepared Ganciclovir niosomes were homogenous in shape with an average size of 144 nm and maximum per-centage drug entrapment was found to be 89 ± 2.13% with NG8. The niosomal formulation showed sustained release characteristics with zero-order drug release. In vivo study in rats discovered 5-fold increase in the oral bioavailabil-ity compared with tablet. Our studies provided evidence that niosomes were valuable as an oral delivery carrier to enhance the bioavailability of Ganciclovir.

Acknowledgment

The authors would like to acknowledge Ranbaxy labo-ratories, Gurgaon, India for providing a gift sample of Ganciclovir.

Declaration of interest

The authors are grateful for financial support from the Department of Biotechnology Govt. of India. The authors state no conflict of interest.

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Table 3. Pharmacokinetic parameters obtained following oral administration of drug-loaded niosomal dispersion and drug suspension to six rats (mean value ± SD; n = 6).

Formulation tmax

(h) Cmax

(µg/mL) AUC 0→t

(µg.h/mL)

Tablet 3 ± 0.32 1.2101 ± 0.4 2120 ± 19.56Niosomal dispersion

1.3512 ± 0.3 1.3512 ± 0.3* 11,200 ± 22.5†

†p < 0.05, compared with oral administration.*p < 0.01, compared with oral administration.

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Pharmacokinetics study revealed that GCV mucoadhesive nanoemulsions (GCV-CNE), Chitosan nanoparticles (GCV-CNP) and GCV mucoadhesive niosome (GCV-CN) provided approximately 6 fold increase in the relative ocular bioavailability compared with GCV solution. This could be attributed to increased pre-corneal retention of the GCV nanoformulations owing to presence of mucoadhesive chitosan and increased corneal penetration of nanosized particles.

In eye irritation test, non-significant redness were seen in the test eyes and only 2ºC rise in temperature above normal (35±0.7ºC) were found on corneal surface on measurement by IR- camera. These results revealed that there were no any sign of local inflammation in eye and the studied formulations were nonirritant and nontoxic in nature.

In conclusion, the achieved physicochemical, pharmacokinetic, gamma scintigraphy and ocular irritation studies results revealed that these nanosystems are potential vehicles for improved ocular delivery of Ganciclovir in ocular viral infections. Furthermore, due to reduced clearance over the cornea and drainage to the systemic circulation, the developed nanocarriers particularly GCV-CNE is expected to reduce the systemic side effects of GCV and also its dosing frequency.

Ganciclovir (GCV) is a synthetic acyclic nucleoside analog of 2’-deoxyguanosine, which exhibits antiviral activity against herpes simplex virus and cytomegalovirus at relatively low inhibitory concentrations [1, 2]. Current conventional treatment involves oral administration of GCV at a dose of 3.0-5g/day. Such a high dose results in dose-related toxicity like bone marrow suppression and neutropenia [2]. Therefore, targeting of ocular viral infections through topical route is valuable, but is limited in case of GCV delivery due to poor ocular availability owing to its hydrophilic character and rapid elimination. Development of mucoadhesive GCV nanoformulations for the treatment of ocular infections is worthwhile since they are expected to prolong the pre-ocular retention and increase the ocular bioavailability[3].

Preparation of GCV nanocarriers GCV mucoadhesive nanoemulsions (GCV-CNE) was prepared by aqueous titration technique using Triacetin, Tween 20 and diethylene glycol monoethylether as oil, surfactant and cosurfactant respectively under chitosan solution as aqueous phase. Chitosan nanoparticles (GCV-CNP) were prepared according to the ionotropic gelation method with slight modification [4]. GCV mucoadhesive niosome (GCV-CN) was prepared by the reverse-phase evaporation technique [5]. Characterization of GCV nanocarriers The mean particle size and zeta potentials to evaluate the stability of the dispersion system of GCV nano-carriers were determined by photon correlation spectroscopy using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Dispersion morphology were established by TEM (Morgagni 268(D) USA) In-vitro transcorneal permeation study was performed on goat eye cornea using Franz Diffusion cell over the period of 24h.

Corneal retention study by γ- scintigraphy and Ocular pharmacokinetic study The pre-corneal retention of GCV nanocarriers were assessed by γ- scintigraphy. 99mTc-labelled GCV loaded nanocarriers was compared with 99mTc-labelled GCV solution. A total of 20 μL of the labelled formulations were instilled into the cul-de-sac of the left eye, and the eye was manually blinked three times to distribute the formulation over the cornea. The rabbit was positioned below the head of the gamma camera and clearance of the formulations from the eye was followed for 30 min by dynamic imaging using gamma camera. Four groups, each having seven rabbits (2.25±0.25 kg), were used in pharmacokinetic study. In both the eyes, a single topical instillation (50 µL) of GCV-solution, GCV nanocarriers dose equivalent to 0.3% w/v of GCV were added and 50 µL of the aqueous humor was and analyzed for GCV by UPLC.

Ocular surface temperatures (ºC) were measured non-invasively through IR-Camera onto the rabbit eyes in which one eye was kept as control. As it was reported earlier that raise in temperature 2ºC above normal is the sign of inflammation in the cornea [3].

Akhter S*1, Ahmad I1, Ahmad M Z1, Anwar M1, Jain N1, Jain G K1, Talegaonkar S1, Khan Z I1., Shamim M1., Bhatnagar A2, Khar R K1, Ahmad F J1. 1Nanomedicine Research Lab, Faculty of Pharmacy, Jamia Hamdard University, N. Delhi - 110062, India 2Institute of Nuclear Medicine and Allied Sciences (INMAS), DRDO, Delhi-110054, India *sohailakhtermph@gmail.com

Abstract: The purpose of present study is to evaluate the ocular retention behavior and enhanced ocular bioavailability of Ganciclovir (GCV) through mucoadhesive nanocarriers. Developed chitosan coated niosomes (GCV-CN), chitosan nanoparticles (GCV-CNP) and mucoadhesive nanoemulsion (GCV-CNE) having the dispersion size of 80.03± 1.02nm (GCV-CN), 59.41± 1.47nm (GCV-CNP), 20.7± 1.11nm (GCV-CNE) with narrow distribution (PDI, < 0.3). Furthermore, the zeta potentials greater than +30mV indicate the stable nature of these nanosized dispersion phases. Sustained release effect of the GCV loaded nanocarriers were investigated goat eye cornea emphasized the increase in permeability parameters with these nanocarriers. Furthermore, the in-vivo performance of the developed carriers were studied through corneal retention behavior and bioavailability study by Gama scintigraphy and UPLC method respectively on rabbits. Results revealed that selected nanocarriers showed excellent retention (> 8hr) over the non mucoadhesive GCV-solution (10min) and more than 3841.9±20.0ng.h/mL ocular bioavailability over the GCV-solution (AUC 0-t, 459.6±11.2 ng.h/mL). In addition, ocular irritation potential of the developed formulation was studied on rabbit eye by IR camera, and it was found that no significant changes in temperature on the corneal surface that confirm its non-irritant nature for ocular delivery. These results suggested that GCV-CN, GCV-CNP and GCV-CNE are potential vehicles for improved ocular delivery of GCV in ocular viral infections.

GCV-CN, GCV-CNP and GCV-CNE were evenly round in shape with mean particle size in the range of 15-200nm with narrow distribution (PI, < 0.3). The zeta potentials which are indicative of stability of dispersion system with value greater than +30mV indicate the stable nature of these nanosized dispersions.

The values of transcorneal flux for selected formulations were observed between 102.59 to 217.32 μg cm-2 h-1 in in-vitro transcorneal permeation study which is 11 fold greater than the control solution (20.213 μg cm-2 h-1).

γ- Scintigraphy study was carried out to study the ocular retention performance of the mucoadhesive Nanocarriers (Figure 2.a, 2.b 2.c & 2d). Results revel that selected nanosystems showed excellent retention (> 8hr) over the non mucoadhesive solution (10min).

INTRODUCTION

METHODOLOGY

Pharmacokinetic Parameter

Formulations

GCV-CNP GCV-CNE GCV-CN

tmax (h) 1.0 1.4 1.0

Cmax (ng/mL) 589±8.9 449±6.5 523±8.2

Ke (1/h) 0.67±0.08 0.08±0.01 0.13±0.02

AUC 0-t ( ng.h/mL ) 1441.9±20.0 3797.7±21.8 3428.1±29.4

AUC 0-∞ ( ng.h/mL) 1738.7±22.80 5622.1±39.5 4160.7±46.9

AUMC0-t ( ng.h/mL) 2763.9±46.1 19402.5±40.4 15543.7±35.8

RESULTS AND DISCUSSION

CONCLUSION

1. J. Colin, Ganciclovir ophthalmic gel, 0.15%: a valuable tool for treating ocular

herpes, Clin Ophthalmo. 1, 441 (2007). 2. J.K. McGavin, K. L. Goa, Ganciclovir: an update of its use in the prevention of

cytomegalovirus infection and disease in transplant recipients, Drugs. 61, 1153 (2001).

3. S. Akhter, S. Talegaonkar Z. I. Khan, G. K. Jain , R. K. Khar, F. J. Ahmad, Assessment of ocular pharmacokinetics and safety of Ganciclovir loaded nanoformulations.. J Biomed Nanotechnol. 7, 144-5 (2011 ).

4. P. Calvo, C. Remunan-Lopez , C.L Vila-Jato, M.J Alonso, Novel hydrophilic chitosan- polyethylene oxide nanoparticles as protein carriers, J of Applied Polymer Sci. 63, 125 (1997).

5. D. Aggarwal, I.P Kaur, Improved pharmacodynamics of timolol maleate from a mucoadhesive niosomal ophthalmic drug delivery system, Int J Pharm. 290,155 (2005).

REFERENCES

Formulation Development

In-vivo study

Ocular irritation test

Formulation Development

In-vitro study

In-vivo study

Ocular irritation test

Table. Pharmacokinetic parameters of GCV after topical instillation of GCV solution and GCV nanoformulations to rabbit eye

Figure. 2d) Radio-image of the whole rabbit 30 min after instillation of 99mTc-labelled GCV-Solution

Figure. 2c) Radio-image of the whole rabbit 30 min after instillation of 99mTc-labelled GCV-CNE

Figure. 2a) Instilled activity remaining on the ocular surface as a function of time after application of 99mTc-labelled GCV-CNE

Figure. 2b) Instilled activity remaining on the ocular surface as a function of time after application of 99mTc-labelled GCV-Solution

The author would like to acknowledge Department of Biotechnology, Government of India, Jamia Hamdard University and INMAS, DRDO, Delhi for providing financial support and adequate infrastructure to carry out the work.

ACKNOWLEDGEMENT

In-vitro study

4th European-Conference for Clinical Nanomedicine (CLINAM 2011), May 23–25, Basel, Switzerland

Figure. 1a) Particle size distribution, 1b) Zeta potential and 1c) TEM images of selected nanocarriers

Figure.3A) Ocular image on 1week application of GCV-CNE, 3B) Ocular image on 1week application of control (Carrageenan)

Corneal surface temperature after 7days application : GCV-CNE = 37±0.710C (20C above normal) Control = 42±1.020C (70C above normal)