Cell Growth Inhibition Effect of DsiRNA Vectorised by

Research ArticleCell Growth Inhibition Effect of DsiRNA Vectorised byPectin-Coated Chitosan-Graphene Oxide Nanocomposites asPotential Therapy for Colon Cancer

Haliza Katas Mohd Cairul Iqbal Mohd Amin NursyafiqahMoideenLi Ying Ng and Puteri Annisa AdhwaMegat Baharudin

Centre for Drug Delivery Research Faculty of Pharmacy Universiti Kebangsaan Malaysia Jalan Raja Muda Abdul Aziz50300 Kuala Lumpur Malaysia

Correspondence should be addressed to Haliza Katas haliz12hotmailcom

Received 31 January 2017 Revised 5 April 2017 Accepted 19 April 2017 Published 25 May 2017

Academic Editor Yan Zou

Copyright copy 2017 Haliza Katas et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Colonic-targeted drug delivery system is widely explored to combat colon-related diseases such as colon cancer Dicer-substratesmall interfering RNA (DsiRNA) has been explored for cancer therapy due to its potency in targeting specific gene of interestHowever its application is limited by rapid degradation and poor cellular uptake To address this chitosan-graphene oxide (CS-GO) nanocomposite was used to deliver DsiRNA effectively into cells Additionally pectin was used as compatibilization agent toallow specific delivery to the colon and protect the nanocomposites from the harsh environment in the stomach and small intestineCS-GO-DsiRNA nanocomposites were prepared by electrostatic interaction between CS and GO prior to coating with pectin Themean particle size of CS-GO-DsiRNA-pectin nanocomposites was 5545 plusmn 1246 nm with PDI and zeta potential of 047 plusmn 019and minus107 plusmn 30mV respectively TEM analysis revealed smooth and spherical shape of CS-GO-DsiRNA nanocomposites andthe shape became irregular after pectin coating FTIR analysis further confirmed the successful formation of CS-GO-DsiRNA-pectin nanocomposites Furthermore the nanocomposites were able to entrap high amount of DsiRNA ( entrapment efficiencyof 926 plusmn 39) with strong binding efficiency CS-GO-DsiRNA-pectin nanocomposites also selectively inhibited cell growth ofcolon cancer cell line (Caco-2 cells) and were able to decrease VEGF level significantly In a nutshell pectin-coated DsiRNA-loadedCS-GO nanocomposites were successfully developed and they have a great potential to deliver DsiRNA to the colon effectively

1 Introduction

Colorectal cancer is ranked as the third most commoncancer worldwide and it contributes to major cause ofdeath in Western country [1] Conventional treatments ofcolon cancer such as chemotherapy and radiotherapy haveconsiderable drawbacks as they could not specifically targetcancer cells and might cause injury to healthy cells [2]Besides patients on conventional treatment often developtolerance to targeted therapy by genemutation Gene therapyhas gained enormous interests recently as it can be used totreat wide range of diseases An effective treatment approachby producing siRNA-based drug may target specific mRNAsdespite of their cellular locations or structures of translatedprotein [3] Kulisch et al [4] reported that Dicer-substrate

siRNA (DsiRNA) displays excellent potency in gene silencingand is able to silence gene for longer time compared to siRNADsiRNAhas several advantages compared to standard 21-mersiRNAsThe advantages include better selectivity of the guidestrand as a consequence of Dicer processing and handoffto RNA-induced silencing complex (RISC) as well as higherpotency attributed to lower effective concentration needed[5]

RNAi technology was reported to provide an alterna-tive strategy in treating cancer by inhibiting overexpressedoncogenes blocking cell division by interfering with relatedgenes or promoting apoptosis by suppressing antiapoptoticgenes [6] Vascular endothelial growth factor (VEGF) is animportant angiogenic factor associated with tumor growthand metastasis [7] According to Ahluwalia et al [8] normal

HindawiJournal of NanomaterialsVolume 2017 Article ID 4298218 12 pageshttpsdoiorg10115520174298218

2 Journal of Nanomaterials

colonic epithelial cells do not produce VEGF or expressits receptors However in colon cancer cells VEGF and itsreceptors are expressed and it promotes colon cancer cell pro-liferation directly Therefore DsiRNA targeted against VEGFgene was used to kill cancer cells by silencing overexpressionof its protein [6]

In a strategy to activate RNAi pathway siRNA moleculesneed to be delivered to the interior of target cells and tobe incorporated into the RNAi machinery However siRNAscannot cross the lipid bilayers of the cell membrane readilyas they have anionic backbone and hydrophilic properties[9] Besides siRNAs are facing some other obstacles toreach their site of action The obstacles include undergoingdegradation by nucleases short half-life due to the uptake bymononuclear phagocyte system off-target effect and rapidrenal clearance [10] As a result of these obstacles the needto develop carriers for siRNA is paramount to deliver it tothe site of interest efficiently An ideal delivery carrier forsiRNA should possess properties such as being nontoxic andnonimmunogenic being able to condense siRNA efficientlyprotecting the integrity of its content before reaching thetarget site having the ability to evade rapid eliminationfrom blood circulation and internalizing and dissociatingin intracellular compartments of the target cells to releasethe adequate amount of siRNA thereby exposing siRNA tomRNA [11]

Nanomedicine-based carriers have been extensivelyexplored as potential candidates to guide siRNAs directlyto the target cells Nanocarriers offer several advantagesthat include providing sustained release and prolonging thetime of siRNA at the target site Nanocarriers also are ableto penetrate capillaries hence they can accumulate at thesite of interest The nanotechnology approach could alsoprotect siRNA against RNase degradation and avoid off-target effects [12] In nanomedicine polycationic polymerssuch as chitosan (CS) were used to condense siRNA intonanoparticles to facilitate cellular uptake [13]

CS in combination with pectin has been widely exploredfor their benefit as colon-specific drug delivery system [14]Apart from being biocompatible less toxic nonimmuno-genic and degradable by enzymes [15] CS can also be used ingene delivery system Tremendous studies on graphene oxide(GO) have made it the gold approach in biomedicine appli-cations GO is a highly oxidised graphene form with oxygenfunctional groups on its surface GO has been combined withCS to form a nanocarrier with better aqueous solubility andbiocompatibility In addition it can exhibit powerful loadingcapacity besides having the ability to condense plasmid DNAinto stable nanosized complexes [16] In an attempt to ensurethat CS-GO-DsiRNA nanocomposites would be deliveredto the target site successfully pectin was used to coat thenanocomposites due to its unique property that will bedegraded by colonic microflora This property enables it tobe used as a specific drug carrier to the colon [17] Besides italso displays antitumor activity [18] that can further enhancegrowth inhibition or cytotoxic effect in colon cancer cells

In this study water-soluble CS was synthesised fromlow molecular weight (LMW) CS to enhance its solubilitywhile GO was synthesised using Hummerrsquos method from

graphite flakes Oxidation of graphite using oxidising agentwill increase interplanar spacing between graphite layersproducing GO [19] Later DsiRNA was adsorbed onto CS-GO nanocomposites Pectin was utilised as coating agent tohinder premature DsiRNA release in gastric and intestinalfluids To confirm successful synthesis of CS-GO-DsiRNA-pectin nanocomposites and their ability to load DsiRNAphysical characterization structural analysis and in vitrodrug release study were conducted The nanocompositeswere also tested for their in vitro cytotoxic effect in normaland cancerous cell lines Finally silencing of VEGF gene byDsiRNA was determined using ELISA

2 Materials and Methods

21 Materials DsiRNA targeting VEGF gene [51015840-rGrGrArGrUrA rCrCrC rUrGrA rUrGrA rGrArU rCr- GrA rGrUAC-31015840 (sense strand) and 51015840-rGrUrA rCrUrC rGrArU rCrUrCrArUrC rArGrG rGrUrA rCrUrC rCrCrA-31015840 (antisensestrand)] was purchased from Integrated DNA TechnologiesUSA Low molecular weight (LMW) CS of 190 kDa with75ndash85 degree of deacetylation was purchased from Sigma-Aldrich (Ireland) Acetic acid and hydrochloric acid wereobtained from RampMChemicals (UK) Hemicellulase enzymefrom Aspergillus niger and pectin with 55ndash70 degree ofesterification were obtained from Sigma-Aldrich USA 10 bpDNA ladder was obtained from Invitrogen Corporation(Carlsbad) while sodium hydroxide was obtained fromJIT (Baker Sweden) Deionised water was produced inthe laboratory using Millipore Milli-Q Phosphate-bufferedsaline (PBS) and dimethyl sulfoxide (DMSO) were obtainedfrom Sigma-Aldrich (St Louis MO) Fetal bovine serum(FBS) Dulbeccorsquos Modified Eaglersquos Medium (DMEM) HighGlucose Eaglersquos Minimum Essential Medium (EMEM)penicillin-streptomycin MTT reagent and trypsin-EDTAwere purchased from Invitrogen (Carlsbad CA) Caco-2 cell line (colorectal adenocarcinoma cell derived fromHomo sapiens human) and CCD-18CO (normal colon cellderived from Homo sapiens human) were obtained from theAmerican Type Culture Collection (ATCC Manassas VA)HumanVEGFELISA kit was purchased from Sigma-AldrichUSA Acetic acid glacial potassium dihydrogen phosphatedipotassium hydrogen phosphate sodium chloride (NaCl)and sodium hydroxide (NaOH) were purchased from RampMChemicals (UK) All chemicals used were of analytical gradeand were used as received

22 Methods

221 Synthesis of Water-Soluble CS 1 g of LMW CS wasdissolved in 2 vv of acetic acid using amagnetic stirrerTheCS solution was adjusted to pH 45 by adding few drops ofNaOH and left overnight A 20 wv hemicellulase enzymesolution was added to CS solution and the mixture was leftin the water bath at 40∘C for 6 h pH of the mixture wasthen adjusted to 55 by adding few drops of NaOH solutionAfter that the mixture was boiled for 10min to denature theenzyme The upper layer of solution containing enzyme wasremoved manually by using a spatula until a concentrated

Journal of Nanomaterials 3

solution formed Then the solution was frozen at minus80∘C andit was lyophilised using a freeze dryer (ScanVac CoolSafeFreeze Drier Lynge Denmark) at minus110∘C for one day toobtain water-soluble CS It was then crushed into powderedform by using mortar and pestle The functional groups ofwater-soluble CS were identified by using Fourier-TransformInfrared Spectrophotometer (FTIR) (Perkin-Elmer Spectrum100 Waltham USA) 3mg of water-soluble CS and 300mgof dried IR-grade potassium bromide (previously heated for2 h at 105∘C) were ground into powder to produce a thindisc The sample was scanned at frequency range of 4000 to400 cmminus1

222 Synthesis of GOUsingHummerrsquosMethod Anamount of110mg graphite and an amount of 55mg NaNO

3were mixed

in 6mL concentrated H2SO4and the mixture was stirred

overnight using a magnetic stirrer An amount of 300mgKMNO

4was added and left overnight while the conical

flask was being surrounded by ice bath Another 300mg ofKMNO

4was added and left overnight on the next day Ice

bath was removed and it was replaced with water bath at40∘Covernight Later 7mL of deionisedwater was added andheated in water bath at 98∘C for 2 h until yellow-brownishsolution was formed 7mL of 30 H

2O2was added and the

mixture was left for 1 h at 98∘C After that themixture was leftto cool down to room temperature and poured into centrifugetube The supernatant was removed and the pellet formedwas resuspended with 10 HCl and deionised water severaltimes Then it was poured into dialysis bag for 5ndash7 daysThe solution was frozen at minus80∘C before it was lyophilisedusing a freeze drier (ScanVac CoolSafe Freeze Drier LyngeDenmark) at minus110∘C for one day The functional groups ofGO were identified using FTIR (Perkin-Elmer Spectrum 100Waltham USA) as mentioned above

223 Preparation of DsiRNA-Loaded CS-GONanocompositesWater-soluble CS was dissolved in distilled water and stirredto produce 01 wv of its solution GO 025 wv was addedto CS solution separately and it was mixed continuouslyfor 12 h using a magnetic stirrer The resultant mixture wasdegassed in a sonicator bath for 30min DsiRNA-loaded CS-GO nanocomposites were prepared by adding 500 120583L of thenanocomposites solution to an equal volume of DsiRNAsolution (15 120583gmL) in deionised water The mixture was

quicklymixed by inverting the reaction tube up and down fora few seconds and incubating for 30min at room temperaturebefore further analysis

224 Preparation of Pectin-Coated CS-GO NanocompositesPectin powderwas dissolved in deionisedwater at 70∘Cundermagnetic stirring to produce 01 wv pectin solution 10 120583Lof pectin solution was added to the DsiRNA-loaded CS-GOnanocomposites and the mixture was vortexed The coatedformulation was harvested by centrifugation (UNIVERSAL320R Benchtop Centrifuge (Hettich Centrifuges UK)) at13000 rpm for 1 h at 10∘C The pellets were resuspended indeionised water

225 Determination of Particle Size PDI and Zeta PotentialA volume of 1mL nanocomposites suspension of CS-GOCS-GO-DsiRNA and pectin-coated CS-GO-DsiRNA wasplaced separately in a glass cuvette using a pipette priorto analysis No dilution was done during the analysis Theparticle size (119911-average) polydispersity index (PDI) andzeta potential of the nanocomposites were characterised intriplicate using Zetasizer Nano ZS (Malvern InstrumentsUK) The measurements were performed at 25∘C with adetection angle of 90∘ All data are expressed as the mean plusmnstandard deviation (SD)

226 Determination of DsiRNA Entrapment Efficiency andNanocomposites Yield DsiRNA entrapment efficiency wasobtained from determination of free DsiRNA concentrationin supernatant recovered from centrifugation process using aUV-1061 UV-Visible Spectrophotometer (Shimadzu Japan)Unloaded nanocomposites formulation was used as blankConcentration of free DsiRNA was determined using BeerrsquosLaw (119860

260= 120576119862119871) and calculations were done using the

following equation

119862 =119860

120576119871 (1)

where 119862 is the concentration of DsiRNA 119860260

is the extinc-tion coefficient and 119871 is the path length of the cuvetteExtinction coefficient of DsiRNA is 518500 Lminus1sdotcmminus1 Thesample was measured in triplicateThe entrapment efficiencywas calculated by using the following equation

concentration of DsiRNA added minus concentration of DsiRNA in supernatantconcentration of DsiRNA added

times 100 (2)

The yield of nanocomposites was calculated by using thefollowing equation

119909 =mass of DsiRNA minus nanocomposites

total mass of composites and DsiRNA added

times 100

(3)

227 Determination of DsiRNA Binding Efficiency The bind-ing efficiency of DsiRNA to the nanocomposites was deter-mined by using E-Gel 4 agarose (GP) stained withethidium bromide (Invitrogen Israel) 20120583L samples ofnanocomposites were loaded into respective wells of the gelNakedDsiRNAwas used as positive control while 10 bpDNAladder was used as size reference Electrophoresis was run for


30minThemigration of DsiRNA in form of bands in the gelwas then visualised by using a real-time UV transilluminator(Invitrogen USA)

228 Morphological Analysis Morphological analysis ofpectin-enveloped CS-GO nanocomposites was characterisedby using a Transmission Electron Microscopy (TEM TecnaiSpirit FEI Eindhoven Netherlands) A drop of sample wasplaced on the copper microgrid that was stained by uranylacetate and evaporated at room temperature (25 plusmn 2∘C) Itwas viewed under a TEM for imaging of samples at differentmagnifying scales

229 ATR-FTIR Spectroscopic Analysis TheAttenuatedTotalReflectance FTIR spectra of pectin CS-GO and CS-GO-pectin nanocomposites were recorded against the back-ground by using a universal ATR sampling assembly (Perkin-Elmer Spectrum 100 Waltham USA) For each sample 16scans were obtained at a resolution of 4 cmminus1 in the rangeof 4000 to 400 cmminus1 Meanwhile LMW CS water-solubleCS and GO powder were characterised by using FTIRSpectrophotometer (Perkin-Elmer Spectrum 100 WalthamUSA)

2210 In Vitro Release Study In vitro drug release study wasperformed in the simulated gastrointestinal (GI) conditionsimulated gastric intestinal and colonic fluid (SGF SIF andSCF resp) Formulations containing 025wt GO coatedwith 01 and 02 pectin were used to determine theinfluence of pectin concentration on DsiRNA release SGFwas prepared by dissolving 2 g of NaCl in sufficient amountof distilled water 7mL of 01 N HCl was added to thesolution and the volume was made up to 1000mL usingdistilled water The pH was finally adjusted to 12 using01 N HCl or 01 N NaOH Meanwhile SIF was prepared bymixing solution containing 68 g of potassium dihydrogenphosphate in 250mL distilled water with 72mL of 02MNaOH Distilled water was added to make up to 1000mLsolution and the pH was adjusted to 68 by using 01 N HClor 02M NaOH The SCF was prepared by mixing 250mL of02Mdipotassiumhydrogen phosphatewith 285mLof 02MNaOH and distilled water was added to make final volumeof 1000mL The pH of the solution was adjusted to 72 using01 NHCl or 02MNaOH For the first 2 h the in vitro releasestudy was conducted in SGF (pH 12) to follow the averagegastric emptying time The medium was then replaced withSIF at pH68 (with addition of fewdrops ofNaOH)The studywas continued for another 3 h After that the medium wasreplaced with SCF at pH 72 (with addition of few drops ofNaOH) in the presence of 03mL pectinolytic enzyme andthe study was extended for another 3 h Each formulation(300 120583L) was added to a dialysis tubing cellulose membrane(Sigma-Aldrich) and immersed in 50mL of simulated fluidsin a beaker The beakers were placed in a shaker water bath(JP Selecta) (37 plusmn 02∘C) with horizontal shaking of 25 rpmAt predetermined time intervals 4mL of the simulated fluidwas withdrawn and the concentration of DsiRNA releasedwas measured using UV-Vis Spectrophotometry at 260 nm

To prevent sink condition 4mL of fresh medium was addedto the beaker to replace the withdrawn fluid The percentageof DsiRNA released was determined by using the followingequation

percentage of DsiRNA released ()

=concentration of DsiRNA from withdrawn fluid

concentration of DsiRNA added

times 100

(4)

2211 Cytotoxicity Study Caco-2 cells (ATCC ManassasVA USA) were cultured in DMEM medium at a cell densityof 1 times 104 per well The cells were supplemented with amedium containing 10 FBS and 1 penicillin-streptomycinand maintained at 37∘C in a humidified 5 CO

295 air

atmosphere Meanwhile CCD-18CO cells (ATCCManassasVAUSA)were cultured in EMEMmedium at a cell density of5 times 103 per well The cells were supplemented with a mediumcontaining 10 FBS 1 penicillin-streptomycin and 1sodium pyruvate and maintained at 37∘C in a humidified 5CO295 air atmosphere After 24 h incubation of untreated

cells CS pectin GO and CS-GO-DsiRNA nanocompositesand CS-GO-DsiRNA nanocomposites coated with pectin at37∘C a final dilution of 110 per cell volume of MTT reagentwas added to all the wells followed by incubation for 4 hprior to analysis Then the MTT containing medium wasaspirated and the formazan crystals formed by the living cellswere dissolved in 200120583L of DMSO solution The absorbanceof each sample at 570 nm was measured using a microplatereader (Varioskan Flash Thermo Scientific Waltham MAUSA) The cytotoxicity effect was calculated as a percentageof the cell viability of untreated cells using the followingequation

cell viability () = absorbance of treated cellsabsrbance of control cells

times 100 (5)

2212 ELISA Analysis of VEGF Protein Caco-2 colon cancercells were cultured in complete medium of DMEM (high glu-cose) which was supplemented with 10 fetal bovine serumand 1 penicillin-streptomycin The cells were incubated in25 cm2 plastic culture flasks at 37∘C and 5 carbon dioxide(CO2) When the cells achieved 70 confluency they were

then dissociated using 1mL of 25 gL-Trypsin1mmolL-EDTA solution and incubated for 3ndash5min at humidified5 CO

295 air atmosphere After trypsinization the cells

were suspended in 1mL fresh medium and centrifugedfor 3min at 1000 g Then the supernatant was discardedand the cell pellet was subsequently resuspended in 1mLcomplete medium Suspension density was measured usinga haemocytometer Once the cell density reached 1 times 105mL100 120583L of suspension was pipetted into each well The cellswere incubated for 24 h at 37∘C with 5 CO

2 After 24 h

the medium was changed to serum-free medium and Caco-2colon cancer cells were exposed to pectin-enveloped CS-GOnanocomposites DsiRNA-loaded CS-GO nanocompositespectin-enveloped DsiRNA-loaded CS-GO nanocomposites


LMW CS

WS CS

4000 3500 3000 2500(cmminus1)

2000 1500 1000 500400

384042

4644

48

5250

5654

58606265

T

454850525456586062646668707276

T

102327 cmminus1

108024 cmminus1115619 cmminus1

140418 cmminus1

156756 cmminus1

292650 cmminus1

341314 cmminus1

108123 cmminus1


346048 cmminus1

Figure 1 FTIR spectra of LMW CS and water-soluble CS

and naked DsiRNA for 24 h at humidified 5 CO295 air

atmosphere At the end of the exposure time the culturemedia were collected and centrifuged to remove cell debrisThe cell culture supernatants were used in ELISA assay100 120583L of each standard and cell culture supernatants wereadded to human EG-VEGF antibody-coated ELISA plateThe wells were covered and incubated in room temperaturefor 25 h with gentle shaking The solution was discardedand washed with 300 120583L of 1x wash solution for each wellfor 4 times After the last wash the 96-well plate wasinverted and blotted against clean paper towels 100120583L ofbiotinylated human EG-VEGF detection antibody was addedto each well and incubated for 1 h at room temperaturewith gentle shaking The solution was discarded and thewashing step was repeated Next 100 120583L of HRP-Streptavidinsolution was added to each well and incubated for 45minat room temperature with gentle shaking The solution wasdiscarded and the washing step was repeated Then 100120583Lof 331015840551015840-tetramethylbenzidine (TMB) One-Step SubstrateReagent was added to each well and incubated for 30minat room temperature in the dark with gentle shaking 50 120583Lstop solution was added to each well and the plate was readat 450 nm immediately by a microplate reader (VarioskanFlash Thermo Scientific Waltham MA USA) The amountsof VEGF secreted from the untreated and treated cellswere determined from the standard curve of optical densityagainst known concentration of VEGF All measurementswere performed in triplicate and data are reported as meanplusmn SD

2213 Statistical Analysis The data obtained were shownas mean plusmn standard deviation (SD) The data were furtheranalysed using analysis of variance (one-way ANOVA fol-lowed by Tukeyrsquos post hoc analysis) using SPSS 230 (SPSS

Inc Chicago IL USA) 119901 value lt 005 was consideredsignificantly statistically different among the groups tested

3 Results and Discussion

31 Preparation ofWater-Soluble CS CShas beenwidely usedfor various purposes especially in biomedical applicationbut it has limited capacity as it is insoluble in water and canonly dissolve in acidic medium In physiological pH 74 CSis insoluble In order to ensure that it can be utilised acrosswide pH range it is important to improve the solubility of CSThus in current study water-soluble CS has been synthesisedfrom LMWCS by mean of enzymatic degradation Katas andAlpar [20] reported that the use of LMW CS can generatesmaller mean particle size as compared to high molecularweight (HMW) CS for the individual CS derivatives Thiswas also supported by Ilyina et al [21] who reported thatpartially hydrolysed CS with LMWhas better water solubilitydue to shorter chain lengths and free amino groups in D-glucosamine unit The nature of the dissolution of CS wasthe degradation of intermacromolecular hydrogen bonds andinterchain hydrogen bonds which modified the structure ofCS reducing its crystallinity and unfolding its molecularchains [22] The LMW CS powder is in yellowish colorwhereas the water-soluble CS produced is in creamy whitecolor powder form

FTIR spectra of water-soluble CS and LMWCS are shownin Figure 1 The broad spectrum observed in LMW CSis 346048 cmminus1 indicating the presence of O-H stretchingbond There is a shift of band measured in water-soluble CSat 341314 cmminus1 also representing O-H stretching bond butthe peak width is narrower C-H stretching can be seen inwater-soluble CS at 292650 cmminus1 The characteristic peaks at


Table 1 Particle Size PDI and zeta potential of CS-GO nanocomposites before and after DsiRNA loading and pectin coating (119899 = 3)

Before DsiRNA loading After DsiRNA loading After pectin coatingPS (nm) plusmn SD PDI plusmn SD ZP (mV) plusmn SD PS (nm) plusmn SD PDI plusmn SD ZP (mV) plusmn SD PS (nm) plusmn SD PDI plusmn SD ZP (mV) plusmn SD3082 plusmn 859 065 plusmn 014 +170 plusmn 14 1252 plusmn 156 045 plusmn 016 +105 plusmn 14 5544 plusmn 1266 047 plusmn 019 minus107 plusmn 30Keynotes PS particle size PDI polydispersity index ZP = zeta potential

GO

3500 3000 2500 2000 1500 1000 6504000(cmminus1)

172025303540455054

T 107885 cmminus1

140037 cmminus1

163740 cmminus1

318601 cmminus1

343448 cmminus1

Figure 2 FTIR spectrum of GO prepared via Hummerrsquos method

163935 cmminus1 and 140064 cmminus1 represent amide 1 (C=O) andamide III (complex vibration of NHCO group) bands of CSrespectively Degree of deacetylation decreases as CS turns tobe water-soluble and this is proven by the presence of peak at156756 cmminus1 due to contribution of amide II band Stretchingof ether (C-O-C) bridge is observed at 115619 cmminus1 In LMWCS C-O-C stretching is assigned at 108123 cmminus1 and thereis a slight shift of the same functional group presence inwater-soluble CS (108024 cmminus1) Difference in FTIR spectraof LMW CS and water-soluble CS suggests that there is acleavage of some glycosidic bonds introduced by hemicel-lulase enzyme This reflects successful production of water-soluble CS from LMWCS

32 Preparation of GO GO was synthesised by using Hum-merrsquos method from graphite prior to preparation of CS-GOnanocomposites In Hummerrsquos method graphite was beingoxidised by an oxidising agent (KMNO

4) and increase in

interplanar spacing between the layers of graphite would laterproduce GO [19] In literature the thickness of single-layerGO was reported in the range of 04 and 17 nm [23] Thisvariation in the thickness of the single graphene layers couldbe attributed to different measurement conditions samplepreparation procedures or other laboratory conditions [24]GO was then characterised by FT-IR analysis to investigatethe bonding interactions in GO Figure 2 depicts that GOhas peak at 343448 cmminus1 and it was attributed to O-Hstretching of H

2O molecules absorbed during GO synthesis

Besides C-H stretching of aromatic grouprsquos peak is shown at318601 cmminus1 In addition since GO has planar structure withbundle of aromatic rings C=C stretching of aromatic groupcan be observed at 140037 cmminus1 The peak at 107885 cmminus1was assigned toC-O stretching of ether functional groupThiswas supported by Paulchamy et al [19] who also reported thatC-O bond was observed at 1081 cmminus1 verifying existence of

oxide functional groups after the oxidation process HenceGO was successfully synthesised from graphite by usingHummerrsquos method

33 Particle Size Surface Charge and PDI of NanocompositesIn current study concentration of CS at 01 wv was usedas it produced nanocomposites with smaller particle sizecompared to higher concentrations of CS (02 and 03 wv)(data was not shown) It was previously reported that lowerconcentrations of CS contributed to smaller particle sizeof nanoparticles owing to their low viscosity that producesefficient gelation procedure [25] In preliminary study for-mulation of 01 wv CS and 025 wv GO had been shownto produce nanocomposites with the most favorable physicalcharacteristics (Supplementary 1 in Supplementary Materialavailable online at httpsdoiorg10115520174298218) Fur-thermore the formulation had shown the desired releaseprofile of DsiRNA because it allowed low release of DsiRNAin the simulated stomach and intestine fluids but with highcumulative release in the simulated colon fluid (Supplemen-tary 2)

Based on these results CS-GO nanocomposites wereprepared from 01 wv CS and 025 wv GO Table 1presents the mean particle size of CS-GO nanocompositesbefore and after DsiRNA loading and pectin coating GOprepared from graphite powder usingHummerrsquos method wasmixed with CS and the mean particle size measured was3082 plusmn 859 nm CS is positively charged due to protonatedamine and it will interact electrostatistically with negativelycharged carboxyl group and phenolic group of GO to formstrong interaction of CS-GO nanocomposites These func-tional groups form intermolecular hydrogen binding andinitiate very fine codispersion in the molecular space [26]

Upon the addition of DsiRNA to CS-GO nanocompos-ites there was a significant reduction in the mean particlesize measured The particle size reduced to 1252 plusmn 156 nm(119901 lt 005 ANOVA followed by Tukeyrsquos post hoc analysis)This was expected to be due to the interaction of oppositelycharged CS and DsiRNA molecules Negatively chargedbackbone of DsiRNA allows electrostatic interaction withcationic CS-GO and this resulted in adsorption of DsiRNA tothe nanocomposites which also condensed DsiRNA throughneutralization into more compact shape Furthermore phos-phate group of DsiRNA further increase negative chargeof this nanocomposite which previously was contributedmainly by GO As a result more negative charges are ableto neutralise the protonated amine group of CS and thiscan be proven by significant particle size reduction of CS-GO-DsiRNA nanocomposites Raja et al [27] reported thatsmaller particle size of the DsiRNA-loaded formulations wasdue to less extended or denser arrangement of CS molecules


CS-GO

Pectin

CS-GO-pectin

2840506070809098

T

3040506070809096

T

2940506070809098

T

3500 3000 2500 2000 1500 1000 6504000(cmminus1)



281133 cmminus1 210476 cmminus1

288930 cmminus1

333066 cmminus1

163708 cmminus128943 cmminus1 177318 cmminus1

332549 cmminus1


234258 cmminus1



334275 cmminus1

Figure 3 FTIR spectra of CS-GO pectin and CS-GO-pectin nanocomposites

Even though CS is a well-known carrier for drug deliveryit has major drawback that is related to its fast dissolutionin the stomach due to solubility in acidic condition [28]To overcome this problem an enteric coating is required toprotect drug from being released in the stomach Pectin hasbeen used to coat the CS-GO-DsiRNA nanocomposites Thenanocomposite had increased particle size after coating withpectin Polyelectrolyte complexes were formed when cationicamino groups on the C2 position of the repeating glucopy-ranose units of CS form electrostatic interaction with thenegatively charged carboxyl groups of pectin [28] The meanparticle size of pectin-coated CS-GO-DsiRNA nanocompos-ites was 5545 plusmn 1246 nm Larger size of nanocompositesafter pectin coating was expected due to the presence ofpectin layer on the surface of CS-GO nanocomposites Onthe other hand PDI value of CS-GO nanocomposites was065 plusmn 014 which indicated that particle size was broadlydistributed Interestingly PDI value of these nanocompositesreduced to 045 plusmn 016 after loading with DsiRNA indicatinghomogeneity of the particles After coating with pectin PDIincreased slightly to 047 plusmn 019 and it was considered withinthe acceptable range of narrow particle size distribution

Zeta potential of CS-GO nanocomposites measured inthis study was +170 plusmn 14mV CS is a very hydrophilicbiopolymer and it has polycationic properties As CS ismixed in aqueous medium the functional group of NH

2

and OH will be protonated to polycationic material Mean-while surface of GO sheets is negatively charged whendispersed in water due to ionisation of carboxylic acid andphenolic hydroxyl groups on the GO sheets [26] The netpositive charge decreases to +105 plusmn 14 after DsiRNA wasloaded Phosphate group of DsiRNA adsorbed onto thesurface of CS-GO nanocomposites was attributed to the

decrease in magnitude of zeta potential The zeta potentialof CS-GO-DsiRNA nanocomposites was further droppedand shifted from positive to negative value (minus107 plusmn 30)after they were coated with pectin This effect was owingto the negatively charged carboxylic acid (COOminus) of pectinthat forms electrostatic interaction with positively chargedamino group (NH3+) of CS [28]This electrostatic interactioncaused pectin to coat on CS-GO nanocomposites and led toreduction in zeta potential [29] Upon addition of DsiRNAfollowed by pectin there were significant changes in zetapotential indicating existence of interactions at each step insynthesising nanocomposites (119901 lt 005 ANOVA followed byTukeyrsquos post hoc analysis)

34 ATR-FTIR Spectroscopic Analysis FTIR peak spectrumcomparison of pectin CS-GO and CS-GO-pectin nanocom-posites provides further verification of successful configu-ration among them as shown in Figure 3 The presence ofoxygen functional groups such as epoxy hydroxyl carbonyland carboxyl groups on the basal plane and edges can be usedto characterise GO Hydroxyl group (-O-H) stretching wasobserved at 334275 cmminus1 332549 cmminus1 and 333066 cmminus1 indifferent formulations consisting of CS-GO pectin and CS-GO-pectin respectively The peaks at 177318 cmminus1 of pectinand 177280 cmminus1 of CS-GO were assigned to C=O stretchingof carbonyl group The peak then shifted to 176715 cmminus1 forCS-GO-pectin nanocomposites and this might be attributedto conjugation that moves absorption to a lower numberThis was further confirmed when Bao et al [16] reportedthat residual carbonyl moieties (C=O) on the basal plane andperiphery of GO can be represented by small absorbancepeak appearing at 1740 cmminus1 The peak then shifted to


Zigzag

Armchair

(a) (b)

(c) (d)

Figure 4 Morphology of GO (a) CS-GO nanocomposites (b) CS-GO-DsiRNA nanocomposites (c) and pectin-coated CS-GO-DsiRNAnanocomposites (d) at magnification of 17000x TEMmicrograph of GO shows ldquoarmchairrdquo and ldquozigzagrdquo configurations

176715 cmminus1 for CS-GO-pectin nanocomposites and thismight be attributed to conjugation thatmoved absorption to alower number Amine group characterised by C-N stretchingin CS-GO and also CS-GO-pectin nanocomposites can beseen clearly at 112892 cmminus1 and 112977 cmminus1 respectively

35 Morphological Analysis The morphology of GO CS-GOCS-GO loadedwithDsiRNA and pectin-coatedCS-GO-DsiRNA nanocomposites was investigated using TEM thatillustrates nanoscale visualisation of an individual particleMost importantly TEM can provide information of bothparticle size and morphology of the nanoparticles GO anal-ysed under an electron microscope appeared to be in layersof lateral dimension sheets with sharp edges (Figure 4(a))It was reported that GO sheets exist with very sharp edgesand flat surfaces [16] that coincided with the GO producedin current study Besides large surface area of GO sheetsis usually utilised as support for growth and stabilisation ofnanoparticles [30]

Upon mixing GO into CS solution irregular shape ofnanocomposites was observed as shown in Figure 4(b)Similar morphology was also reported by Bao et al [16] inwhich CS-GO appear to be coarse and some protuberancescould be seen on the surface which mainly were generatedfrom the polymer wrapping and folding Figure 4(c) shows

formation of small rounded and smooth surface of CS-GO-DsiRNA nanocomposites Small particle size reflectsthat DsiRNA is well adsorbed onto CS-GO nanocompositesHowever after the nanocomposites were coated with pectinthey appeared to fuse with one another and form aggregatesThis could be seen from Figure 4(d) Generally the estimatedsizes for CS-GO CS-GO-DsiRNA and pectin-coated CS-GO-DsiRNA nanocomposites were 26- 28- and 55-foldsmaller than those measured using dynamic light scattering(DLS) The difference in measured particle size using TEMand DLS might be attributed to different principles appliedThe equivalent diameter used in particle size analysis for TEMand DLS is projected area and hydrodynamic diameter ofdiffusion area respectively Despite that DLS provides amoreaccurate measurement as it represents the whole sample

36 DsiRNA Entrapment and Binding Efficiencies and Nano-composites Yield High entrapment efficiency of 926 plusmn 39was measured for CS-GO-DsiRNA (01 wv CS and 025wv GO) nanocomposites besides its high nanocompositesyield (771plusmn46) which is considered suitable for deliveringa therapeutically active dose [31] High entrapment efficiencyplays significant role in ensuring that drug is being deliveredto its target successfully CS-GO sheets have powerful capac-ity to entrap plasmid DNA to form compact complexes [16]Besides graphene nanosheets have wide surface area that


1 2 3 4 5

(1) DNA ladder (10 bp) (2) Naked DsiRNA(3) CS-GO-DsiRNA(4) Pectin-coated DsiRNA CS-GO

nanocomposites (01 wv pectin)(5) Pectin-coated DsiRNA CS-GO

nanocomposites (02 wv pectin)

330 bp

100 bp

40 bp

27 bp

Figure 5 Binding efficiency of CS-GO-DsiRNA nanocomposites

enables them to be functionalised maximally besides havinggreat loading capacity They also allowed encapsulation ofmolecules together for protection purpose and sustainedrelease of loadedmolecules which becomes a trend in currentdelivery applications [3] Pectin was further added as a coat-ing agent to ensure thatDsiRNAwas entrapped on the surfaceof CS-GO sheets and protected from the harsh environmentof the stomach and small intestine Moreover the coatinghas no significant effect on the entrapment efficiency as thepercent was maintained after coating with 01 pectin ( EE= 902 plusmn 54 refer to Supplementary 1) Similar finding wasobserved for the yield of nanocomposites after coating withpectin (yield = 749plusmn14)On the other hand Figure 5 showsthe absence of a trailing band of DsiRNA This indicatedthat DsiRNA has strong binding interaction with CS-GOnanocomposites and it was well protected by pectin layerThese results suggested that pectin coating did not affect thebinding efficiency of anionic DsiRNA with cationic CS-GOnanocomposites or causing premature release of DsiRNAThe results also further supported the finding of highDsiRNAentrapment efficiency for CS-GO DsiRNA was found to beefficiently and tightly bound to CS nanoparticles as reportedby Raja et al [27] However the binding of DsiRNA can bereversed and it will be released when the polymeric matrixdegrades [27] In this situation dissociation of pectin bythe action of pectinase enzyme will also allow the release ofDsiRNA in colon

37 In Vitro Drug Release Two formulations with constantGO wt but different pectin concentrations were testedto demonstrate the influence of pectin on drug release(Figure 6) From the results obtained the amount of DsiRNA

01 pectin02 pectin

pH 12 pH 68 pH 72

0

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

2 3 4 5 6 7 81Time (h)

Figure 6 Release profile of DsiRNA from pectin-enveloped CS-GOnanocomposites of different pectin concentration at different pHs(12 68 and 72) (119899 = 3) CS-GO nanocomposites were preparedusing 01 wv CS and 025 GO

released was slightly higher in formulation coated with 01wv pectin It could be due to the surface of nanocompositethat was coated with lower amount of pectin and hencewould be degraded faster than the formulation with higherpectin amount Nevertheless the difference was insignificant(119901 gt 005 independent 119905-test) In contrast to that a studyconducted by Kushwaha et al [32] demonstrated that the rateof drug released was mainly affected by pectin concentrationused to coat the drug The percentage of cumulative releaseof DsiRNA in SGF (pH 2) for 2 h was approximately 1indicating a slow drug release pattern Moving to SIF (pH68) the percentage increased to approximately 2 Onlysmall amount of DsiRNA was detected in the medium owingto the properties of pectin which is insoluble in acidic pH[32] In contrast to that a remarkable increase in releaseof DsiRNA was observed in SCF (pH 72) (from sim2 tosim52) The maximum release was achieved after 3 h in SCFand the percent measured was 656 and 639 for 01and 02 wv pectin respectively The exposure to SCFand pectinase enzyme causes pectin to be degraded thusreleasing greater amount ofDsiRNA Inside the human colonnumerous bacteria secrete enzymes that break down thepolymer backbone resulting in decrease in its molecularweight thus losing their mechanical strength and ending upwith the release of drug entity Besides pectin CS is alsoable to shield drugs from harsh stomach and small intestineenvironment [33] A successful colon drug delivery requiresthe triggering mechanism in the delivery system that onlyresponds to the physiological conditions particular to thecolon [34] The in vitro release study showed that pectinwas not much degraded in the stomach and small intestinebut was susceptible to enzymatic breakdown in the colon bythe natural microflora Therefore it is a suitable candidateto deliver and target drugs to the colon A high release rateof DsiRNA in SCF with the presence of pectinolytic enzyme


Caco-2CCD-18CO

Con

trol

CS GO

Pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

020406080

100120

Cel

l via

bilit

y (

)

Figure 7 Cytotoxicity effect of Caco-2 and CCD-18CO cellsexposed to nanocomposites and their parent compounds at 24 hafter incubation (119899 = 3)

pointed out the ability of pectin coating to hinder prematurerelease of drug in upper GI tract

38 Cell Growth Inhibition Effect and VEGF DownregulationThe cytotoxic effects of nanocomposites developed in thisstudy were determined in normal and cancerous cell linesThe cytotoxic effects of CS-GO-DsiRNA nanocompositescoated with pectin and its individual component were inves-tigated against human colon adenocarcinoma cells (Caco-2cells) and human colon normal cells (CCD-18CO cells) Thepercentage of cell viability of nanocomposites in Caco-2 andCCD-18CO is shown in Figure 7 CS-GO-DsiRNA nanocom-posites coated with pectin caused significant reduction in cellviability towards Caco-2 cells in which the percent reducedto almost half from the untreated cells (119901 lt 005 ANOVAfollowed by Tukeyrsquos post hoc analysis) In normal cell linethe cell viability recorded was 85 and it was higher than incancer cells

Moreover 33 loss of cell viability was measured afterCaco-2 cells were treated with pectin followed by 13 lossby GO Apart from being utilised as specific drug carrierto the colon pectin also has been studied for its antitumoractivity According toGlinsky andRaz [18] antitumor activityof pectin was attributed to abundant 120573-galactose present inmodified citrus pectin (MCP) The primary mechanism ofaction for MCP is by inhibiting a 120573-galactoside binding pro-tein galectin-3 (Gal-3) which is responsible for angiogenicactivity Interestingly induction of pectin in healthy cellsCCD-18CO did not display any reduction in cell viabilitySimilar finding was reported elsewhere [2] in which pectinacts more destructively against Caco-2 colon cancer cellsbut not against healthy VERO cells The selective killingeffect towards cancer cells makes it a precious naturalpolysaccharide In addition GO used in this study has alsobeen shown to enhance cytotoxicity effect displayed by finalformulation Recent study claimed that negatively chargedoxygen group of GO can form electrostatic interaction withpositively charged lipids present on cell membranes thusdestroying the cell membrane [30] In contrast to that GOdid not cause decreased cell viability in normal colon cellsThis might be due to low amount of GO which was used

CS-G

O-

pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

DsiR

NA

Neg

ativ

eco

ntro

l05

101520253035404550

Con

cent

ratio

n of

VEG

F (p

gm

L)

Figure 8 Human EG-VEGF level of Caco-2 cells treated withpectin-coated CS-GO CS-GO-DsiRNA and pectin-coated CS-GO-DsiRNA nanocomposites and naked DsiRNA and nontreated cells(negative control) (119899 = 3)

in this assay (250120583gmL) Insignificant loss of peritonealmacrophages viability was also reported previously whenthe cells were exposed to GO at low concentration [30] Inaddition both cell lines treated with uncoated formulationdid not show any decrease in cell viability The absence ofpectin that is responsible for enhancing antitumor activityfurther eliminates the inhibitory effect of DsiRNA and GO

In this study DsiRNA targeting VEGF gene was usedas the gene is excessively expressed in Caco-2 cells It wasreported that increased metastasis risk in colon cancer cor-relates well with VEGF expression [35] VEGF is most com-monly associated with tumor angiogenesis which involvedtumor growth andmetastasis in human colon cancer [36] andit is supported elsewhere [37] The VEGF concentration levelin Caco-2 colon cancer cells was determined by ELISA afterbeing treatedwith pectin-envelopedCS-GODsiRNA-loadedCS-GO and pectin-enveloped CS-GO-DsiRNA nanocom-posites and naked DsiRNA for 24 h From Figure 8 therewas no significant change in VEGF concentration levelobserved after Caco-2 colon cancer cells were treated withpectin-envelopedCS-GO (blank nanocomposites) and nakedDsiRNA for 24 h as compared to negative control (untreatedcells) indicating the absence of a gene silencing effectHowever a lower VEGF concentration level was significantly(119901 value lt 005) measured for the cells treated with pectin-enveloped DsiRNA-loaded CS-GO nanocomposites after 24incubation as shown in Figure 8 This finding demonstratedthat pectin was able to protect and facilitate DsiRNA toaccumulate at and act on the site of actionwhich subsequentlyresulted in significant decrease of VEGF level [38]

4 Conclusions

In summary pectin-coated CS-GO-DsiRNA nanocompos-ites were successfully developed via electrostatic interac-tion FTIR analysis further confirmed successful synthe-sis of GO and CS-GO-pectin nanocomposites The resultsrevealed significant difference in particle size at each stage


of nanocomposites preparation which signifies productionof CS-GO-DsiRNA-pectin nanocomposites Moreover highentrapment efficiency of these nanocomposites indicated thatDsiRNA was effectively adsorbed onto CS-GO carrier CS-GO-DsiRNA nanocomposites coated with pectin selectivelykilled cancer cells which could be a promising therapeuticagent for cancer treatment Moreover VEGF concentrationlevel significantly decreased after 24 h incubation with thepectin-enveloped CS-GO-DsiRNA nanocomposites thus itindicated the ability of nanocomposites to deliver DsiRNAeffectively into the cells and subsequently cause gene silencingon the target gene Further studies in animalmodel in vivo arenecessary to study the safety and efficacy of CS-GO-DsiRNAnanocomposites coated with pectin

Conflicts of Interest

The authors report no conflicts of interest in this work

Acknowledgments

The authors greatly acknowledge Universiti KebangsaanMalaysia (UKM) (GUP-2014-061) for supporting and provid-ing resources to complete this research project

References

[1] International Agency for Research on Cancer ldquoEstimated can-cer incidence mortality and prevalence worldwide in globo-canrdquo World Health Organization 2012 httpwwwiarcfr

[2] E A M S Almeida S P Facchi A F Martins et al ldquoSynthesisand characterization of pectin derivative with antitumor prop-erty against Caco-2 colon cancer cellsrdquo Carbohydrate Polymersvol 115 pp 139ndash145 2015

[3] M S Draz B A Fang P Zhang et al ldquoNanoparticle-mediatedsystemic delivery of siRNA for treatment of cancers and viralinfectionsrdquoTheranostics vol 4 no 9 pp 872ndash892 2014

[4] S Kulisch T Esch C Whitman-Guliaev and T RubioldquoDicer-substrate siRNA technology advances in siRNA designsimprove gene-specific silencingrdquo BioRadiations vol 120 pp 1ndash6 2006

[5] M Amarzguioui and J J Rossi ldquoPrinciples of Dicer substrate(D-siRNA) design and functionrdquo RNAi Design and Applicationpp 3ndash10 2008

[6] M Izquierdo ldquoShort interfering RNAs as a tool for cancer genetherapyrdquo Cancer Gene Therapy vol 12 no 3 pp 217ndash227 2005

[7] N Yamagishi S Teshima-Kondo K Masuda et al ldquoChronicinhibition of tumor cell-derived VEGF enhances the malignantphenotype of colorectal cancer cellsrdquo BMC Cancer vol 13article 229 2013

[8] A Ahluwalia M K Jones S Szabo and A S TarnawskildquoAberrant ectopic expression of VEGF and VEGF receptors1 and 2 in malignant colonic epithelial cells Implications forthese cells growth via an autocrine mechanismrdquo Biochemicaland Biophysical Research Communications vol 437 no 4 pp515ndash520 2013

[9] J Conde AAmbrosone YHernandez et al ldquo15 years on siRNAdelivery beyond the state-of-the-art on inorganic nanoparticlesfor RNAi therapeuticsrdquo Nano Today vol 10 no 4 pp 421ndash4502015

[10] R Kanasty J R Dorkin A Vegas and D Anderson ldquoDeliverymaterials for siRNA therapeuticsrdquo Nature Materials vol 12 pp967ndash977 2013

[11] A Falamarzian X-B Xiong H Uludag and A LavasanifarldquoPolymeric micelles for siRNA deliveryrdquo Journal of Drug Deliv-ery Science and Technology vol 22 no 1 pp 43ndash54 2012

[12] R K Tekade M Tekade P Kesharwani and A DrsquoEmanueleldquoRNAi-combined nano-chemotherapeutics to tackle resistanttumorsrdquo Drug Discovery Today vol 21 no 11 pp 1761ndash17742016

[13] Y Ping C D Liu G P Tang et al ldquoFunctionalization of chitos-an via atom transfer radical polymerization for gene deliveryrdquoAdvanced Functional Materials vol 20 pp 3106ndash3116 2010

[14] V R Sinha and R Kumria ldquoPolysaccharides in colon-specificdrug deliveryrdquo International Journal of Pharmaceutics vol 224no 1-2 pp 19ndash38 2001

[15] F Liu and L Huang ldquoDevelopment of non-viral vectors forsystemic gene deliveryrdquo Journal of Controlled Release vol 78no 1 pp 259ndash266 2002

[16] HQ Bao Y Z Pan Y Ping et al ldquoChitosan-functionalized gra-phene oxide as a nanocarrier for drug and gene deliveryrdquo Smallvol 7 no 11 pp 1569ndash1578 2011

[17] E Bernstein A A Caudy S M Hammond and G J HannonldquoRole for a bidentate ribonuclease in the initiation step of RNAinterferencerdquo Nature vol 409 no 6818 pp 363ndash366 2001

[18] VVGlinsky andARaz ldquoModified citrus pectin anti-metastaticproperties one bullet multiple targetsrdquo Carbohydrate Researchvol 344 no 14 pp 1788ndash1791 2009

[19] B Paulchamy G Arthi and B Lignesh ldquoA simple approach tostepwise synthesis of graphene oxide nanomaterialrdquo Journal ofNanomedicine amp Nanotechnology vol 6 no 1 article 1 2015

[20] H Katas andHOAlpar ldquoDevelopment and characterisation ofchitosannanoparticles for siRNAdeliveryrdquo Journal of ControlledRelease vol 115 no 2 pp 216ndash225 2006

[21] A V Ilyina N Y Tatarinova and V P Varlamov ldquoThe prepara-tion of low-molecular-weight chitosan using chitinolytic com-plex from streptomyces kurssanoviirdquo Process Biochemistry vol34 no 9 pp 875ndash878 1999

[22] S Lu X Song D Cao Y Chen and K Yao ldquoPreparation ofwater-soluble chitosanrdquo Journal of Applied Polymer Science vol91 no 6 pp 3497ndash3503 2004

[23] C J Shearer AD Slattery A J Stapleton J G Shapter andC TGibson ldquoAccurate thickness measurement of graphenerdquo Nano-technology vol 27 no 12 Article ID 125704 2016

[24] P Nemes-Incze Z Osvath K Kamaras and L P Biro ldquoAnoma-lies in thickness measurements of graphene and few layergraphite crystals by tapping mode atomic force microscopyrdquoCarbon vol 46 no 11 pp 1435ndash1442 2008

[25] S S Omar Zaki H Katas and Z A Hamid ldquoLineage-relatedand particle size-dependent cytotoxicity of chitosan nanopar-ticles on mouse bone marrow-derived hematopoietic stem andprogenitor cellsrdquo Food and Chemical Toxicology vol 85 pp 31ndash44 2015

[26] N I Syuhada N M Huang S Vijay Kumar et al ldquoEnhancedmechanical properties of chitosanedta-go nanocompositesthin filmsrdquo Sains Malaysiana vol 43 no 6 pp 851ndash859 2014

[27] M A G Raja H Katas Z Abd Hamid and N A RazalildquoPhysicochemical properties and in vitro cytotoxicity studiesof chitosan as a potential carrier for dicer-substrate siRNArdquoJournal of Nanomaterials vol 2013 Article ID 653892 10 pages2013


[28] J H Hamman ldquoChitosan based polyelectrolyte complexes aspotential carrier materials in drug delivery systemsrdquo MarineDrugs vol 8 no 4 pp 1305ndash1322 2010

[29] R K Dutta and S Sahu ldquoDevelopment of diclofenac sodiumloadedmagnetic nanocarriers of pectin interactedwith chitosanfor targeted and sustained drug deliveryrdquo Colloids and SurfacesB Biointerfaces vol 97 pp 19ndash26 2012

[30] L A V de Luna A C M Moraes S R Consonni et al ldquoCom-parative in vitro toxicity of a graphene oxide-silver nanocom-posite and the pristine counterparts toward macrophagesrdquoJournal of Nanobiotechnology vol 14 no 1 article 1 2016

[31] B T AL-Quadeib M A Radwan L Siller B Horrocks and MC Wright ldquoStealth amphotericin B nanoparticles for oral drugdelivery in vitro optimizationrdquo Saudi Pharmaceutical Journalvol 23 no 3 pp 290ndash302 2015

[32] P Kushwaha S Fareed S Nanda and A Mishra ldquoDesign ampfabrication of tramadol HCL loaded multiparticulate colontargeted drug delivery systemrdquo Journal of Chemical and Phar-maceutical Research vol 3 no 5 pp 584ndash595 2011

[33] A K Philip and B Philip ldquoColon targeted drug delivery sys-tems a review on primary and novel approachesrdquo Oman Med-ical Journal vol 25 no 2 pp 70ndash78 2010

[34] L Yang J S Chu and J A Fix ldquoColon-specific drug deliverynew approaches and in vitroin vivo evaluationrdquo InternationalJournal of Pharmaceutics vol 235 no 1-2 pp 1ndash15 2002

[35] Y Takahashi Y Kitadai C D Bucana K R Cleary and LM Ellis ldquoExpression of vascular endothelial growth factor andits receptor KDR correlates with vascularity metastasis andproliferation of human colon cancerrdquo Cancer Research vol 55no 18 pp 3964ndash3968 1995

[36] L M Ellis Y Takahashi W Liu and R M Shaheen ldquoVascularendothelial growth factor in human colon cancer biology andtherapeutic implicationsrdquo Oncologist vol 5 no 1 pp 11ndash152000

[37] L F Brown B Berse RW Jackman et al ldquoExpression of vascu-lar permeability factor (vascular endothelial growth factor) andits receptors in adenocarcinomas of the gastrointestinal tractrdquoCancer Research vol 53 no 19 pp 4727ndash4735 1993

[38] M A G Raja H Katas and Z Abd Hamid ldquoPenetration andsilencing activity of VEGF dicer substrate siRNA vectorized bychitosan nanoparticles in monolayer culture and a solid tumormodel in vitro for potential application in tumor therapyrdquoJournal ofNanomaterials vol 2016Article ID7201204 16 pages2016

Submit your manuscripts athttpswwwhindawicom

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NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

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CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of



colonic epithelial cells do not produce VEGF or expressits receptors However in colon cancer cells VEGF and itsreceptors are expressed and it promotes colon cancer cell pro-liferation directly Therefore DsiRNA targeted against VEGFgene was used to kill cancer cells by silencing overexpressionof its protein [6]

In a strategy to activate RNAi pathway siRNA moleculesneed to be delivered to the interior of target cells and tobe incorporated into the RNAi machinery However siRNAscannot cross the lipid bilayers of the cell membrane readilyas they have anionic backbone and hydrophilic properties[9] Besides siRNAs are facing some other obstacles toreach their site of action The obstacles include undergoingdegradation by nucleases short half-life due to the uptake bymononuclear phagocyte system off-target effect and rapidrenal clearance [10] As a result of these obstacles the needto develop carriers for siRNA is paramount to deliver it tothe site of interest efficiently An ideal delivery carrier forsiRNA should possess properties such as being nontoxic andnonimmunogenic being able to condense siRNA efficientlyprotecting the integrity of its content before reaching thetarget site having the ability to evade rapid eliminationfrom blood circulation and internalizing and dissociatingin intracellular compartments of the target cells to releasethe adequate amount of siRNA thereby exposing siRNA tomRNA [11]

Nanomedicine-based carriers have been extensivelyexplored as potential candidates to guide siRNAs directlyto the target cells Nanocarriers offer several advantagesthat include providing sustained release and prolonging thetime of siRNA at the target site Nanocarriers also are ableto penetrate capillaries hence they can accumulate at thesite of interest The nanotechnology approach could alsoprotect siRNA against RNase degradation and avoid off-target effects [12] In nanomedicine polycationic polymerssuch as chitosan (CS) were used to condense siRNA intonanoparticles to facilitate cellular uptake [13]

CS in combination with pectin has been widely exploredfor their benefit as colon-specific drug delivery system [14]Apart from being biocompatible less toxic nonimmuno-genic and degradable by enzymes [15] CS can also be used ingene delivery system Tremendous studies on graphene oxide(GO) have made it the gold approach in biomedicine appli-cations GO is a highly oxidised graphene form with oxygenfunctional groups on its surface GO has been combined withCS to form a nanocarrier with better aqueous solubility andbiocompatibility In addition it can exhibit powerful loadingcapacity besides having the ability to condense plasmid DNAinto stable nanosized complexes [16] In an attempt to ensurethat CS-GO-DsiRNA nanocomposites would be deliveredto the target site successfully pectin was used to coat thenanocomposites due to its unique property that will bedegraded by colonic microflora This property enables it tobe used as a specific drug carrier to the colon [17] Besides italso displays antitumor activity [18] that can further enhancegrowth inhibition or cytotoxic effect in colon cancer cells

In this study water-soluble CS was synthesised fromlow molecular weight (LMW) CS to enhance its solubilitywhile GO was synthesised using Hummerrsquos method from

graphite flakes Oxidation of graphite using oxidising agentwill increase interplanar spacing between graphite layersproducing GO [19] Later DsiRNA was adsorbed onto CS-GO nanocomposites Pectin was utilised as coating agent tohinder premature DsiRNA release in gastric and intestinalfluids To confirm successful synthesis of CS-GO-DsiRNA-pectin nanocomposites and their ability to load DsiRNAphysical characterization structural analysis and in vitrodrug release study were conducted The nanocompositeswere also tested for their in vitro cytotoxic effect in normaland cancerous cell lines Finally silencing of VEGF gene byDsiRNA was determined using ELISA

2 Materials and Methods

21 Materials DsiRNA targeting VEGF gene [51015840-rGrGrArGrUrA rCrCrC rUrGrA rUrGrA rGrArU rCr- GrA rGrUAC-31015840 (sense strand) and 51015840-rGrUrA rCrUrC rGrArU rCrUrCrArUrC rArGrG rGrUrA rCrUrC rCrCrA-31015840 (antisensestrand)] was purchased from Integrated DNA TechnologiesUSA Low molecular weight (LMW) CS of 190 kDa with75ndash85 degree of deacetylation was purchased from Sigma-Aldrich (Ireland) Acetic acid and hydrochloric acid wereobtained from RampMChemicals (UK) Hemicellulase enzymefrom Aspergillus niger and pectin with 55ndash70 degree ofesterification were obtained from Sigma-Aldrich USA 10 bpDNA ladder was obtained from Invitrogen Corporation(Carlsbad) while sodium hydroxide was obtained fromJIT (Baker Sweden) Deionised water was produced inthe laboratory using Millipore Milli-Q Phosphate-bufferedsaline (PBS) and dimethyl sulfoxide (DMSO) were obtainedfrom Sigma-Aldrich (St Louis MO) Fetal bovine serum(FBS) Dulbeccorsquos Modified Eaglersquos Medium (DMEM) HighGlucose Eaglersquos Minimum Essential Medium (EMEM)penicillin-streptomycin MTT reagent and trypsin-EDTAwere purchased from Invitrogen (Carlsbad CA) Caco-2 cell line (colorectal adenocarcinoma cell derived fromHomo sapiens human) and CCD-18CO (normal colon cellderived from Homo sapiens human) were obtained from theAmerican Type Culture Collection (ATCC Manassas VA)HumanVEGFELISA kit was purchased from Sigma-AldrichUSA Acetic acid glacial potassium dihydrogen phosphatedipotassium hydrogen phosphate sodium chloride (NaCl)and sodium hydroxide (NaOH) were purchased from RampMChemicals (UK) All chemicals used were of analytical gradeand were used as received

22 Methods

221 Synthesis of Water-Soluble CS 1 g of LMW CS wasdissolved in 2 vv of acetic acid using amagnetic stirrerTheCS solution was adjusted to pH 45 by adding few drops ofNaOH and left overnight A 20 wv hemicellulase enzymesolution was added to CS solution and the mixture was leftin the water bath at 40∘C for 6 h pH of the mixture wasthen adjusted to 55 by adding few drops of NaOH solutionAfter that the mixture was boiled for 10min to denature theenzyme The upper layer of solution containing enzyme wasremoved manually by using a spatula until a concentrated




3were mixed















following equation

119862 =119860

120576119871 (1)




times 100 (2)




times 100

(3)











times 100

(4)


295 air




times 100 (5)





2 After 24 h



LMW CS

WS CS

4000 3500 3000 2500(cmminus1)

2000 1500 1000 500400

384042

4644

48

5250

5654

58606265

T

454850525456586062646668707276

T

102327 cmminus1


140418 cmminus1

156756 cmminus1

292650 cmminus1

341314 cmminus1

108123 cmminus1


346048 cmminus1












GO

3500 3000 2500 2000 1500 1000 6504000(cmminus1)

172025303540455054

T 107885 cmminus1

140037 cmminus1

163740 cmminus1

318601 cmminus1

343448 cmminus1




4) and increase in









CS-GO

Pectin

CS-GO-pectin

2840506070809098

T

3040506070809096

T

2940506070809098

T

3500 3000 2500 2000 1500 1000 6504000(cmminus1)




288930 cmminus1

333066 cmminus1


332549 cmminus1


234258 cmminus1



334275 cmminus1




2





Zigzag

Armchair

(a) (b)

(c) (d)








1 2 3 4 5




330 bp

100 bp

40 bp

27 bp




01 pectin02 pectin

pH 12 pH 68 pH 72

0

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

2 3 4 5 6 7 81Time (h)




Caco-2CCD-18CO

Con

trol

CS GO

Pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

020406080

100120

Cel

l via

bilit

y (

)





CS-G

O-

pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

DsiR

NA

Neg

ativ

eco

ntro

l05

101520253035404550

Con

cent

ratio

n of

VEG

F (p

gm

L)




4 Conclusions






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of





3were mixed















following equation

119862 =119860

120576119871 (1)




times 100 (2)




times 100

(3)











times 100

(4)


295 air




times 100 (5)





2 After 24 h



LMW CS

WS CS

4000 3500 3000 2500(cmminus1)

2000 1500 1000 500400

384042

4644

48

5250

5654

58606265

T

454850525456586062646668707276

T

102327 cmminus1


140418 cmminus1

156756 cmminus1

292650 cmminus1

341314 cmminus1

108123 cmminus1


346048 cmminus1












GO

3500 3000 2500 2000 1500 1000 6504000(cmminus1)

172025303540455054

T 107885 cmminus1

140037 cmminus1

163740 cmminus1

318601 cmminus1

343448 cmminus1




4) and increase in









CS-GO

Pectin

CS-GO-pectin

2840506070809098

T

3040506070809096

T

2940506070809098

T

3500 3000 2500 2000 1500 1000 6504000(cmminus1)




288930 cmminus1

333066 cmminus1


332549 cmminus1


234258 cmminus1



334275 cmminus1




2





Zigzag

Armchair

(a) (b)

(c) (d)








1 2 3 4 5




330 bp

100 bp

40 bp

27 bp




01 pectin02 pectin

pH 12 pH 68 pH 72

0

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

2 3 4 5 6 7 81Time (h)




Caco-2CCD-18CO

Con

trol

CS GO

Pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

020406080

100120

Cel

l via

bilit

y (

)





CS-G

O-

pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

DsiR

NA

Neg

ativ

eco

ntro

l05

101520253035404550

Con

cent

ratio

n of

VEG

F (p

gm

L)




4 Conclusions






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of











times 100

(4)


295 air




times 100 (5)





2 After 24 h



LMW CS

WS CS

4000 3500 3000 2500(cmminus1)

2000 1500 1000 500400

384042

4644

48

5250

5654

58606265

T

454850525456586062646668707276

T

102327 cmminus1


140418 cmminus1

156756 cmminus1

292650 cmminus1

341314 cmminus1

108123 cmminus1


346048 cmminus1












GO

3500 3000 2500 2000 1500 1000 6504000(cmminus1)

172025303540455054

T 107885 cmminus1

140037 cmminus1

163740 cmminus1

318601 cmminus1

343448 cmminus1




4) and increase in









CS-GO

Pectin

CS-GO-pectin

2840506070809098

T

3040506070809096

T

2940506070809098

T

3500 3000 2500 2000 1500 1000 6504000(cmminus1)




288930 cmminus1

333066 cmminus1


332549 cmminus1


234258 cmminus1



334275 cmminus1




2





Zigzag

Armchair

(a) (b)

(c) (d)








1 2 3 4 5




330 bp

100 bp

40 bp

27 bp




01 pectin02 pectin

pH 12 pH 68 pH 72

0

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

2 3 4 5 6 7 81Time (h)




Caco-2CCD-18CO

Con

trol

CS GO

Pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

020406080

100120

Cel

l via

bilit

y (

)





CS-G

O-

pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

DsiR

NA

Neg

ativ

eco

ntro

l05

101520253035404550

Con

cent

ratio

n of

VEG

F (p

gm

L)




4 Conclusions






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of



LMW CS

WS CS

4000 3500 3000 2500(cmminus1)

2000 1500 1000 500400

384042

4644

48

5250

5654

58606265

T

454850525456586062646668707276

T

102327 cmminus1


140418 cmminus1

156756 cmminus1

292650 cmminus1

341314 cmminus1

108123 cmminus1


346048 cmminus1












GO

3500 3000 2500 2000 1500 1000 6504000(cmminus1)

172025303540455054

T 107885 cmminus1

140037 cmminus1

163740 cmminus1

318601 cmminus1

343448 cmminus1




4) and increase in









CS-GO

Pectin

CS-GO-pectin

2840506070809098

T

3040506070809096

T

2940506070809098

T

3500 3000 2500 2000 1500 1000 6504000(cmminus1)




288930 cmminus1

333066 cmminus1


332549 cmminus1


234258 cmminus1



334275 cmminus1




2





Zigzag

Armchair

(a) (b)

(c) (d)








1 2 3 4 5




330 bp

100 bp

40 bp

27 bp




01 pectin02 pectin

pH 12 pH 68 pH 72

0

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

2 3 4 5 6 7 81Time (h)




Caco-2CCD-18CO

Con

trol

CS GO

Pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

020406080

100120

Cel

l via

bilit

y (

)





CS-G

O-

pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

DsiR

NA

Neg

ativ

eco

ntro

l05

101520253035404550

Con

cent

ratio

n of

VEG

F (p

gm

L)




4 Conclusions






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of





GO

3500 3000 2500 2000 1500 1000 6504000(cmminus1)

172025303540455054

T 107885 cmminus1

140037 cmminus1

163740 cmminus1

318601 cmminus1

343448 cmminus1




4) and increase in









CS-GO

Pectin

CS-GO-pectin

2840506070809098

T

3040506070809096

T

2940506070809098

T

3500 3000 2500 2000 1500 1000 6504000(cmminus1)




288930 cmminus1

333066 cmminus1


332549 cmminus1


234258 cmminus1



334275 cmminus1




2





Zigzag

Armchair

(a) (b)

(c) (d)








1 2 3 4 5




330 bp

100 bp

40 bp

27 bp




01 pectin02 pectin

pH 12 pH 68 pH 72

0

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

2 3 4 5 6 7 81Time (h)




Caco-2CCD-18CO

Con

trol

CS GO

Pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

020406080

100120

Cel

l via

bilit

y (

)





CS-G

O-

pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

DsiR

NA

Neg

ativ

eco

ntro

l05

101520253035404550

Con

cent

ratio

n of

VEG

F (p

gm

L)




4 Conclusions






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of



CS-GO

Pectin

CS-GO-pectin

2840506070809098

T

3040506070809096

T

2940506070809098

T

3500 3000 2500 2000 1500 1000 6504000(cmminus1)




288930 cmminus1

333066 cmminus1


332549 cmminus1


234258 cmminus1



334275 cmminus1




2





Zigzag

Armchair

(a) (b)

(c) (d)








1 2 3 4 5




330 bp

100 bp

40 bp

27 bp




01 pectin02 pectin

pH 12 pH 68 pH 72

0

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

2 3 4 5 6 7 81Time (h)




Caco-2CCD-18CO

Con

trol

CS GO

Pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

020406080

100120

Cel

l via

bilit

y (

)





CS-G

O-

pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

DsiR

NA

Neg

ativ

eco

ntro

l05

101520253035404550

Con

cent

ratio

n of

VEG

F (p

gm

L)




4 Conclusions






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of



Zigzag

Armchair

(a) (b)

(c) (d)








1 2 3 4 5




330 bp

100 bp

40 bp

27 bp




01 pectin02 pectin

pH 12 pH 68 pH 72

0

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

2 3 4 5 6 7 81Time (h)




Caco-2CCD-18CO

Con

trol

CS GO

Pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

020406080

100120

Cel

l via

bilit

y (

)





CS-G

O-

pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

DsiR

NA

Neg

ativ

eco

ntro

l05

101520253035404550

Con

cent

ratio

n of

VEG

F (p

gm

L)




4 Conclusions






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of



1 2 3 4 5




330 bp

100 bp

40 bp

27 bp




01 pectin02 pectin

pH 12 pH 68 pH 72

0

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

2 3 4 5 6 7 81Time (h)




Caco-2CCD-18CO

Con

trol

CS GO

Pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

020406080

100120

Cel

l via

bilit

y (

)





CS-G

O-

pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

DsiR

NA

Neg

ativ

eco

ntro

l05

101520253035404550

Con

cent

ratio

n of

VEG

F (p

gm

L)




4 Conclusions






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of



Caco-2CCD-18CO

Con

trol

CS GO

Pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

020406080

100120

Cel

l via

bilit

y (

)





CS-G

O-

pect

in

CS-G

O-

DsiR

NA

CS-G

O-

DsiR

NA-

pect

in

DsiR

NA

Neg

ativ

eco

ntro

l05

101520253035404550

Con

cent

ratio

n of

VEG

F (p

gm

L)




4 Conclusions






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of






Acknowledgments


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of





















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of









CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Documents

Cell Growth Inhibition Effect of DsiRNA Vectorised by