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Colloids and Surfaces B: Biointerfaces 109 (2013) 10– 19

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

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

elative efficacies of insulin and poly (lactic-co-glycolic) acidncapsulated nano-insulin in modulating certain significantiomarkers in arsenic intoxicated L6 cells

smita Samadder, Sreemanti Das, Jayeeta Das, Anisur Rahman Khuda-Bukhsh ∗

ytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India

a r t i c l e i n f o

rticle history:eceived 7 November 2012eceived in revised form 12 March 2013ccepted 21 March 2013vailable online xxx

eywords:rsenic

nsulin

a b s t r a c t

The present study evaluates relative efficacies of insulin and PLGA-loaded-nano-insulin (NIn) in combat-ing arsenic-induced impairment of glucose uptake, insulin resistance and mitochondrial dysfunction inL6 skeletal muscle cells. L6 cells were treated with 0.5 mM sodium arsenite for 30 min and then the cellswere further treated with either 100 nM insulin (standardized dose) or either of the two doses, 50 nM and100 nM of nano-insulin. Various biomarkers like pyruvate-kinase and glucokinase, ATP/ADP ratio, mito-chondrial membrane potential, cytosolic release of mitochondrial cytochrome c, cell membrane potentialand calcium-ion level were studied and analyzed to ascertain the status of mitochondrial functioning inall experimental and control sets of L6 cells. The size, morphology and zeta potential of formulated NIn

LGA encapsulationitochondrial signalling

6 skeletal muscle cells

were determined by using dynamic light scattering, scanning electronic and atomic-force microscopies.Expression of signalling cascades like GLUT4, IRS1, IRS2, UCP2, PI3, and p38 was critically analyzed. Overallresults suggested that both insulin and NIn improved mitochondrial functioning in arsenite-intoxicatedL6 cells, NIn showing better effects at a much lower dose (at nearly 10-fold decreased dose) than thatproduced by insulin. Nano-insulin being non-toxic and effective at a much lesser dose, therefore, haspotential for therapeutic use in the management of arsenic induced diabetes.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

Arsenic-exposure through contaminated drinking water haseen suggested to play an etiologic role in the development of var-

ous dreadful diseases. Increasing risk of development of diabetesnd its associated complications in high arsenic areas of Taiwannd Bangladesh [1] have become a serious issue of mankind. Theossibility of an association between chronic arsenic exposure andiabetes has great implications warranting extensive research as it

nvolves public health to a great extent.In general, diabetes onsets with a mark of impairment of glu-

ose tolerance caused by dysfunction of pancreatic � cells thatometimes produce severe symptoms needing immediate medicalttention. It is believed that the initial change in diabetes melli-

us (DM) leads to decreased production of insulin hormone and/orncreased resistance to insulin action in the peripheral tissues (par-icularly skeletal muscle and adipose tissue) [2]. The incidence of

∗ Corresponding author at: Department of Zoology, University of Kalyani, Kalyani41235, W.B., India. Tel.: +91 33 25828750x315.

E-mail addresses: prof arkb@yahoo.co.in, khudabukhsh 48@rediffmail.comA.R. Khuda-Bukhsh).

927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfb.2013.03.028

diabetes and insulin resistance is known to increase with age [3].Mitochondrial dysfunction has also been correlated with increasein age [4]. Since diabetes is a metabolic problem recent researchershave focussed their work on mitochondria, the major site of fuelmetabolism in the body.

Skeletal muscles, portraying various signalling events involvedin the occurrence of diabetes, are being widely used as one of thebest models to study diabetes [5–7]. They are the predominant sitefor disposal of glucose and fatty acids. Thus, skeletal muscles playa key role in whole body metabolism, thereby contributing to glu-cose homeostasis. They are capable of expressing the kinetics ofglucose transporters. Insulin resistance in skeletal muscles has alsobeen well established as starting events of diabetes [8]; however,the underlying causal relationship between muscle mitochondrialdysfunction and insulin resistance, if any, still remains to be under-stood.

The potential bio-molecules involved in arsenic induced dia-betes include phosphorous substitution, high affinity for sulphydrylgroups forming covalent bonds with the disulfide bridges in

the molecules of insulin, insulin receptors, glucose transporters(GLUTs), increased oxidative stress and interference with geneexpression [9]. Arsenic has been shown to alter the expressionsof genes related to the maintenance of glucose homeostasis, alter

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he adenosine tri-phosphate/adenosine di-phosphate (ATP/ADP)alance ratio leading to disruption of membrane polarity of mito-hondria, impairment of influx of calcium ions dependent anduel-stimulated (ATP-dependent) insulin secretion [9,10]. There-ore, studies associated with the glucose uptake capability bypecific cells/tissues have been the focus of many researchers tostablish the level of occurrence/prevalence of diabetes.

In recent years, several approaches have been made by utilizinganotechnology as a basic as well as applied science tool [11,12] inherapeutic applications. It is also necessary to ensure that nano-articles focusing on possible enhancement of drug bioavailabilityhould be less toxic to target organism or organ system. Poly (lactic-o-glycolic) acid (PLGA) polymers have gained attention for thereparation of a biodegradable and biocompatible non-toxic andfficient carrier system for the delivery of drugs within the cells. Its an obvious fact that the secretion of insulin can play a critical rolen the development and progression of diabetes. Therefore, keepingace with time and advanced drug formulation strategies, our studyttempts for the first time to assess the mechanism of action anditochondrial association of formulated PLGA loaded insulin and

n-encapsulated insulin in arsenic-stressed skeletal muscle cells.Therefore, in the present study the main objectives were: (i) to

xamine the increase in fold of efficacy of nano-insulin in relationo un-encapsulated insulin in combating As-induced stress in L6keletal muscle cells and (ii) to track down the signalling eventsodulated by the nano-insulin and insulin during the process of

melioration of As-induced stress.

. Materials and methods

.1. Reagents

All the chemicals used were of analytical grade and procuredrom Sigma Chemical Co., St. Louis, MO, USA.

.2. Formation of blank and drug loaded nanoparticles

The solvent displacement technique [13] was deployed for PLGAncapsulation of insulin under optimal conditions. 50 mg of PLGAnd 20 mg of dried Insulin (bovine origin) were dissolved in 3 mlcetone. To this organic phase mixture 20 ml of aqueous solutionontaining the stabilizer (1% polyoxyethylene–polyoxypropylene)F68; w/v) was added in a drop-wise manner (0.5 ml/min). The mix-ure was stirred continuously at room temperature until completevaporation of the organic solvent. Removing the redundant stabi-izer from the nanoparticles by centrifugation at 2500 × g at 4 ◦C for0 min the pellet was re-suspended in Milli-Q water, and washedhree times; the obtained nanoparticles loaded suspensions weretored at 4 ◦C until further use. For the preparation of blank nano-articles, the same method was deployed but without addition ofhe insulin particles. These blank nano-particles were initially useds one arm of control, but when tested on arsenic-stressed L6 cells,hese blank nanoparticles did not show any attenuative efficacy,or which this control was abandoned and further studies wereonducted only with the drug-loaded-nano-particles.

.3. Scanning electron microscopic (SEM) study

To determine the surface morphology of the PLGA formulatedano-insulin (NIn), scanning electron microscopic (SEM) study was

ndertaken (Hitachi S 500, USA equipped with 15 kV, SE detectorith a collector bias of 300 V), the lyophilized samples being spread

ver the double-sided conductive tape (12 mm) fixed onto metallictud.

B: Biointerfaces 109 (2013) 10– 19 11

2.4. Atomic force microscopic (AFM) studies of nano-insulin

The samples for AFM imaging were prepared by placing a dropof NIn-suspension on a freshly cleaned mica sheet and allowed todry in the air. The observation was recorded through AFM (Veecodi CP-11) imaging in amplitude and tapping modes.

2.5. Particle size determination by dynamic light scattering (DLS)method

To determine the average size and distribution of the poly-meric micelles, DLS study using a Zetasizer, Nano-ZS instrument(Malvern Instruments, Southborough, UK) was conducted. Thesizes were estimated and presented as the average value of 20runs, with triplicate measurements within each run. Zeta poten-tial of the nanoparticles was measured in the same instrumentand with the same procedure. Additionally, the zeta potential ofun-encapsulated/free insulin was also measured.

2.6. Insulin loading, encapsulation efficiency, burst release profileof nano-insulin during its formulation

To estimate the entrapment efficiency (E%) of nano-insulin (NIn)loaded in PLGA and the release of insulin from the nano-particles,the method of De Rosa et al. [14] was followed. In brief, insulin stan-dard curve was made by estimating the absorbance of differentconcentrations of insulin (10–200 nM range) at 280 nm wave-length. The encapsulation efficiency (%, w/w) of insulin, inside theencapsulated-nano-particles, was calculated from the insulin stan-dard curve by following standard protocol [10,11], according to theequation as given below:

Encapsulation efficiency (%) = E(%) = [Drug]tot − [Drug]free[Drug]tot

× 100

2.7. Cell culture procedure

L6 cells (from rat skeletal muscle cell line) were obtained fromNational Centre for Cell Science, Pune, India and grown at 37 ◦Cin 5% carbon dioxide atmosphere in Dulbecco’s modified Eagle’sMedium (DMEM) supplemented with 10% foetal bovine serumand 1% antibiotic (PSN). For experimental studies, the cells wereallowed to grow to 80–90% confluence, harvested in ice-cold buffersaline (PBS) and plated at desired density. The cells were allowedto re-equilibrate for 24 h before any treatment [12].

2.8. Introduction of arsenic induced stress

Sodium arsenite (NaAsO2) was diluted in sterilized Milli-Qwater at a concentration of 100 mM and was stored at 4 ◦C. Cellswere exposed to freshly prepared NaAsO2, 0.5 mM in DMEM mediafor 30 min [4]. Earlier in our previous study a range finding trialwas undertaken with different concentrations of sodium arsenite(0.1 mM, 0.3 mM, 0.5 mM. 0.7 mM and 1 mM at different timeintervals. On the basis of the obtained results [15], 0.5 mM SA for30 min exposure was selected as the most suitable dose [15] to beadopted for experimentation with all parameters of the presentstudy.

2.9. Dose of insulin and NIn

Stock solutions at a concentration of 1 mM insulin and PLGAnano-insulin were prepared in DMEM media and stored at 4 ◦C.Cells were exposed to freshly prepared insulin at a dose of 100 nMand nano-insulin at a dose of 50 nM, 100 nM (the dose had earlier

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een standardized for optimum effect) [4]. After 30 min of treat-ent different experiments were performed.

.10. Cytotoxicity assessment of nano-insulin in L6 cells

To check whether the formulated nano-insulin render anyytotoxicity to normal L6 cells, the MTT [3(4,5-dimethylthiazol-2-l)-2,5-diphenyltetrazolium bromide] assay [15] was conducted.nsulin (In) and nano-insulin (NIn) were added to the cultured L6ells (106 cells/ml) in 96-well micro plates and incubated for dif-erent periods of time. At the termination of incubation period,

TT was added into each well. After 4 h, the formazon crystalshus formed were dissolved with DMSO and the absorbance of theolution was measured at 595 nm using ELISA reader.

.11. Entrapment efficiency of nanoparticles by cellular uptaketudy

The entrapment efficiency of nanoparticles of the L6 cells wasuantified by measuring the amount of intracellular and extracel-

ular protein before and after treatment of the drugs.

.12. In vitro release kinetics of nano-insulin

The in vitro release kinetics of insulin from its nano-ncapsulated form after being administered in L6 cells wastudied by indirect ELISA method [16]. To quantify insulin, anti-nsulin antibody (Santa Cruz Biotechnology, USA) and alkalinehosphatase-conjugated secondary antibody (Sigma, USA) weresed.

.13. Glucose uptake

Glucose uptake by L6 cells was estimated after treatment withnsulin and nano-insulin following a standard procedure [17,18].he amount of glucose in the media was then estimated usingnthrone reagent [17]. To measure non-specific glucose uptake wencubated the cells parallely in the presence of 10 �M cytochalasin, to block transporter-mediated glucose uptake, and subtractedhem from total uptake in each assay [18].

.14. Localization of GLUT4 expression in L6 cells

To detect the localization of GLUT4 in L6 cells at different exper-mental sets the immunofluorescence study was undertaken byollowing the standard procedure [19].

.15. Analysis of several proteins in L6 cells by immunoblotting

To analyze the expressions of different proteins immunoblotas conducted by using anti-GLUT2, GLUT4, IRS1, IRS2, PI3, PPAR�,YP1A1 and p38 antibodies (Santa Cruz Biotechnology, USA) andlkaline phosphatase-conjugated secondary antibody (Sigma, USA)20] for the purpose. For quantitative analysis of each band, theand intensity was determined using the Total Lab software (Ultraum, USA).

.16. RNA extraction and quantitative reverseranscriptase-polymerase chain reaction (RT-PCR) analysis

The total RNA was extracted from the L6 cells using Tri-ol reagent according to the manufacturer’s instructions

nd converted to cDNA by reverse transcriptase. This cDNAas used as template for PCR amplification with the aid of

aq polymerase as per the standard practice [20]. Syntheticligonucleotide primers were procured from Chromus Biotech,

B: Biointerfaces 109 (2013) 10– 19

Bangalore, India. The sequences (Bioserve Biotech, Hyderabad,India) of the forward and reverse primers used for specificamplifications were as follows: G3PDH (5′-ATGGGGAAGGTGAA-GGTCGG-3′ and 5′-GGATGCTAAGCAGTTGGT-3′); IRS1 (F:5′-AGAGTGGTGGAGTTGAGTTG-3′ and R: 5′-GGTGTAACAGA-AGCAGAAGC-3′), IRS2 (F: 5′-GGATAATGGTGACTATACCGAGA-3′

and R: 5′-CTCACATCGATGGCGCGATATAGTT-3′), GLUT2 (5′-ATG-TCAGAAGACAAGATCACCGG-A-3′ and 5′-CCTACTGGACGGATTT-TGGCTC-3′); GLUT4 (5′-AAGATGGCCACGGAGA-GAG-3′ and5′-GTGGGTTGTGGCAGTGAGTC-3′); Glucokinase (5′-CACCCA-ACTGCGAA-ATCACC-3′ and 5′-CATTTGTGGGGTGTGGAGTC-3′);PPAR� (F: 5′-GCGGAGATCTCCAGTGATATC-3′ and F: 5′-TCAGCGA-CTGGCACTTTTCT-3′), PI3 (F: 5′-TTAAACGCGAAGGCAACGA-3′

and R: 5′-CAGTCTCCTCCTGCTGCTGAT-3′), PDK1(F: 5′-CCCGCTACTACGTTTCTTATCTTCC-3′ and R: 5′-CCGAGAGACAC-ATCAAAATCTGG-3′), CYP1A1 (F: 5′-CCCACAGCACCACAAGAGATA-3′ and R: 5′-AAGTAGGAGGCAGGCACAATGTC-3′), UCP2 (F:5′-GTACAGGAATTCAGCATCATGGTTGGGTT-3′ and R: 5′-AGCAGCT-CTAGAGGCTCAGAAGGCAGC-3′).

After PCR amplification, the DNA bands were photographed anddensitometrically analyzed using Total Lab software (Ultra Lum,USA).

2.17. Analysis of glucokinase (GK) and pyruvate kinase (PK)activities and measurement of ATPase level

Enzymatic assays for GK and PK activities were undertaken onthe basis of standard protocol described in EC 2.7.1.2 [21] and EC2.7.1.40 [22], respectively. The level of ATPase enzyme was mea-sured by the standard procedure [23] using Phenol red indicator.

2.18. Analysis of mitochondrial membrane potential (MMP)

The changes in mitochondrial membrane potential of L6 cellswere recorded from different groups of mice, using a fluorescentprobe, Rhodamine 123 (1 mmol) under fluorescence microscope(Leica, Germany) and analyzed by using a flow cytometer (BD FACS,ARIA) [19].

2.19. Detection of cytochrome c from mitochondrial andextracellular cytosolic fraction of L6 cells to ascertain changes inMMP

Mitochondria from L6 cells were isolated following the proto-col of Frezza et al. [24] with slight modifications as described inSamadder et al. [10].

2.20. Quantitative estimation of ATP/ADP ratio

ATP/ADP was measured using the Enzylight ADP/ATP ratio-bioluminescence-assay-kit (ELDT100) and assessed in a lumi-nometer (Thermo-Scientific-Varier-Skan). The result obtained wasinversed to get the ATP/ADP ratio [25].

2.21. Cell membrane potential

Changes in membrane potential of L6 cells were determined byusing the fluorescent dye 3,3′-diphenylthiocarbocyanine iodide inspectroflurometer (Thermo-Scientific Varier Skan) [26].

2.22. Quantitative estimation of intracellular calcium ion content

Different sets of L6 cells were incubated overnight in 1 ml PBSand then sonicated (Sonics vibra) at an amplitude of 56 Hz, pulse60 s with an interval of 5 s; the intracellular calcium ion (Ca2+) was

A. Samadder et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 10– 19 13

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average (particle size), PDI, zeta potential and % encapsulation effi-ciency, over a long period of time. The results revealed no suchchanges in their physico-chemical character even after 180 days ofstorage of the nano-particles (Supplementary Table 1c).

ig. 1. Scanning electron microscopic image (a), atomic force microscopic image [article size (Z average value) obtained from DLS data (c) of PLGA-encapsulated-ins

stimated following the standard titration method in chemistrysing Eriochrome black T indicator [27].

.23. Statistical analysis

All data were presented as mean values of three independentxperiments after statistically analyzing them by Student’s t-testnd one-way ANOVA.

. Results

.1. Characterization of Nano insulin (NIn)

As observed under SEM (Fig. 1a), the surface morphology ofIn was spherical in shape; AFM (2D) and the corresponding 3D

mage (Fig. 1b and c) displayed smooth surface without any pin-oles or cracks. DLS data showed that the mean diameter of NInas 93.67 nm with polydispersity index (PDI) of 0.265 (Fig. 1d).

he zeta potential of insulin was 0.171 mV while that of NIn was18.2 mV (Fig. 2a).

.2. Assessment of insulin-loading, encapsulation-efficiency andurst-release of PLGA-nano-insulin

The drug-loading and encapsulation-efficiency of insulin insideLGA to form nano-encapsulated-insulin was calculated to be0.2%. Insulin burst-release (i.e. loss of insulin during theormulation-process of nano-insulin) from the PLGA-encapsulated-nsulin was found to be minimum (Supplementary Table 1a andb).

.3. Assessment of storage stability of the formulated

ano-insulin

The stability of the formulated nano-particles was ascertainedy checking certain physico-chemical parameters of Nin, like Z

imension and three-dimension (b and c)] and graphical representation of averageano-particles.

Fig. 2. Zeta potential of PLGA-encapsulated-insulin-nano-particles and un-encapsulated/free insulin (a). Percentage drug entrapment efficiency of L6 cells (b).The means values of three independent experiments (each time n = 5) are presentedwith their respective standard error (SE).

14 A. Samadder et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 10– 19

Fig. 3. Graphical representation of the mean activities of glucose uptake (a), glucokinase, pyruvate-kinase (b), ATPase (c), intracellular calcium ion content (d), in differentgroups of L6 cells before and after the treatment of drugs. ***p < 0.001, **p < 0.01, *p < 0.05, vs. sodium arsenite (SA)-treated group. The mean values of three independente E).

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xperiments (each time n = 5) are presented with their respective standard error (S

.4. Drug entrapment efficiency and in vitro release kinetics

The drug entrapment/cellular uptake of the drugs, insulin andano-insulin, after being administered in L6 cells, was evaluatedy determining the protein content (i.e. insulin content in eachase, whether in un-encapsulated free form, or in PLGA nano-ncapsulated form). Results revealed that there was a significantncrease in the percentage of cellular uptake of the drugs, insulinnd nano-insulin, within L6 cells, with the increase in the concen-ration of the drugs, up to an optimal concentration (the highesteing at 5 �l of the drugs in each case). Beyond this concen-ration of the drugs, there was a slight decrease in the cellularptake/entrapment efficiency within L6 cells. Therefore, on theasis of this result the doses of the drugs were chosen accordinglyor our further study. The doses were: 5 �l (=100 nM) dose of insulinnd 2.5 �l and 5 �l (=50 nM and 100 nM) of NIn (Fig. 2c).

Constant release of insulin from its nano-encapsulated form wasbserved from 0 to 30 min of post treatment of NIn in L6 cellsSupplementary Graph 1d).

.5. % cell viability/cellular cytotoxicity of nano-insulin

Result of % cell cytotoxicity assessment suggests that the nano-articles formulated had no effect on modulating % cell viabilitynormal: 98.89 ± 1.75 and at highest dose and longest treatmentnterval of NIn: 95.98 ± 2.14) (Supplementary Graph 1e).

3.6. Analysis of glucose uptake by L6 cells

A significant inhibition of glucose uptake was observed insodium arsenite (SA)-treated L6 cells. Treatment with In and NIntends to elevate the level of glucose uptake significantly (Fig. 3a);the NIn had greater efficacy than the un-encapsulated form.

3.7. Analysis of glucokinase (GK) and pyruvate kinase (PK)activities and measurement of ATPase level

Activity of both the enzymes GK, the first enzyme in con-verting glucose to glucose-6-phosphate, and PK, the last enzymeof glucose metabolism, decreases rapidly in arsenic treated cells(Fig. 3b). But on administration of the In and NIn, a significantincrease in their levels was observed. The modulations of thesebiomarkers were more pronounced in the latter case than that ofthe former. In ATPase level analysis just the reverse modulationwas observed after SA treatment and further treatment with In anNIn (Fig. 3c).

3.8. Quantitative estimation of intracellular calcium ion andATP/ADP ratio

A marked decrease in the intracellular calcium ion (Fig. 3d) andthe ATP/ADP ratio (Fig. 4a) was observed on SA treatment, but theratio was increased in insulin and NIn administered groups.

A. Samadder et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 10– 19 15

Fig. 4. Analysis of ATP/ADP ratio (a), spectroflurometric analysis of cell membrane potential (b), in different groups of L6 cells before and after the treatment of drugs.* f mitom time n

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**p < 0.001, **p < 0.01, *p < 0.05, vs. sodium arsenite (SA)-treated group. Analysis oental and control sets. The mean values of three independent experiments (each

.9. Spectroflurometric analysis of cell membrane potential (CMP)

The membrane potential of L6 cells was less towards negativityn SA treated cells when compared to that of normal. The potential

as re-established, i.e., it was again brought towards negativityffectively on administration of insulin and NIn (Fig. 4b).

.10. Analysis of mitochondrial membrane potential (MMP)

The dye Rhodamine 123 gives negative intensity of thereen fluorescence for mitochondrial membrane depolarization.s observed from our study the L6 cells, treated with SA showedarked decrease in the intensity of the greenish stain indicating a

epolarization state of the MMP, when compared to that of con-rol. However, after the administration of insulin and NIn in L6ells pre-treated with SA, the greenish fluorescence reappeared.he intensity of the fluorescence was more pronounced in case of

chondrial membrane potential by fluorescence microscopy (c) in different experi- = 5) are presented with their respective standard error (SE).

NIn, indicating that NIn could re-establish the hyper-polarizationstate (Fig. 4c) of the SA-stressed L6 cells in a better way than that ofun-encapsulated insulin. The quantitative data generated by flow-cytometry corroborated well with this finding (Fig. 5).

3.11. Immunofluorescence of GLUT4 protein expression

FITC-tagged secondary antibody for localization of GLUT4 pro-teins (rendering green fluorescence for positive localization) indifferent sets of L6 cells revealed that there was a sharp depletionin fluorescence intensity in SA-stressed L6 cells when compared tocontrol set. But cells pre-treated with SA, when exposed to insulinand NIn, showed a significant elevation in the fluorescence intensity

in these experimental sets, revealing an increase in the localizationof GLUT4 proteins. This increase in the localization was more strik-ing in NIn treated L6 cells (Fig. 6a) when compared to insulin treatedones.

16 A. Samadder et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 10– 19

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.12. Western blot and RT-PCR analyses

The expression levels of different proteins by immunoblotFig. 6b) and mRNAs by RT-PCR (Fig. 7a) were analyzed in differentroups of L6 cells. The results are presented below individually:

.12.1. Disruption of GLUT4 glucose transporterA gradual decrease was observed in the expressions of GLUT4 in

A-treated cells. Administration of In and NIn increased the level ofLUT4 in them, NIn bringing more pronounced modulations than

hat of In.

.12.2. Loss of glucokinase activity and integrity of IRS1 and IRS2n arsenic stressed condition

There was a down-regulation of the mRNA expression of glucok-nase and protein and mRNAs of IRS1 and IRS2 on arsenic treatment.ignificant up-regulation in the expression of the receptors wasoted on the administration of In and NIn, the latter showingreater up regulation.

.12.3. Role of PI3, and p38 as glucose transport mediators andxpression of UCP2 mRNA

The protein and mRNA expressions of the PI3 glucose transportediator were observed to be down-regulated in the arsenic-

reated L6 cells, as compared to that of normal control. On theontrary, its expression in In and NIn administered series increasedignificantly. A reverse condition was observed in case of UCP2xpression whereas no significant change was observed in thexpression of P38 protein in all the groups of experimental cells.

.13. Analysis of mitochondrial and cytosolic cytochrome-crotein

The expression of cytochrome c was down-regulated in mito-

hondrial fraction and up-regulated in cytosolic fraction ofA-treated L6 cells. This condition was reversed significantlyn In and NIn administration, the latter proving better in effi-acy. The result was normalized with the expression of the

ifferent groups of L6 cells. A: Normal; B: SA; C: SA + In; D: SA + NIn(I); E: SA + NIn(II).

mitochondrial control gene COX-4, which showed no significantchange in its expression in different groups of treatment (Fig. 7b).

4. Discussion

Results of the present study suggested that arsenic inducesinsulin resistance through modulations of the insulin receptorsubstrate molecule, resulting in alteration of the glucose uptakecapability and gene expressions of L6 skeletal muscle cells (in vitro).This result is in agreement with that of earlier researchers whohave shown the prevalence of diabetes in inhabitants of arseniccontaminated areas that implicated an association between arsenicexposure and diabetes [8], either leading to decreased productionof the insulin hormone and/or increased resistance to the action ofinsulin in the skeletal muscles.

The designing and testing of organ/tissue/cell-specific drugson biological system demands the formulation and usage ofbiodegradable nanoparticles with better efficacy, availability, rapiddelivery, faster and targeted action in minimum dosage. The prepa-ration of poly (lactide-co-glycolide) nano-insulin formulation hasbeen used by earlier researchers to encapsulate a wide variety ofhydrophobic drugs [28], estradiol [29], but none of the reportsshowed modulatory changes brought about by nano-encapsulatedinsulin (NIn) in As-induced stress conditions in L6 cells. Nano-encapsulation of drug not only decreases the size of drug moleculesentering the body thereby enhancing their penetrance, but theamount of drug entering the body is also decreased in this processby several folds. Nano-particles also have the potential to modulateseveral biomarkers by several folds, depending on their amounttaken for the encapsulation (20 mg insulin in this case) and thefinal yield after formulations of encapsulated form (∼200 mg nano-insulin). Hence, the actual amount of the original drug substance innano-encapsulated form gets minimized by about 10-fold (approx-imately in this case) but nonetheless provides similar efficacy asthat of their un-encapsulated counterpart.

Glucose transport is the rate limiting step for glucose disposal.Insulin-induced GLUT4 translocation in the plasma membrane isthe hallmark of glucose transport in skeletal muscles which regu-lates glucose uptake and alter mitochondrial energy output [30].

A. Samadder et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 10– 19 17

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ig. 6. Immunofluorescence detection of GLUT4 (a) in different groups of L6 cells. Ay immunoblot technique (b) in different series of L6 cells before and after drug tre

herefore, slightest of imbalance due to arsenic stress could bebserved primarily in the glucose uptake level. In our study, theevel was found to be decreased after arsenic intoxication, anddministration of In and NIn caused an increase in glucose uptake

evel.

It is well accepted that activation of the insulin receptorubstrate (IRS/PI3K axis) is indispensable to insulin stimulatedLUT4 translocation and glucose uptake. In skeletal muscle, insulin

al; B: SA; C: SA + In; D: SA + NIn(I); E: SA + NIn(II). Expression of different proteinst. Ln1: Normal; Ln2: SA; Ln3: SA + In; Ln4: SA + NIn(I); Ln5: SA + NIn(II).

binding to insulin receptor complex causes auto-phosphorylationof tyrosine residues [31] leading to the employment of insulinreceptor substrate (IRS1 and IRS2) proteins via activation of thephosphatidyl inositol 3-kinase (PI-3 kinase) pathway [32]. Our

observation revealed the involvement of PI3 signalling moleculesin glucose transport via insulin secretion by the modulations in thePI3 protein and mRNA expressions in different experimental set up.Introduction of In and NIn resulted in an elevation in the ability of

18 A. Samadder et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 10– 19

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ig. 7. Expression of different mRNA by RT-PCR techniques (a), in different serieA + NIn(I); Ln5: SA + NIn(II). Mitochondrial and cytosolic cytochrome c protein exp

he IRS molecules to dock with receptor and interact with down-tream signalling cascade via PI3 kinase pathway to restore theormal glucose uptake, NIn showing a better ability. This implieshat nano-insulin has greater potential to stabilize the PI3 activityn glucose transport via serine phosphorylation of IRS1 and IRS2.

GLUT4 facilitates rapid equilibration across the skeletal muscleell membrane, and the hexokinase isoform, “Glucokinase (GK)”,llows the generation of a proportionate signal through glycolyticetabolism of glucose [33]. Our findings suggest that the exposure

o arsenic possibly restricts the GLUT4 transporter from workingormally, resulting in blockage of GK activity, thereby setting therst step of disparity in glucose metabolism pathway. Introduc-ion of nano-insulin and insulin results in an elimination of thelockage, thus allowing GK to re-incorporate glucose in the glycol-sis pathway. The pyruvate derived from glycolysis is transportednto the mitochondria, where it is oxidized by the tricarboxyliccid (TCA) cycle leading to transfer of reducing equivalents to theespiratory chain, hyperpolarization of the mitochondrial mem-rane, and ATP generation (via oxidative phosphorylation by thelectron-transport chain). Not only mitochondrial but also cyto-lasmic membrane potentials play crucial roles in fuel-stimulated

nsulin secretion and glucose uptake. The membrane potential ofhe L6 cells, when subjected to arsenic insult, changed along with

odulation of glucose uptake; the condition was brought backlmost to normal on administration of In and NIn. This implicateshat NIn and In were capable of eliciting a significant stimulationf glucose transport in L6 cells.

Maintenance of ATP/ADP ratio or rather production/synthesisf ATP also plays important role in insulin secretion. Uncoupling

rotein 2 (UCP2), a negative regulator of insulin secretion [34]as reported to divert energy away from ATP synthesis, therebyecreasing the rate of formation of ATP. From our present study,e ascertained that expression of UCP2 gene was down-regulated

6 cells before and after drug treatment. Ln1: Normal; Ln2: SA; Ln3: SA + In; Ln4:n (b).

in the stressed cells. This might lead to lowering of the ATP/ADPratio causing impairment in glucose-stimulated insulin secretion.Introduction of NIn promotes a decrease in the UCP2 protein muchmore effectively than un-encapsulated form, thereby restoring itsfunction almost back to normal.

The present study also supports the fact that cytosolic Ca2+

mediates insulin stimulated glucose transport. It was reported ear-lier that chelation of intracellular Ca2+ in L6 cells reduces glucoseuptake [35]. Arsenic-induced reduction in cytosolic free Ca2+, asobserved from our study, imposes a lowering in glucose uptake.Introduction of NIn brings about a positive modulation in main-taining optimum glucose balance inside the muscle cells in a Ca2+

dependant phosphorylation of insulin receptor.Mitochondrial dysfunction may contribute to insulin resistance

[2,36–38], impaired insulin secretion and diabetic complications[33]. Stump et al. [3] further characterized the role of insulin in theregulation of skeletal muscle mitochondrial function. Infusion ofNIn in stressed cells led to significant increase of ATP productionrate and protein level of cytochrome c in mitochondria. Thereforethis study supports the theory that insulin is a stimulant of musclemitochondrial biogenesis and function.

The therapeutic arsenals that include modern ways of pharma-ceutical interventions, by nano-encapsulation of drugs, are nowbeing explored in various fields of research. The prime objectiveof our work was therefore to examine if the efficacy of commonlyused drugs could be enhanced through nano-encapsulation. Skele-tal muscle, an emerging target of acute insulin effects on musclemitochondria, interestingly revealed that the increments in mito-chondrial transcripts were positively related to those of In and NIn

mediated glucose disposal. The results also suggested that the zetapotential of insulin (in its free/un-encapsulated form) was 0.171 mV(positive) and that of PLGA encapsulated nano-insulin was found tobe −18.2 mV (negative). Earlier reports suggest that negative zeta

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mparts an increase in the rate of absorption and transport of theano-particles across various cells and tissues [39]. Therefore thisay be one of the major possible reasons of PLGA encapsulated

ano-insulin to show nearly similar effect but in a much lower dosenearly 10-fold lower) than that of insulin in its un-encapsulatedorm. Thus, this would surely decrease the severe side-effects ofnsulin administration in hyperglycaemic condition, especially inrsenic-intoxicated victims who has to suffer a sequel of ailmentsue to arsenic-infestation. Further animal trials are therefore essen-ial to explore if the experimental use of nano-insulin with testedncreased ability could be used in future drug designing for arsenicictims suffering from diabetes and its complications.

onflict of interest

None to declare.

cknowledgements

Grateful acknowledgements are made to Boiron Laboratory,yon, France, for financial grant provided to Prof. A.R. Khudaukhsh. We are thankful to Mr. Ashim Kumar Chakraborty, Chiefcientist & Head, Materials Characterization Division and Mrs.wati Samanta, Materials Characterization Division, CSIR-Centrallass and Ceramic Research Institute, Kolkata, for allowing us tose DLS machine in their laboratory. We are also thankful to Dr.. Basu, Department of Biophysics and Biochemistry, University ofalyani, for his help. Sincere thanks are also due to Mr. R. Banik,

ACS, India, for his kind help in AFM study.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.colsurfb.2013.03.028.

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