Anti-HIV efficacyreverse transcriptase inhibitors delivered as squalenoylated prodrug nanoassemblies

Anti-HIV efficacy and biodistribution of nucleoside reverse transcriptase inhibitors delivered as squalenoylated prodrug nanoassembliesHervéHillaireau a,b,1,Nathalie Dereuddre-Bosquet c,d,1,Rym Skanji a,b,Fawzia Bekkara-Aounallah a,b,e,Joachim Caron a,b,Sinda Lepêtre a,b,Sébastien Argote f, Laurent Bauduin g,Rahima Yousfic,Christine Rogez-Kreuz c,Didier Desmaële a,b, Bernard Rousseau f,Ruxandra Gref a,b,Karine Andrieux a,b,Pascal Clayette c,Patrick Couvreur a,b,*a Institut Galien Paris-Sud,UMR8612,UniversitéParis-Sud,Châtenay-Malabry F-92290,Franceb CNRS,Châtenay-Malabry F-92290,Francec Bertin Pharma,Neurovirology Department,CEA,Fontenay-aux-Roses F-92265,Franced Present address:CEA,Division of Immuno-Virology,iMETI,Fontenay-aux Roses F-92265,Francee UniversitéDjillali Liabès,LCOPM,BP89,Sidi Bel Abbès22000,Algeriaf CEA/Saclay,Department of Radiolabeled Molecules,Gif-sur-Yvette F-91191,Franceg Bertin Pharma,Bioanalysis Department,CEA,Gif-sur-Yvette F-91191,Francea r t i c l e i n f oArticle history:Received29December2012 Accepted9March2013 Available online2April2013Keywords:Squalenoylation NanoassembliesProdrugDidanosineZalcitabine a b s t r a c tDue to their hydrophilic nature,most nucleoside reverse transcriptase inhibitors(NRTIs)display a var-iable bioavailability after oral administration and a poor control over their biodistribution,thus hampering their access to HIV sanctuaries.The limited cellular uptake and activation in the triphosphate form of NRTIs further restrict their efficacy and favour the emergence of viral resistance.We have shown that the conjugation of squalene(sq)to the nucleoside analogues dideoxycytidine(ddC)and didanosine (ddI)leads to amphiphilic prodrugs(ddC-sq and ddI-sq)that spontaneously self-organize in water as stable nanoassemblies of100e300nm.These nanoassemblies can also be formulated with polyethylene glycol coupled to either cholesterol(Chol-PEG)or squalene(sq-PEG).When incubated with peripheral blood mononuclear cells(PBMCs)in vitro infected with HIV,the NRTI-sq prodrugs enhanced the antiviral efficacy of the parent NRTIs,with a2-to3-fold decrease of the50%effective doses and a nearly2-fold increase of the selectivity index.This was also the case with HIV-1strains resistant to ddC and/or ddI.The enhanced antiviral activity of ddI-sq was correlated with an up to5-fold increase in the intracellular concentration of the corresponding pharmacologically active metabolite ddA-TP.The ddI-sq prodrug was further investigated in vivo by the oral route,the preferred route of administration of NRTIs.Pharma-cokinetics studies performed on rats showed that the prodrug maintained low amounts of free ddI in the plasma.Administration of3H-ddI-sq led to radioactivity levels higher in the plasma and relevant organs in HIV infection as compared to administration of free3H-ddI.Taken together,these results show the potential of the squalenoylated prodrugs of NRTIs to enhance their absorption and improve their bio-distribution,but also to enhance their intracellular delivery and antiviral efficacy towards HIV-infected cells.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionHighly active antiretroviral therapies(HAART)have significantlydecreased the mortality associated to HIV/AIDS since the late1990s.In2012,reference HAART treatments are based on a boostedprotease inhibitor regimen associated with several reverse*Corresponding author.Institut Galien Paris-Sud,UMR8612,Univ.Paris-Sud,5 rue J.-B.Clément,F-92290Châtenay-Malabry,France.Tel.:þ33146835396; fax:þ33146619334.E-mail address:patrick.couvreur@u-psud.fr(P.Couvreur).1Have equally participated to thisstudy.Contents lists available at SciVerse ScienceDirectBiomaterialsjournal homepage:w ww.elsevi/locate/biomaterials0142-9612/$e see front matterÓ2013Elsevier Ltd.All rights reserved./10.1016/j.biomaterials.2013.03.022Biomaterials34(2013)4831e4838transcriptase inhibitors,either nucleoside/nucleotide or non-nucleoside analogues.Nucleoside reverse transcriptase inhibitors (NRTIs)were thefirst molecules described for this purpose and remain today a cornerstone of the treatments.However,despite their efficacy and widespread use,these molecules still suffer from important limitations leading to an incomplete suppression of viral replication and a failure to eradicate HIV from the organism.Failure of antiretroviral therapy involves poor adherence,viral resistance and pharmacological issues[1],such as:(i)a short plasma half-life, (ii)a variable bioavailability after oral administration,(iii)a variable cell uptake due to their hydrophilic nature and(iv)a restricted intracellular activation into their active triphosphorylated form by cellular kinases[2].To overcome these limitations(and beside the synthesis of new NRTIs),two main strategies have been pursued.First,several NRTI prodrugs have been synthesized in an effort to increase plasma stability and cellular uptake[3,4].Second,NRTIs were loaded onto liposomes or biodegradable polymeric nanocarriers in order to modulate the drug biodistribution[5,6].To date,despite some successes,both strategies have faced important limitations:most prodrugs display a poor solubility,restricting their administration, while the clinical use of nanocarriers is limited by their low drug loading capacity,which requires the administration of excessive amounts of transporter material(ie.polymers or lipids).We have recently shown that the conjugation of nucleoside analogues to squalene(sq),a natural terpene,lead to amphiphilic prodrugs able to self-assemble spontaneously in water as nano-particles(nanoassemblies)[7].Such bioconjugates possess the advantages of both the prodrugs(high drug loading and ease of formulation)and the nanocarriers(high dispersibility in water and drug targeting potential).This concept,termed“squalenoylation”, has been applied to natural nucleosides such as thymidine,to anticancer nucleoside analogues such as gemcitabine and cytar-abine,as well as to the antiviral NRTIs azidothymidine(AZT,zido-vudine),didanosine(ddI)and dideoxycytidine(ddC,zalcitabine).At the pre-clinical stage,gemcitabine-squalene nanoassemblies were already found to dramatically improve the anticancer efficacy of the parent drug on experimental mice leukaemia[8]and on human pancreas cancer models[9].In this study,we investigated the antiviral efficacy of the squa-lenoylated ddI(ddI-sq)and ddC(ddC-sq)nanoassemblies on HIV by exploring(i)anti-HIV effects on human peripheral blood mono-nuclear cells(PBMCs)infected with sensitive and resistant HIV strains,(ii)the ability of ddI-sq nanoassemblies to intracellularly produce the active ddA-TP metabolite and(iii)the impact of the squalenoylation on the NRTI absorption and biodistribution after oral administration to rats.2.Materials and methods2.1.Chemical synthesis2.1.1.NRTI-sq synthesisSqualenoylated NRTI(NRTI-sq)were synthesized by addition of tris norsqualenic acid[7]onto the50NRTI hydroxyl group(ddI-sq)or onto the amino group of the NRTI heterocycle(ddC-sq).ddI-sq was obtained as follows.To a stirred solution of tris norsqualenoic acid (0.84g,2mmol)in anhydrous DMF(7mL),was added under nitrogen,the1-hydroxybenzotriazole hydrate(0.38g, 2.4mmol),O-(7-azabenzotriazol-1-yl)-N,N,N,N’-tetramethyluronium hexafluoroborate(0.95g, 2.4mmol),didanosine (0.50g,2.05mmol)and diisopropylethylamine(0.62g,4.6mmol).The reaction was stirred for84h at room temperature and the reaction mixture was then concentrated in vacuo.Aqueous sodium hydrogen carbonate was added and the mixture was extracted with ethyl acetate(3Â100mL).The combined extracts were washed with water,dried on MgSO4,and evaporated.The crude product was purified by chromatography on silica gel eluting with1e5%methanol in dichloromethane to give pure50-squalenoyl-didanosine as a colourless oil(0.82g, 63%)(Fig.1A).Tritiated ddI-sq(3H-ddI-sq)was synthesized similarly but using tritiated ddI (3H-ddI).3H-ddI(1.8m g,250m Ci,obtained from Hartmann Analytic)was mixed with cold ddI(10mg).The coupling reaction was then performed as described above, leading to pure3H-ddI-sq(8.35mCi/mmol).3H-ddI-sq was dissolved in ethanol (26m Ci/mL)and stored atÀ20 C.In the case of ddC-sq,to a stirred solution of tris norsqualenoic acid(0.50g, 1.2mmol)in anhydrous DMF(7ml),was added under nitrogen,1-hydroxybenzotriazole hydrate(0.23g, 1.4mmol),O-(7-azabenzotriazol-1-yl)-N,N,N,N’-tetramethyluronium hexafluoroborate(0.56g,1.4mmol),dideoxycytidine (0.29g,1.3mmol)and diisopropylethylamine(0.35g,2.6mmol).The reaction mixture was stirred for84h at room temperature and was then concentrated in vacuo.Aqueous sodium hydrogen carbonate was added and the mixture was extracted with ethyl acetate(3Â100mL).The combined extracts were washed with water,dried on MgSO4,and evaporated.The crude product was purified by chro-matography on silica gel eluting with1e5%methanol in dichloromethane to give pure4-N-squalenoyl-dideoxycytidine as a colourless oil(0.59g,50%)(Fig.1B).2.1.2.Thymidine-sq synthesisTo a stirred solution of tris norsqualenoic acid(0.10g,0.24mmol)in anhydrous DMF(3.2mL),was added under nitrogen1-hydroxybenzotriazole hydrate(0.044g, 0.29mmol),O-(7-azabenzotriazol-1-yl)-N,N,N,N’-tetramethyluronium hexa-fluoroborate(0.11g,0.29mmol),thymidine(0.117g,0.48mmol)and diisopropy-lethylamine(0.0944g,0.73mmol).The reaction mixture was stirred84h at room temperature and was then concentrated in vacuo.Aqueous sodium hydrogen car-bonate was added and the mixture was extracted with ethyl acetate(3Â100mL). The combined extracts were washed with water,dried on MgSO4,and evaporated. The crude product was purified by chromatography on silica gel eluting with1e5% methanol in dichloromethane to give pure50-squalenoyl-thymidine(T-sq)as a colourless oil(0.15g,50%)(Fig.1C).2.1.3.Sq-PEG synthesisThe sq-PEG was obtained by alkylation of the sodium alkoxide of MePEG by trisnorsqualenyl methanesulfonate as previously reported[10].Briefly,90mg of sodium hydride(60%dispersion in mineral oil,2.24mmol)was suspended in2mL of cyclohexane and stirred for2min.The cyclohexane was drawn off via syringe and the process was repeated.Anhydrous THF(2mL)was added and a solution of MePEG (weight-average molar mass M w¼2000g mol-1,Fluka)(1.49g,0.74mmol)in THF (2mL),was added drop-wise to the stirred suspension.After completed gas evo-lution,a solution of1,10,2-tris norsqualenyl methanesulfonate(695mg,1.49mmol) dissolved in THF(2mL)was added and the resulting mixture was stirred at room temperature for18h.Water was added and the mixture was extracted with CH2Cl2 (3Â20mL).The combined organic extracts were washed with brine,dried over MgSO4and concentrated in vacuo.The crude residue was triturated with Et2O,the solid wasfiltered over a sintered glass funnel,and washed with dried ether to give trisnorsqualene-PEG as a white amorphous solid(1.09g,62%)(Fig.1D).2.2.NRTI-sq nanoassemblies preparation and characterizationNRTI-sq nanoassemblies were prepared by nanoprecipitation.Briefly,NRTI-sq was dissolved in acetone(ddC-sq)or ethanol(ddI-sq)(1mL,2e10mg mL-1)and added drop-wise under stirring(500rpm)into2mL of MilliQÒwater.Precipitation of nanoassemblies occurred spontaneously.After thorough solvent evaporation,the final volume was adjusted to2mL,leading to a1e5mg mL-1aqueous NRTI-sq posite PEGylated nanoassemblies(noted NRTI-sq-PEG)at afinal concentration of1mg mL-1were prepared by co-nanoprecipitation of NRTI-sq with either Chol-PEG or sq-PEG using acetone as organic solvent with different NRTI-sq:Chol-PEG(sq-PEG)w/w ratios,ie.1:0;1:0.002;1:0.008;1:0.05;1:0.1;1:0.3; 1:0.5;1:0.7.Other squalenoylated nanoassemblies used as control formulations(T-sq,T-sq-PEG and sq-PEG)were prepared similarly.Radiolabeled3H-ddI-sq nano-assemblies were prepared similarly from an isotopic dilution of ddI-sq in ethanol, obtained by mixing ethanol solutions of3H-ddI-sq(0.2mL,5m Ci)and cold ddI-sq (0.5mL,52mg/mL).For all experiments,the nanoassemblies were freshly pre-pared and used within24h(conservation at4 C).The storage stability of the nanoassemblies was assessed at room temperature using a50m g mL-1 concentration.The mean particle size and polydispersity index were determined at20 C by quasi-elastic light scattering(QELS)(Nano ZS,Malvern Instruments Ltd.,UK).The selected angle was173 and the measurement was made using a50m g mL-1con-centration in MilliQÒwater or PBS.The zeta potential was determined with the same equipment using a50m g mL-1nanoassemblies concentration in a1mM NaCl so-lution.Measurements were performed in triplicate on at least three independent experiments.The morphology of the nanoassemblies and composite nanoassemblies was investigated using transmission electron microscopy(TEM),without or after freeze-fracture(FF).For TEM observations,10m L of aqueous nanoassemblies suspension (1mg mL-1)was deposited on a thin copper grid,allowed to evaporate for2min,and observed using a Philips EM208electron microscope operating at80kV.For FF-TEM observations,1mL of aqueous nanoassemblies suspension(1mg mL-1)was incu-bated with glycerol(30%v/v),used as a cryoprotectant.A drop of each sample wasH.Hillaireau et al./Biomaterials34(2013)4831e4838 4832placed on a copper support and immediately frozen in liquid propane and then kept in liquid nitrogen.Fracturing and shadowing were performed in a Balzers BAF 400freeze-etching unit.The replicas were washed in THF and distilled water and placed on copper grids.Observations were made under a transmission electron microscope JEOL 100SX.2.3.In vitro biological evaluation of NRTI-sq nanoassembliesNRTI-sq nanoassemblies were tested for their anti-HIV activity,comparatively to the parent NRTIs,using phytohemaglutinin (PHA)-P-activated PBMCs in vitro infected with the HIV-1-LAI strain,a methodology previously described [11e 13].Brie fly,PBMCs obtained from healthy donors were incubated with six concentra-tions of each treatment and infected with one hundred 50%tissue culture infectious doses (TCID50)of HIV-1-LAI per 100,000cells.Treatments were maintained throughout the culture,and cell supernatants were collected on day 7post-infection and stored at À20 C.Viral replication was measured by quantifying reverse tran-scriptase (RT)activity in cell culture supernatants using the RT RetroSys kit,ac-cording to the manufacturer ’s (Innovagen)instructions.In parallel,cytotoxicity of the nanoassemblies and their corresponding parent NRTI was evaluated in unin-fected PHA-P-activated PBMC by MTT assay on day 7.Experiments were performed in triplicate and 50%,70%and 90%effective doses (ED 50,ED 70and ED 90respectively)as well as 50%cytotoxic doses (TC 50)were calculated using SoftMaxPro software.Evaluation against resistant HIV-1strains was performed similarly using cells of the MT2lineage.HIV resistant viruses (HIV-1-144and HIV-1-146)were generously provided by NIH AIDS Reagent Program.The HIV-1-144strain is mutated in position74(mutation 74V)and is known to be resistant to ddI and ddC.The HIV-1-146strain is mutated in positions 41(L),74(V),106(A)and 215(Y)and is known to be resistant to ddI.Intracellular concentrations of ddA-TP,the active triphosphate metabolite of ddI,was assessed as previously described [14]in PBMCs after incubation with ddI-sq or ddI at 1,10and 20m M.Natural dA-TP was also quanti fied as a control.2.4.In vivo biological evaluation of NRTI-sq nanoassembliesThe animal experiments were carried out according to the principles of labo-ratory animal care and European legislation (recommendation 2007/526/EC)after the protocol ethics were institutionally approved.Wistar rats weighing around 220g were purchased from Charles River laboratory (Domaine des Oncins,l ’Arbresle,France).Animals had ad libitum access on water and food but were fasted 16h before treatment.For pharmacokinetics and biodistribution studies,two treatments were compared (ddI vs .ddI-sq)and untreated animals were used as a control group.ddI and ddI-sq were administered by oral gavage to rats as a 3e 4mL drug solution or prodrug suspension in a pH ¼8phosphate buffer,to administer a dose equivalent to 45mg/kg ddI.All animals were sacri ficed after anaesthesia with 50mg/kg pento-barbital by the intraperitoneal route.2.4.1.PharmacokineticsThe pharmacokinetics study was conducted on rats catheterized on the jugular vein.7rats per group were used.Blood samples (0.4mL)were collected at various times (0.25,0.5,1,2,4,8and 24h)in heparinized test tubes andimmediatelyFig.1.Chemical structures of (A)ddI-Sq,(B)ddC -Sq,(C)T-Sq and (D)Sq-PEG.The positions of tritium double radiolabeling are indicated for ddI-Sq.H.Hillaireau et al./Biomaterials 34(2013)4831e 48384833centrifuged at 4,000rpm at room temperature (20 C)using a microcentrifuge (Minispin,Eppendorf).Plasma was aspirated and frozen at À80 C until further analysis by liquid chromatography-mass spectrometry (LC-MS/MS).ddI plasmatic concentrations were determined as follows:brie fly,after defrost,plasma samples obtained from treated animals were completed to 500m L (validated dilution up to 1/10)with blank plasma and were centrifuged (18,000Âg ,at room temperature for 10min)and the internal standard (2-chloro-adenosine)was added to supernatant.The extraction of ddI and its internal standard was performed by a solid phase technique using cartridges ATH according to several steps:conditioning (methanol,water),sampling,washing (water)and elution (methanol).The fraction of interest was then rescued and the solvent was evaporated under nitrogen flux.The residue was then resuspended in water/methanol (95/5).After centrifugation (9000Âg for 20min),the supernatants were analysed by LC-MS/MS.Calibration and quality control samples were prepared and treated in the same way.80m L of the extracted samples were injected into the HPLC system Agilent HP100(Agilent technologies).The separation was performed using a Zorbax SB C18(150Â2.1)column at a flow rate of 300m L/min,with a gradient elution (methanol/acetonitrile/water).Detection was performed with a mass spectrometer API 3000(AB Sciex).ddI and chloro-adenosine were detected in negative mode using the transitions,m /z 234.8>135and m /z 284>168,respectively.The quanti fication process was performed using Analyst software 1.4(AB Sciex,Les Ulis,France).2.4.2.BiodistributionTritiated (3H-)versions of ddI or ddI-sq were used to study drugs absorption and biodistribution following oral administration.Practically,animals (3rats per group)were housed in individual metabolic cages up to the end of feces collection.5m Ci of either 3H-ddI-sq or 3H-ddI were administered by oral feeding to rats as a 3e 4mL drug solution or prodrug suspension in a pH ¼8phosphate buffer,to administer a dose equivalent of 45mg/kg ddI.After 24h,animals were sacri ficed by cardiac puncture under anaesthesia and the blood samples were collected.Blood samples were centrifuged at 2000Âg for 10min at room temperature,and the supernatant plasma was isolated.To 700m L of plasma,10mL of Ultima Gold scintillation liquid was added,vigorously vortexed for 1min and counted after 1h in a beta counter (Beckmann LS 6000TA,Beckman Coulter).Organs including liver,rate,kidneys,testis,heart,lungs,thymus,brain,intestine,muscle,bone marrow were dissected and weighed.2mL Solvable Òwas added into the vials containing the tissues and the feces and maintained overnight at 50 C to allow complete dissolution.200m L of hydrogen peroxide was then added to each vial and kept for 1h at 50 C to decol-orize solutions.After cooling,10mL Hionic-Fluor scintillation liquid was added to the vials and vigorously vortexed for 1min.Separately,the urine samples (1mL)were mixed with 10mL of Ultima Gold,vortexed for 1min,and set aside for 1h.The radioactivity in the samples was counted using a beta counter.The results were expressed as percentage of administered dose per gram of tissue.Urine and feces were interpreted as percentage of administered dose.The statistical comparisons were made using Student ’s test of an unequal variance.3.Results3.1.Preparation and characterization of NRTI-sq nanoassemblies When dissolved into ethanol or acetone and added dropwise to an aqueous phase,the squalenoylated NRTI prodrugs (NRTI-sq)self-assembled as nanoassemblies with a size in the range of 100e 250nm and a homogeneous distribution (polydispersity index around 0.2or lower)(Table 1).The squalenoylated bioconjugate based on the natural thymidine nucleoside (T-sq),used as a control for antiviral activity experiments,also leads to nanoassemblies,similarly to the NRTI-sq prodrugs.The addition of sq-PEG as well as Chol-PEG to the NRTI-sq formulations resulted in a signi ficant decrease of the size of the nanoassemblies.Noteworthy,the size of the NRTI-sq nanoassemblies was stable over at least 5weeks,which was also true for NRTI-sq-PEG,regardless of the PEGylation method (either sq-PEG or Chol-PEG)and the PEGylation ratio (Fig.2).Transmission electron microscopy (TEM)observations of ddI-sq aqueous suspensions con firmed the size range of the nano-assemblies displaying a spherical shape (Fig.3A).TEM observations of ddC-sq formulations performed after freeze-fracture revealed a solid and rather matrix type core (Fig.3B)and a smooth surface (Fig.3C)of the nanoassemblies.3.2.Antiviral activity of the NRTI-sq nanoassembliesWhen assessed on HIV-1-LAI-infected PBMCs,the two NRTI-sq prodrug nanoassemblies were found to improve signi ficantly the antiviral ef ficacy of the corresponding parent molecule,as shown by a 2-to 3-fold decrease of the ED 50values (Fig.4A).Noteworthy,since their cytotoxicities were similar to the corresponding parent molecules (supplementary data,table S1),the squalenoylated prodrugs had increased selectivity as compared to the NRTIs (by 1.9-fold for the ddI and 1.8-fold for the ddC;Fig.4B).In parallel to nucleoside derivatives,a squalenoylated natural nucleoside (thymidine-squalene,T-sq)was tested and displayed a cytotoxicity similar to ddI-sq,with no antiviral ef ficacy,as expected (selectivity index:1.5).The PEGylation of the NRTI-sq (NRTI:Chol-PEG ratio of 1:0.7)further increased their antiviral ef ficacy (Fig.4A)but also their cytotoxicity (supplementary data,table S1),resulting in only slightly increased (ddC)or even decreased (ddI)selectivity indexes (Fig.4B).The PEGylated T-sq (T-sq-PEG;selectivity index:1.6)also displayed an increased cytotoxicity compared to T-sq,suggesting a potential deleterious effect of the PEGylated moieties at theTable 1Size and polydispersity of the NRTI-Sq prodrug nanoassemblies as measured by dynamic light scattering.Mean diameter (nm)Polydispersity index ddI-Sq 217Æ470.220Æ0.079ddC-Sq 204Æ220.020Æ0.010T-Sq130Æ20.075Æ0.018250200150100500250200150100500d (n m )d (n m )1we ek 2we ek s3we ek s4we ek s5we ek s1we ek2we ek s3 we ek s4w e e k s 5 we e k sTime (weeks)Time (weeks)ABFig.2.Stability of the NRTI-Sq and NRTI-Sq-PEG nanoassemblies.The ddC-Sq:Chol-PEG (A)and ddC-Sq:Sq-PEG (B)nanoassemblies were prepared using different weight ratios of ddC-Sq and Chol-PEG,resp.Sq-PEG:1:0(black);1:0.1(dark grey);1:0.3(light grey)and 1:0.7(white).H.Hillaireau et al./Biomaterials 34(2013)4831e 48384834Fig.3.Morphology of the NRTI-Sq nanoassemblies.Transmission electron microscopy (TEM)images of (A)a ddI-Sq suspension without freeze-fracture,and (B,C)of a ddC-Sq suspension after freeze-fracture showing the inner (B)and outer (C)structures.NRTI NRTI-Sq NRTI-Sq-PEGNRTI NRTI-Sq NRTI-Sq-PEGNRTINRTI-SqABCFig.4.In vitro anti-HIV activity of the NRTI-Sq prodrug nanoassemblies towards HIV-1-LAI (A,B)and resistant HIV-1strains (C).Results are expressed as 50%or 90%effective dose (ED 50or ED 90)or selectivity index (TD 50/ED 90ratio)after 7-day incubation with NRTIs or NRTI prodrugs (*,<0.04m M).Results are representative of 3independent experiments (n ¼3).H.Hillaireau et al./Biomaterials 34(2013)4831e 48384835concentrations tested,which was likely due to their surfactant properties.3.3.Antiviral activity towards resistant HIV strainsIn our experimental conditions,we have con firmed the resis-tance of the 144-and 146-HIV-1strains towards ddI and/or ddC in MT-2cells.The HIV-1-144strain was found 34Æ18times more resistant to ddI and 11Æ4times more resistant to ddC as compared to the sensitive HIV-1-LAI strain;the HIV-1-146strain was 14Æ1times more resistant to ddI and 7Æ3more resistant to ddC than the sensitive HIV-1-LAI.When assessed towards the HIV-1-144strain (resistant to both ddI and ddC),the NRTI-sq nanoassemblies signi ficantly improved the antiviral ef ficacy of the parent molecules:the ED 90decreased by 2.7fold for ddI and by 2.8fold for ddC (Fig.4C).For the HIV-1-146strain (mainly resistant to ddI),the effect of the squalenoylation was even more pronounced for ddI (the ED 90dropped by 8-fold),whereas no change in ED 90was observed for ddC.3.4.Intracellular concentrations of the ddA-TP active metabolite The improved antiviral activity of ddI-sq nanoassemblies versus ddI,in both sensitive and resistant HIV-infected cells prompted us to quantify the intracellular levels of dideoxyadenosine triphos-phate (ddA-TP),the metabolite accounting for the anti-HIV activity of ddI.ddA-TP (as well as the natural nucleoside dA-TP)were intracellularly quanti fied after 2e 24h incubation with either ddI or ddI-sq at concentrations of 1(z ED 50),10and 20m M (z ED 90).In all cases,the maximal ddA-TP/dA-TP ratios increased with the ddI orddI-sq concentration,and were maximal 24h after treatment ini-tiaion.For each of the concentrations tested,the values of the ddA-TP/dA-TP ratios were found to be 3-to 5-times higher after 4e 24h incubation with ddI-sq vs.ddI (Fig.5).After 3days the ratio became similar for ddI-sq and ddI.3.5.Pharmacokinetics and biodistribution of ddI-sq nanoassemblies Plasma ddI concentrations were monitored by LC-MS/MS over 24h following oral administration to rats of ddI as a solution or as squalenanoylated prodrug nanoassemblies at equivalent dose in ddI.The pharmacokinetic parameters (Table 2)show that the treatment with ddI-sq prodrug decreased the plasma ddI concen-trations,comparatively to ddI free.However,ddI-sq nano-assemblies also decreased the elimination rate of ddI and doubled the t max ,as compared to the drug administered as a single solution,suggesting a reservoir-like behaviour of the prodrug nanoassemblies.In order to further investigate the biodistribution of ddI-sq vs.ddI,we have radiolabeled both ddI-sq and ddI using tritium iso-topic exchange (see materials and methods).After oral adminis-tration,3H-ddI-sq led to 4-times higher radioactivity concentrations in the plasma than 3H-ddI administered as a solu-tion,con firming that the ddI-sq prodrug acted as a reservoir in the blood circulation (Fig.6).Additionally,the biodistribution of the tritium into the tissues clearly showed that 3H-ddI-sq allowed (comparatively to 3H-ddI)a higher fraction of the administered dose to concentrate in all the investigated tissues,especially those of the mononuclear phagocyte system (up to 4.7-fold in the liver,spleen and bone marrow)and other tissues relevant for HIV infection (3-fold in thymus and 2.7-fold in brain)(Fig.6).Fig.5.In vitro intracellular ddA-TP levels in PHA-P-activated PBMCs treated with ddI or ddI-Sq at (A)1m M,(B)10m M and (C)20m M.Results are expressed as ddA-TP/dA-TP ratios.H.Hillaireau et al./Biomaterials 34(2013)4831e 48384836。