Pure Drug and Polymer Based Nanotechnologies for the Improved Solubility, Stability

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27presents discussion on the contribution of pure drug and polymer based nanotechnologies for the delivery28

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Advanced Drug Delivery Reviews xxx (2009) xxxxxx

ADR-11952; No of Pages 12

Contents lists available at ScienceDirect

Advanced Drug D

j ourna l homepage: www.e

ARTICLE IN PRESSCOR

1. Introduction

Human immunodeciency virus (HIV) related acquired immunedeciency syndrome (AIDS) has claimed over 25 million lives since itsdiscovery in 1981. Based on the profound knowledge gained about theHIV replication cycle, several drug targets have been identied over

treatment or HAART, is considered as one of the most signicantadvances in the eld of HIV therapy [2]. Since mid 1990s, HAART hasmade a remarkable contribution towards reducing the mortality inpatients.

Recommended HAART regimens include at least three actives [3].Since majority of antiretrovirals are administered orally, theirUNthe years and effective treatment options arThe current clinical therapy, known as hig

This review is part of the Advanced Drug DelivNanotechnology Solutions for Infectious Diseases in De

Corresponding author. School of Pharmacy, The Univ92019, Auckland, New Zealand. Tel.: +64 9 373 7599x8

E-mail address: [email protected] (S. Garg).

0169-409X/$ see front matter 2009 Elsevier B.V. Adoi:10.1016/j.addr.2009.11.019

Please cite this article as: P. Sharma, S.bioavailability and targeting of anti-HIV drR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . .

1. Introduction . . . . . . . . . .2. Pure drug nanoparticles . . . .3. Polymer based nanotechnologies

3.1. Polymeric micelles. . . .3.2. Polymeric nanoparticles .3.3. Dendrimers . . . . . . .

4. Conclusion and future directionsECTE

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adequate systemprerequisite forBiopharmaceutisolubility and peoral bioavailabilconsidered highin 250 ml or less[5]. Likewise, a d

ery Reviews theme issue onveloping Nations.ersity of Auckland, Private Bag2836; fax: +64 9 367 7192.

ll rights reserved.

Garg, Pure drug and polymer based nanoteugs, Adv. Drug Deliv. Rev. (2009), doi:10.101D 2009 Elsevier B.V. All rights reserved.anti-HIV drugs.improvement of water solu

PROOF

a b s t r a c ta r t i c l e i n f o

Article history:Received 14 June 2009Accepted 14 September 2009Available online xxxx

Keywords:HIVPolymerNanoparticle

The impact of human immunodeciency virus (HIV) infection has been devastating with nearly 7400 newinfections every day. Although, the advent of highly active antiretroviral therapy (HAART) has made atremendous contribution in reducing the morbidity and mortality in developed countries, the situation indeveloping countries is still grim with millions of people being infected by this disease. The newadvancements in the eld of nanotechnology based drug delivery systems hold promise to improve thesituation. These nanoscale systems have been successfully employed in other diseases such as cancer, andtherefore, we now have a better understanding of the practicalities and technicalities associated with theirclinical development. Nanotechnology based approaches offer some unique opportunities specically for the

bility, stability, bioavailability and targeting of antiretroviral drugs. This reviewPure drug and polymer based nanotechnobioavailability and targeting of anti-HIV d

Puneet Sharma, Sanjay Garg gies for the improved solubility, stability,gs

elivery Reviews

l sev ie r.com/ locate /addr59ic absorption from gastrointestinal (GI) tract is a60successful therapy. Amidon et al. [4] suggested a61c Classication System (BCS) that identies the62rmeability of drug substances as markers for their63ity. Following BCS guidelines, a drug substance is64ly solublewhen its highest dose strength solubilizes65of aqueous media over a pH range of 1.07.5 at 37 C66rug substance is considered highly permeablewhen

chnologies for the improved solubility, stability,6/j.addr.2009.11.019

CF

67 the extent of the intestinal absorption (parent drug plus metabolites)68 in humans is determined to be 90% of an administered dose based69 on a mass balance determination or in comparison to an intravenous70 (i.v.) reference dose [5]. However, there are other factors such as71 effect of efux and absorptive transporters, intestinal metabolizing72 enzymes (CYP34A and UDP-glucuronosyltransferases (UGTs)) and73 food that have to be taken into consideration for the appropriate74 estimation of the bioavailability [6]. Considering properties such as75 highest dose strength, dose number, solubility and log P, Kasim et al.76 [7] have classied twelve antiretrovirals (12th edition of World77 Health Organization Essential Core Drug List) in different BCS classes78 (Table 1). Out of these twelve antiretrovirals, nine have either79 solubility (BCS Class 2) or permeability (BCS Class 3) related issues.80 Consequently, oral absorption and, in turn, bioavailability is variable81 (Table 2) and mainly dependent on (i) stability in stomach acid82 conditions, (ii) aqueous solubility and dissolution from dosage form83 and (iii) permeability through the intestinal membrane. Most of the84 protease inhibitors (Pis) have high molecular weight (N500 Da), pH85 dependent solubility (high solubility at acidic pH) and high log P (high86 lipophilicity) (Table 3), and all these properties adversely affect the87

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112affects the quality of patient's life but also signicantly adds to the113economic burden of the health care system.114In the context of oral drug delivery, the important characteristics115of a molecule that needs to be considered for positive anti-HIV effects116are (i) solubility and ionization, (ii) lipophilicity and permeability,117(iii) stability in biological uids, (iv) gastrointestinal metabolism and118(v) viral reservoir targeting. When these properties are unfavorable119for drug development, alternative processing and formulation specic120approaches can be employed to attain maximum therapeutic gains.121The nanometer size and high surface area to volume ratio which affect122the pharmacokinetics and biodistribution of the associated drug123molecule are main features of nanotechnology based drug delivery124systems. The nanotechnology based approaches discussed in this125review for the delivery of anti-HIV drugs include pure drug126nanoparticles, polymeric micelles, dendrimers and polymeric nano-127particles. Several reviews are available on various pharmaceutical128aspects of these nanoparticulate systems such as preparation129methods, physicochemical properties, toxicity and others ([1117]).130This review presents information pertaining to their applicability131specically for anti-HIV drugs.

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2 P. Sharma, S. Garg / Advanced Drug Delivery Reviews xxx (2009) xxxxxx

ARTICLE IN PRESSUNCORRE

oral bioavailability [8]. In addition, they are substrates of P-glycoprotein efux transporters. Similarly, although the majority ofnucleoside reverse transcriptase inhibitors (NRTIs) show goodsystemic absorption (N70% at clinically relevant doses), didanosine(ddI) and zidovudine (AZT) show variable bioavailability. ddI isunstable at acidic pH and is subjected to rst-pass metabolism; whichmay be responsible for its lower bioavailability. AZT also undergoesrst-pass metabolism and additionally shows dose dependent effecton bioavailability.

HAART is a lifelong necessity and any non-compliance leads to arapid increase in the viral load [2]. The reason for this relapse is relatedto the poor targeting ability of the antiretroviral agent to the latentsites of infection [9]. Another major limitation of the current HIVtreatment is linked to the short residence time and resulting lowconcentration of antiretroviral drugs at certain inaccessible viralreservoir sites such as lymphatic system, macrophages, lymphocyte,central nervous system (CNS) and lungs [10]. Thus, administration ofhigher doses is required for prolonged duration to eradicate the virus.This prolonged therapy is often associated with the emergence ofresistant HIV strains. In addition, there are major issues of adversedrug reaction and drugdrug interaction associated with antiretro-viral therapy. Although newer regimens in HAART have signicantlylower toxicity, modulation of lipid and glucose metabolism is still amajor issue [2]. After chronic treatment even moderate toxicity maylead to serious complications. The resulting treatment failure not only

Table 1Antiretroviral drugs in the WHO essential core drug list classied according to BCS (ada

Drug Highest dosestrength (mg)

Solubility(mg/ml)

Dose numb(Do)a

Indinavir sulfate 400 1000 0.0016Nelnavir mesylate 250 4.5 0.22Saquinavir mesylate 200 2.22 0.36Efavirenz 200 0.01 80Lopinavir (with ritonavir) 133.3 0.01 53.3Nevirapine 200 0.1 8Ritonavir 100 0.01 40Abacavir sulfate 300 77 0.016Didanosine 200 27.3 0.03Lamivudine 150 70 0.0086Stavudine 40 83 0.002Zidovudine 300 20.1 0.06

Classication criteria: dose number1=high solubility and N1=poor solubility. Estimpermeability.BCS classication: class 1 high solubility, high permeability; class 2 low solubility, high pe

a Do is the ratio of drug concentration in the administered volume (250 ml) to the sa

highest dose strength (mg), Cs is the solubility (mg/ml), and Vo=250 ml.

Please cite this article as: P. Sharma, S. Garg, Pure drug and polymbioavailability and targeting of anti-HIV drugs, Adv. Drug Deliv. Rev. (2TEDPROO2. Pure drug nanoparticlesIn a pure drug nanoparticle formulation, submicron size particles

(mean particle size b1 m and 99th percentile particle size of b5 m)of drugs are stabilized in aqueous medium with GRAS (generallyrecognized as safe) stabilizers. Such formulation can be used for drugswith properties such as insolubility in bothwater and oil, highmeltingpoint, high log P and high dose [18]. Following NoyesWhitneyequation, progressive size reduction of the drug particles leads to anincrease in the surface area resulting in an increased dissolution rate.Additionally, particle size reduction results in the decrease of thediffusion layer thickness surrounding the particles [19] and anincreased concentration gradient between the surface of the particleand bulk solution, which facilitates particle dissolution by increasingdissolution velocity. Therefore, nanosizing is a suitable approach forincreasing bioavailability of those drugs where dissolution is the ratelimiting step in systemic absorption.

Van Eerdenbrugh et al. investigated the dissolution and in vitroabsorption of a poorly water soluble non-nucleoside reverse tran-scriptase inhibitor (NNRTI), loviride (water solubility 0.1 mg/l), afternanonization [20]. In addition to poor water solubility, loviride has ahigh melting point (225 C) and poor solubility in oils. Suchphysicochemical properties make loviride a brick-dust moleculewith low oral bioavailability. Media milling led to mean particle sizes

d from [7]).

CLogP Log P pKa(s)

BCS class

Log P-based CLogP-based

3.68 2.49 1 15.84 4.62 1.2 1 14.73 2.73 1 14.95 3.68 10.2 2 26.1 4.56 2 22.42 2.05 2.8 2 24.94 5.98 2 20.58 0.22 5.01 3 3

1.92 1.1 9.12 3 31.46 0.06 3 30.73 0.47 3 30.04 3

log P and CLogP values1.72 and 1.35=high permeability and b1.72 and 1.35=low

ability; class 3 high solubility, low permeability; class 4 low solubility, low permeability.tion solubility of the drug in water and calculated as Do=(Mo/Vo)/Cs where Mo is theQ1

er based nanotechnologies for the improved solubility, stability,009), doi:10.1016/j.addr.2009.11.019

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167nanosized loviride formulation which was freeze-dried with sucrose168(nanopowder) did not show amorphization as indicated by powder X-169ray diffraction (PXRD) and Differential Scanning Calorimetry. How-170ever, there was a peak broadening in PXRD and a decrease in the Tm of171the nanosized drug, indicating the presence of nanosize crystalline172particles. Further, in a medium containing 3% sodium lauryl sulfate173(SLS) at 37 C, nanopowders showed enhanced dissolution (104%)174over (i) the freeze-dried nanosuspension without sucrose (58.1%), (ii)175the physical mixture containing sucrose (54.8%), (iii) the physical176mixture without sucrose (14.5%) and (iv) the pure loviride (64.7%)177(Fig. 1A). The increased dissolution of the nanopowder also resulted178in the enhanced transport of the drug in Caco-2 cells (Fig. 1B) as179shown by the high value of cumulative amount transported (1.591800.02 g) compared with the physical mixture with sucrose (0.931810.01 g) and pure loviride (0.740.03 g). Together, these results182indicate advantages of a nanocrystal formulation for delivery of183loviride, despite its poor aqueous solubility.184Following a similar approach, pure drug nanoparticles of a poorly185water soluble investigational anti-HIV agent (BMS-488043) were186prepared to investigate the effect on oral bioavailability in dogs [21].187BMS 488043 is a BCS class II compound with poor solubility and188optimum permeability. Moreover, the solubility is pH dependent189(0.04 mg/ml in pH 48). Nanosuspensions of this compound were190prepared using media milling. To prevent particle growth and191

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Table 2t2:1Bioavailability of various commercially available dosage forms of antiretroviral drugs(adapted from [63]).

t2:2t2:3 Class of drug Name Dosage form F (%)a

t2:4 Nucleoside reversetranscriptaseinhibitors (NRTI)

Zidovudine Capsule, liquid 60t2:5 Lamivudine Tablet, liquid 86t2:6 Didanosine Tablet, capsule (EC), liquid 3040t2:7 Zalcitabine Tablet 85t2:8 Stavudine Capsule, powder for

reconstitution80

t2:9 Abacavir Tablet, liquid 83100t2:10 Emtricitabine Capsule 93t2:11 Nucleotide reverse

transcriptaseinhibitors (NtRTI)

Tenofovir Tablet 2539

t2:12 Non-nucleosidereverse transcriptaseinhibitors (NNRTI)

Nevirapine Tablet, syrup N90t2:13 Efavirenz Tablet, capsule, solution 4280t2:14 Delavirdine Tablet 85t2:15 Etravirine Tablet Unknownt2:16 Protease inhibitors (PI) Amprenavir Capsule, solution No datat2:17 Indinavir Capsule 65t2:18 Saquinavir Tablet, capsule Erratic, 4t2:19 Nelnavir Tablet, powder 2080t2:20 Ritonavir Tablet, capsule, liquid 65t2:21 Atazanavir Capsule No datat2:22 Darunavir Tablet 37t2:23 Entry and fusion

inhibitors (FI)Enfuvirtide Powder for subcutaneous

injection84.3

t2:24

t2:25

t2:26

3P. Sharma, S. Garg / Advanced Drug Delivery Reviews xxx (2009) xxxxxx

ARTICLE IN PRESSb300 nm. In the process, the drug suspension containing macro ormicro size particles along with milling media (e.g., small spheres ofglass, zirconium oxide or polystyrene resin) is placed in an enclosedcylindrical milling device. Milling occurs through the movement ofmilling media facilitated by a rotating agitator. During the process,mechanical energy is applied to generate sufcient strain on the solidparticles resulting in the disruption of the crystal lattice and particle

Maraviroc Tablet 2333Integraseinhibitors (II)

Raltegravir Tablet No data

a Bioavailability.UNCORREC

fracture. Under the stress conditions generated there is a possibility ofphysical or chemical change in the crystalline solid, includingpolymorphic transitions, chemical degradation, complexation orformation of an amorphous phase and therefore, post nanosizingsamples are characterized for process induced transformation. A

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Table 3Physicochemical properties of different protease inhibitors (PIs) (adopted from [8]).

HIV protease inhibitor Molecular weight Log P(o/w)a

Saquinavir mesylate 767.0 4.1

Ritonavir 721.0 5.2

Indinavir sulfate 711.9 2.9

Nelnavir mesylate 663.9 4.0(pH 7.4)4.1(pH 6.0)

Amprenavir mesylate 601.7 3.3 or 4.2d

Aq: puried water. NR: not reported.a Log partition coefcient (octanol/water).b Apparent Caco-2 cell monolayer permeabilities in the apical to basolateral (absorptive) d

indinavir base.c Values reported for nelnavir base.d Conicting reports.

Please cite this article as: P. Sharma, S. Garg, Pure drug and polymbioavailability and targeting of anti-HIV drugs, Adv. Drug Deliv. Rev. (2TEDPRO

enhance the stability, hydroxypropylcellulose was used as thestabilizer along with a surfactant (SLS, sodium docusate andpolyvinylpyrrolidone). The formulation was found to be stable at4 C and room temperature for 4 weeks and the mean cumulativeparticle size was 120 nm. As compared with the conventional capsuledosage form, the nanosuspension yielded 4.7- and 4.6-fold increase inCmax and AUC values, respectively. This indicates that nanonization ofBMS 488043 resulted in enhanced dissolution and bioavailability.

Antiretrovirals are administered in high dose. Thus, their bioavail-ability is affected by the presence of food in the GI tract; food alters thevolume and composition of gastric and intestinal uids. For instance,both lopinavir and ritonavir are water insoluble drugs and increaseduid volume and bile salt concentrations coupled with delayed gastricemptying time lead to enhanced dissolution and bioavailabilitycompared to fasted state. Therefore, a lopinavir/ritonavir combination(Kaletra capsule and oral solution) is recommended to be taken withfood. Administration of water insoluble drugs in the form of pure drug

Solubility pKa Papp AP to BLb (cm/s)

Aq (2.22 mg/ml) 7.0 or 5.5d 1.5106

pH 7.4 (36 g/ml)pH 6.5 (73 g/ml)Aq (1 g/ml) NR 3.5106

pH 7.4 (5.3 g/ml)pH 4.0 (6.9 g/ml)Aq (N100 mg/ml) pKa1=3.7

pKa2=5.93.0106

pH 7.4 (70 g/ml)pH 4.8 (0.3 mg/ml)pH 3.5 (60 mg/ml)Aq (4.5 mg/ml) pKa1=6.0c

pKa2=11.1cNR

pH 7.4 (very low)pH 3.5 (0.5 mg/ml)pH 2.6 (4.5 mg/ml)Aq (0.19 mg/ml) NR NRpH 7.4 (60 g/ml)pH 6.8 (190 g/ml)

irection, donor concentrations range from 10 to 200 mM, the indinavir value representser based nanotechnologies for the improved solubility, stability,009), doi:10.1016/j.addr.2009.11.019

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nanoparticles reduces the bioavailability variability brought by thepresence or absence of food [22,23]. However, to elicit this effect,nanoparticles should re-disperse homogeneously in the GI uids afteradministration. Further, as expected, this is a dose dependent effectand at higher doses of poorly water soluble drugs some variation maybe observed in fed and fasted state bioavailability [22].

As an i.v. formulation, the major advantage of nanosuspension isthe avoidance of precipitation following administration. Since theformulation contains a poorly water soluble drug as nanosizedparticles, their biodistribution depends upon the solubility of thedrug in the blood [24]. After administration, if the particles dissolverapidly due to dilution in the blood circulation, the biodistributionwillbe similar to that of the solution formulation. However, in case of slowdissolving particles, macrophage uptake occurs. Other advantages ofnanosuspension as i.v. formulation include (i) high dose per volume(high drug loading), thus, requiring low volumes for injection and(ii) reduced toxicity due to elimination of co-solvents and solubilisingagents (such as cremophor) along with scale up feasibility.

In a proof of concept study, Dou et al. demonstrated theconcentration-dependent in vitro uptake of indinavir nanocrystals(mean particle size 1.6 m, stabilized with phospholipid stabilizer) inmonocyte-derived macrophages [25]. Following incubation, 50%macrophages contained nanocrystals after 2 h, which increased to95% after 8 h. The authors reported that after cellular uptake, indinavirnanocrystals were exposed to the low pH of phagolysosomes,

Fig. 1. (A) Dissolution proles: freeze-dried nanosuspension without sucrose (), phyuntreated loviride (). Dissolution of the nanopowders is complete within minutes. (Bnanopowder (), physical mixture (), untreated loviride () (adopted from [20]).UNCORresulting in dissolution and release of indinavir in the extracellularmatrix. Furthermore, incubation of HIV infected macrophages withindinavir nanocrystals showed signicantly reduced reverse tran-

scriptase activity and expression of HIV-1p24 antigens comparedwithsoluble drug in culture uids. Considering that monocyte-derivedmacrophages are HIV reservoirs, results of this study indicated theviability of transporting nanocrystals engulfed by the macrophagesacross the blood brain barrier.

Nanosuspensions can also be used as a long acting parenteralformulation, as reported recently for rilpivirine (TMC278) [26]. A longacting formulation has a major advantage of minimizing the dosingfrequency, thereby, increasing compliance to the HIV therapy. Rilpivir-ine is a potent NNRTI and is poorly soluble in both water and oil. Afterintramuscular (i.m.) and subcutaneous (s.c.) administration of a singledose of 5 mg/kg of 200 nm size particles in dogs, a sustained plasmaconcentration was achieved up to 3 months. Compared with i.m., s.c.administration resulted in sustained release of rilpivirine (Cmax, IM:173 ng/ml and Tmax, IM: 24 h versus Cmax,SC: 38 ng/ml and Tmax, SC:144 h). This recent study is the rst to provide a proof of concept for ananosuspension as a long acting formulation. In addition tominimizingthe dose frequency, the parenteral route of administration also avoids

Please cite this article as: P. Sharma, S. Garg, Pure drug and polymbioavailability and targeting of anti-HIV drugs, Adv. Drug Deliv. Rev. (2TEDPROOF

rst-pass metabolism and results in dose reduction which is highlysignicant considering the adverse effects associated with the anti-HIVdrugs.

It is also worth mentioning that media milling, developed byLiversidge et al., is a proprietary wet milling technology (Nanocrystal)of the Elan Corporation [12,27] and there are at least four oral dosageforms in the market containing pure drug nanoparticles; i.e., Rapa-mune (Wyeth), Emend (Merck), Tricor (Abbott Laboratories) andMegace ES (Bristol-Myers Squibb) [28]. Thus, Nanocrystal is avalidated technology with well established process controls and scaleup feasibility. The choice of stabilizer duringmedia milling signicantlyaffects the nal size of the particles. The process of milling generatesnumerous nanosize particles with a very high surface area. An increasein the surface area (A) is associated with an increase in the surfacefree energy (G) by the equation G=As/l where, s/l is theinterfacial tension between the surface of solid and the surroundingliquid phase. In order to reduce the free energy, particles tend toagglomerate resulting in an increased particle size and reducedsurface area. Under these circumstances, inclusion of a surfacestabilizer leads to a decrease in the G by decreasing the interfacialtension s/l. The overall effect of the addition of a stabilizer isreduction in the particle growth, thereby conferring physicalstabilization to the nanosuspension [29]. Further, because thebenets of nanosized particles (such as high surface area) can beexploited only by keeping particles dispersed, it is possible to convert

l mixture without sucrose (), nanopowder (), physical mixture with sucrose (),mulative transported amount of loviride as a function of time in Caco-2 experiments:279nanosuspension obtained after media milling to solid products for280stability enhancement and protection against aggregation using281processes such as spray drying, lyophillization and wet granulation282following media milling. Such solid products can be powder for283reconstitution, pellets or powders for capsule lling or tablet284compression which ultimately improves patient compliance.

2853. Polymer based nanotechnologies

2863.1. Polymeric micelles

287Polymeric micelles are nanostructures of b100 nm diameter that288have been utilized for improving aqueous solubility, intestinal289permeability and disease site targeting of several drug molecules.290Compared to the conventional surfactant based micelles, polymeric291micelles are composed of block copolymers. Although the structural292coreshell arrangement is similar to surfactant based micelles,293polymer micelles self associate at much lower concentration (low294critical micelle concentration, CMC), typically 106107 M com-295pared with 103104 M for surfactant based micelles [30,31].296Consequently the thermodynamic and in vivo stability of polymeric


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3630.001% and 0.01% showed signicant reduction in HIV replication.364When compared with control (no treatment), HIV replication was365reduced to 55% and 44% at 0.001 and 0.01% concentrations. The366activity was further enhanced in the presence of nelnavir compared367to either P85 or nelnavir alone (Fig. 2). In vivo, the antiretroviral368combination without P85 (0.2%) reduced the HIV-1 p24 positive cells369to 35%. P85 alone and in combination with the antiretroviral showed370greater effect, 13.4 and 7.0% respectively, compared to the control371(68.5%) (Fig. 3). This inhibition of the HIV replication by P85 alone372could be due to its direct interaction with P-gp or glycolipid373membrane of HIV [55] which results in membrane disruption. In a374recent study, inuence of P85 on the interaction of nelnavir and375saquinavir with P-gp expressed on MDR1-transfected epithelial376MadinDarby canine kidney (MDCKII) and MDR1-transfected porcine377kidney epithelial cells (LLC-PK1) cells was investigated [36]. P-gp378utilizes the energy released from the hydrolysis of ATP. This action is379mediated by ATPase enzyme [56]. PIs have been shown to stimulate380the ATPase activity leading to their efux. In the study, P85 was found381to inhibit the basal activity of ATPase, thereby, inhibiting the efux382action of P-gp. Moroever, P85 (0.01%) was also found to inhibit MRP1383and MRP2, in addition to P-gp, leading to enhanced accumulation of384saquinavir in both MDCKII and LLC-PK1 wild type and MDR1 cells385[36].386

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micelles is relatively high [16,32]. Polymeric micelles consist of ahydrophobic block which constitutes the core and a hydrophilic blockconstituting the shell of the micelle. This coreshell arrangementfacilitates their utilization as a drug delivery nanocarrier wheredepending upon the polarity the drug molecule can be entrapped inthe (i) core (non polar molecule), (ii) shell (polar molecule) and(iii) in-between core and shell (intermediate polarity). As a result,micelles are suitable for (i) solubilization of poorly water solubledrugs, (ii) protection against chemical degradation and metabolismand (iii) controlled release. Additionally, surface properties ofmicellescan be tailored by attaching hydrophilic blocks to antibodies or otherligands specic for the type of receptors present on the disease site.For example, micelles of polyethylene (PEG)polylactide copolymerwith a surface modied with galactose and lactose units specicallyinteract with lectins [33]. Lectin receptors are present on HIV viralreservoirs such as T lymphocytes, dendritic cells and macrophages,and therefore, this can be a promising approach for viral reservoirtargeting.

The use of polymeric micelles for oral delivery has been recentlydiscussed by Bromberg [32]. Various polymers have been used ashydrophilic shell forming blocks in polymeric micelles such as PEG(molecular weight 1 to 15 kDa), poly(N-vinyl-2-pyrrolidone) (PVP),poly(vinyl alcohol) (PVA), polyethyleneimine and poly(ethyleneoxide) (PEO). Examples of hydrophobic core forming blocks includeL-lysine, aspartic acid, caprolactone, D,L-Lactic acid, propylene oxideand others [16,34]. Studies have been reported where polymericmicelles composed of different block copolymers were used for oraldelivery [35]. Among these, block copolymers of poly(ethyleneoxide)poly(propylene oxide) (PEOPPOPEO), also known as Pluro-nics, have been used to investigate their potential as intestinalpermeability enhancers of antiretroviral drugs [36]. Pluronic, P85modulates the activity of ATP binding cassette transporters [3639]that have been shown to reduce the oral bioavailability [4045] andCNS permeability [10,4547] of many PIs and NRTIs. For example, theP-gp mediated efux transport of the PI saquinavir has been reportedto be a key factor for its exceptionally low bioavailability [44].Similarly, among other reasons such as poor water solubility, CYP3A4metabolism and systemic clearance, P-gp mediated efux has alsobeen reported to play a role in the low bioavailability of indinavir,atazanavir and lopinavir; concomitant administration of a low dose P-gp and CYP 3A4 inhibitor, ritonavir (also a PI), results in theirenhanced bioavailability [4852]. Inhibition of P-gp by P85 was rstobserved in SKVLB multidrug resistant (MDR) cancer cells where 0.01to 1% P85 concentration resulted in 700 times more cytotoxicity (orMDR reversal) of otherwise MDR drug daunorubicin [53]. In addition,since the effect was also observed at the 0.01% concentration at whichP85 was present as unimers, it was proposed that the observed effecton P-gp was not due to micelles. Batrakova et al. showed a similareffect of P85 on Caco-2 monolayers (in vitro model for intestinalepithelium) over-expressing P-gp [54]. Below CMC, presence of P85unimers caused a signicant uptake and efux inhibition of P-gpdependent probe rhodamine 123 in Caco-2 cells, whereas, the effectobserved with other nonionic detergents, Cremophor EL and Tween60 was signicantly lower.

The effect of different P85 concentrations (0.01 to 1%) on thepermeability of the NRTI AZT in bovine brain microvessel endothelialcells (BBMEC, in vitro model for blood brain barrier) and Caco-2 cellwas studied among other drugs. At all P85 concentrations, AZTpermeability enhancement was signicant due to P-gp and MRPmediated efux inhibition [37]. Further, the effect of P85 mediatedinhibition of P-gp efux on the efcacy of antiretrovirals was assessed[55]. P-gp inhibition by P85 could enhance the in vitro efcacy ofnelnavir in HIV-1 infected monocyte-derived macrophages and thein vivo efcacy of AZT, lamivudine (3TC), and nelnavir combinationtherapy in HIV 1 encephalitis (HIVE) severely combined immunode-

ciency (SCID) mice. Interestingly, in the in vitro studies P85 alone at

Please cite this article as: P. Sharma, S. Garg, Pure drug and polymbioavailability and targeting of anti-HIV drugs, Adv. Drug Deliv. Rev. (2TEDPROComparative studies of P85 with other Pluronic (F 127, F 68) have

reected the importance of structural requirements of PEOPPOPEOunits for the biological effects; molecules having intermediate lengthof PPO and hydrophobicity, such as P85, being more effective [36,56].For oral delivery of antiretroviral molecules, for example saquinavir,whose absorption is limited by the efux action of multidrugtransporters, inclusion of P85 in the formulation, at a concentrationless than the expected CMC after dilution in GI uid, may lead toenhanced bioavailability.

3.2. Polymeric nanoparticles

Polymeric nanoparticles (NPs) are b1000 nm in size and arecomposed of biodegradable or biostable polymers and copolymers.The active agent can be (i) entrapped or encapsulated within theparticle, (ii) physically adsorbed on the surface, or (iii) chemicallylinked to the surface of the particle. There aremany studies where NPshave been explored for the delivery of anti-HIV molecules [5761].Recently Govender et al. and Ojewole et al. have reviewed this subject

Fig. 2. Pluronic block copolymer P85 inhibits HIV-1 replication in monocyte-derivedmacrophages (MDM) (adopted from [55]). Effect of combined treatment with(nelnavir) NEL (0.01 and 0.001 mol/L) and P85 (0.01% and 0.001%) on HIV-1replication in MDM as detected by reverse transcriptase (RT) activity at day 12,normalized to control cells. Pb0.01, #Pb0.05 NEL+P85 as compared withNEL (0.01 mol/L) or P85 (0.01%) alone. Results are presented as meanstandard

error (SE).


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ARTICLE IN PRESS[62,63]. In most cases, polymeric nanoparticles have been used totarget antiretrovirals to (i) macrophages/monocytes [59,60,64,65]and (ii) CNS [57,58,61] which act as viral reservoir sites during HIVinfection [6669]. Macrophages have been reported to be a majorcause of dissemination of the infection in the body [69] in the laterstages of the disease during which there is a continuous depletion ofCD4+ T lymphocytes. During this period, virus production from thesemature non-proliferating macrophages/ monocytes is dramaticallyenhanced without being affected by the lethal effect of the replicatingvirus.

Nanoparticulatemediated targeting of macrophages is well known

Fig. 3. Effect of P85 (0.2%) and triple drug combination (antiretroviral-ART) on viralreplication in HIVE SCID mice (adopted from [55]). At day 7, the combined ART-P85 andART alone groups showed a signicant decrease of HIV-1 p24 expressing MDM (9% and13%, respectively) while the P85 alone was not different from control. At day 14, mostnotable effects were seen in the P85 alone and ART-P85 groups (6% to 15% of HIV-1 p24-positive MDM), which were superior to the ART group (35% p24-positive cells). Barvalues represent meanSE.UNCORRECand has been reported by several authors [64,7072]. Following i.v.administration, nanoparticles are removed from the blood circulationby macrophages [73,74]. The recognition of particles by macrophagesis mediated by a process called opsonization [75,76]. When thedistance between the particles and the opsonins is sufciently small,

they can bind to the surface of particles by any of the interactions suchas van der Waals, electrostatic, ionic etc. After binding to the surface,particles become recognizable by macrophages and phagocytosistakes place [75]. Size and surface characteristics of nanoparticles arethe determinants of their clearance kinetics and tissue distribution[73,74,76]. These properties interfere with the opsonization processand affect the macrophage uptake.

In 1992, Schaffer et al. showed the monocyte/macrophagetargeting of nanoparticles using macrophages separated from HIVinfected individuals [64]. In addition, these authors also showed theeffect of particle size, concentration and surface characteristics on themacrophage uptake. Subsequently, other studies also reported themacrophage targeting of nanoparticles [65,77].

HIV infection of brain macrophages, i.e. perivascular macrophages,meningeal macrophages, the macrophages of choroid-plexus andmicroglial cells, is directly linked to HIV associated dementia complex(HAD) [9]. Several works reported on the targeting of entrapped orconjugated drugs to the CNS. For example, Zensi et al. recently studiedthe brain uptake of human serum albumin nanoparticles coated withcovalently bound apolipoprotein E after i.v. administration in mice[61]. The presence of apolipoprotein bound nanoparticles in theendothelial cells of the brain capillaries and neurons demonstratedtheir preferential brain uptake over non-apolipoprotein bound

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nanoparticles. In addition, transportation of apolipoprotein boundnanoparticles through tight junctions of the endothelial cells and theluminal membrane was not observed, and therefore, a transcytosismechanism of transport was suggested for the internalization. Anextensive discussion of this application is out of the scope of thereview and it is covered by another review of this Theme Issue.

Biodegradable polymeric nanoparticles have been used for thevaginal delivery of unconventional anti-HIV agents such as chemo-kines [77] and siRNA (small interfering ribose nucleic acid) [78]. Theaim behind the development of a vaginal formulation (known asmicrobicide) is to prevent transmission of HIV during sexualintercourse. A microbicide formulation is intended for intravaginalapplication before sexual exposure. The effort needed in this eld ofresearch is also supported by the fact that nearly half of the HIVinfected population is female, and majority of them acquire theinfection during intercourse. The major advantage of using nanopar-ticles includes protection of the actives against degradation in thevaginal environment. Also, nanoparticulate delivery may assist bydirect action on one or more of the target sites such as submucosalepithelium (Fig. 4). Nanoparticles have been reported to permeatethrough the interstitial spaces in tissue due to their nanosize whichmay result in enhanced cellular uptake [79]. Recently, Lai et al. haveshown that physicochemical properties of polyethyleneglycol (PEG)-conjugated nanoparticles (carboxyl-modied polystyrene) such assize, surface charge and particle concentration signicantly affecttheir diffusion through cervico-vaginal mucus [80]. Specically, it wasreported that 200500 nm size particles (b0.01% by weight) linkedwith 2 kDa PEG diffuse through the mucus faster than 100 nm sizeparticles. This could be considered as signicant because an effectivemicrobicide should act on the luminal as well as basal side of thevaginal epithelium. In order to reach the basal side, traversing throughmucus is a prerequisite for nanoparticles. Further, Cu et al. havereported similar results of higher diffusion rate of PEG modied(copolymer polylactic acid-co-glycolic acid) nanoparticles [81]. Thediffusion rate was dependent upon PEG type and concentration withrates 10 times higher compared with non-PEG modied nanoparti-cles. In addition to the prophylactic application, these ndings alsowiden the nanoparticle horizons towards diagnostic applications inHIV.

Compliance of some protease inhibitors (ritonavir, indinavir) inpediatric population is low due to the poor palatability (extremebitterness) of the liquid formulation [52]. In order to mask thebitterness of indinavir sulfate, Chiappetta et al. [82] prepared pHsensitive microparticles of Eudragit E 100 which is a cationiccopolymer consisting of dimethylaminoethyl methacrylate andneutral methacrylic esters. Three different microparticle formula-tionsMP20, MP40 and MP60containing three different theoreticalindinavir loadings, 20%, 40% and 60%, respectively, were prepared. ForMP20 and MP40, 80% (by weight) of the particles were between 105and 210 m, whereas, for MP60 the mean particle size was higher(210420 m) with broad distribution. The in vitro release studies ofall the formulations showed 100% drug release in 5 min at acidic pH1.5; in contrast, at pH 6.8 slow release of indinavir from themicroparticles was observed (Fig. 5). This shows that in saliva likepH conditions (pH 6.8), the polymer remained unprotonated resultingin poor solubility, and therefore, most of the bitter drug remainedinside the microparticles. However, following exposure to the acidicpH (gastric pH), indinavir was immediately released due to thesolubilization of polymer matrix mediated through protonation of theamine side chains of the polymer. When the MP20 and MP40microparticles were dispersed in tap water (simulation of extempo-raneous reconstitution), indinavir concentrations after 1 min werewithin the threshold limits (2045 g/ml) that are consideredpalatable. However, MP60 produced higher indinavir concentrations(1215 g/ml) which were unpalatable. Moreover, after 5 min com-507pared with MP40 and MP60, only MP20 produced palatable indinavir


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ARTICLE IN PRESSFig. 4. Mode of transmission of human immunodeciency virus (HIV) through female gtract through submucosal tissue (consisting of squamous stratied epithelium (left) andoccur through various pathways [110]: following entrapment inmucus (A) and diffusioncells present in stratied epithelium may also get infected with virus (C). Virus can permthese pathways, HIV can infect T cells (E), macrophages (F) and dendritic cells (G). Throudissemination in the body. To effectively counter HIV infection, microbicides must be eUNCORREC

concentrations. Taste masking studies were conducted on healthyhuman volunteers using indinavir solutions having concentrationsequivalent to that produced after keeping the formulations in tapwater for 1 and 5 min. MP20 scores were within the limits (b2) set forpalatability. However, MP40 and MP60 formulations scored morethan 2 and, therefore, were unpalatable. These results indicated thatmicroparticles of Eudragit E100 polymer could efciently entrap 20%indinavir (theoretical loading) with acceptable palatability. Eventhough this work focused on microparticles, the results of this studyshowed the utility of a polymer based technology in improving thepalatability of indinavir formulations.

Fig. 5. Released indinavir (expressed as %) versus time for MP20 and MP40 systems(adopted from [104]). Microparticles (100 mg) were dispersed in the test medium(500 mL, pH values 1.5 and 6.8) and assayed in a dissolution test using the USPdissolution apparatus 2, at 37 C.

Please cite this article as: P. Sharma, S. Garg, Pure drug and polymbioavailability and targeting of anti-HIV drugs, Adv. Drug Deliv. Rev. (2PROOF

al mucus and target sites for microbicides (adapted from [109]). HIV can enter genitalical columnar epithelium (right)). Transmission of HIV across the submucosal tissue canards single layer of columnar cells throughwhich endocytosis (B) can occur. Langerhansthrough ulceration or lesions in the stratied epithelium (D). Following entry throughhese cells, HIV can travel via lymphatics to regional lymph nodes leading to further viralive against these multiple sites shown above.TED

5193.3. Dendrimers

520A dendrimer is a polymeric nanostructure (b100 nm) built around521a core unit. There are several branching units around the core units in522a layer by layer fashion which denes the growth, size and the523microenvironment within the dendrimer [83]. Dendrons are formed524after removal of core units and can be divided into (empty) core, the525interior (branching units) and the periphery (end groups). The empty526core of dendrons can be utilized for the entrapment of drug molecules527for solubilization, controlled release, targeting or protection from528surrounding degrading environment. Dendrimers offer unique prop-529erties such as uniform particle size (especially for lower generation i.e.530up to G-3), poly-valency of the end groups which helps in binding to531diverse receptors and an ability to bind a variety of targeting agents to532their high density peripheral functional groups.533The potential of the oral delivery of dendrimer has been explored534with cationic and anionic PAMAM dendrimers [8490]. Various535reports have indicated that the permeability of PAMAM dendrimers536across Caco-2 monolayers and everted rat intestinal sac is dependent537on their size, surface charge, charge concentration and surface538modication [91]. Additionally, conjugation of propranolol (a P-gp539susbtrate) to G-3 PAMAM dendrimer was shown to bypass the efux540action of P-gp [92]. The proposed mechanisms for the dendrimer541passage across the GI epithelium are energy-dependent adsorptive542mediated endocytosis and paracellular pathway [91]. Wiwattanapa-543tapee et al. studied the tissue uptake and serosal transport of 125I544labelled rst and half generation PAMAM dendrimers across the545everted rat intestinal sac [93]. Rate of tissue uptake for G-5.5 was546signicantly higher than lower generations G-2.5 and 3.5 whereas the547rate of serosal transfer was relatively similar between the three548generations. Moreover, contrary to anionic dendrimers, the rate of549tissue uptake of G-3 and G-4 cationic dendrimers was higher than550serosal transfer and both showed similar rates for tissue and serosal


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transfer for the rst 60 min [93]. Also, anionic PAMAM dendrimershave shown to bemore permeable in Caco-2 monolayers compared tocationic PAMAM dendrimers [84,89]. The success of dendrimers asdelivery systems lies in their biocompatibility and acceptability.PAMAM and PPI dendrimers are non-biodegradable which afterchronic administration can cause unpredictable toxicity. Therefore,the size of the dendrimers must be controlled to facilitate renal orhepatic clearance [94]. Dendrimers with cationic (amino) end groupssuch as PAMAM and PPI have been shown to cause concentration-dependent toxicity and hemolysis, whereas, neutral or anionicdendrimers are less toxic and less hemolytic [82,94]. In addition,modication of the terminal amino surface groups with anionic orneutral groups (for example PEG 2000) reduces the toxicity[84,95,96]. Results of in vitro studies correlate well with in vivostudies and intraperitoneal administration of doses above 10 mg/kg ofcationic melamine dendrimers caused liver toxicity [97]. Administra-tion of a 160 mg/kg dose caused 100%mortality within 12 h. However,for a structurally similar dendrimer, modication of the terminalcationic groups with neutral polyethylene oxide reduces the toxicityand i.v. or intraperitoneal administration of doses more than 1 g/kgproduced no toxic effects [98].

Interfering ribonucleic acid (siRNA) are small pieces of doublestranded RNA used to halt gene expression by participating in RNAinterference pathway. Such gene silencing activity is sequence specicand results in degradation of messenger RNA complementary tosiRNA. Noticeably, many siRNA based therapeutics have recentlyentered clinical trials [99,100] and are promising approaches forsilencing the genes involved in pathological conditions. Studies haveshown these approaches to be effective against HIV [101,102].However, the in vivo stability of siRNA is poor and it undergoesdegradation under physiological conditions. Moreover, poor cellularuptake and transfection efciency coupled with endosomal degrada-tion are the major drawbacks associated with siRNA delivery. Beingpolyanionic, complexation of siRNA to polycationic biodegradablepolymers is a promising approach for efcient delivery [104]. Weberet al. developed dendriplexes of positively charged carbosilanedendrimer and short negatively-charged siRNA [105]. The G-2ammonium-terminated carbosilane dendrimers were 2G-NN8 (2G-CBS-(OCH2CH2NMeCH2CH2N+Me3+ I)8) and 2G-NN16 (2G-CBS-(OCH2CH2N+Me2CH2CH2N+Me3I)8) carrying 8 and 16 positivecharges, respectively. These dendrimers were water soluble, efcientin binding siRNA and able to release their load in a time dependentmanner as a result of carbon-silicon bond hydrolysis [105]. Specicpolyanionic siRNA sense and antisense sequences coding for P24,GAG1 and NEF HIV proteins were used for binding to dendrimers.Heparin competition assay showed that compared to 2G-NN16, siRNAwere strongly bound to 2G-NN8. Moreover, after addition of 2G-NN16to 500 nM siGAG1 zeta potential increased from20 to +5 for +/charge ratio 1 up to 3, with a further increase in +/ charge ratioresulting in a gradual increase in zeta potential as well as size (Fig. 6).For charge ratios up to 2, the measurements showed a negative zetapotential and size b300 nm. Results of agarose gel electrophoresisshowed that attachment of siRNA to the quaternary ammoniumgroups of carobosilane dendrimer increased the stability of siRNAagainst RNase-mediated degradation. Further, tests performed tomeasure the membrane rupture, cell viability, metabolic activity andcell proliferation showed decreased cytotoxicity of the dendriplex inlymphocytic cell line SupT1 compared with dendrimer alone becauseof the shielding of the positive charges of the dendriplex which wereotherwise exposed in the absence of siRNA in the dendrimers. Thecytotoxicity was dependent on +/ charge ratios of dendriplexes,with charge ratios b8 being non-toxic. Similarly, results of owcytometry performed with a uorochrome-labeled siRNA either aloneor in dendriplexes showed that the transfection efciency of thedendriplexes was charge-dependent and highest for either siRNA

alone or with dendriplexes with +/ charge ratio of 1 and 2. These


results were corroborated with confocal microscopy, where uoro-chrome-labeled siRNA either naked or complexed with dendrimerwere observed in the interior of the cells of lymphocytic cell lineSupT1 (Fig. 7). Contrary to SupT1 cells, the transfection efciency ofsiRNA alone was poor (1%) in HIV infected peripheral bloodmononuclear cells (PBMC) after 3 h; however, following complexa-tion with 2G-NN16 the dendriplex showed signicantly higher(40%) transfection efciency. After 24 h, the transfection efcienciesof siRNA and dendriplexes were more or less similar with minimalcytotoxicity (80% cell viability) (Fig. 8). In addition, dendriplexeswere efcient in reducing HIV replication in SupT1 and PBMC.Compared with siNEF and siCocktail (containing siP24, siNEF andsiGAG1) alone, treatment with 2G-NN16 dendriplexes showed 40%HIV inhibition for +/ charge ratio of 2 (Fig. 9). The low cytotoxicitycoupled with high transfection efciency and stability of siRNAprovided by carbosilane dendrimer demonstrates the importance oftheir development as a delivery system for siRNA.

Dutta et al. prepared G-5 PPI dendrimers for targeting of efavirenz(EFV) [106] and lamivudine (3TC) [107] to human monocytes/macrophages and MT-2 cells, respectively. For EFV targeting, the Cterminus of a tetrapeptide (ThrLysProArg)tuftsin (tu)wasconjugated to the amino end group of the PPI. Tetrapeptide tu bindsspecically to the macrophages/monocytes and polymorphonculearnucleocytes and also activates the immune system. EFV loading wasfound to be 37.4% in PPI and 49.3% in tuPPI dendrimers. The higherdrug loading efciency for tuPPI was attributed to the availability ofmore functional groups for complexation with EFV. The presence of tualso signicantly delayed the release of EFV from the dendrimer(144 h for tuPPI as compared with 24 h for PPI) due to the presenceof dense steric groups of tu at the surface of dendrimer. Presence ofbulky tu also shielded the positive charges of the PPI dendrimer andled to a signicantly decreased cytotoxicty in macrophage/monocytecells compared with the unmodied PPI dendrimer. The cellularuptake of EFV was signicantly (34.5 times) higher when present intuPPI dendrimer compared with free drug which was attributed tothe targeting effect of tuftsin present on the periphery of PPIdendrimer. Moreover, HIV infected macrophages/monocytes showedhigher uptake of tuPPI as compared to uninfected cells possibly dueto their activated state. tuPPI loaded with or without EFV also led toinhibition of HIV growth at low concentrations as determined by p24antigen assay.

For the targeting of 3TC, Dutta et al. prepared G-5 mannosylatedPPI dendrimers (MPPI) by divergent synthesis [107]. The aim was totarget lectin receptors (molecular target for sugar molecules such asmannose). Mannose receptor is a transmembrane glycoproteinexpressed by macrophages and has the ability to phagocytosenanoparticles coated with saccharides [70]. This has been reportedfor mannan-coated nanoparticles which show enhanced uptake inmannose receptor-positive mouse macrophage cell line (J-774E)[108]. Puried mannose receptor shows specic interaction withmonosaccharide ligands such as mannose and fucose [70]. Comparedwith PPI dendrimer, there was an increase in the drug entrapmentefciency from 35.7 to 43.2% for MPPI due to the steric hindranceprovided by the presence of mannose on the surface of PPI end groups.Cytotoxicity studies of blank dendrimer in MT2 cell line showeddecreased toxicity for MPPI (0.156 nM/ml) compared with PPI(0.039 nM/ml) due to shielding of the positive charges of PPI bymannose. Cellular uptake studies in MT2 cells showed 21 and 8.3times higher uptake for MPPI compared to free 3TC and PPI,respectively, indicating ligand specic internalization of MPPI den-drimers. Furthermore, MPPI dendrimers containing 3TC also showedsuperior anti-HIV activity (determined by p24 antigen assay)compared with free 3TC and PPI containing 3TC. The anti-HIV activityof MPPI dendrimers at 0.156 ng/ml was 1.5 fold higher than free 3TCat 0.625 ng/ml suggesting superior targeting ability of MPPI682dendrimers.


DPROOF

683 4. Conclusion and future directions

684 The growing number of HIV infected individuals and related685 deaths reects upon the efforts required to tackle this pandemic. To686 date there are over twenty ve drugs approved for the treatment. The687 effect of antiretroviral therapy on the quality of life of HIV patients has688 been enormously positive; however, toxicity, adverse drug reactions,689 suboptimal bioavailability due to poor physicochemical properties,

690impaired biodistribution in HIV reservoirs, emergence of drug691resistance, requirement of drug monitoring and lifelong adherence692are the problems associated with antiretroviral treatment.693The studies discussed in this review provide the proof of concept694that rational use of nanoscale drug delivery systems has the potential695to tackle the water solubility, stability, bioavailability and viral696targeting issues associated with antiretroviral therapy. Most of these697studies have utilized cell cultures for in vitro and small animals for in

Fig. 6. Zeta potential and particle size of dendriplex. (A) Zeta potential of 500 nM siGAG1 upon addition of 2G-NN16 at varying +/ charge ratios. (B) Particle size (estimated byintensity) of the same dendriplex formulations. Dendriplex formed in 0.15 mM HEPES buffer, pH 7.4, 25 C (adopted from [105]).


ARTICLE IN PRESSECUNCORR

Fig. 7. Complete internalization of siRNA into T cells (adopted from [105]). Confocal microslabeled siRNA (red) alone (b) or complexed with 2G-NN16 at a +/ charge ratio of 1 (c) orpositive cells for siRNA uptake are indicated. (For interpretation of the references to colour

Please cite this article as: P. Sharma, S. Garg, Pure drug and polymbioavailability and targeting of anti-HIV drugs, Adv. Drug Deliv. Rev. (2TEcopy images of SupT1 cells after 20 h incubation with mock treatment (a) or with Cy3-2 (d). Cell membranes are labeled with CD45-FITC antibodies (green). Percentages ofin this gure legend, the reader is referred to the web version of this article.)


UNCORREC

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Fig. 8. Transfection and toxicity proles of dendriplexes on HIV infected T cells(adopted from [105]). Dendriplex at varying +/ charge ratios after 3 h incubation (A)and 24 h incubation (B). Toxicity was measured by LDH enzyme release, whiletransfection and cell viability were measured by ow cytometry. Lipofectin was usedas a comparative control for both toxicity and transfection.

Fig. 9. Sequence specic inhibition of HIV in PBMC after 24 h incubation withdendriplexes at varying +/ charge ratios with siNEF (black bars) and siCOCKTAIL(grey bars). Toxicity values as measured by LDH enzyme release after 3 h incubation areshown (white bars). Data are shown as meanSEM; n=3 (adopted from [105]).


ARTICLE IN PRESS


vivo studies.With the establishment of the proof of concept, there is aneed to test efcacy of these delivery systems in higher animalmodelssuch as rhesus macaquemonkeys, which are routinely used in the HIVdrug development. The unavailability of references on the efcacy ofnanoscale delivery system in higher animal models and humansindicates that the research in this eld is still in the early stages.

Amajor factor that would govern the applicability of the nanoscaledrug delivery systems is the cost of the treatment compared to thestandard antiretroviral therapy. Development of nanoscale deliverysystems frequently requires specialty chemicals and unique manu-facturing procedures which signicantly add to the cost of the nalproduct. In addition, complexities involved in scale up and qualitycontrol of the nal formulation need to be considered in advance.Majority of the studies published in the literature have focused ondeveloping a drug delivery system suitable for a single drug only.Although, most of the antiretroviral drugs can reduce the viral loadand increase CD4 count individually, to sustain the levels and avoiddrug resistance, a combination of three or more drugs is required.Therefore, further studies on nanoscale delivery systems should focuson combinational therapy. The loading efciency of a nanoparticulatesystem would dictate the size and route of administration of thedosage form. For example, for a 500 mg dose, 15% loading efciencywould require 3.3 g of the nanoparticulate system to be administered.Such an amount can only be administered through oral route. In theabsence of HIV eradication approach, the major challenge associatedwith current treatment is poor patient compliance. In this regard,development of a cost effective dosage form which can be self-administered would be preferred.

The advantages associated with nanoscale drug delivery systemsare enormous. At the same time, one has to judiciously consider thepracticality of the approach within clinical settings. In this context,insights gained from the application of nanoscale drug deliverysystems in other diseases, such as cancer, will be useful in assisting therational development of delivery systems for anti-HIV drugs. With anumber of challenges associated with HIV prophylaxis and treatment,there is an ever-growing need for the development of new drugdelivery systems. Nanotechnology based drug delivery systems holdpromise to alleviate the problems associated with anti-HIV drugs andimprove the quality of life of the HIV infected patients.

5. Uncited reference

[103]

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Pure Drug and Polymer Based Nanotechnologies for the Improved Solubility, Stability

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