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Colloids and Surfaces B: Biointerfaces 112 (2013) 400– 407

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

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

valuation of clay/poly (l-lactide) microcomposites as anticancerrug, 6-mercaptopurine reservoir through in vitro cytotoxicity,xidative stress markers and in vivo pharmacokinetics

havesh D. Kevadiyaa,c,2, Shiva Shankaran Chettiarb,1, Shalini Rajkumarc,2,ari C. Bajaja,∗, Kalpeshgiri A. Gosaia, Harshad Brahmbhatta

Discipline of Inorganic Materials and Catalysis, Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), Council of Scientific and Industrialesearch (CSIR), Gijubhai Badheka Marg, Bhavnagar 364 002, Gujarat, IndiaDepartment of Biotechnology, Shree Ramkrishna Institute of Computer Education and Applied Sciences, Veer Narmad South Gujarat University, Surat,

ndiaInstitute of Science, Nirma University, Ahmedabad 382481, Gujarat, India

r t i c l e i n f o

rticle history:eceived 7 February 2013eceived in revised form 5 June 2013ccepted 3 July 2013vailable online xxx

a b s t r a c t

Intercalation of 6-mercaptopurine (6-MP), an antineoplastic drug in interlayer gallery of Na+-clay (MMT)was further entrapped in poly (l-lactide) matrix to form microcomposite spheres (MPs) in order to reducethe cell toxicity and enhance in vitro release and pharmacokinetic proficiency. The drug–clay hybridwas fabricated via intercalation by ion-exchange method to form MPs from hybrid. In vitro drug releaseshowed controlled pattern, fitted to kinetic models suggested controlled exchange and partial diffusion

eywords:a+-clayytotoxicity-Mercaptopurinexidative stress

through swollen matrix of clay inter layered gallery. The in vitro efficacy of formulated composites drugwas tested in Human neuroblastoma cell line (IMR32) by various cell cytotoxic and oxidative stressmarker indices. In vivo pharmacokinetics suggested that the intensity of formulated drug level in plasmawas within remedial borders as compared to free drug. These clay based composites therefore have greatpotential of becoming a new dosage form of 6-MP.

euroblastoma

. Introduction

Since 1950s, 6-MP has been used to cure human leukemiand many other diseases such as inflammation of colon andmall intestine e.g. Crohn’s disease and ulcerative colitis, systemicutoimmune disease and rheumatoid arthritis [1–4]. It has beenstablished that 6-MP and its metabolites exert their primary cyto-oxicity through incorporation of deoxythioguanosine into DNAhroughout anabolism pathway and inhibit the function of RANase-

in DNA–RNA heteroduplex molecules [5]. The drug is waternsoluble and the free sulfhydryl group can easily form a disulfideond with the plasma protein. The drug has a short plasma half-life0.5–1.5 h) and lower bioavailability (about 16%) and plummeting

hemotherapeutic effect [6,7]. Drug carriers proficient of sustainedelease with low toxicity are urgently required for providing andaintaining desired concentration of drug without letting it reach

∗ Corresponding author. Tel.: +91 278 2471793; fax: +91 278 2567562.E-mail addresses: scheti@gmail.com (S.S. Chettiar), shalini.rjk@nirmauni.ac.in

S. Rajkumar), hcbajaj@csmcri.org (H.C. Bajaj).1 Tel.: +91 0261 2240172; fax: +91 0261 2240170.2 Tel.: +91 2717 241900–04, 241911–15; fax: +91 2717 241916x17.

927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfb.2013.07.008

© 2013 Elsevier B.V. All rights reserved.

a higher toxic level or drop below the least efficient level for longperiod [8–15]. In order to address these issues, carriers such as lipo-somes, cement, dendrimers and nanoparticles have been typicallyexplored, as they represent excellent carriers for the integration ofhydrophobic 6-MP [3,16–19] but several limitations, such as expen-sive or conservative synthesis procedure, lack of bioavailability andbiodegradation with precursor material toxicity.

One of the possible approaches for overcoming these disad-vantages and improving the oral chemotherapeutic activity, thelayered silicate material is used as drug carriers. During the lastdecade, incorporation of anticancer drugs into a variety of lay-ered silicate materials as carriers is gaining popularity for thepreparation of controlled release dosage forms [8–11]. MMT (mont-morillonite) is one of the most commonly used medical clay thatconsists of a lamellar stack of crystalline, 1 nm thick aluminosili-cate sheets. Its crystalline lattice consists of an aluminum–oxygenand aluminumhydroxyl octahedral sheet sandwiched by twosilicon–oxygen tetrahedral sheets [10–15]. Naturally occurringcation (i.e., Na+) reside between the sheets to balance the over-

all negative surface charge of MMT. In water, the lamellar stackswells to an electrostatically stabilized dispersion of nanoscalesheets. MMT has swelling capability by the stepwise hydrationof the interlayer cations and intercalation with positively charged

faces B

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B.D. Kevadiya et al. / Colloids and Sur

iomolecules [20,21]. The ion exchange capacity of MMT enableseplacement of Na+ with other organic and inorganic cations tonsert functionality, spurring research into the use of MMT andther clay species as drug delivery and tissue regeneration agentor molecules such as docetaxel, 5-fluorouracil, paclitaxel, ibupro-en, timolol maleate, temoxifen citrate, procainamide, buspiron,nd epidermal growth factor [9–15,22–28]. For example, whenrganic modified silicate nanoparticles (cloisite clay) were addedo poly (ethylene-co-vinyl acetate) and study the release kinet-cs of dexamethasone, the authors reported increase in silicateanoparticle concentration resulting in higher mechanical strengthf the polymer nanocomposite and sustained release of dexa-ethasone [29]. Lin et al. [30] reported use of MMT with cationic

exadecyltrimethylammonium (HDTMA) and preparation of var-ous DNA–HDTMA–MMT complexes. DNA also was successfullyransfected into the nucleus of human dermal fibroblast whichxpressed enhanced green fluorescent protein (EGFP) gene withreen fluorescence emission. MMT was also investigated as a novelector for oral gene delivery by Kawase et al. [31]. The complex ofMT and plasmid DNA encoding the EGFP gene was prepared with

arious ratios. Gene expression was detected in cultured cells andn the small intestine of mice with oral administration of plasmidNA complex with MMT, while no gene expression was detected

or naked plasmid DNA. Wang et al. [32], prepared quaternizedhitosan-montmorillonite (HTCC/MMT) complex nanocompositesnd applied as protein drug carrier. Katti et al. [33] studied thentercalation mechanisms of amino acids arginine and lysine innterlayer space of montmorillonite and its mechanical behaviorsing molecular dynamics simulations. Overall, the ion exchangeature, null toxicity, biodegradation potential with polymersnd biocompatibility of clays make it a versatile carrier for 6-P.Herein we focused on the layered aluminosilicate clay, montmo-

illonite (MMT)/poly (l-lactide) microcomposite spheres (MPs) as delivery systems for 6-mercaptopurine (6-MP). 6-MP-MMT andP composites were prepared under optimal reaction conditions

y ion-exchange method and characterized. The 6-MP-MMT andP composites were evaluated for in vitro release characteristics in

imulated gastric juice and simulated phosphate buffer and releaseata was fitted to kinetic models (Higuchi, Korsmeyer–Peppas,lovich equation and parabolic diffusion). In the present study thexperiments have been designed to understand the fate of 6-MP-MT in the human neuroblastoma cell line (IMR32) by various

ytotoxic, oxidative stress markers and in vivo pharmacokineticsn rats.

. Experimental

.1. Starting materials and reagents

For the present study, 6-mercaptopurine monohydrate (6-MP),oly (l-Lactide) (PLLA) with (molecular weight = 1,52,000 Da and

nherent viscosity: 2.0 d/g), cellulose acetate dialysis tube (Cutoffolecular weight at 7014 Da) were purchased from Sigma–Aldrich,SA. RPMI-1640 (Roswell Park Memorial Institute 1640), Trypanlue, MTT (3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyl tetra-olium bromide), trypsin, streptomycin, penicillin, amphotericin,BS (fetal bovine serum), sodium dodecyl sulphate (SDS), thiobar-ituric acid (TBA), digitonin, phenazine methosulphate, nitroblueetrazolium, nicotinamide adenine, 2,4-dinitrophenyl hydrazine2,4-DNPH), guanidine hydrochloride and DMSO were procuredrom Himedia laboratory, India. HPLC grade methanol, acetoni-

rile, potassium di-hydrogen phosphate and analytical grade EDTA,ween-80, PVA (molecular weight = 1,25,000 Da) were purchasedrom S.D. Fine Chemicals Pvt. Ltd. All the other HPLC grade reagentsere used as received. Millipore water was prepared by a Milli-Q

: Biointerfaces 112 (2013) 400– 407 401

plus System (Millipore Corporation, USA). The MMT rich bentonitewas collected from Akli mines, Barmer district, Rajasthan, India,and purified as explained in our earlier report [12,13].

2.2. The 6-MP-MMT composite preparation

For preparing the 6-MP-MMT composite hybrids, 2 g MMT in100 mL methanol was vigorously stirred for 1 h. 2 g of 6-MP solu-tion (2 wt.% in methanol, pH 7.5) was added drop wise (2 mL/min)into the suspension of MMT within 1 h at room temperature usingperistaltic pump (Master flex L/S 7518-00, Cole–Parmer, USA). Themixed solution was further stirred (600 rpm) for 12 h in sealed flask,filtered, washed several times with methanol to remove the non-intercalated 6-MP, dried at 60 ◦C and ground with mortar and pestleto obtain fine powder. This sample was designated as 6-MP–MMThybrid. The remaining concentrations of 6-MP in the filtrates weremeasured by UV absorbance at �max = 323 nm using UV–visiblespectrophotometer UV 2550 (Shimadzu, Japan), equipped with aquartz cell having a path length of 1 cm. All the intercalation stud-ies were performed in triplicates and the values were averaged fordata analysis.

2.3. Preparation of 6-MP-MMT/PLLA microcomposite spheres(MPs)

The 6-MP-MMT/PLLA microcomposite spheres (MPs) were pre-pared with the oil in water (o/w) solvent evaporation method. 2 gof PLLA was dissolved in 100 mL dichloromethane, to which 6-MP-MMT hybrid (PLLA: 6-MP-MMT = 1:0.5 w/w) was added andfurther sonicated for 10 min. The organic phase was added dropwise (0.5 mL/min) into the external aqueous phase containing 0.5%w/v of polyvinyl alcohol (500 mL) with stirring till dichloromethaneevaporation. The MPs were collected using filter paper, washed 5times with Milli-Q water and re-suspended in 25 mL Milli-Q water,frozen in liquid nitrogen and lyophilized. The samples were desig-nated as MPs. Scheme 1 represents steps and mechanism involvedin preparation of intercalates and composites.

2.4. Characterization

X-ray diffraction (XRD) analysis was carried out on Phillipspowder diffractometer X’ Pert MPD using PW3123/00 curvedNi-filtered Cu-K� radiation with a scanning of 0.5◦/min in 2�range of 2–10◦. Fourier transform infrared spectra (FT-IR) wererecorded on PerkinElmer, GX-FTIR as KBr pellet in 4000–400 cm−1

range. Thermo gravimetric analysis (TGA) was carried out within30–600 ◦C at the heating rate 10 ◦C/min under nitrogen flow(20 mL/min) using by TGA/SDTA 851e, Mettler-Toledo, Switzerland.The morphology of MPs was observed by scanning electron micro-scope (SEM), LEO-1430VP, UK. The UV–visible absorbance of drugsolutions were measured at �max = 323 nm using UV–visible spec-trophotometer UV2550 (Shimadzu, Japan), equipped with a quartzcell having a path length of 1 cm.

2.5. In vitro release behavior of 6-MP

In vitro release of 6-MP was carried out with the help of USPeight stage dissolution rate test apparatus (Veego, Mumbai, India)using dialysis bag method [14]. The 6-MP release was tested insimulated gastric juice (pH 1.2) and simulated phosphate buffersolutions (pH 7.4) consisting of 1% w/v, Tween-80 at 37 ± 0.5 ◦C.Tween-80 was used to augment the solubility of 6-MP in the buffer

solution and prevent binding of 6-MP to the bowl wall. The dialysisbags were soaked in release medium for 2 h prior to release studies.The weighed amount of hybrids and MPs (equivalent to 20 mg ofentrapped 6-MP) were suspended in dialysis bag containing 5 mL

402 B.D. Kevadiya et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 400– 407

xchan

ohr1vafywp

2

2

Csof(1caapct8el

2

Mo0adtmwGot

2

Iatu

Scheme 1. Cartoon representation of drug–clay intercalates formation by ion e

f the release medium. The dialysis bags were then placed in theolding baskets and were immersed in vessel containing 500 mL ofelease medium. The revolving rate of basket was kept at 100 rpm.

mL aliquots were withdrawn at regular time interval and the sameolume was restored with fresh release medium. Samples werenalyzed by RP-HPLC. These studies were performed in triplicatesor each sample and the average values were used in data anal-sis. The release behaviors of the drug from the 6-MP-MMT/MPsere fitted in Higuchi, Korsmeyer–Peppas, Elovich equation andarabolic diffusion kinetic models [12,15].

.6. Cell culture assays

.6.1. Cell cultureHuman neuroblastoma cell line (IMR32) procured from National

entre for Cell Science (NCCS), India, was used in the presenttudy. IMR32 has been reported to be sensitive to a broad rangef anticancer drugs [34] and this characteristic makes it suitableor our study. The cells were grown in 12 well cell culture plateTarson–980020, 12 well plates, Tarson India) in RPMI 1640 in

mM of non-essential amino acid supplemented with 10% fetalalf serum (FBS), 100 �g/mL of streptomycin sulfate, 0.25 �g/mL ofmphotericin, 100 units/mL of penicillin incubated under 5% CO2t 37 ◦C (N-Biotek NB203XL, Korea). Cells were trypsinized fromarent stock of 5th passage and subjected to viability and totalount. Cells were re-suspended in fresh medium to make dilu-ion of 0.5 × 103 cells/well. Cells were grown to achieve more than0% confluence (which was achieved normally by 72 h) and werexposed to pristine drug, carrier materials and drug loaded formu-ations along with media change for a duration of 24 h.

.6.2. Cell culture and drug-microcomposite exposureThe MMT was suspended in Milli-Q water and 6-MP, 6-MP-

MT, MPs and PLLA were dissolved/suspended in DMSO. A stockf 1 mg/ml was prepared and sterilized by syringe filtration with.22 �m pore size filter (Whattmann filters). Cultures as mentionedbove were exposed for duration of 24 h with 10 ppm equivalentrug concentration of each test composite. Concentration and dura-ion of the test formulations were selected based on the preliminary

etabolic stress indices monitored by MTT assay. Culture wellsere designated as one to six groups like, G1 = control, G2 = MMT,3 = PLLA, G4 = 6MP-MMT, G6 = MPs and two wells were run withnly media and another with media and test formulations as con-rols to monitor contamination from either source.

.6.3. Cell viability assayCell viability was assessed by 0.1% trypan blue dye exclusion.

n all experiments, the medium was gently removed from the wellfter 24 h of exposure to test compounds and washed with PBSo remove residual compounds. The number of cells was countedsing a light microscope (Unilab-XS, India) on a hemocytometer.

ge mechanism and further entrapment in PLLA matrix by solvent evaporation.

Cell viability was expressed as the percentage of the cell numberaccording to the following formula:

Cell viability (%) = (Live cells/Total number of cells) × 100

2.6.4. MTT assay for cell cytotoxicityMedia from control and experimental cultures exposed to pris-

tine 6-MP, MMT, PLLA, 6-MP-MMT and MPs was aspired out andwashed with sterile PBS couple of times to remove any traces oftest composites in culture. 100 �l of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye (5 mg/ml PBS) wasadded to each well and further incubated for 4 h under similarconditions [35]. After incubation the cultures were gently shakenand media with excess non reacted dye was removed and 1 mlof DMSO was added to the cells, so as to solubilize the coloredformazan formed. The absorbance was measured using spectropho-tometer (Systronics-2201, Digital spectrophotometer, India) at�max = 490 nm against DMSO as blank. The percentage growth inhi-bition was calculated using the formula below:

Cell inhibition (%) = 100 − [(At − Ab)/(Ac − Ab)] × 100

where, At = absorbance value of test formulations, Ab = absorbancevalue of blank and Ac = absorbance value of control.

2.6.5. Thiobarbituric acid reactive substance (TBARS) assayThe extent of lipid peroxidation was evaluated by measuring

malonyl di-aldehyde as 2-thiobarbituric acid-reactive substances(TBARS). The measurement of TBARS was estimated from the cul-tures by modified method of Ohkawa et al. (1979) [36]. Briefly,cultures were subjected to trypsinization and cells were lysed with8.0% of sodium dodecyl sulphate (SDS) and allowed to react with1% thiobarbituric acid (TBA) in presence of 20% acetic acid solu-tion. The solution was mixed and heated in water bath at 95 ◦C for60 min and cooled followed by addition of 10% trichloroacetic acid(TCA). This solution was mixed and centrifuged at 1000 rpm for15 min and aliquot of the complex formed with 2-thiobarbituricacid was read against blank on Systronics Digital Spectropho-tometer at �max = 523 nm. Lipid peroxide level was expressed asnanomoles of TBARS/1000 cells.

2.6.6. SOD activity assayThe activity of superoxide dismutase was assayed by the mod-

ified spectrophotometrical method of Kakkar et al. (1984) [37].The cells after trypsinization were subjected to 0.01% digitonintreatment to yield the enzyme extract. Enzyme fraction was iso-lated by centrifuging at 3000 rpm for 15 min and the supernatantwas mixed with phosphate buffer, phenazine methosulphate(186 �M), nitroblue tetrazolium (300 �M) and nicotinamide ade-

nine (780 �M). The reaction was terminated precisely at 90 s withaddition of acetic acid and formazan was extracted in n-butanol.The tubes were centrifuged and upper phase with dissolved form-azan were measured for optical density at �max = 560 nm. The

faces B: Biointerfaces 112 (2013) 400– 407 403

at

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2

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2ZtUaotlmatrrMu6g(uhf2vad(sTt

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100

200

300

400

6 MP-MM T 2θ = 6.4 °d =1.4 nm

MMT 2θ = 7.3 °d =1.20 n m

Inte

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2θ

B.D. Kevadiya et al. / Colloids and Sur

ctivity of the enzyme was expressed as units SOD/mg pro-ein.

.6.7. GSH assayThe cell supernatant after cell lysis was utilized for GSH assay,

here GSH reacts with Ellmans reagent to give formazan [38].he supernatant was precipitated with 0.1% TCA and subjected toentrifugation. The supernatant thus obtained was treated withllmans reagent (19.8 mg of 5, 5′-dithiobis-2-nitrobenzoic acid in00 ml of 1% sodium citrate) and incubated for 30 min at 37 ◦C.bsorbance was measured at �max = 412 nm with standards treated

n similar way to make a linear plot. Absorbance of unknown sam-le was plotted against concentration with respect to the standardnd glutathione levels were expressed as �g/mg protein.

.6.8. Protein carbonyl assayThe protein carbonyl content was measured according to the

ethod described by Levine et al. (2000) [39]. Protein carbonylerivatives readily reacted with 2,4-dinitrophenylhydrazine (2,4-NPH) to form hydrazone derivatives that could be measured

pectrophotometrically. The 150 �L supernatant of assaying SODas reacted with 600 �L of 10 mM DNPH (in 2.5 M HCl) for

h with occasional mixing and TCA precipitates were washedhree times with 1 mL of ethanol/ethyl acetate (1:1). The pel-ets were broken up mechanically, dissolved in 1 mL of 6 Muanidine hydrochloride and 0.5 M potassium phosphate (pH 2.5)nd the absorbance was measured at A370. A blank with therotein reacted with 2 M HCl containing no DNPH was keptor each sample and its absorbance was subtracted from thenal reading. The carbonyl content was determined as nmol/mgrotein.

.7. In vivo pharmacokinetics (PK)

.7.1. Animals and dosingTen- to twelve-week-old healthy female wistar rats weighing

00–250 g were procured from the Laboratory Animals Center ofydus Research Center (ZRC), Ahmedabad, India, and were main-ained at the Animal Holding Unit of Institute of Pharmacy, Nirmaniversity. The animal caring, handling and the protocols werepproved by the Institutional Animal Ethics Committee (IAEC)f the department. The animals were acclimatized at tempera-ure of 25 ± 2 ◦C and relative humidity of 50–60% under 12 h/12 hight/dark conditions for one week before experiments. All ani-

als were fasted for 24 h before the studies and water was madevailable ad libitum. The animals were arbitrarily distributed intohree groups each containing six animals. First group of animalseceived oral pristine 6-MP, while the second group of animalseceived 6-MP-MMT hybrid (suspension) and third group receivedPs (suspension). All the formulations were administered orally

sing a feeding tube attached to a hypodermic syringe at a dose of-MP (50 mg/kg) body weight. All animals were observed for theireneral condition, clinical signs and mortality. The blood samplesapproximately 0.3 ml) were collected from the retro orbital plexusnder mild anesthesia into the micro-centrifuge tubes containingeparin (500 units/ml blood) as an anticoagulant. The time breaks

or blood collection were kept at 0 (pre dose), 1, 3, 6, 9, 12 and4 h after administration of the drug. Plasma samples were har-ested by centrifugation (Kubota-6500, Kubota Corporation, Japan)t 10,000 rpm for 15 min at 5 ◦C. The plasma samples, 200 �l, wereeproteinized with 500 �l of methanol and acetonitrile mixture

1:1, v/v %), mix for 5 min, centrifuged at 12,000 rpm for 15 min andupernatants were collected and stored at −20 ◦C for HPLC analysis.he pharmacokinetic parameters assayed were (a) total area underhe curve (AUC)0–∞, (b) the mean residence time (MRT), (c) peak

Fig. 1. XRD patterns of pristine MMT and 6-MP-MMT hybrid.

plasma concentration (Cmax) and (d) time to reach the maximumplasma concentration (Tmax).

2.8. The 6-MP quantification by HPLC

The quantification of 6-MP from release media and plasma wasdetermined by using a validated HPLC method [40]. In short, subse-quent to the preparation of samples, analysis by high-performanceliquid chromatography (HPLC) system consisting of photodiodearray detector (Waters Alliance model: 2695 separation modulewith Waters 2996 Photo diode Array Detector, Waters Corpora-tion, Milford, MA, USA) and a reverse-phase C18 column (WatersXBridgeTM C18 HPLC column with length = 100 mm, ID = 2.1 mm,particle size = 5.0 �m, Waters Corporation, Milford, MA, USA) wascarried out. 6-MP containing samples were transferred to auto sam-pler vials, capped and placed in cassettes of the HPLC autosampler.Mobile phase employed for the analysis was the mixture of 0.01 MKH2PO4 buffer and acetonitrile (80:20% v/v). The injection volumewas 10 �l with flow rate of 0.2 ml/min and the detection wave-length (�max) for 6-MP was 325 nm. Drug concentration in sampleswas determined using the standard curve obtained from knownconcentrations of 6-MP in release medium/plasma processed sim-ilarly.

3. Results and discussion

3.1. Confirmation of intercalation of6-mercaptopurine-montmorillonite

3.1.1. X-ray diffractionFig. 1 shows the XRD patterns of the starting untreated clay

and clay after treatments with 6-MP in methanol. Pristine MMTshowed a typical XRD pattern with the basal spacing of 1.20 nm(2� = 7.3◦) and intercalation of 6-MP which lead to a significantincrease in interlayer space and decrease of 2� values of MMT(1.4 nm at 2� = 6.4◦). The increased basal spacing after the reactionof 6-MP with MMT was evidence of 6-MP intercalation into MMT.However, the intensity of the XRD characteristic peak increased in

6-MP-MMT, which indicated an impervious ordering of sheet struc-ture caused by the cation exchange and the higher restoration ofcharge density during drug loading.

404 B.D. Kevadiya et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 400– 407

P-MM

3

cdaws

3

s1et∼w[FawwaTdtwt

atwt

F

Fig. 2. SEM images of (A) pristine MMT (B) 6-M

.1.2. IR spectroscopyThe FT-IR spectra of the 6-MP loaded clay hybrids and MPs were

ompared with that of the pristine clay and drug (Supplementaryata; Fig. S1). The spectrum of MMT revealed the characteristicbsorption bands [12–14]. The characteristic purine ring bandsere observed between 770 and 490 cm−1 in 6-MP-MMT/6-MPs

pectra which confirmed intercalation of 6-MP into MMT.

.1.3. Thermal analysis and SEM studyTGA patterns of dried MMT (Supplementary data; Fig. S2)

howed weight loss in three steps at the temperatures around00 ◦C, 270 ◦C and 430 ◦C. We could trace one strong and two weakndothermic peaks on the DTA pattern of pristine MMT at the sameemperature range. The first weight loss and endothermic peak at100 ◦C in MMT corresponded to the loss of adsorbed water. Theeight loss at 430 ◦C was due to the dehydroxylation of the MMT

9]. The TGA curves of 6-MP-MMT and MPs (Supplementary data;ig. S2) showed three steps for weight loss at the temperatureround 140 ◦C, 300–320 ◦C and 450–500 ◦C. One strong and twoeak endothermic peaks were observed in DTA patterns. The firsteight loss and the strong endothermic peak at the temperature

round 140 ◦C were due to the free water evaporation from MMT.he second weight loss at the temperature around 300–320 ◦C wasue to the removal of the 6-MP from intercalated MMT and disin-egration of PLLA structure. The third endothermic peak was due toeight loss at the temperature around 450–500 ◦C, corresponding

o the complete degradation of 6-MP/PLLA from 6-MP-MMT/MPs.The SEM images of the pristine MMT, 6-MP-MMT and MPs

re shown in Fig. 2, where MMT (Fig. 2A) exhibits layered struc-ure with platelet morphology consisting of stacked silicate sheetshich are approximately 1 nm thick and 200 nm long. Fig. 2B shows

he surface morphology of 6-MP-MMT hybrids with slight swelling

0.0 0.5 1. 0 1.5 2.0 2.5 3.0

0

3

6

9

12

15

6-MP-MM T

MP s

Time ( h )

6-M

P R

elea

se (

%)

[a]

ig. 3. In vitro release behaviors of 6-MP in (a) simulated gastric fluid (pH 1.2) and (b) sim

T hybrid (C) microcomposites spheres (MPs).

of the layered matrix structure with clear drug deposition seen ininterlayer gallery of MMT. The SEM images (Fig. 2C) show MPs assolid spheres. This confirmed the advantage of using MMT overPLLA matrices which are fragile and exhibit uncontrollable solidity.

3.2. In vitro release study

The release patterns of drug from 6-MP-MMT hybrids and MPs inbuffer solutions with two different pH 1.2 and pH 7.4 were carriedout at physiological temperature by dialysis bag method (Fig. 3aand b). Approximately 15% of the intercalated 6-MP was releasedfrom 6-MP-MMT and ∼12% from MPs within 3 h in the gastric envi-ronment (Fig. 3a). At pH 7.4, the formulation exhibited controlledrelease profile up to 68 h (Fig. 3b). The 6-MP release from MPs hadcontrolled pattern with ∼22% of drug released in 10 h followed bysustained release >68 h (52%). No initial burst was observed fromthe MPs. The initial release of the drug from 6-MP-MMT hybridwas somewhat faster compared to that from MPs, where ∼36% ofthe intercalated drug was released in 10 h and ∼52% the drug wasreleased in 68 h. The prolonged delay in drug release from MPs canbe explained on the basis of the differences in the distribution of6-MP-MMT in hybrid plates within the PLLA matrix and wrappingof the 6-MP-MMT hybrid plates by PLLA. This was probably due tolow permeability of the water in interior of MPs due to PLLA. Theabrupt release must not be considered negative in all cases as inthe later phase, the 6-MP release from 6-MP-MMT hybrid was con-trolled and the rate was determined by the de-intercalation of drugfrom the clay plates.

To understand the release mechanism of drug moleculesfrom the 6-MP-MMT and MP carriers better, the Higuchi,Korsmeyer–Peppas, Elovich equation and parabolic diffusionkinetic models were employed (Table 1 and Supplementary data

0 10 20 30 40 50 60 70

0

10

20

30

40

50

6- MP-MMT

MP s

Time ( h )

6-M

P R

elea

se (

%)

[b]

ulated intestinal fluid (pH 7.4) at 37 ± 0.5 ◦C; data represent mean ± SD (n = 3).

B.D. Kevadiya et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 400– 407 405

Table 1Linear correlation coefficient (r2) and rate constant (K) of the diffusion kinetic models applied to 6-MP release from 6-MP-MMT and MPs (pH 7.4, data considered from first15 h of release experiments) .

Kinetic models Parameters Formulations

pH 1.2 pH 7.4

6-MP-MMT MPs 6-MP-MMT MPs

Higuchi equation r2 0.964 0.893 0.988 0.993KH 9.12 7.01 11.15 7.581

Korsmeyer–Peppas r2 0.991 0.975 0.989 0.996n 0.721 0.875 0.471 0.611KHP 0.461 0.353 0.238 0.106

Elovich equation r2 0.946 0.867 0.978 0.965KE 2.882 2.430 5.325 3.943

Parabolic diffusion r2 0.964 0.893 0.988 0.993

Flkdm

3

t

Fi

Kp 0.592

ig. S3 (a–d) from pH 1.2 and Fig. S4 (a–d) from pH 7.4). The bestinearity was obtained in the all model (r2 = 0.965 − 0.996). Thus, theinetics of 6-MP release in intestinal environment was governed byiffusion controlled exchange and partial diffusion through swollenatrix of the MMT/MPs.

.3. Cell culture experiments

In experiment to understand the fate of 6 MP-MMT and MPs inhe cell lines was determined. We followed cytotoxic and oxidative

ig. 4. In vitro cell cytotoxic and oxidative stress marker indices: (A) percentage growth in control and groups exposed to test formulations data represent mean ± SD (n = 6).

0.547 0.214 0.145

stress markers to report the efficacy of drug intercalation in reduc-ing the toxic effects of the drug without affecting the activity ofdrug against cancer cells, which was attained by controlled releaseof the 6-MP. All groups of cells were exposed for duration of 24 hto the drug formulations and pristine compounds.

3.4. Cell count, viability and MTT assay

Cultures exposed to 6-MP alone showed significant reductionin cell count (1.5 × 103) and viability of (76%), while, 6-MP-MMT

nhibition (C) thiobarbituric acid reactive species (D) superoxide dismutase activity

406 B.D. Kevadiya et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 400– 407

0 5 10 15 20 25

0

20

40

60

80

100

120

6- MP

6- MP-MMT

MPs

Time (h)

6-M

P C

onc

in p

lasm

a (n

g/m

l)

Fag

acTitictt2itiMecta

3

moMcacopepMcewFdciI

Table 2Pharmacokinetics of pristine 6-MP, 6-MP-MMT hybrids and MPs in wistar rats aftersingle oral administration of same drug dose; data represent mean ± SD, (n = 6).

PK parameter 6-MP 6-MP-MMT MPs

Cmax (ng/ml) 105 ± 11.15 97.2 ± 12.04 92.0 ± 8.10Tmax (h) 1 1 6

ig. 5. In vivo pharmacokinetics study of 6-MP-MMT and MPs in female wistar ratsnd compared to pristine 6-MP, results are shown as means ± SD of six animals perroup (p < 0.05).

nd MPs showed enhancement in viability (88% and 82%) as well asell count (2.1 × 103 and 2.8 × 103) (Supplementary data; Fig. S5A).he cultures with pristine MMT/PLLA treatment showed no signif-cant decrease in count and viability and interestingly the culturesreated with 6-MP-MMT/MPs also showed similar results, imply-ng reduced effect of drug on cells. However, 6-MP exposure to cellultures significantly inhibited (49.0%) cell growth as comparedo pristine MMT (16.79%) (Fig. 4A) which it indicated that pris-ine 6-MP had toxic effect on cells. The cell growth inhibition was2.3% and 11.5% respectively in 6-MP-MMT and MP exposed cells

ndicated that MMT provided significant protective and preserva-ive role. The cell viability and MTT studies revealed that theres augmentation in cell death on exposure of 6-MP, while 6-MP-

MT/MPs increased viability and reduced cytotoxicity. MTT assayndowed as excellent representation of total metabolic status ofell, as it measures total dehydrogenase activity in cell. Thus reduc-ion in formazan production signified metabolic stress induced by

compound which attributed to cytotoxicity in cell.

.5. Oxidative stress indices

Oxidative stress markers like lipid peroxide, super oxide dis-utase, Glutathione-SH and protein carbonyl were measured to

verview the metabolic stress in cells due to exposure of 6-MP, 6-P-MMT and MPs. The LPO levels in 6-MP-MMT and MPs exposed

ells were significantly reduced (0.843 and 0.817 nmol/1000 cells)s compared to pristine 6-MP treated cells (1.329 nmol/1000ells) (Fig. 4B). SOD activity was similar in 6-MP-MMT (3.97 U/mgf protein) and MPs (3.85 U/mg of protein) as compared to theristine 6-MP (3.67 U/mg of protein) treated cells (Fig. 4C). How-ver, elevation in protein carbonyl was expressed (8.12 nmol/mgrotein) in pristine 6-MP treated cells while, 6-MP-MMT andPs proved to be somewhat less toxic in production of protein

arbonyl (Supplementary data; Fig. S5B). Intracellular glutathionestimation also revealed similar results that 6-MP-MMT and MPsere less toxic to cells than pristine drug (Supplementary data;

ig. S5C). Anticancer drugs are known to be responsible for cell

eath mediated by several pathways, but all of them share oneommon physiological characteristic of elevated oxidative stressn all type of cells which is of major concern in cancer therapy.t has been documented in several reports that anticancer drugs

AUC 0-∞ (ng h/ml) 443.17 461.83 419.61MRT (h) 3.7 3.9 6.5

enhance oxidative stress in targeted and normal cells [41–44]. Bythis experiment we have attempted to reduce toxicity of 6-MP byintercalating it in MMT and further entrapments with PLLA whileallowing controlled release of drug in the system.

3.6. In vivo pharmacokinetics (PK)

Fig. 5 shows the in vivo pharmacokinetics, i.e., the drugconcentration in plasma with respect to time after single oraladministration of pristine 6-MP, 6-MP-MMT and MPs to femalewistar rats at the concentration of 6-MP (50 mg/kg) (n = 6). Thekey PK parameters were analyzed and the results are listed inTable 2, which include Cmax (in ng/ml) and Tmax (h) – the maximumdrug concentration encountered after the drug administration andthe time at which Cmax is reached, MRT (h) – the mean residencetime of the drug in the plasma and AUC0–∞ (ng h/ml) – the totalarea under the curve that represents the in vivo therapeutic effectsof drug. A few significant advantages of the 6-MP-MMT and MPscould be concluded from PK data. The oral administration of pris-tine 6-MP caused high peak value of the drug concentration (Cmax)(∼105 ng/ml) with Tmax = 1 h. When drug was captured inside thegallery of MMT and entrapped in PLLA matrix prior to oral adminis-tration to rats, the drug concentrations could be detected in plasmaup to ∼24 h. Upon comparing the MRT of pristine 6-MP and 6-MP-MMT, it was observed that the MPs increased the residence time ofthe drug in the plasma by ∼6.5 h. The MPs significantly reduced AUC0–∞ values and Cmax values of drug release while improving MRT ofdrug in plasma which might be responsible for reduced drug toxic-ity as compared to 6-MP/6-MP-MMT. Thus, oral administration of6-MP-MMT and MPs in rats leads to significant reduction in drugtoxicity.

4. Conclusion

We have successfully intercalated 6-MP into MMT gallerieswhich was further entrapped in PLLA matrix to form MPs fororal drug delivery system by ion exchange and solvent evapo-ration methods. The XRD patterns, TGA/DTA and FT-IR analysesindicated intercalation of 6-MP into the MMT interlayer throughelectrostatic interaction. No significant change in structural andfunctional properties of 6-MP was observed in the MMT layers. Theintercalates (6-MP-MMT) and microcomposites (MPs) had superiordrug entrapment potential and showed controlled release pat-terns ∼70 h in intestinal environment. The kinetic data showeddrug release from clay/PLLA by partial diffusion through swollenmatrix/de-intercalation of layers of carriers to its individual com-ponents or nanostructures of different compositions. In vitro cellcytotoxic and oxidative stress marker indices (lipid peroxide, superoxide dismutase, glutathione-SH and protein carbonyl) were sig-nificantly reduced in 6-MP-MMT and MPs treated cells with time.In vivo pharmacokinetics of formulated drug in plasma was withinremedial limits as compared to pristine drug. We believe this

experimental work will be valuable for the drawing of commercialattention to layered nanomaterials where intercalation of anti-cancer drug in the layered nanostructures could lead to prolongeddrug release with null toxicity.

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B.D. Kevadiya et al. / Colloids and Sur

cknowledgments

Authors are thankful to Directors, CSMCRI, Bhavnagar, andnstitute of Science, Nirma University, Ahmedabad, for providingecessary infrastructure facilities and the Council of Scientific and

ndustrial Research (CSIR), Government of India, New Delhi, India,or financial support under the project “Speciality Materials basedn Engineered Clays” (SPEC, CSC-0135) and Senior Research Fellow-hip to Mr. B.D. Kevadiya. Authors are also thankful for help ando-operation rendered by Mr. V. Agarwal (FT-IR), Mr. J.C. Chaud-ari (SEM) and Mrs. S. Patel (TGA) of the central analytical facilityf CSMCRI.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.013.07.008

eferences

[1] L.W. Law, Proc. Soc. Exp. Biol. Med. 84 (1953) 409.[2] J.J. Coffey, C.A. White, A.B. Lesk, W.I. Rogers, A.A. Serpick, Cancer Res. 32 (1972)

1283.[3] P. Podsiadlo, V.A. Sinani, J.H. Bahng, N.W.S. Kam, J. Lee, N.A. Kotov, Langmuir

24 (2008) 568.[4] O.H. Nielsen, B. Vainer, Aliment Pharmacol. Ther. 15 (2001) 1699.[5] J.A. Nelson, J.W. Carpenter, M.L. Rose, D.J. Adamson, Cancer Res. 35 (1975) 2872.[6] M. Zacchigna, F. Cateni, G. Di-Luca, S. Drioli, Bioorg. Med. Chem. Lett. 17 (2007)

6607.[7] M.H. Cheok, W.E. Evans, Nat. Rev. Cancer 6 (2006) 117.[8] S.J. Choi, G.E. Choi, J.M. Oh, Y.J. Oh, M.C. Park, J.H. Choy, J. Mater. Chem. 20 (2010)

9463.[9] S.S. Feng, L. Mei, P. Anitha, C.W. Gan, W. Zhou, Biomaterials 30 (2009) 3297.10] B.D. Kevadiya, T.A. Patel, D.D. Jhala, R.P. Thumbar, H. Brahmbhatt, M.P. Pandya,

S. Rajkumar, G.V. Joshi, P.K. Gadhia, C.B. Tripathi, H.C. Bajaj, Eur. J. Pharm. Bio-pharm. 81 (2012) 91.

11] Y. Dong, S.S. Feng, Biomaterials 26 (2005) 6068.12] B.D. Kevadiya, G.V. Joshi, H.C. Bajaj, Int. J. Pharm. 388 (2010) 280.13] G.V. Joshi, H.A. Patel, B.D. Kevadiya, H.C. Bajaj, Appl. Clay Sci. 45 (2009) 248.

[

[[

: Biointerfaces 112 (2013) 400– 407 407

14] B.D. Kevadiya, G.V. Joshi, H.M. Mody, H.C. Bajaj, Appl. Clay Sci. 52 (2011) 364.15] G.V. Joshi, B.D. Kevadiya, H.C. Bajaj, Microporous Mesoporous Mater. 132 (2010)

526.16] M. Umrethia, P.K. Ghosh, R. Majithya, R.S. Murthy, Cancer Invest. 25 (2007) 117.17] M. Otsuka, Y. Matsuda, Y. Suwa, J.L. Fox, W.I. Higuchi, J. Pharm. Sci. 83 (1994)

1565.18] V. Agrawal, M.K. Paul, A.K. Mukhopadhyay, J. Liposome Res. 15 (2005) 141.19] J.B. Wolinsky, M.W. Grinstaff, Adv. Drug Deliv. Rev. 60 (2008) 1037.20] J.D.F. Ramsay, S.W. Swanton, J.J. Bunce, J. Chem. Soc. Faraday Trans. 86 (1999)

3919.21] T. Szabo, R. Mitea, H. Leeman, G.S. Premachandra, C.T. Johnston, M. Szekeres, I.

Dekany, R.A. Schoonheydt, Clays Clay Miner. 56 (2008) 494.22] G.V. Joshi, R.R. Pawar, B.D. Kevadiya, H.C. Bajaj, Microporous Mesoporous Mater.

142 (2011) 542.23] G.V. Joshi, B.D. Kevadiya, H.M. Mody, H.C. Bajaj, J. Polym. Sci. Part A: Polym.

Chem. 50 (2012) 423.24] G.V. Joshi, B.D. Kevadiya, H.A. Patel, H.C. Bajaj, R.V. Jasra, Int. J. Pharm. 374 (2009)

53.25] B.D. Kevadiya, R.P. Thumbar, M.M. Rajput, S. Rajkumar, H. Brambhatt, G.V. Joshi,

G.P. Dangi, H.M. Mody, P.K. Gadhia, H.C. Bajaj, Eur. J. Pharm. Sci. 47 (2012) 265.26] B. Sun, B. Ranganathan, S.S. Feng, Biomaterials 29 (2008) 475.27] D. Depan, A.P. Kumar, R.P. Singh, Acta Biomater. 5 (2009) 93.28] C.A. Vaiana, M.K. Leonard, L.F. Drummy, K.M. Singh, A. Bubulya, R.A. Vaia, R.R.

Naik, M.P. Kadakia, Biomacromolecules 12 (2011) 3139.29] S.H. Cypes, W.M. Saltzman, E.P. Giannelis, J. Control. Release 90 (2003)

163.30] F.H. Lin, C.H. Chen, W.T.K. Cheng, T.F. Kuo, Biomaterials 27 (2006) 3333.31] M. Kawase, Y. Hayashi, F. Kinoshita, E. Yamato, J.I. Miyazaki, J. Yamakawa, T.

Ishida, M. Tamura, K. Yagi, Biol. Pharm. Bull. 27 (2004) 2049.32] X. Wang, Y. Du, J. Luo, Nanotechnology 19 (2008) 065707.33] D.R. Katti, P. Ghosh, S. Schmidt, K.S. Katti, Biomacromolecules 6 (2005) 3276.34] S. Karin, L. Christer, BMC Cancer 3 (10) (2003) 1471.35] T. Mosmann, J. Immunol. Methods 65 (1983) 55.36] H. Ohkawa, N. Ohishi, K. Yagi, Anal. Biochem. 95 (1979) 351.37] P. Kakkar, B. Das, P.N. Viswanathan, Indian J. Biochem. Biophys. 4 (1984) 130.38] J. Sedlak, R.H. Lindsay, Anal. Biochem. 25 (1968) 192.39] R.L. Levine, N. Wehr, J.A. Williams, E.R. Stadtman, E. Shacter, Methods Mol. Biol.

99 (2000) 15.40] M.L. Umrethia, P.K. Ghosh, R.J. Majithiya, R.S.R. Murthy, J. Liq. Chromatogr. Relat.

Technol. 29 (2006) 55.41] M.B. Wolf, J.W. Baynes, Biochim. Biophys. Acta 1760 (2006) 267.

42] J. Alexandre, C. Nicco, C. Chereau, Laurent Alexis, B. Weill, F. Goldwasser, F.

Batteux, J. Natl. Cancer Inst. 98 (2006) 236.43] K.A. Conklin, Integr. Cancer Ther. 3 (2004) 294.44] Y. Sun, L. Huang, G.G. Mackenzie, B. Rigas, J. Pharmacol. Exp. Ther. 338 (2011)

775.