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Colloids and Surfaces B: Biointerfaces 93 (2012) 161– 168

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

jou rn al h om epage: www.elsev ier .com/ locate /co lsur fb

rotein based nanoparticles as platforms for aspirin delivery for ophthalmologicpplications

aikat Dasa,1, Jayesh R. Bellareb, Rinti Banerjeea,∗

Department of Biosciences and BioEngineering, Indian Institute of Technology Bombay, IndiaDepartment of Chemical Engineering, Indian Institute of Technology Bombay, India

r t i c l e i n f o

rticle history:eceived 20 September 2011eceived in revised form7 December 2011ccepted 27 December 2011

a b s t r a c t

Most conventional ophthalmic dosage forms, though simplistic are limited by poor bioavailability in theposterior chamber of the eye. Application of nanotechnology has the potential to overcome this problem.By varying aspirin albumin ratios from 0.06 to 1.0, we obtained electrokinetically stable, pharmacolog-ically active albumin based aspirin nanoparticles of <200 nm diameter with low polydispersity. In vitrorelease study showed nanoparticle formulation can release aspirin at a sustained rate for prolonged dura-

vailable online 3 January 2012

eywords:anoparticlesspirincular drug delivery

tion (90% at 72 h) and 11% drug release in the posterior chamber over a period of 72 h under simulatedcondition. Stability of the formulation was well maintained on storage for six months and after recon-stitution for 24 h. The formulation showed no hemolysis in contrast to the high hemolysis due to thefree drug. This study shows that aspirin loaded albumin nanoparticles prepared by coacervation holdspromise as a formulation for topical delivery in diabetic retinopathy.

iabetic retinopathy

. Background

Diabetic retinopathy resulting from long standing uncontrollediabetes mellitus is one of the major causes of loss of vision [1].latelet aggregation, changes in surface adhesiveness of leuco-ytes and the capillary endothelial cells causing capillary occlusionlay an important role in the pathogenesis of diabetic retinopa-hy [2]. Aspirin inhibits the cycloxygenease enzyme and reduceshe prostaglandin production. A variety of ocular pathologies are

ediated by prostaglandins, so aspirin has wide therapeutic poten-ial in eye. Various randomized controlled trials have investigatedhe potential role of aspirin in diabetic retinopathy. The numberf macular microaneurysm is related to deteriorating ocular signsn diabetic retinopathy. A multicentric randomized controlled trialonducted by the DAMAD study group demonstrated that aspirinlone or in combination with dipyridamole reduces the develop-ent of macular microaneurysm and progression of early diabetic

etinopathy [3]. Based on animal studies it has been shown that oralspirin can inhibit diabetic induced increase in NF-�b DNA binding

ffinity, translocation of P-50 and P-65 in the nuclei of retinal vas-ular endothelial cells, ganglionic cell layer and inner nuclear layer

∗ Corresponding author. Tel.: +91 22 25767868.E-mail address: rinti@iitb.ac.in (R. Banerjee).

1 Present address: Department of Radiation Oncology, Christian Medical College,ellore 632004, India.

927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2011.12.033

© 2012 Elsevier B.V. All rights reserved.

[4]. Studies suggest that high dose aspirin could play an importantrole in the prevention of early diabetic retinopathy [5].

Systemic administration of such high dose of aspirin is asso-ciated with significant side effects. The blood ocular and bloodretinal barrier reduces the bioavailability of systemically admin-istered drug in retina [6]. Vitreoretinal administration of the drugis an invasive procedure which requires technical expertise andis associated with serious complications like retinal detachmentand endophthalmitis [7]. It may also lead to complications such asvitreous hemorrhage, infection, lens and retinal injury [8,9].

Therefore, a clinically viable option is development of a top-ical formulation with increased precorneal residence time andbioavailability in vitreoretinal tissues or posterior segment of theeye. Nanoparticle systems have the potential to improve the reachof aspirin in the posterior chamber while masking its unwantedtoxic effects. Nanoparticles are colloidal particles having size lessthan 1 �m in diameter and are made of macromolecular materi-als in which the active drug is dissolved, entrapped, encapsulatedadsorbed or attached. Reduction of the particle size causes mini-mal irritation. They are administered in the form of nanospheres,nanocapsules; which allows higher surface to volume ratio andincrease in precorneal residence time, overcomes poor solubilityof drugs, reduces the therapeutic dose requirement and chances oftoxicity than the conventional formulations [10–13]. Biodegradable

and biocompatible albumin nanoparticles have proved to be par-ticularly effective for administration of various ocular drugs [14].Drug loaded polymeric nanoparticles based on various polymerslike albumin, chitosan, polylactide, poly lactide co glycolide, poly

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caprolactone have been investigated [7]. Albumin is an integralomponent of plasma protein and most of the drugs bind to albu-in in plasma. Albumin nanoparticles prepared by coacervation

echniques [15] have been evaluated for various drugs includinganciclovir and Paclitaxel [16,17]. Among this Paclitaxel loadedlbumin nanoparticles are already in clinical use for breast andvarian cancer [18]. Protein nanoparticles of 100 nm diameter canscape phagocytosis and reach relatively inaccessible sites [19]. Weave already optimised the process parameters for the develop-ent of stable aspirin loaded nanoparticles [20]. In view of these

enefits the present study aimed at evaluation of aspirin loadedlbumin nanoparticles for their suitability as ocular carriers toeliver aspirin to the posterior chamber of the eye.

. Materials and methods

.1. Materials

The following commercially available materials were used aseceived. Bovine serum albumin (Sisco Research Lab., Bombay),5% glutaraldehyde (Loba Chemie, Bombay), 99% absolute alcoholChangshu Yangyuan Chemical, China), mannitol (S K Lab., Ahmed-bad), phosphate buffer (prepared from NaH2PO4 obtained fromerck, India, and Na2HPO4 obtained from Qualigen fine Chemi-

als), lysozyme (15,000 units/mg, SRL Limited, Bombay), Tear PlusAllergen, India), xanthan gum (food grade, Loba Chemie). Aspirinowder and glucose used for the experiments were of pharmaceu-ical grade.

.2. Methods

.2.1. Preparation of the formulationAspirin loaded albumin nanoparticles with various aspirin pro-

ein ratios (w/w, from 0.06 to 1) were prepared by desolvationased coacervation method of microencapsulation, followed byrosslinking with glutaraldehyde [21]. We have described the pro-ess of preparation of the formulation, determination of particleize, drug entrapment and in vitro release kinetics in our previ-us publication [20]. In brief, aspirin was incubated with requiredmount of protein solution (2%, w/v) for 1 h at room temperature (atH 5.5) and then ethanol was added to the solution in 2:1 ratio (v/v).he coacervate so formed was hardened with 25% glutaraldehyde1.56 �g/mg of protein) for 2 h to allow cross-linking of protein.rganic solvents were then removed under reduced pressure and

hen the solution was purified by centrifugation at 4 ◦C for 45 mino obtain the nanoparticle pellets. Three batches of nanoparticlesere centrifuged at different speeds corresponding to 27,000 × g,

7,200 × g and 10,000 × g. However, the first batch (27,000 × g) wassed for further studies as the particle diameters were found to bemallest in this group. Pellets of nanoparticles were suspended inhosphate buffer (pH 7.4; 0.1 M) and each sample was lyophilizedith mannitol (2%, w/v) at −48 ◦C and 28 × 10−3 Mbar pressure for

4 h in Freeze Dry System/Free zone, 4.5 Labconco Model 117 (Lab-onco, USA). Albumin nanoparticle controls were also prepared byhe same procedure without drug loading.

.2.2. Characterization of the formulationLyophilized nanoparticles were suspended in phosphate buffer

o prepare 1% solution. Particle size and zeta potential was deter-ined using Photon Correlation Spectroscopy; which is based

n the principle of dynamic light scattering (BI MAS, Multiangleizing option on Zetaplus, Brookhaven Instruments, USA). In addi-ion, particle size was also characterized by transmission electron

icroscopy (TEM).

iointerfaces 93 (2012) 161– 168

2.3. Determination of drug entrapment

Based on small particle diameter and stable zeta potential val-ues as obtained from the results of PCS and TEM, the formulationwith aspirin protein ratio of 0.4 was chosen for further study. Thesupernatant obtained after centrifugation of drug loaded albuminnanoparticles at (27,000 × g), was analyzed for the drug content(w) at 230 nm on Lambda 25 UV-vis spectrophotometer (PerkinElmer, USA). A standard calibration curve of concentration versusabsorbance was plotted for this purpose. The amount of drugpresent in the pellet was calculated as difference from the totaldrug used in the preparation (W − w). The amount of drug encap-sulated in the pellet was expressed as a fraction of the total drugused in the formulation to provide the percentage entrapment as((W − w) × 100)/W.

2.4. In vitro release studies

2.4.1. In vitro release kineticsThe release kinetics of the drug was studied in a specialized

diffusion chamber with two compartments, and having dimen-sions 8 cm × 3 cm × 2.5 cm; which was designed at the IndustrialDesign Center (IDC) of the institute. The two compartments areseparated by a septum with an opening of 2 cm diameter, whichwas covered with spectrapor membrane to allow diffusion of thedrug molecules. In vitro release kinetics study across spectra-por membrane (cut off 3500 Da) precluding albumin (molecularweight 66,432 Da) was performed in triplicate at 37 ◦C in the dif-fusion chamber, under constant slow magnetic stirring. 20 mg ofdrug-loaded nanoparticles, suspended in 15 ml phosphate buffer(pH 7.4), was placed in the donor compartment and the othercompartment was filled with phosphate buffer of same volume.To determine the concentration of aspirin in the receiving com-partment, samples 1 ml each were withdrawn from the receiversolution at prefixed times and the absorbance was measured at230 nm on Lambda 25 UV-vis spectrophotometer (Perkin Elmer,USA) [22]. After each measurement, same amount of phosphatebuffer was reintroduced into the receiver chamber to account forthe change in concentration. Finally the concentration correspond-ing to the absorbance was determined from the concentration vsabsorbance calibration curve. Aspirin free drug cumulative releasewas calculated in similar manner.

2.4.2. Release kinetics in presence of viscoelastic coatingsThe residence time of the formulation can be increased by incor-

porating viscosity enhancers like xanthan gum in the formulation.Therefore, 20 mg of aspirin loaded albumin nanoparticles weresuspended in 15 ml 0.5% solution of xanthan gum made in phos-phate buffer. The preparation was kept in the donor compartmentof the diffusion chamber and the other compartment was filledwith phosphate buffer. Diffusion of the drug was studied acrossspectrapor membrane with cut off 3500 Da. 1 ml solution from thereceiving compartment was withdrawn at different time intervalsand replaced with same volume of phosphate buffer. Absorbance ofthe sample at 230 nm was measured spectrophotometrically usingLambda 25 UV-visible spectrophotometer (Perkin Elmer, USA) andamount of drug released in terms of percentage entrapment wascalculated.

2.4.3. Release kinetics in simulated ocular conditionsIn literature, the drug release of particulate carriers in ophthal-

mology has been measured under sink conditions by determining

the transport through synthetic membranes using dialysis bags orby applying in vitro diffusion cells. The reported data offer verylimited information relative to the realistic conditions of the eye. Inorder to simulate ocular conditions in vivo, the method chosen here

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akes into account the ratio of the amount of preparation tested andhe tear solution expected to release because of application of therug topically. However, this method may not take into account theynamics of tear fluid in the eye and the loss of drug by drainage.ommercially available artificial tear solution, Tear Plus (contain-

ng Polyvinyl Alcohol 14 mg, Povidone 6 mg, Chlorbutol IP, 5 mger ml, Allergan) was used for this purpose. Aspirin loaded albu-in nanoparticles 20 mg was added to 15 ml phosphate buffer in

he donor compartment of the diffusion chamber. Normal tear vol-me in conjunctival cul de sac is 10 �l. In presence of any foreignody there is 300% increase in the tear secretion. Keeping this fact inind 600 �l of artificial tear was added to the donor compartment

nd the release kinetics was studied at 37 ◦C at pH 7.4.It may be mentioned that there is change of the profile of pro-

eins in the tear of diabetic patients. However, electrophoreticatterns obtained by SDS PAGE show that there is no significantifference in the quantity of four major tear proteins – lactofer-in, lysozyme, lipocalin, and albumin, in diabetic and non diabeticatients [23]. As a further step towards simulation, lysozymeas added to the tear fluid (mean tear lysozyme concentration

.4 ± 0.5 mg/ml) and the release profile was observed [24].

.4.4. Simulation of drug delivery in retrovitreal tissueA specialized diffusion chamber consisting of three chambers;

ith surrounding water jacket was designed to simulate the pos-erior chamber drug delivery. Each compartment is separated fromhe other by a septum that contains a hole of 2 cm diameter. Thisas used to simulate the delivery of the drug in the retrovitreal

issue and on the anterior part of vitreous. Normally the drug aftereing applied topically has to pass through the cornea to anteriorhamber of the eye, which is separated from the retrovitreal tis-ue including the retina by vitreous humor. However it should beentioned here that, the pharmacokinetic model of ocular drug

elivery to the posterior chamber of the eye is complex as the num-er of anatomical barriers and sources of removal of the drug fromhe site of application are multiple. Therefore, a proper in vitro

odel taking all the factors into consideration is difficult to con-eive. However, the three-compartment release kinetics may be atep closer towards the simulation of drug delivery to the posterioregment of the eye. To study the release kinetics, 25 mg of aspirinoaded albumin nanoparticles were added to 20 ml of 0.5% solutionf xanthan gum in phosphate buffer. 600 �l of Tear Plus solutionontaining 1.4 mg/ml lysozyme and glucose (120 mg/100 ml) wasdded to the xanthan gum solution. The entire preparation wasoured in the left compartment of the chamber. Normal fastinglood glucose level is 60–90 mg/100 ml. Since diabetic retinopa-hy is a long-term complication of diabetes associated with poorlycemic control, it may be assumed that the glucose level in thecular fluids will be higher than normal. 20 ml of vitreous humorooled from four goat eyes (obtained from butcher’s shop, ani-als not sacrificed for this experiments) was kept in the middle

ompartment and 20 ml phosphate buffer was kept in the rightompartment (receiving compartment). Glucose (120 mg/100 ml)as added to both middle and receiving compartment to equal-

ze the chemical gradient due to glucose. In another experimentitreous humor in the middle compartment was replaced withhosphate buffer, other conditions being remaining unaltered. This

s to simulate the drug reaching the anterior portion of vitreous.he UV absorbance spectrum of glucose was obtained to confirmts negligible interference in UV range. The separating membraneetween the left and the middle compartment was goat’s corneand that between the middle and right compartment was spectra-

or membrane. Temperature of the circulating water around thehamber was 37 ◦C. 1 ml solution from the receiving compartmentas withdrawn at different time intervals and replaced with phos-hate buffer. Absorbance of the sample at 230 nm was measured

iointerfaces 93 (2012) 161– 168 163

by UV spectrophotometry and amount of drug released in terms ofpercentage entrapment was calculated.

2.4.5. Pharmacological efficacy by platelet aggregometryIn vitro, platelet aggregation occurs when aggregating agents

are added to platelet rich plasma (PRP), under constant stirring.As aggregation proceeds, platelet clumping occurs. In plasma thisreduces the optical density of the PRP allowing more light (infrared) to pass through. If all the platelets are clumped, the resultantoptical density would be equal to that of the platelet poor plasma(PPP). The light transmittance through the PPP represents 100%aggregation and that through PRP represents 0% aggregation. Theinstrument develops a voltage proportional to the transmittanceof light through plasma. This voltage is recorded on a strip chartrecorder as a function of time.

To measure the platelet aggregation with the formulation, 15 mlof blood was collected from a healthy volunteer and platelet richplasma (PRP) was obtained by centrifugation at 100 × g for 10 min.PRP was incubated with 0.18 mg/ml of aspirin loaded albuminnanoparticles at 37 ◦C for 1 h. This corresponds to the concentrationof aspirin in blood for its standard therapeutic dose for pre-venting thromboembolism (75 mg/day). The minimum inhibitoryconcentration of aspirin for prevention of thromboembolism can becalculated taking into consideration the volume of distribution ofaspirin after administration, which is 0.17 l/kg body weight. Aggre-gation of platelets was studied after adding 5 �M of ADP to 450 �lof PRP in a Platelet Aggregometer (Chronolog Corporation) after1/2 h of incubation. Results were compared with PRP control with-out aspirin addition. In all cases a baseline of platelet poor plasmawas obtained as a measure of the supernatant during 100% aggre-gation.

2.4.6. Evaluation of formulation stabilityThe stability study included short-term stability after recon-

stitution and long-term storage stability. A stable formulationshould be able to maintain small size with lesser degree ofaggregation even after reconstitution or storage. The physicalstability of the formulation with aspirin protein ratio 0.4, wasanalyzed by PCS after six months of storage. To evaluate thestability of the formulation after reconstitution, 1 mg/ml solu-tion of aspirin loaded albumin nanoparticles in phosphate buffer(pH 7.4) was kept at room temperature. Particle size was deter-mined after 1, 2 and 4 h. To assess the stability after storage,dynamic light scattering was carried out to determine the parti-cle size of lyophilized nanoparticles stored in screw capped glassbottles in the refrigerator at 4 ◦C, away from direct light for 6months. Transmission electron microscopy of the formulationswas also carried out after six months of storage in refrigera-tor at 4 ◦C. Lyophilized nanoparticles 5 mg/ml were suspended inphosphate buffer (pH 7.4) and were observed using transmissionelectron microscope (Philips, CM 200, acceleration voltage 200 kV,resolution 0.23 nm).

2.4.7. In vitro hemolysis with formulationThe protocol for the In vitro hemolysis of the formulation was

based on the procedure developed by Ali and Tayyab [25]. Thedesired concentration of the aspirin free drug and nanoparticleseach was prepared in phosphate buffer. Whole blood 5 ml wastaken from a healthy volunteer and centrifuged at 1000 × g for20 min, to isolate the red blood cells. The buffy coat was removedand the blood cells were washed with normal saline (0.9% NaCl)solution for 3 times. The final packed cell volume was diluted with

normal saline in a ratio of 1:1 to get 50% hematocrit value of bloodcells. Next, 250 �l of these cells was added to 250 �l of the free drugsolution and nanoparticles each prepared in phosphate buffer; andthe volume was made to 1.5 ml with normal saline solution. For

164 S. Das et al. / Colloids and Surfaces B: Biointerfaces 93 (2012) 161– 168

Fig. 1. Comparison between the kinetics of free drug and nanoparticles. (i) Time required for 50% drug release. (ii) Drug released at 30 min. At ½ h almost 75% of the freed , erro

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00% hemolysis or positive control, 250 �l of the blood cells wasdded to deionized water and made up to 1.5 ml. The blood samplesontaining the free drug, nanoparticles and deionized water werehen incubated for 1 h in a water bath maintained at 37 ◦C. After 1 h0 �l of 2.5% glutaraldehyde solution was added to the samples toerminate the reaction. The blood samples were then centrifugedt 1000 × g for 15 min and the absorbance of the supernatant waseasured at 540 nm.The percentage of hemolysis was calculated as:

ercent hemolysis = absorbance of sampleabsorbance at 100% hemolysis

× 100

bsorbance of sample: absorbance of blood sample containing freerug/drug nanoparticles at 540 nm. Absorbance at 100% hemolysis:bsorbance of blood cells + deionized water at 540 nm.

The concentration of the albumin nanoparticles/free drugequired to produce 50% hemolysis, HC50 was estimated.

. Results

.1. Results of PCS and TEM

Results of PCS and TEM were described in our previous publica-ion [20]. To summarize, by varying aspirin albumin ratios from.06 to 1.0, we obtained stable nanoparticles of sizes 12.4 nmo 190.8 nm respectively, with low polydispersity. The range ofolydispersity was 0.005–0.281, indicating uniformity in size dis-ribution. For most of the samples sample qualities were above 8n a scale of 10, which suggests no turbidity and homogenous sam-le. In vitro release kinetics study shows, unlike the free drug, whicheaks at 1/2 h and saturates at 1 h (Fig. 1), nanoparticles are capablef releasing the drug for as long as 72 h with 50% release occurringt 12 h. Therefore this system overcomes the burst effect of the freerug.

.2. Release kinetics in presence of viscoelastic coating

One of the challenges in ophthalmic drug delivery is to increase

he residence time of the formulation without causing irritationr blurring [27]. This can be achieved by addition of biodegrad-ble polymers as viscosity enhancers. We employed xanthan gum0.5%) to enhance the viscosity of the system. The release kinet-cs was studied in presence of xanthan gum (0.5%) solution inhosphate buffer which showed that though the release kinetics

r bar denotes standard deviation).

remains the same (two plateau), the release is more sustained withnear hundred percent release achieved even after 80 h and 50%release obtained around 40 h (Fig. 2).

3.3. Release study in the presence of artificial tear

The release kinetics in presence of artificial tear and artificialtear containing lysozyme was studied to simulate the ocular con-ditions. Fig. 3 represents the release kinetics of nanoparticles inthe presence of artificial tear and tear solution reconstituted withlysozyme (1.4 mg/ml). The drug released is plotted as percentagereleased from the entrapped drug on a time scale. It is evident thatthe release profile in both the cases is almost similar. Fifty per-cent drug release is observed around 30 h in presence of tear andin 40 h in presence of lysozyme. Release close to 100% is attainedaround 80 h. The release profiles in the presence of artificial tear andlysozyme show that nanoparticles are capable of releasing aspirinin slow and sustained manner even in presence of tear fluid. How-ever, comparison with the release profiles of control (in absenceof tear fluid) shows that though the pattern of release is same theyare not identical. More specifically slightly slower release is noticedin the presence of tear solution. It may be because of the presenceof charged moieties in the system, which affects the release of thedrug.

3.4. Simulation of posterior chamber delivery

In order to simulate the posterior chamber drug delivery,the pharmacokinetic pathway was simulated through the three-compartment diffusion chamber. When a drug is administeredtopically, the first significant barrier it encounters is the cornea.It then passes through the aqueous humor and reaches the ante-rior plane of the vitreous. Crossing the thick vitreal gel it reachesthe posterior plane of vitreous and retrovitreal tissue. Thereforethe drug, which reaches the third compartment with phosphatebuffer in the middle, may be considered equivalent to the drugreaching anterior plane of posterior chamber. Similarly, the drugreaching the third compartment crossing the vitreous in the middleis equivalent to the drug coming to the posterior plane of vitreousand retrovitreal structures. Fig. 4 shows the release profile stud-

ied in the three-chambered compartment with 0.5% xanthan gumand tear in presence of vitreous. It is observed that the amount ofdrug reaching the receiving compartment through vitreous is lowercompared to that in the presence of phosphate buffer in the middlecompartment. Only around 11% entrapped drug was detected at the

S. Das et al. / Colloids and Surfaces B: Biointerfaces 93 (2012) 161– 168 165

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nd of 72 h (5% at 24 h) in presence of vitreous, whereas 75% drugeached the third compartment in presence of phosphate buffer at2 h (30% at 24 h).

.5. Stability of formulations

Stability of the formulation implies retention of physicochem-cal parameters and functional efficacy after storage. Analysis ofarticle size based on dynamic light scattering is one of the standard

ig. 3. Release kinetics in the presence of nanoparticles and in the commercially availableear and lysozyme (n = 3, error bar denotes standard deviation).

lease obtained around 90 h (n = 2, error bar denotes standard error).

ways of assessment of the shelf life of pharmaceutical formulationsand has also been employed for topical ocular drugs [29].

The electron micrograph of the formulation after six monthsstorage (Fig. 5) showed discrete particles with uniform size distri-bution and minimum aggregation. The photograph was analyzedby Image J software for particle size and the summary statistics

is presented as an inset. The modal distribution is in the classinterval 107–143 nm. PCS measurements of the formulation werecarried out after six months of storage, which showed narrow

artificial tear [tear plus] solution with nanoparticles and in the presence of artificial

166 S. Das et al. / Colloids and Surfaces B: Biointerfaces 93 (2012) 161– 168

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ig. 4. Release kinetics in three chamber model with (i) vitreous in the middle com.5% xanthan gum with drug, artificial tear. Middle compartment: 20 ml of pooled anrror bar denotes standard deviation; for vitreous, 4 animal vitreous pooled, repres

ize distribution, unimodal pattern and average size less than00 nm.

It is observed that the particle size of the formulation withspirin protein ratio 0.4 reconstituted in phosphate buffer is around2 nm after reconstitution and storage at room temperature for 4 h.

.6. Pharmacological efficacy and dosage requirement

Aspirin significantly inhibits retinal hemorrhage and the for-ation of cellular capillaries. The less prevalence of retinopathy in

iabetic subjects with arthritis led to the belief that aspirin mighte an effective therapy against diabetic retinopathy. Aggregationf platelets plays an important role in the pathogenesis of diabeticetinopathy. Aspirin inhibits the aggregation of platelets and thisroperty of aspirin can be used to demonstrate the pharmacological

fficacy of the formulation.

The pharmacological efficacy of the formulation was deter-ined by measuring the total inhibition of platelet aggregation

roduced when PRP (platelet rich plasma) is incubated for 1 h

ig. 5. TEM micrograph after six months and results of analysis by Image J software. Graphost of the particles are less than 200 nm in diameter.

ent and (ii) 20 ml phosphate buffer in the middle compartment. Left compartment:itreous right compartment: phosphate buffer (n = 3 for release in phosphate buffer,

ve release curve depicted).

with 0.18 mg/ml of aspirin loaded albumin nanoparticles. Asseen from Fig. 6, almost total inhibition of platelet aggregationis attained with aspirin nanoparticles. Similar results were alsoobtained at 1/3rd of the therapeutic dose of the free drug. Aspirinreleased from nanoparticles inhibit the platelet aggregation in atime dependent manner. Prevention of platelet aggregation wasalmost 50% at the end of 1 h compared to control PRP. Thisindicates that drug loaded nanoparticles are pharmacologicallyactive, and sustained release of aspirin from the carrier preventsplatelet aggregation. The result also implies that nanoparticlesare capable of inhibiting platelet aggregation at a much lowerdose.

3.7. In vitro hemolysis of the formulation

The RBC eye irritation classification model [30,31] shows thatthe test material is non-irritant if its HC50 is greater than 10 mg perml of red blood cells. The HC50 value of the albumin nanoparti-cles/free drug would indicate whether it is non-irritant.

ical representation of frequency distribution of particle sizes in the form histograms.

S. Das et al. / Colloids and Surfaces B: Biointerfaces 93 (2012) 161– 168 167

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ig. 6. Platelet aggregation at therapeutic dose horizontal axis: time; vertical axis:f platelet aggregation [right panel]. Left panel: control PRP aggregation.

The In vitro hemolysis of the aspirin nanoparticles was per-ormed to verify that the formulation was non-irritant. It wasbserved that the aspirin nanoparticles did not produce any hemol-sis till a concentration of 60 mg/ml whereas the aspirin free drug

ave 100% hemolysis at 30 mg/ml of red blood cells (Fig. 7). TheC50 (concentration of test material needed to produce 50% hemol-sis) of aspirin nanoparticles was above 60 mg/ml and aspirinree drug was 24 mg/ml of red blood cells. It was observed that

ig. 7. Hemolysis. Percentage hemolysis produced when different concentrations0.02, 0.2, 8, 16, 30, 60 mg/ml] of aspirin free drug/aspirin nanoparticles weredded to the blood cells (representative curve, each error bar represents maximumxpected error, dotted line represents the sigmoid non-linear fit of aspirin free drug;edian concentration (50% hemolysis) = 24.10).

e of platelet aggregation [in arbitary unit] on addition of ADP. Complete inhibition

the aspirin nanoparticles and free drug both are non-irritantsince their HC50 values are higher than 10 mg/ml red blood cells;which is based on the RBC eye irritation classification model[30,31].

4. Discussion

The coacervation method resulted in stable albumin nanopar-ticles of sizes less than 200 nm in diameter. The formulation withaspirin protein ratio of 0.4 showed small diameter 64.6 nm, stablezeta potential (25 mV) and 81% drug entrapment. This formulationshowed favorable release kinetics, which was sustained andprolonged. The total cumulative release of albumin nanoparticlesat prefixed time intervals in terms of percentage entrapment wasdetermined by spectrophotometry, which shows that the releasekinetics has two distinct phases. The early phase correspondsto the release of drugs physically bound to the surface of theprotein and the delayed phase conforms release of covalentlybound drug. This is in accordance with the finding from literaturethat protein nanoparticles have ability to bind to a large numberof drugs in a relatively nonspecific manner [13,26]. It is foundthat the bioavailability of the second half is more than the firsthalf. In contrast to simple drug solution, which attains peakconcentration within 1/2–1 h, nanoparticle formulation releasesaspirin at a sustained rate for a longer duration of time (50% totalcumulative release at the end of 20 h, 90% release at 72 h). In thepresence of artificial tear fluid, it is capable of maintaining sus-tained release kinetics overcoming the burst effect of the free drug

over a period of 80 h. Increasing the viscosity of the formulationby coating with 0.5% xanthan gum is capable of achieving evenhigher residence time. Therapeutic implications of this finding areimperative. It would mean the longer stay of the formulation at the

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dministration site releasing the drug in slow and sustained man-er, increasing the bioavailability of the drug. Secondly it wouldeduce the frequency of doses of administration and chances ofoxicity will also be much less. All these will lead to better patientompliance.

Under simulated ocular conditions in vitro, it is observed that1% drug is reaching the posterior plane of vitreous and retrovit-eal tissues. This evidence suggests that the drug release throughlbumin nanoparticles is much higher than the 1–2% drug reachinghe deeper tissues after topical administration in conventional for-

ulations [14]. It further indicates that the concentration achieveds much higher than the concentration at which acetyl salicylic acidnhibits the cyclooxygenase enzyme (ID50 = 0.9 �g/ml), as demon-trated by the antagonism of the fibroblastic growth promotingctivity of the intraocular fluid in proliferative vitreoretinopathy28].

This results of the stability studies signify that the nanoparti-les demonstrated a stable system when monitored after storageor six months and upon reconstitution at room temperatureor 24 h. The fact that nanoparticles with particle size less than00 nm can escape phagocytosis and can accumulate at the sitef inflammation provides distinct therapeutic advantage to thisormulation.

In vitro hemolysis and platelet aggregometry showed that theormulation is non irritant and pharmacologically effective at ther-peutic doses. The results suggest the feasibility of using aspirinoaded albumin nanoparticles <200 nm in size with or without aoating of 0.5% xanthan gum, in the eye for treatment of diabeticetinopathy with better tolerance than the free drug. Further, in vivotudies are required to confirm the clinical relevance of these find-ngs.

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