Pharmaceutical nanotechnology

Adenosine conjugated lipidic nanoparticles for enhanced tumortargeting

Rajan Swami a, Indu Singh a, Manish Kumar Jeengar b, V.G.M. Naidu b, Wahid Khan a,*,Ramakrishna Sistla c,*aDepartment of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500037, IndiabDepartment of Pharmacology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500037, IndiacMedicinal Chemistry and Pharmacology Division, Indian Institute of Chemical Technology (IICT), Hyderabad 500007, India

A R T I C L E I N F O

Article history:Received 24 February 2015Received in revised form 24 March 2015Accepted 27 March 2015Available online 31 March 2015

Keywords:AdenosineAdenosine receptorsSolid lipid nanoparticlesConjugationCell cycle analysis

A B S T R A C T

Delivering chemotherapeutics by nanoparticles into tumor is impeded majorly by two factors:nonspecific targeting and inefficient penetration. Targeted delivery of anti-cancer agents solely to tumorcells introduces a smart strategy because it enhances the therapeutic index compared with untargeteddrugs. The present study was performed to investigate the efficiency of adenosine (ADN) to target solidlipid nanoparticles (SLN) to over expressing adenosine receptor cell lines such as human breast cancerand prostate cancer (MCF-7 and DU-145 cells), respectively. SLN were prepared by emulsification andsolvent evaporation process using docetaxel (DTX) as drug and were characterized by various techniqueslike dynamic light scattering, differential scanning calorimeter and transmission electron microscopy.DTX loaded SLNs were surface modified with ADN, an adenosine receptors ligand using carbodiimidecoupling. Conjugation was confirmed using infrared spectroscopy and quantified using phenol–sulfuricacid method. Conjugated SLN were shown to have sustained drug release as compared to unconjugatednanoparticles and drug suspension. Compared with free DTX and unconjugated SLN, ADN conjugated SLNshowed significantly higher cytotoxicity of loaded DTX, as evidenced by in vitro cell experiments. TheIC50 was 0.41 mg/ml for native DTX, 0.30 mg/ml for unconjugated SLN formulation, and 0.09 mg/ml forADN conjugated SLN formulation in MCF-7 cell lines. Whereas, in DU-145, there was 2 fold change in IC50of ADN–SLN as compared to DTX. IC50 was found to be 0.44 mg/ml for free DTX, 0.39 mg/ml forunconjugated SLN and 0.22 mg/ml for ADN–SLN. Annexin assay and cell cycle analysis assay furthersubstantiated the cell cytotoxicity. Fluorescent cell uptake and competitive ligand-receptor binding assaycorroborated the receptor mediated endocytosis pathway indicated role of adenosine receptors ininternalization of conjugated particles. Pharmacokinetic studies of lipidic formulations depictedsignificant improvement in pharmacokinetic parameters than marketed formulation. ADN conjugatedSLN proved to be an efficient drug delivery vehicle. Hence, ADN can be used as a potential ligand to targetbreast and prostate cancer.

ã 2015 Elsevier B.V. All rights reserved.

1. Introduction

Identifying cancer hallmarks helps in targeting cancer cells inbetter ways. Over expression of certain receptors on tumorsurface enables them to become hyper responsive to the ambientlevels of growth factor that normally would nottrigger proliferation (Hanahan and Weinberg, 2011; Wang,

2014). With this advancement of understanding of receptor’srole in cancer biology it becomes mandatory to use suchinformation to target and eradicate cancer. There are manyreceptors such as transferrin receptors, folic acid receptors,estrogen receptors and adenosine receptors etc., which are overexpressed on tumor surface. Their respective ligands can be usedto actively target drug encapsulated nanoparticles to cancer cellswhich further increases the efficacy of these nanoparticles(Sagnella et al., 2014).

Adenosine receptors are over expressed on many tumor cellsurfaces like prostate, breast melanoma and brain tumors etc.(Fishman et al., 2002; Jajoo et al., 2009; Merighi et al., 2003; Weiet al., 2013). Adenosine (ADN) is a purine nucleoside composed of a

* Corresponding authors. Tel.: +91 8790037913 (W. Khan)/+91 4027193753 (R.Sistla).

E-mail addresses: wahid@niperhyd.ac.in (W. Khan), sistla@iict.res.in,rksistla@yahoo.com (R. Sistla).

http://dx.doi.org/10.1016/j.ijpharm.2015.03.0650378-5173/ã 2015 Elsevier B.V. All rights reserved.

International Journal of Pharmaceutics 486 (2015) 287–296

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molecule of adenine attached to a ribose sugar molecule(ribofuranose) moiety via a b-N9-glycosidic bond, has beenreported to stimulate or inhibit the release of angiogenic factorsdepending on the cell type examined. It was documented that ADNand other agonists of adenosine receptors may be useful foreradicating or suppressing the growth of tumor malignancies(Fishman et al., 2002; Merimsky et al., 2003).

In different approaches, many polymer chains were synthe-sized, where ADN is part of the basic backbone of polymer to play amajor role in internalization of DNA cargo in tumor cell viaadenosine receptors mediated endocytosis (Chung et al., 2011) anddecreases in endocytosis was evident when cells were pre-treatedwith ADN. Decrease was attributed to blockage of adenosinereceptors. Hence, considering these reports as basis, it can beassumed that ADN can be used as ligand to target over expressedadenosine receptors on cancer cells and this present studypioneers to investigate the targeting potential of ADN for breastand prostate tumors.

Solid lipid nanoparticles (SLN) are efficient way to deliver suchdrugs because SLN offers advantages of polymeric nanoparticles,fat emulsion or liposomes with simultaneously avoiding theirdisadvantages (Akbarzadeh et al., 2013; Mehnert and Mader, 2001;Mueller et al., 2000). SLN found renowned attention in tumortargeting because of their lymph targeting potential which furtherhelps in increasing bioavailability and targeting to breast cancersetc. (Singh et al., 2014). Modification of large surface area of SLNwith charge modifying lipids such as stearic acid (SA), stearylamineetc. can aid in conjugation of ligand on surface (Sood et al., 2013).Consequently, contributes another advantage to SLN for sitespecific drug delivery.

Docetaxel (DTX) is a semi-synthetic, taxane derived, highlypotent anticancer drug. It has shown broad spectrum antitumoractivity against prostate, breast, pancreatic, lung, gastric andhepatic carcinomas (Xu et al., 2009; Zhao and Astruc, 2012). DTXbinds irreversibly with actin and stabilizes the microtubuleassembly which is responsible for inhibition of cell division andfinally cell death (Musumeci et al., 2006). Taxotere1 marketedformulation for DTX considered to be first line drug for prostateand breast cancer, however their side effects; that may preclude orat least limit their potential clinical application (Schrijvers et al.,1993).

The present study was conducted to establish the efficiency ofadenosine receptor ligand namely ADN to target adenosinereceptors over expressed cancer cell lines. Prepared ADN nano-conjugates were physicochemically characterized studied for invitro cytotoxicity and their pharmacokinetic parameters.

2. Materials and methods

2.1. Materials

Docetaxel (DTX) was a kindly provided by Therdose Pharma,Hyderabad, India as a gift sample. Adenosine (ADN) and glycerylmonostearate (GMS) were procured from Alfa Aesar, USA and soyalecithin (SL), stearic acid (SA), Tween 80, 1-ethyl-3-[3-dimethyla-minopropyl] carbodiimide hydrochloride (EDC), N-hydroxysucci-nimide (NHS), fluorescein isothiocyanate (FITC), octadecylamine(ODA) and cellulose dialysis tubing (molecular weight cut off14,000 Da) were procured from Sigma–Aldrich (Germany). Chlo-roform, methanol, acetonitrile were of HPLC grade (Merck, India).MCF-7, DU-145 cell lines were obtained from National Center forCell Science (NCCS), Pune, India. MEM, MTT [3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyl tetrazolium bromide], trypsin, EDTA werepurchased from Sigma Chemicals Co. (St. Louis, MO), Dulbecco’smodified Eagle’s Medium (DMEM), fetal bovine serum waspurchased from Gibco, USA, 96 well flat bottom tissue culture

plates were purchased from Tarsons Products Pvt., Ltd., Mumbai,India.

2.2. Preparation of SLN

DTX loaded unconjugated SLN (DSLN) were prepared usingemulsification and solvent evaporation method (Trotta et al., 2003).Briefly, DTX and lipid phase containing SA, SL and GMS (1:2:6, w/wratio) were dissolved in chloroform to get oil phase in organicsolvent. Aqueous phase was prepared by dissolving 1.5% w/w Tween80. Lipid phase containing organic solvent was added to aqueousphase and homogenized (Ultra Turrax T25, Germany) for 5 min at11,000 rpm. Emulsion, thus obtained, was sonicated (Vibra cell,Sonics, USA) for 20 min. The nanoemulsion was kept under stirringfor 3 h followed by characterization for size, zeta potential. Thesenanoparticles were further used for conjugating ADN on surface.

2.3. Surface conjugation

ADN was conjugated on the surface of preformed DSLN usingcarbodiimide chemistry (Kulhari et al., 2014). 20 mg DSLN wasdispersed in phosphate buffer (pH 7.4) and were incubated with NHSand EDC (1:5 w/w ratios). The dispersion was kept under gentlestirring for 2 h at room temperature. To this, 600 mg ADN was added,mixed well and kept for further stirring for 4 h. ADN conjugated DTXloaded SLN (ADN–SLN) were collected after centrifugation (SigmaLaborentrifugen GMBH, Germany) at 12,000 rpm for 30 min andwashed thrice with distilled water to remove unconjugated ADN insupernatant. Prepared ADN–SLN pellets were recollected and freezedried (Skadi, Europe). Freeze dried nanoparticles were characterizedfor qualitative estimation of conjugation using Fourier transforminfrared spectroscopy (FTIR).

Conjugation efficiency was deduced from estimating the free orunconjugated ADN in supernatant (indirect method). Saturatedphenol sulfuric acid method (Dubois et al., 1956) was employed tocalculate amount of adenosine attached to DSLN. Briefly, 2 mlaliquot of carbohydrate solution (supernatant ADN–SLN conjuga-tion process) was mixed with 1 ml of 5% aqueous solution of phenolin a test tube. Subsequently, 5 ml of concentrated sulfuric acid wasadded rapidly to the mixture. After allowing the test tubes to standfor 10 min, they were vortexed for 30 s and placed for 20 min in awater bath at 37 �C for color development. Reference solutionswere prepared in identical manner as above, except 2 ml aliquot ofcarbohydrate solution, which was replaced with water. Absor-bance of samples was taken at 480 nm using UV spectrophotome-ter (Jasko, Japan). Conjugation efficiency was expressed aspercentage of ADN bound to DSLN.

2.4. In vitro characterization of nanoparticles

2.4.1. Particle size and zeta potentialParticle size and zeta potential of blank SLN, DSLN and ADN–

SLN were measured by photon correlation spectroscopy usingZetasizer, Nano ZS (Malvern Instruments, UK). Furthermore,transmission electron microscope (TEM, JEOL, Japan) was usedto evaluate the surface morphology of ADN–SLN.

2.4.2. Entrapment efficiencyEntrapment efficiency was determined by measuring the

concentration of unloaded drug in supernatant collected aftercentrifugation of nanoparticle dispersion and analysis was doneusing high performance liquid chromatography (HPLC) withphotodiode array detector (Waters, USA). An octadecylsilanecolumn (Inertsil, 250 mm � 4.6 mm, 5 mm) was used for analysisand column temperature was maintained at 25 � 5 �C. The mobilephase, acetonitrile (60%) and water (40%), was pumped at a flow

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rate of 1.2 ml/min and monitored at a wavelength of 229 nm(Andersen et al., 2006). Calibration graphs plotted were linear witha correlation coefficient of 0.999. The inter–intraday accuracy andprecision were within a relative standard deviation (RSD) of �5%.

2.4.3. Differential scanning calorimetry (DSC)The physical state of DTX inside the final formulation (ADN–

SLN) was investigated by DSC. DSC measurements were carried outon the samples: (a) DTX, (b) physical mixture of lipids and DTX; (c)ADN–SLN. DSC studies were performed in a (DSC822e, MettlerToledo, Switzerland), using aluminium open pans. The heating ratewas 10 �C/min, with a temperature range of 30–250 �C and a 50 ml/min nitrogen flow.

2.5. In vitro release studies

To study the release profile of drug pure DTX, DSLN andADN–SLN (containing 1 mg of DTX) were suspended in 1 mldistilled water and placed in a dialysis tubing (14,000 molecularweight cut off, Sigma–Aldrich, USA). The tubing was placedindividually into 50 ml release media at 37 �C and kept for stirringat 100 rpm. The phosphate buffer saline (PBS, pH 7.4) containing0.5% Tween 80 were used as release media (Venkateswarlu andManjunath, 2004). An aliquot of 1 ml was withdrawn from therelease medium at different time intervals and was replaced withsame volume of fresh medium. Released samples were analysed byHPLC as described in previous sections. Data obtained in triplicatewere analysed graphically. Drug release data were expressed as thepercentage of the cumulative amount of drug release.

2.6. Cytotoxicity evaluation

Two cell lines (MCF-7 and DU-145) were selected on the basis ofover expression of adenosine receptors (Aghaei et al., 2012;Fishman et al., 2012). Cell lines were grown as adherent in DMEMmedium supplemented with 10% fetal bovine serum, 100 mg/mlpenicillin, 200 mg/ml streptomycin, 2 mM L-glutamine, and culturewas maintained in a humidified atmosphere with 5% CO2. Stocksolution of DTX at 10 mg/ml concentration in DMSO was preparedand from the stock solutions (10 mg/ml) different dilutions weremade with sterile PBS to get required concentration for treatmentof cells.

2.7. MTT assay and competitive ligand-receptor binding assay

Cytotoxicity of formulations was determined by MTT assaybased on mitochondrial reduction of yellow MTT tetrazolium dyeto a highly colored blue formazan product. 1 �104 Cells (counted bytrypan blue exclusion dye method) in 96-well plates wereincubated with formulations and positive control DTX with seriesof concentrations for 48 h at 37 �C in DMEM with 10% FBS medium(Twentyman and Luscombe, 1987). Then the above media wasreplaced with 90 ml of fresh serum free media and 10 ml of MTTreagent (5 mg/ml) and plates were incubated at 37 �C for 4 h, thereafter the above media was replaced with 200 ml of DMSO andincubated at 37 �C for 10 min. The absorbance at 570 nm wasmeasured on a spectrophotometer (SpectraMax, Molecular devi-ces) IC50 values were determined from plot: % inhibition (fromcontrol) vs. concentration.

For competitive ligand-receptor binding assay, cell line showinglower IC50 for formulations was selected. 1 �104 Cells werecoincubated with an 800 M excess of ADN for 30 min followedby washing and treatment with formulations and positive controlDTX with series of concentrations for 48 h at 37 �C in DMEM with10% FBS medium. Thereafter MTT assay was followed as describedabove. Obtained IC50 values from competitive ligand-receptor

binding assay compared with previously obtained IC50 values ofMTT assay.

2.8. Cell uptake study

FITC was used as fluorescent marker to tag formulations. ODA–FITC was prepared as described by Yuan et al. (2008) with minormodifications (Xing-guo et al., 2008). Briefly, ODA was dissolved inethanol and the same molar amount of FITC was added understirring. After 24 h of reaction in darkness, ODA–FITC was mixedwith a tenfold volume of distilled water and filtered. Distilledwater was used to wash the product five times. The obtainedconjugate (ODA–FITC) was freeze dried and stored in the dark forfurther use. Prepared ODA–FITC was incorporated in blank SLN tomake drug free FITC tagged unconjugated SLN and FITC taggedADN–SLN for evaluating cellular uptake. Supernatant of ODA–FITCincorporated lipidic formulations (SLN and ADN–SLN) was used tocheck the degree of incorporation of ODA–FTC into theseformulations. Around 20% ODA–FITC was found entrapped inthe nanoparticles.

Cells were seeded on 24 well plate at a density of 5 �104 cells/well in the respective media and allowed to adhere for 24 h. After24 h, the cells were incubated with various FITC labeledformulations for two hours and phase contrast and fluorescentimages were taken (Nikon Eclipse Microscope, Japan). Finally toquantify the cell uptake the media was removed and cells werewashed twice with PBS. Cell lysis was carried out using TritonX-100. The cell uptake was quantified by measuring the fluores-cence in the cell lysate (Yuan et al., 2008). Results were expressedas% cellular uptake.

2.9. Cell cycle analysis and apoptosis studies

Cell cycle is an important phenomenon that plays a crucial rolein developmental pathways, and is frequently deregulated in manycancer diseases. The distribution of DNA in the cell cycle wasstudied by flow cytometer (BD FACSVerseTM, USA) (Syam et al.,2011). To determine the effect of formulations on the cell cycle,cells were seeded in two sets, one set for a cell cycle analysis studyand one set for apoptosis studies. 6-Well plates at a density of1 �105 cells/ml for 24 h. After incubation, cells were treated with400 ng/ml and 442 ng/ml of drug loaded formulations for 24 h inMCF-7 and DU-145 cell lines respectively. Concentration wasselected on the basis of IC50 of the drug on particular cell lines.After treatment period, cells were collected, washed and fixedovernight in 70% ethanol in PBS at �20 �C. Fixed cells were pelletedand stained with cell cycle analysis reagent for 30 min at 37 �C indark according to the manufacturer’s instruction, and about10,000 events were analysed on a flow cytometer (BD FACSVer-seTM, USA).

The study was performed according to the method given byShrivastava et al. (2014). Briefly, 1 �105 cells were seeded in 6 wellplates treated with 400 ng/ml and 442 ng/ml of drug loadedformulations for 24 h in MCF-7 and DU-145 cell lines respectively.Then cells were analyzed by MuseTM Cell Analyzer (MerckMillipore, Germany) according to the manufacturer’s instructionsusing MuseTM, Annexin-V and Dead Cell reagent (Merck-Millipore,Germany). Apoptosis and necrosis were analyzed with quadrantstatistics on propidium iodide-negative cells, fluorescein positivecells and propidium iodide (PI)-positive cells, respectively.

2.10. Pharmacokinetics in plasma

Animal experiments were carried out in accordance with theguidelines of CPCSEA, India and the protocol was approved by theInstitutional Animal Ethics Committee (IAEC) of National Institute

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of Pharmaceutical Education and Research, Hyderabad, India(Protocol No.: NIP/11/2014/PE/124). Mice (25 � 5 g) were dividedinto three groups for each formulation. Each group received eitherADN–SLN or DSLN or Taxotere1 at dose equivalent to 10 mg/kg ofDTX i.v. Blood samples were collected at predetermined intervalsup to 12 h and plasma was immediately separated and stored at�80 �C until further analysis. The pharmacokinetic data obtained

after i.v. administration of lipidic formulations was compared withthat obtained with Taxotere1. An HPLC method was used for thedetermination of DTX in plasma using paclitaxel as an internalstandard. Calibration curve was made by spiking drug solutionwith required amount of internal standard in plasma or tissuehomogenate (tissue:normal saline, 1:3). 500 ml acetonitrile:meth-anol (50:50) was used as precipitating and extraction solvent.

Fig. 1. Schematic representation of conjugation and characterization process of ADN–SLN.

Fig. 2. Characterization of lipidic nanoparticles. (A) Size distribution of ADN–SLN nanoparticles analysed by zeta sizer, illustrating size range of nanoparticles afterconjugation of ADN to DSLN. (B) Appearance of ADN–SLN nanoparticles image acquired from TEM indicating spherical shape of particles. (C) FTIR spectra of ADN–SLN, ADN,and DSLN. FTIR spectra showing formation of amide bond between carboxylic group of DSLN and amine group of ADN changes the carbonyl stretching band from 1724 cm�1 to1658 cm�1. (D) Differential scanning calorimetry (DSC) thermogram of DTX showing melting endotherm at 173 �C but absence in physical mixture and ADN–SLN signifyingthe change of crystalline nature of drug to amorphous form.

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Calibration graphs plotted were linear with a correlation coeffi-cient was 0.999. The inter–intraday accuracy and precision werewithin a relative standard deviation (RSD) of �5%.

Plasma and tissue pharmacokinetic parameters were calculatedfrom free version of Kinetica – Adept Scientific Software. Cmax

values were obtained from visual inspection of obtained data.

2.11. Statistical analysis

The pharmacokinetic data were analyzed for statistical signifi-cance using one-way ANOVA followed by Bonferroni multiplecomparison procedure (Prism Software, version 2.0). Significancewas evaluated at p < 0.05.

3. Results and discussion

3.1. Optimization and characterization of SLN

Formulation variables, such as quantity of GMS, SL, SA andconcentration of Tween 80 were optimized based on the particlesize and entrapment efficiency. Schematic presentation ofconjugation process and characterization is shown in Fig. 1. At1.5% Tween 80, blank-SLN formed were of small particle size i.e.,55.57 � 3.51 nm and incorporation of drug increased the size ofDSLN i.e., 76.43 � 6.05. To evaluate ADN conjugated lipidic nano-particles, size and zeta potential of these particles were evaluatedand were compared with unconjugated lipidic nanoparticles. Theparticle size of ADN–SLN was considerably increased to98.77 � 2.50 nm (Fig. 2A), which was further confirmed by TEMimages. As it is observed from TEM images ADN–SLN were almostspherical in shape (Fig. 2B). Zeta potential which is another crucialcharacteristic, can influence particle stability and may also suggestsurface modification (Shah et al., 2014; Zhang et al., 2006). The zetapotential of ADN–SLN was decreased to �19 mV indicating surfacegroup modification. The entrapment efficiency of DTX in ADN–SLNwas decreased notably in comparison with DSLN (Table 1). The lossof drug could be due to release of drug in conjugation process.

3.2. FTIR spectroscopy

The formation of conjugate was confirmed by FT-IR spectralanalysis (Fig. 2C). The peak at 1658 cm�1 (C¼O stretching) ischaracteristic peak of amide group, which clearly indicates theformation of conjugate between SA and ADN. Absence of strongacid peak at 1734 cm�1 (C¼O stretching) due to SA in DSLN andamine doublet of ADN at 3337 cm�1 also indicate the formation ofamide bond in ADN–SLN.

3.3. Conjugation efficiency

ADN conjugation on DSLN surface was proved using FTIRspectroscopy analysis in previous section but quantification wasdone to determine the exact amount of ADN coupled to DSLNsurface. Estimation was done using phenol–sulfuric acid method asdescribed previously. The basic principle of this method is thatcarbohydrates, when dehydrated by reaction with concentrated

sulfuric acid, produce furfural derivatives. Further reactionbetween furfural derivatives and phenol develops detectablecolor. Conjugation efficiency was estimated to be around 80%.

3.4. DSC analysis

DSC thermograms in Fig. 2D clearly illustrates that meltingendothermic peak of pure DTX appeared at 173 �C. However, nomelting peak was detected for ADN–SLN. The probable reasoncould be that DTX has completely solubilised in the lipid matrix orchange in crystallinity (Padhye and Nagarsenker, 2013).

3.5. In vitro release studies

Both DSLN and ADN–SLN exhibited sustained release of DTXfrom the nanoparticles which is evident by the in vitro cumulativepercentage drug release profile obtained from formulations. PureDTX exhibited 81% drug released in first 12 h only. Whereas drugrelease from lipidic nanoparticle formulations were very slow.DSLN showed 32% release and ADNkSLN also exhibited around 24%release of drug in PBS in 24 h (Fig. 3). Conjugation may haveattributed to this decreased drug release from ADN–SLN.

3.6. Cytotoxicity study

The cytotoxicity of conjugated and unconjugated formulationwas evaluated in MCF-7 and DU-145 cell lines. With increase in thedrug concentration, the cell viability was decreased. A quantitativeevaluation of in vitro therapeutic effect of a dosage form is IC50,which is defined as the drug concentration needed to kill 50% of theincubated cells in a designated time. It can be calculated from the invitro cellular viability data after the treatment, the IC50 was0.41 mg/ml for free DTX, 0.30 mg/ml for DSLN formulation, and0.09 mg/ml for ADN–SLN formulation in MCF-7 cell lines (Fig. 4A

0

10

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30

40

50

60

70

80

90

0 5 10 15 20 25

Cu

mu

lati

ve %

Rel

ease

Time (h)

DTXDSLNADN-SLN

Fig. 3. In vitro drug release studies. In vitro drug release studies of different DTXformulations were done in PBS pH 7.4 and presented as cumulative drug release vs.time graph (mean � SD; n = 3). Pure DTX (DTX) showed fast release of about 80% in12 h whereas release from DSLN and ADN–SLN showed sustain release.

Table 1Particle size, zeta potential and encapsulation efficiency of DTX formulations (n = 3).

Formulations Particle size (nm) (mean � SD) Zeta potential (mV) (mean � SD) Encapsulation efficiency(%) (mean � SD)

Blank SLN 55.57 � 3.51 �17 � 2.0 –

DSLN 76.43 � 6.05 �24.0 � 1.0 97.22 � 2.17ADN–SLN 98.77 � 2.50 �19 � 2.0 66.0 � 5.20

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and B), whereas, in DU-145, there was 2 fold change in IC50 ofADN–SLN as compared to DTX. IC50 was found to be 0.44 mg/ml forfree DTX, 0.39 mg/ml for DSLN and 0.22 mg/ml for ADN–SLN

(Fig. 5A and B). Significantly higher cytotoxicity (p < 0.05) or lowerIC50 of ADN–SLN formulation in both the cell lines indicate thatconjugating ADN on the surface of DSLN increases the targeting

Fig. 4. In vitro cell line studies on MCF-7 cell line. (A) Results showing percentage inhibition on MCF-7 cell line challenged with different concentration of DTX, DSLN, andADN–SLN for 48 h, ADN–SLN showed significantly higher cytotoxicity then unconjugated lipidic nanoparticle or plain docetaxel drug solution. (B) IC50 obtained from aftertreating MCF-7 cells with different tested formulations and comparison of these with that obtained with competitive ligand-receptor binding assay. IC50 value got increasesdue to saturation of adenosine receptors with free ADN. (C) Quantitative cell uptake of FITC tagged formulations in MCF-7 cells (mean � SEM, n = 6). (D) Phase contrast images(FITC, DSLN, ADN–SLN) and fluorescent images (FITC’, DSLN’, ADN–SLN’) of cell uptake studies taking FITC as control, FITC tagged uncojugated SLN, FITC tagged ADN–SLNrespectively. ADN–SLN showed enhanced cellular uptake than unconjugated or control dye. (E) Annexin V binding assay by MuseTM cell analyzer after 24 h of treatment withDTX and lipidic nanoparticle formulations in MCF-7. Results explained that ADN–SLN showed better apoptosis than other tested formulations. (F) Cell cycle analysis by flowcytometer after 24 h of treatment with control (PBS), DTX solution, DSLN and ADN–SLN in MCF-7 cell line showing maximum percentage of cells were in G2/M phase whentreated with ADN–SLN. ###p < 0.001 IC50 of ADN–SLN (with free ADN) vs. ADN–SLN (without free ADN), IC50 of ADN–SLN (without free ADN) vs. DSLN (without free ADN),*p < 0.05 ADN–SLN (without free ADN) vs. DTX (without free ADN), ***p < 0.001 ADN–SLN uptake vs. SLN uptake.

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efficiency of DTX in mentioned cells, caused by the enhancedendocytosis mediated by the adenosine receptors. The significantincrease in toxicity of DSLN compared to DTX can be attributed topassive targeting of nanoparticles. Hence, these studies justified

the use of ADN as targeting ligand to target tumors over expressingadenosine receptors.

Mechanism involved in cellular uptake of ADN–SLN was studiedby competitive ligand-receptor binding assay. IC50 were

Fig. 5. In vitro cell line studies on DU-145 cell line. (A) Results showing percentage inhibition on DU-145 cell line challenged with DTX, DSLN, and ADN–SLN for 48 h, ADN–SLNshowed significantly higher cytotoxicity then unconjugated lipidic nanoparticle or plain docetaxel drug solution. (B) IC50 obtained after treating DU-145 cells with differenttest formulations. (C) Quantitative cell uptake of FITC tagged formulations in DU-145 cells (mean � SEM, n = 6). (D) Phase contrast images (FITC, DSLN, ADN–SLN) andfluorescent images (FITC’, DSLN’, ADN–SLN’) of cell uptake studies taking FITC as control, FITC tagged uncojugated SLN, FITC tagged ADN–SLN respectively. ADN–SLN showedenhanced cellular uptake than unconjugated or control dye. (E) Annexin V binding assay by MuseTM cell analyzer after 24 h of treatment with DTX and lipidic nanoparticleformulations in DU-145. Both cell cycle analysis and annexin assay results explained that ADN–SLN showed better apoptosis than other tested formulations. (F) Cell cycleanalysis by flow cytometer after 24 h of treatment with control (PBS), DTX solution, DSLN and ADN–SLN in DU-145 cell line. (*p < 0.05 ADN–SLN vs. lipidic formulation (DSLNor SLN), #p < 0.05 IC50 ADN–SLN vs. DTX.

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significantly increased (p < 0.001) after saturation of receptorswith free ADN. IC50 of ADN–SLN was found to be 0.390 � 0.011mg/ml after saturation of MCF-7 cell line with free ADN. Thecomparison of IC50 obtained is presented in Fig. 4B. Increase inIC50 was attributed to blockage of adenosine receptors by freeADN, hence hinders endocytosis of ADN–SLN. Once all thetransporters are saturated with free ligand, conjugated ADN onlipidic nanoparticles becomes ineffective. Hence, fate of ADN–SLNwill be decided on its physical properties, which are similar toDSLN. This study delineated the importance of ADN conjugation onthe surface of DSLN which aids in internalization of ADN–SLNthrough adenosine receptors in cancer cells.

3.7. Cellular uptake studies

MCF-7 and DU-145 cell lines express adenosine receptors ontheir cell surface. Targeting effect of ADN decorated lipidicnanoparticles can be evaluated by investigating the cellular uptakeof the formulations. Cellular uptakes of lipidic formulations werequalitatively and quantitatively evaluated by using ODA–FITC (inplace of drug) incorporated SLN and ADN–SLN and employingfluorescence microscopy and the measurement of fluorescenceintensity. Quantitative cellular uptakes of FITC (control marker),drug free FITC tagged unconjugated SLN and FITC tagged ADN–SLNafter 2 h were determined to be 0.31 �0.18, 8.3 � 2.07 and31.1 �4.61%, respectively in MCF-7 cells (Fig. 4C), while inDU-145, cellular uptake was found to be 0.24 � 0.057, 1.2 � 0.03and 4.2 � 0.23%, respectively (Fig. 5C). Conjugated ADN–SLNformulation showed higher uptake than unconjugated SLNformulation as shown in the fluorescent images of both thementioned cell lines (Figs. 4 and 5D). As a control, negligiblefluorescence inside MCF-7 or DU-145 cells was observed afterincubating with FITC solution, which clearly indicates the uptake ofADN coupled lipidic nanoparticles by the adenosine receptorswhich are present on current studied cancer cells. These studiesalso corroborated the higher cell cytotoxicity of ADN–SLN as foundfrom MTT cell viability assay.

3.8. Apoptosis determinations and cell cycle analysis

Apoptosis is considered to be the main cell death mechanism inresponse to taxanes. DTX targets tubulin, stabilizing microtubulesand thereby inducing cell-cycle arrest and apoptosis (Herbst andKhuri, 2003). We therefore studied these well-documented effectson cell cycle distribution and apoptosis in the tumor cells. Annexinassay takes advantage of the fact that Annexin V shows a strongand specific natural affinity for phosphatidylserine, which isusually translocated from the inner (cytoplasmic) leaflet of theplasma membrane to the outer (cell surface) leaflet soon after theinduction of apoptosis (Koopman et al., 1994). It is thereforepossible to distinguish between intact cells (stained negative forboth annexin V-FITC and propidium iodide (PI), early apoptosis(stained positive for annexin V-FITC and negative for PI), lateapoptosis or cell death (stained positive for both annexin V-FITCand PI), and necrosis (stained positive for PI). Annexin V can be

accurately estimated quantitatively by MuseTM as a very specificapoptotic marker. Apoptotic activity of formulations was estimatedin cancerous cell line MCF-7 (Fig. 4E) and DU-145 (Fig. 5E) stainedpreviously by Annexin V-FITC and PI staining, after treatment withstudy formulations. From results, it can be concluded thatADN–SLN showed significant apoptotic effect (81.85% apoptoticcells) in comparison to DSLN (58.27%), DTX (33.25%) and controlgroup (5.95%) in MCF-7 cell line (Fig. 4E) (p < 0.05). Whereas in DU-145 (Fig. 5E) too, ADN–SLN (51.40%) illustrated improved apoptoticeffect in comparison to DSLN (38.75%), DTX (22.6%) and controlgroup (7.2%) (p < 0.05). It can be illustrated from the results thatmaximum numbers of cells were in apoptosis stage indicatingbetter efficacy of the conjugated formulation over unconjugated orfree drug. This study substantiated the lower IC50 obtained in cellcytotoxicity assay.

It has also been reported that DTX acts at molecular level byimpairing mitosis and inducing cell-cycle arrest. Cell cycle analysisstudies were done to determine differences in cell cycle distribu-tion among the tested formulations (ADN–SLN, DSLN and DTXsolution). Figs. 4 and 5F depicts the cell cycle analysis results inMCF-7 and DU-145 cells respectively, for 24 h with free drug orlipidic nanoparticle formulations at IC50 values. All treatments inboth cell lines showed substantial decrease in G0/G1 phase vs.control cells and induced accumulation in G2/M and S phases. G0/G1 phase cell cycle arrested population was considerably greater incells treated with ADN–SLN than in cells treated with DSLN or freedrug, which signified better efficacy of ADN–SLN as a carrier forDTX among other tested formulations (DSLN and DTX solution).

3.9. Pharmacokinetic studies

Pharmacokinetic studies were conducted to evaluate the fate ofdrug through nanoparticles via i.v. administration. Mean plasmaconcentration-time profiles and the pharmacokinetic parametersof Taxotere1 (marketed formulation) and lipidic formulations areshown in Fig. 6 and Table 2, respectively. Compared with

Fig. 6. Graphical Illustration of mean plasma drug concentration w.r.t time. Plasmadrug concentration (mean � SD) w.r.t time after administration of differentformulations of same dose of 10 mg/kg animal weight in mice. Data suggestedthat administering lipidic formulation leads to higher plasma concentration of DTXin plasma than marketed formulation, Taxotere1. Data shown in mean � SD.

Table 2Pharmacokinetic parameters obtained after administering different DTX formulation in mice. AUC0�t got enhanced with lipidic formulation as compared to DTX marketedformulation. Similarly other parameter like T1/2 and MRT also showed marked improvement. #p < 0.05 Taxotere1 vs. ADN–SLN; *p < 0.05, ADN–SLN vs. SLN; p<0.05, SLN vs.Taxotere1,t refers final time point 12 h.

Parameters Units Taxotere1 DSLN ADN–SLN

Peak plasma concentration (Cmax) mg/ml 19.9 � 1.4# 32.5 � 2.8 36.4 � 1.9AUC(0�t) mg-h/ml 44.1 � 1.8# 68.2 � 1.5 90.4 � 1.9*

Elimination half life (T1/2) h 1.9 � 0.5 2.3 � 0.10 2.5 � 0.4Mean residence time (MRT) h 2.2 � 0.2# 3.1 � 0.1 3.5 � 0.2

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Taxotere1, DSLN and ADN–SLN resulted in a higher plasmaconcentration and longer retention time of DTX post i.v. injectionin mice. As shown in Table 2, AUCs after administration of DSLNand ADN–SLN were increased approximately 1.5 and 2.1 times thanthat of marketed formulation, respectively. These might be due tothe small total body clearance for both DSLN and ADN–SLN,indicating that DTX incorporated in lipidic nanoparticles removedslowly from circulating system. The initial plasma concentration at15 min is lower for Taxotere1 than lipidic nanoparticles following i.v. administration due to rapid distribution of the drug, which is inaccordance with previously reported data (Gulyaev et al., 1999).DTX released slowly from lipidic nanoparticle formulations forextended period of time, thus free DTX is not readily available fordistribution hence prolonged retention period in systemiccirculation. It may confer time to lipidic nanoparticle formulationsto approach site of action hence aids in active targeting.

Other adjuvant used in making SLN such as poloxamer orTween 80 help to stabilize fat emulsions and other nanoparticles,surface amended by adsorption of Tween 80 were found to havelesser uptake by phagocytes in vitro and after their i.v. injection invivo. This can reduce the uptake by the reticuloendothelial system(RES) and confer longer circulation time in blood (Alyautdin et al.,1997; Goppert and Muller, 2003). The results of the pharmacoki-netics studies suggested that the pharmacokinetic behavior oflipidic formulations were statistically significant (p < 0.05) ascompared to that of formulation in clinical use (Taxotere1).

4. Conclusions

Ligand-targeted nanoparticles have been the main impetusbehind the progress of targeted nanomedicines toward the clinic.ADN conjugated lipidic nanoparticles were designed with a view totarget the delivery of anticancer drug docetaxel to treat breast andprostate tumor. These systems were characterized for particle size,zeta potential and in vitro drug release. Preliminary efficacy studiesin representative culture cell lines MCF-7 and DU-145 indicatedenhanced cytotoxicity of doctetaxel in ADN–SLN and it wascompared with DSLN. Cellular uptake studies employing fluores-cent dye and competitive ligand-receptor binding assay corrobo-rated the receptor mediated endocytosis pathway provingreceptor's role in uptake of nanoparticles. Moreover, the encapsu-lated docetaxel maintained its complete activity and conserved itsmechanism of action, characterized by apoptosis and prematurecell cycle progression from G1 to G2-M phase. Our resultssuggested that the use of lipidic nanoparticles may allow dosesof docetaxel to be decreased without loss of therapeutic effect,thereby reducing the drug toxicity. Conjugating ADN furtherincreases the efficacy of unconjugated lipidic nanoparticles.Improved pharmacokinetic parameters substantiated the potentialof using ADN–SLN as possible drug delivery carrier for breast andprostate cancer. Further, more in vivo investigation should becarried out to determine the therapeutic efficacy of ADN as ligand.

Conflict of interest

The authors state no conflict of interest in preparation of thismanuscript.

Acknowledgements

Authors are thankful to Director, IICT and Project Director,NIPER-Hyderabad for providing required facilities, support andencouragement throughout the project. This work is supported bygrant (ADD-CSC 0302) from CSIR-IICT.

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