Research Article Mannosylated Chitosan Nanoparticles for...

Research ArticleMannosylated Chitosan Nanoparticles for Delivery ofAntisense Oligonucleotides for Macrophage Targeting

Gyati Shilakari Asthana12 Abhay Asthana1 Dharm Veer Kohli2 and Suresh Prasad Vyas2

1 Maharishi Markandeshwar College of Pharmacy Maharishi Markandeshwar University Mullana Ambala Haryana 133207 India2Department of Pharmaceutical Sciences Dr Hari Singh Gour Vishwa Vidyala Sagar Madhya Pradesh 470003 India

Correspondence should be addressed to Dharm Veer Kohli drdvkohalirediffmailcom

Received 28 February 2014 Accepted 2 June 2014 Published 26 June 2014

Academic Editor Kok Tat Tan

Copyright copy 2014 Gyati Shilakari Asthana et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The therapeutic potential of antisense oligonucleotides (ASODN) is primarily dependent upon its safe and efficient delivery tospecific cells overcoming degradation andmaximizing cellular uptake in vivoThe present study focuses on designingmannosylatedlow molecular weight (LMW) chitosan nanoconstructs for safe ODNs delivery by macrophage targeting Mannose groups werecoupled with LMW chitosan and characterized spectroscopically Mannosylated chitosan ODN nanoparticles (MCHODN NPs)were formulated by self-assembled method using various 119873119875 ratio (moles of amine groups of MCH to phosphate moieties ofODNs) and characterized for gel retardation assay physicochemical characteristics cytotoxicity and transfection efficiency andantisense assay Complete complexation of MCHODN was achieved at charge ratio of 11 and above On increasing the119873119875 ratioof MCHODN particle size of the NPs decreased whereas zeta potential (ZV) increased MCHODN NPs displayed much highertransfection efficiency into Raw 2647 cells (bears mannose receptors) than Hela cells and no significant toxicity was observedat all MCH concentrations Antisense assay revealed that reduction in lipopolysaccharide (LPS) induced serum TNF-120572 is due toantisense activity of TJU-2755 ODN (sequence complementary to 31015840-UTR of TNF-120572) These results suggest that MCHODN NPsare acceptable choice to improve transfection efficiency in vitro and in vivo

1 Introduction

Gene therapy is a potential approach to cure or preventdiseases through gene expression before the transcriptionstage [1] It includes approaches based on utilization ofdeoxyribonucleic acid (DNAs) oligodeoxyribonucleotides(ODNs) small interference RNAs (SiRNA) and so forthfor treatment and mitigation of diseases Oligonucleotidesbased therapeutic techniques have attracted much attentionbecause they provide a rational way to design antisenseoligonucleotides (As-ODN) decoy oligonucleotides that bindto DNA-binding regulatory proteins and siRNAmiRNAthat suppress specific gene expressions ODNs with basesequences complementary to a specific RNA have offeredthe exciting potential to selectively modulate the expressionof an individual gene and thus possess the potential foractivity in the treatment of viral infections or cancer ODNsinclude a new generation of antiviral antitumoral and

anti-inflammatory agents in various categories [2ndash4] As-ODNs are anionic large molecular weight (5ndash10 kDa) 10ndash30nucleotide bases sequence of short single strands of DNARNA their analogs molecules designed to modulate geneexpression [5] and possessing high hydrophilicity [6] Forantisense applications ODNs interact and form a duplexwith their complementary target mRNAs or pre-mRNAsthrough Watson-Crick base pairing in a sequence-specificmanner and then inhibit their translation [7 8]Therefore thegenetic code in the RNA cannot be read which consequentlyinhibits production of the disease-causing protein [1] At themolecular level severalmechanisms of antisense actions havebeen proposed for antisense drugs These include inhibitionof transcription inhibition of splicing and inhibition ofmRNA maturation [9] Among them activation of RNaseH is by far one of the most recognized theories accepted bymany researchers After entering cells ODNs hybridize to thetarget mRNA and form a sense-antisense RNA DNA duplex

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 526391 17 pageshttpdxdoiorg1011552014526391

2 BioMed Research International

For therapeutic application antisense technology promisesgreater advantages over drugs currently on the market byoffering new types of drugs that are easy to design andhave a very high molecular target selectivity and efficacyinhibiting a specific gene expression and an expected lowtoxicity due to metabolism to natural nucleotide componentsby the endogenous systems [5 10] Vitravene is only ODNbased therapeutic (antisense ODN) approved by FDA andseveral others are in Phase 3 clinical trials However ODNstherapeutics have been associated with multiple obstaclesthat is poor physiological stability and cellular uptake [1112] inadequate intracellular ODNs concentration [13 14]inability to target specific cell [14 15] lack of tissue specificity[16] and nuclease degradation [14 17] Thus intracellulardelivery and therapeutic applications in diseases of ODN-based therapeutics have been compromised Although vari-ous chemicalmodifications ofODNs can be used to overcomethese problems [18] they offer other limitations such aslow binding affinity nonsequences specific biological effectstoxicity and acute homeostatic changes after in vivo adminis-tration [19ndash23] Therefore development of effective deliverysystems that are capable of protecting and efficiently deliverODNs intracellularly in target cells becomes essential toexploit the promising applications offered by successful ODNdelivery Among the gene delivery vectors nonviral vectorsare preferred due to its ease of synthesis low immunogenicityand unrestricted gene materials size in addition to potentialbenefits in terms of safety [24ndash27]

Nanoparticles (NPs) were introduced as nonviral genevectors to solve issues related to ODNs delivery [28ndash36]Other carriers such as microspheres matrices complexeswith polycations and starburst dendrimers have beenrecently investigated [10 37ndash40] The major advantage ofusing nanocarriers resides in the possibility of conjugatingligands to them as to direct them to a desired site for localizeddelivery in cells tissue or organ [41] ODNs in complexstate with nanocarriers avail protection against nucleasedegradation [38 42ndash44] Further their cell uptake could beincreased as carrier-ODNs complexes are taken up throughactive endocytosis process

Recently biodegradable nanoparticles have been studiedas potential inert and biocompatible carriers for geneticmaterials for example polylactide polyethylene glycol (PLA-PEG) cationic polystyrene spongelike alginate polyalkyl-cyanoacrylate poly isohexylcyanoacrylate or poly isobutyl-cyanoacrylate and albumin chitosan nanoparticles [36 45ndash51]

Among the large number of cationic polymers describedchitosan is shown to be an effective vector because of con-densing and delivered DNA siRNA in vitro and in vivo [5253] and ODN in vitro [54] It is a natural positively chargedpolymer that can be utilized for preparation ofnanopar-ticulate carriers which represents a novel strategy for thesafe and efficient delivery of gene It has been extensivelyexamined for its potential in the development of controlledrelease drug delivery formulation due to its unique poly-meric cationic characteristic gel forming and film formingproperties [55 56] Chitosan has beneficial qualities such aslow toxicity low immunogenicity excellent biodegradability

and biocompatibility [57 58] It is a suitable candidate forgene delivery system due to its ability to spontaneously forminterpolyelectrolyte stable complexes with genetic material(ODNs or DNA) as a result of cooperative electrostaticinteractions between the positive amino groups of chitosanand the negative phosphate groups ofDNAODN [54 56 59]

Consequently several groups have conducted studiesusing chitosanDNA nanoparticles including use ofgalactosylated chitosan [60] galactosylated chitosan-graft-poly(vinylpyrrolidone) (PVP) [61] mannosylatedchitosanDNA nanoparticles [62] trimethylated chitosanoligomers [63] N-dodecylated chitosan [64] deoxycholicacid modified chitosan [65] galactosylated chitosanODNvector [54] or ligand attached chitosans for targeting cellmembrane receptors [66] However chitosan was difficultto solubilize in water and was dissolved in acidic solutionLow molecular weight water soluble chitosan has beenemployed which is highly water soluble and forms complexwith plasmid DNAODN at physiological pH [54 67 68]They can display high transfection efficiency along withthe higher plasmid DNAODN loading protection againstthe nuclease degradation and being less toxic [54 69 70]Ligands are molecular extension that pave fate of deliverysystem for active targeting and increase overall therapeuticpotential Thus the receptor mediated gene targeting is apromising approach to obtain cell-selective gene transfectionA number of receptor mediated gene delivery systems havebeen developed to introduce the foreign DNAODN intospecific cell types Ligands currently being investigatedinclude galactose [71ndash80] mannose [66 81ndash85] lactose[86] transferrin [87 88] and epidermal growth factor [89]Active targeting using receptor mediated interaction hasbeen effective in gene delivery [90]

Among the various receptors asialoglycoprotein recep-tors and mannose receptors are the most promising for genetargeting because they exhibit high affinity and are rapidlyinternalized [91] Wide variety of macrophages (includingKupffer cells) express mannose specific membrane receptorswhich internalize glycoprotein bearing mannose residue viaclathrin-coated vesicles to allow a delivery into the endoso-mal system [91ndash94]

In gene delivery systems the mannosylation of cationicpolymers such as mannosylated polylysine and PEI wereused to achieve the delivery of gene to macrophages [8285] Mannosylated chitosan for plasmid DNA has also beenreported earlier for macrophage targeting [95]

Although chitosan has been studies for more than adecade as a gene vector for DNA ODN and siRNA [54 96]so for to our knowledge there is no study that has beencarried out to investigate the use of mannosylated chitosannanoparticles to determine ODN in vitro In the presentstudy we formulate mannosylated LMW-water soluble chi-tosan formulations by self-assembled method characterizedfor gel retardation assay particle size particles shape andparticles surface morphology and zeta potential complexingcapacity protection ability and MCHODN NPs stabilityCytotoxicity studies transfection efficiency and antisenseassay were also assessed for developed nanoparticles formu-lations

BioMed Research International 3

2 Materials and Methods

The phosphorothioate modified AS ODN used in this studyTJU-2755 designed to target the 31015840-UTR of the primaryRNA transcript of TNF-120572 was synthesized by Sigma AldrichThe sequence of TJU-2755 is 51015840-TGATCCACTCCCCCC-TCCACT-31015840 random sequence of ODN is 51015840-CCTCCA-CTGCTACCTCACCTC-31015840 FITC-ODN was also receivedfrom Sigma Aldrich Water-soluble LMWC with a molecularweight of 22KDa with degree of deacetylation of 725was received from Kittolife Co Seoul Republic of KoreaOligreen reagent was purchased from Molecular Probs Inc(Netherlands) RAYBio rat TNF-alpha ELISA kit was pur-chased from RAYBio Ltd Mannopyranosyl phenyl isothio-cyanate dimethyl sulfoxide (DMSO) and Tris Borate EDTAwere purchased from Sigma Aldrich All other chemicalswere obtained commercially as reagent grade products

3 Preparation of MannosylatedChitosan (MCH)

Mannosylated chitosan (MCH) was prepared by previouslyreported method with slight modifications [61] Briefly120mg of accurately weighed water-soluble LMWC wasdissolved in 2mL of double distilled water (2mL) andmixed with solution of mannopyranosyl phenyl isothio-cyanate (Sigma Co St Louis India) in 1mL of DMSO Thesolution was stirred for 24 h at room temperature (24∘C)TheMCH was precipitated by adding 10 volumes of isopropanoland centrifuged at 13000 rpm for 15min After repeatingthis process five times the pellets were dried in vacuumoven The MCH composition was determined by FT-IR andproton nuclear magnetic resonance spectroscopy (1H NMR600MHz Bruker Germany) The degree of sugar substitu-tion was determined by the sulfuric acid micromethod [97]

4 Preparation of MCHODNNanoparticles (MCHODN NPs)

MCHODN nanoparticles were prepared by self-assemblingmethods as reported earlier with slight modifications [62]MCHODN charge ratio (119873119875) was expressed as the ratioof moles of the amine groups of MCH to those of thephosphate moieties of ODNs Nanoparticles were induced toself-assemble in phosphate buffer (pH 74) by mixing ODNwith required polymer solution at the desired ratio Thecomplexes were allowed to stand at room temperature for1 h The complex formation was confirmed by gel retardationassay with 1 agarose gel

5 Characterization ofMCHODN Nanoparticles

51 Gel Retardation Assay The formation of MCHODNNPswas determined by using gel retardation assay Agarose gelwas prepared with 1 agarose solution in Tris-borate-EDTA(TBE 445mM Tris base 1mM sodium EDTA 445mMboric acid pH 83) buffer A series of different 119873119875 ratio of

MCHODN nanoparticles were loaded in the well (20 120583L ofthe sample equivalent to 02 120583g of ODN) A 1 6 dilution ofloading dye was added to each well and electrophoresis wascarried out at a constant voltage of 55V for 2 h in Tris BorateEDTA (TBE) running buffer containing 05120583gmL ethidiumbromideThe bands ofODNwere then visualized under aUVilluminator

52 Measurement of Particle Size and Zeta Potential Size ofthe nanoparticles was determined in triplicates by photoncorrelation spectroscopy (PLS) using Zetasizer Nano ZS(Malvern Instruments Ltd Malvern Worcestershire UK)The particle size analysis of the nanoparticles was performedat 25∘C an angle of 90∘ for the photomultiplier and awavelength of 633 nm The surface charge (zeta potential) ofthe nanoparticles was determined from the electrophoreticmobility The zeta potential measurements were performedat pH 74 in triplicates using the 100 120583L aqueous dip cellby Zetasizer Nano ZS (Malvern Instruments Ltd MalvernWorcestershire UK) The samples were diluted 1 100 withdouble distilled water before measuring

53 Shape and Surface Morphology

531 Transmission ElectronMicroscopy (TEM) Morphologi-cal examination ofMCHODNnanoparticles at119873119875 ratio 3 1was investigated by transmission electron microscopy (TEMJEM 1200 EX 11 JEOL Japan) One drop of sample wasplaced on a copper grid and negatively stained with uranylacetate (1 wv) solution for 30 s The grid was allowed todry further for 10min and examined under the transmissionelectron microscope [98 99]

532 Scanning Electron Microscopy (SEM) A drop of theMCHODN nanoparticles preparation was dried on a pol-ished aluminium surface and the sample was sputtered withgold for 30 s under argon atmosphere (Agar sputter coater)Thereafter SEM was performed with S-4500 Hitachi fieldemission electron microscope (Krefeld Germany) with theupper detector at 25 KV [100]

54 Determination of ODN Association Efficiency ODNassociation efficiency of the MCHODN NPs was evaluatedusing the method as reported by Huang et al [99] withslight modification To measure the association efficiencyof ODN 16 120583L of the MCHODN NPs was spun down at15000 rpm for 30min (Avanti J-25 centrifuge BeckmanCoulter Inc Fullerton CA USA) The supernatant wasmixed with 1mL working OliGreen reagent and ODN con-tent was estimated spectrofluorimetrically at 480 nm and a520 nm excitation and emission respectively [101 102] ODNassociation efficiency () of NPs is the amount of condensedODN (difference between the total amount of ODN used forthe NPs preparation and the amount of nonentrapped ODNremaining in the supernatant) calculated as a percent of thetotal amount of ODN used

55 Resistance of MCHNPODN against DNase I Digestionand Release Assay Protection and release assay of ODNin nanoparticles were carried out by gel electrophoresis


following the reported procedure [103] To assess the sta-bility against nuclease digestion free ODN and MCHODNnanoparticles (02 120583g) with various 119873119875 ratios were sepa-rately incubated with DNase I (10U) in DNase digestionbuffer containing 50mM Tris-Cl pH 76 and 10mMMgCl

2

at 37∘C for 1 h After DNase I digestion all the samples weretreated with 4 120583L of 25mMEDTA for 10min to inactivate theDNase I and mixed with 20 120583L sodium dodecyl sulfate (SDS10 by dissolving in 01 N NaOH pH 72)) To dissociate theODN from MCH the reaction mixtures were incubated inBOD incubator at 60∘C overnight After phenolchloroformextraction the ODN was precipitated with cold isopropanolThe pellets were dissolved in 10mL of TBE buffer and appliedto a 10 agarose gel electrophoresis for 40min at 100V [103]

56 Stability of MCHODN NPs

561 Dispersive Stability of Nanoparticles The procedurereported by Kim et al [103] with slight modifications wasfollowed to estimate dispersing stability of NPs Brieflythe nanoparticles formulations were dispersed in 10mMphosphate buffer containing 150mMNaCl and the dispersivestability of nanoparticles was evaluated by turbidity measure-ment spectrophotometrically at 340 nmThe results are givenin Figure 7 Also serum stability of each of the formulationswas observed in the presence of fetal bovine serum (18120583LDMEM medium was taken with 2120583L of FBS) The sampleswere incubated at 37∘C with mild agitation and evaluated byagarose gel electrophoresis at 025 05 1 15 and 2 days Theresults are shown in Figure 8

57 In Vitro Cell Line Study

571 Cell Lines and Cell Culture Raw 2647 (murinemacrophage cell line expressing moderate mannose recep-tors) [62] and Hela (human cervix epithelial g cells) cellswere procured from the NCCS Pune (MHA India) Both celllines were maintained in Dulbeccorsquos modified Eagle medium(DMEM Sigma India) supplemented with 10 fetal bovineserum (FBS) streptomycin (100 120583gmL) and penicillin (100UmL) The cells were incubated at 37∘C in a humidifiedatmosphere containing 5 CO

295 relative humidity (CO

2

Incubator Binder Germany) TrypsinEDTA medium wasused to split the cells

572 Cell Viability Study In vitro cytotoxicity assay wasperformed as per reported method [54] to evaluate theeffect ofpolymer (mannosylated chitosan) concentration andoligonucleotide concentration on cell viability of Raw 2647cells and Hela cells The cell growth inhibition activity ofsamples was evaluated by 3-(45-dimethylthiazol-2yl)-25-diphenyl tetrazolium bromide (MTT) colorimetric assayBriefly both cell lines were maintained in Dulbeccorsquos mod-ified Eagle medium (DMEM) under suitable conditionsas mentioned above When the cells were confluent theywere seeded into 96-well culture plates at a cell densityof 1 times 104 cellswell and the plates were maintained underthe conditions previously mentioned for twenty-four hoursTherefore the old medium was carefully aspirated and

the cells were incubated in 100 120583L serum free medium withvarious amounts of polymer (concentrations ranging from 5to 200120583gmL of and man-chitosan)

After 16 h the medium was removed and the cells wererinsed twice with 1x PBS The wells were refilled withcomplete medium and cells were cultured for another 24 h10 120583L of MTT dye solution (5mgmL) was added into eachwell and the plate was incubated for 4 h at 37∘C allowingviable cells to reduce the MTT into purple colored formazancrystal [104] The medium was later removed and 150 120583L ofdimethylsulfoxide (DMSO) was added to each well and theplate was incubated for 30min at room temperature Theoptical density was measured at 550 nm DMSO was used todissolve the formazan crystals

The cell viability was expressed as percentage comparedto a control that had not been treated with polymers usingthe following equation

Cell Viability () = (119873119894

119873119888

) times 100 or

Cell Viability () = (OD590

sampleOD590

control) times 100

(1)

where 119873119894and 119873

119888are the number of surviving cells in the

group treated with ODN associated formulation and in theuntreated cell group respectively or where the OD

590sample

represents the measurement from the wells treated withpolymer and OD

590control represents the wells treated with

PBS buffer onlyIn order to investigate the effect of ODN concentration

on cell viability another set of experiments was performed inthe same manner as above After 24 h of incubation of cells(1 times 104 cellswell) 100 120583L serum free medium containingdifferent amount of ODN (1 5 and 10 120583g) either free or inMCHODN NPs was added into each well and cells wereincubated for same period of time under the conditionsmentioned earlier and cell viability was also determined

573 Transfection with Complexes In Vitro In a transfec-tion assay Raw 2647 cells and Hela cells were seeded atthe density of 50 times 105 cellsdish in 60mm culture disheswith 5mL of complete medium (Dulbeccorsquos modified Eaglecontaining 10 serum DMEM) and incubated for 24 hprior to transfection Transfection was performed on cellsthat were approximately 70 confluent Before transfectionthe complete medium was removed and cells were rinsedonce with 1x PBS The naked ODN and MCHODN NPs(containing 10 120583g of ODN) were diluted in 2mL DMEMmediumand thenwere used to refill the dishes and incubatedat 37∘C for 6 h Thereafter serum and DMEM mediumwere added to these dishes to make up the final volumeof medium to 5mL containing 10 serum Transfectionwith Lipofectin (GIBCO BRL) was carried out as positivecontrol according to manufacturerrsquos protocol After another18 h the medium containing nanoparticles was removedThe cells were rinsed twice with 1x PBS harvested andresuspended in 1x PBS ODN concentration in cell lysatewas determined spectrofluorimetrically at excitation andemission wavelengths of 495 nm and 520 nm respectively


574 Competition Assay Competition assay was carried outto confirm the uptake of MCHODN nanoparticles mediatedbymannose receptor by adding different amount of mannose(10 20 and 50mM of the final volume) as a competitorfor mannose in MCHODN NPS Raw 2647 cells werepreincubated for 15min with different amount of mannoseand transfection assays by using MCHODN NPs preparedat charge ratio 3 1 were conducted as described previouslyAll experiments were performed in triplicate [62]

58 Quantification of ODNs in Cells ODN concentrationsin cell lysate were determined spectrofluorimetrically asreported earlier [105ndash107] After incubation period trans-fection medium was removed and cells were washed threetimes with ice cold PBS (8 gL NaCl 02 gL KCl 156 gLNa2HPO4and 02 gL KH

2PO4in water at pH 72) The cell

lysate was obtained after cells lysis with 02mL of 1 TritonX-100 in 50mM Tris-HCl buffer (pH 80) The cell lysatesolutions were determined by LB05 spectrofluorophotometer(Shimadzu Japan 120582ex 492 nm 120582em 519 nm) to quantifycell associated FITC ODN concentrations The unknownconcentration ofODNwas determined by extrapolation fromstandard curve Transfection efficiency is expressed as theamount (ng) of ODN per well of cells

59 Cell Uptake Study

591 Fluorescence Microscopy Cellular accumulation ofFITC-SODN was examined using fluorescence microscopyas reported earlier [108] In these studies Raw 2647 cellswere plated on 24-well plates at a density of 5 times 105 cellswellin 3mL complete medium (DMEM containing 5 FBS)and incubated for 24 h The complete medium was removedand cells were rinsed once with 1x PBS Then cells wereincubated with free FITC-ODN or FITC-MCHODN NPs at3 1 MCHODN ratio for 4 h at 37∘C washed three timeswith ice-cold PBS and fixed with 4 (vv) paraformaldehydeinPBS for 15min at room temperature and again washedfour times with PBS The cells were analyzed by fluorescentmicroscope (Nikon Japan)

592 Confocal Laser Scanning Microscopy (CLSM) Raw2647 cells were plated on six-well microplates at a densityof 5 times 105 cellswell and incubated with free FITC-ODNor FITC-ODNnanoparticles at 3 1 MCHODN ratio for4 h at 37∘C washed three times with ice-cold PBS fixedwith 4 (vv) paraformaldehyde in PBS for 15min at roomtemperature and again washed four times with PBS Forstaining nuclei fixed cells were permeabilized with 02Triton X-100-PBS for 20min stained with DAPI washedoncewith 1mLof cold PBS andmounted on slide glasseswith50 glycerol-25 DABCO (14-diazabicyclo-[222] octane)(Sigma Chemical Co Inc St Louis MO)-PBS Then thefluorescence images were acquired with a Zeiss 510 Metaconfocal microscope

510 Antisense Efficacy of ODN Delivered by NanoparticulateFormulations (MCHODN NP2 and Free ODN Solution) Thein vitro suppressive effect of ODN in Raw 2647 cells was

determined using a method reported earlier with slightmodifications [62 109] Raw 2647 cells were seeded at adensity of 3 times 105 cells in a 24-well plate and cultured for 18 htransfected with MCHODN NPs and free ODN solution byaddition into a 24-well plate and incubated for 6 h To thisserum and serum-free growth medium (DMEM) was addedand incubated for another 18 hours at 37∘CAfter 24 h the cellswere challenged with 100 ngmL lipopolysaccharide (LPS)Two hours later the culture mediumwas removed and storedat minus70∘C The content of TNF-120572 in cell culture supernatantwas evaluated using ELISA

6 Results and Discussion

Despite its superiority as a biomaterial chitosan is not fullysoluble in water however it is readily soluble in acidic solu-tion Aqueous solubility of chitosan only in acidic solutionlimits its application to bioactive agents such as gene deliverycarriers peptide carriers and drug carriers

Water-soluble chitosan is easily soluble in aqueous solu-tion with neutral pH Its advantages are ease of modificationuseful as genepeptide drug carriers anddrug carriers [67 110111]

61 Spectroscopic Analysis Chitosan exhibits the charac-teristic bands of ndashNH

2scissoring vibrations at 1657 cmminus1

carbonyl asymmetric stretching vibrations at 1564 cmminus1 andCndashO stretching vibrations of the pyranose ring at 1071 cmminus1(Figure 1) NMR spectrum of chitosan is shown in Figure 2The solvent used was D

2O and 120575 value from 1H NMR at 32

to 31 shows characteristic peaks of ndashCH3ndash second carbon of

chitosanMannosylated chitosan (MCH) was synthesized by the

reaction between amine groups of chitosan and isothio-cyanate groups of 120572-D-mannopyranosyl phenyl isothio-cyanate as shown in Scheme 1 The composition of MCHwas determined by FTIR (Figure 3) and 1H NMR (Figure 4)The MCH exhibited the characteristic band of mannose at808 cm1 and 890 cmminus1 COOasymmetric stretching vibrationat 1626 cmminus1 and COO symmetric stretching at 1417 cmminus1and characteristic 120575 value from 1H NMR at 71 (ndashCHndashof mannopyranosyl phenyl isothicyanate) and 21 (ndashCH

3

of chitosan) (Figure 4) The substitution value of mannosecoupled with water soluble chitosan was 59mol


71 Gel Retardation Assay of MCHODN Nanoparticles Thepositive charge ofmannosylated chitosan and negative chargeon ODN have tendency to enforce electrostatic interactionsbetween them to formMCHODN nanoparticles The confir-mation of nanoparticles formation between MCH and ODNwas evaluated by gel retardation assayThenanoparticles wereanalyzed in 10 agarose gel electrophoresis Figure 5

It shows the influence of charge ratios onODN condensa-tion by MCH from gel retardation assay The results indicatethat migration of ODN in gel was retarded with increasing


O

CH2OH CH2OHCH2OH

OH

NH2

CH3

OO OH

O O

NHOC

CH2OH

OOH OH

OH OHHOH H

H

O N C S

OO

OH O

NH

SC

HN H

O

OCH2OH

OHHH

H

CH2OH

OH OH OO

CH2OH

CH3

NH

OC

OO

NH2

Chitosan

+

Stirring

Mannopyranosylphenylisothiocyanate

Mannosylated chitosan (MC)

M

K M

X

25∘C24hKminusX

Scheme 1 Reaction scheme for MCH Preparation

10000

0003500 3000 2500 2000 1500 1000 500

(cmminus1)

3439

32506

23556 21920

19042

16579

16009

16409

15642

15390 14301

14000

13550

12625

12364

11922

10710

10230

9621 9039

8718

8266

7656

7364

6766 6042

5586

5069

4827

T (

)

Figure 1 FTIR spectrum of chitosan

80 70 60 50 40 30 20 10

(ppm)

Figure 2 1H NMR spectrum of chitosan

the proportion of MCH to ODN In present assay the 119873119875ratio of MCH to ODN was determined at which a givenamount of ODN is completely complexed byMCHThis ratiowas visualized by the gel retardation assay after subsequentstaining with ethidium bromide

Under the electric field free negative ODNs moved tothe positive electrode and displaced in the gel as a visual

10000

0003500 3000 2500 2000 1500 1000 500

(cmminus1)

30847

29215

23724

17312

16514

16261

15621

15234

14174 13

212

12834

11844

11122

10204 9548

8908

8085

7684

7204

6990

6800

6301

6071

5831 55365274 4548

T (

)

Figure 3 FTIR spectrum of mannosylated chitosan

80 70 60 50 40 30 20 10

(ppm)

Figure 4 1H NMR spectrum of mannosylated chitosan

band (Figure 5 Lane A) As the proportion of MCH in thesamples was increased a decrease or absence of stainingintensity of ODN as seen in lanes B to F was observed Thissuggests that progressively increasing amount of ODN wasretained by MCH in the wells as the proportion of MCH


Table 1 The composition and characterization of MCHODN NPs (119899 = 3)

Formulation codes 119873119875 ratio of MCH to ODN Particle size (nm) Zeta potential (mV) Polydispersity index (PI)MCHODN NP1 1 1 26712 plusmn 110 minus62 plusmn 002 038MCHODN NP2 3 1 19248 plusmn 48 +89 plusmn 082 012MCHODN NP3 5 1 18716 plusmn 56 +126 plusmn 064 016MCHODN NP4 7 1 17824 plusmn 74 +142 plusmn 046 020Values expressed as Mean plusmn SD

A FEDCB

Figure 5 Gel retardation assay of MCHODN NPs lane A freeODN lanes BndashF MCHODN NPs at 119873119875 ratio of 05 1 1 1 3 15 1 and 7 1

to ODN increased in the sample As the 119873119875 ratio reached1 1 or above ODN was completely complexed with MCH asindicated by the lack of electrophoretic mobility hence thereis no free ODN available to move toward positive electrodeleading to absences of visible ODNband in the gelThereafterat the charge ratio of 1 1 to 7 1 no bandwas visible indicatingthe enhanced efficiency of complexation (Lane CndashF)

The electrophoretic mobility of ODN was retarded withincreasing amount of MCH and retained at the top of thegel at ratio of 1 1 to 7 1 This suggests that MCH formedcomplexes completelywithODNat this ratio range Althoughsome amino groups are mannosylated leading to reducedcharge density of MCH slightly its ability to condense ODNwas retained (Figure 5 lanes CndashF) This characteristic is anessential requirement for condensing ODN and transfectingto the cells

72 Particle Size Physicochemical properties of MCHODNnanoparticles with various charge ratios were characterizedin terms of size and zeta potential (ZV) Literature reveals thatthe biological activities of the transfection reagents are highlyassociated with their physicochemical properties [112 113]

Specific properties such as particle size and surfacecharge of the complex are necessary to assure its uptakeby cells In particular the particle size of a complex is animportant factor that influences the access and passage ofthe complex through the targeting site [103] Mean particlediameter of MCHODN nanoparticles with different 119873119875ratios was determined by photon correlation spectroscopy(PCS) using Zetasizer as shown in Table 1

It was observed that with increase in119873119875 ratio (from 1 1to 7 1) that the mean particle size of MCHODN nanopar-ticles was decreased It can be inferred that the particle sizewhich was not more than 267 nm is indirectly proportionalto the charge ratio (Table 1) When 119873119875 ratio of MCHODNNPs was around 1 the nanoparticles size was above 250 nmIf the 119873119875 ratio of MCH and ODN was increased from1 to 7 the mean particle size was found to decrease from26712 plusmn 110 nm to 17824 plusmn 74 nm The possible reasoncould be that the polymer complexes with ODN throughionic interactions and at higher 119873119875 ratio there are netelectrostatic repulsive forces that can prevent aggregationamong complexes Similar trend was observed in the earlierreport with galactosylated chitosan NPs carrying DNA [54]

Particle size can play an important role in influencing thetransfection efficiency The NPs with larger particle diameterbear disadvantage in the process of cell endocytosis andthus the corresponding transfection efficiency may be poor[114 115] Further with MCHODN NPs the data representssuitable PI value for 119873119875 ratio from 3 to 7 inferring to theirhomogenous nature

73 Zeta Potential (ZV) The zeta potential of the MCHODNNPs at various119873119875 ratios is given in Table 1 With increasing119873119875 ratio of MCH to ODN from 1 to 7 the zeta potentialof the nanoparticles rapidly increased to positive values Itis inferred that the ZV is directly proportional to the chargeratio of nanoparticles The increase in ZV was due to anincrease in the positive charge density of the mannosylatedchitosan At charge ratio of 1 1 MCHODN NPs displayednegative ZV (minus62mV) however with an increase in 119873119875ratio of MCH to ODN upto 7 1 NPs displayed positive zetapotential Further with increase in charge ratio from 3 1 to7 1 the ZV raised from 89mV to 145mV Complete shield-ing of negative charge of ODN was displayed in formulationwith charge ratio 3 1 The results obtained are in accordancewith the earlier reports [54 62] Upon self-assembly ofODNs and cationic polymer the highly negative charge ofODNs gets neutralized rapidly and the surface charge of thecomplex becomes positive at higher 119873119875 ratio A positivesurface charge of polyplexes is necessary for binding toanionic cell surfaces which consequently facilitates uptakeby the cell [116 117] The surface charge of delivery systemscarrying genetic material (DNAODNs) is one of the majorfactors influencing the biodistribution [118] and transfectionefficiency [119] Surface charge of polymerODN complexesplays an important role in the uptake of the complexes bycells and their physical stability in blood Serumprotein tends


A FEDCB

(a)

A FEDCB

(b)

Figure 6 (a) MCHODN NPs that were separately incubated with DNase I (b) 10 SDS was added to DNase I treated samples in theMCHODN NPs Lane A free ODN lanes BndashF MCHODN NPs with increasing ratio (05 1 3 5 and 7)

to adsorb strongly onto positively charged particles surfacesThis opsonization would lead to severe aggregation of thecomplexes and subsequent opsonization It was reportedthat the slightly positive zeta potential resulted in the besttransfection efficiency [120] MCHODN NPs charge ratioabove 1 is thought to be profitable for the ODN transfer intocells

These results also indicate that it is possible to formulatenanoparticles of a specific particle diameter by adjusting con-densing conditions such as the 119873119875 ratio of the complexesconcentration of ODN and concentration of MCH

74 Shape and SurfaceMorphology The formulated nanopar-ticles were morphologically examined by TEM at magni-fications at 8000 (Figure 15(a)) TEM images reveal solidconsistent and compact structure having a spherical shapeAs depicted from earlier studies the formulations were innanometric range Nanoparticles had a dense and sphericalshape which is in good agreement with the SEM photomi-crograph (Figure 15(b)) Percent ODN association of selectedMCHODN NP2 formulation was determined to evaluatethe amount of ODN associated with MCH in nanopartic-ulate formulation The optimum transfection efficiency ofMCHODN NP formulations was achieved at charge ratio ofMCHODN at 3 1 On the basis of transfection efficiencyresults MCHODN NP2 formulation was opted for furtherin vitro and in vivo studies The percent ODN associationof MCH ODN NP2 was found to be 985 This suggests thatalmost all the ODN loads were condensed into NP

75 Resistance of MCHNPODN against DNase I Digestionand Release Assay The plasmid DNA is readily degraded byendonucleases such as DNase I [121] which is one of theobstacles for the delivery of gene in vitro or in vivoThereforethe stability in the presence of DNase I is one of the essentialparameters of systemic gene delivery The chitosan was usedto condense plasmid DNAODN to protect them againstDNase digestion [54 67 103 122] To investigate whethernaked ODN as well as complexed ODN with mannosylatedchitosan was stable against nuclease digestion DNase I pro-tection assay was carried outThe results indicated that nakedODN remained intact after DNase I treatment indicated

by the presence of visual band in the gel and maintainedits integrity (Figure 6(a) lane A) because phosphorothioateODN is resistant to nuclease degradation

Figure 6(a) lanes AndashF show that the free ODN as wellas ODN associated with MCHODN NPs at all 119873119875 ratioremained intact after treatment with DNase I enzyme andstaining intensity in the well was increased on increasing the119873119875 ratio of MCHODN Only single band was observedwith free ODN in the gel which reveals that the processof complexation with MCH does not affect the integrityof the ODN adversely rather it seems to offer protectionThis suggests that ODN either in free form or in complexedform with mannosylated chitosan at various 119873119875 ratiosmaintained their integrity and resistant to DNase I enzymedegradation

In release assay DNase I digested NPs suspension wastreated with SDS to release ODN from complexes that wasevaluated by agarose gel electrophoresis The released ODNfrom nanoparticulate carriers displayed visual bands in thegel (Figure 6(b) lanes AndashF) The results indicate that at05 1 to 7 1 ratios of MCHODN ODN remained intactafter DNase and SDS treatment This demonstrates that theprocess of complexation of mannosylated chitosan does notaffect the ODN integrity adversely rather it seems to offerprotection Under the physiological conditions where thenuclease concentration is markedly lower than the testedconcentration a formulation should render a significantprotection to the ODNs The results indicate that binding ofODNs to MCH was sufficient to provide protection againstenzymatic degradation

76 Stability of MCHODN NPs Formulation

761 Dispersive Stability The study was conducted with anobjective to evaluate whether the formulations form self-aggregate or precipitate in phosphate buffer at room tempera-ture It is expected that the decrease in turbidity with durationof storage would indicate precipitate formation From thepresent data (Figure 7) it can be inferred that the formulationMCHODN NP2 displayed steady values of absorbance till15th day and thereafter slight decrease on 2nd day indicatesprecipitate formation The results suggested that MCHODN


033

035

025 05 1 15 2Time (days)

Dispersive stability

Turb

idity

at340

nm

Figure 7 Stability of MCHODN NP2 formulations in 10mMphosphate buffer containing 150mM NaCl

A FEDCB

Figure 8 Gel electrophoresis of MCHODN NP2 after incubationat 37∘C under mild agitation with increase storage period Lane Ananoparticles prepared immediately prior to gel analysis (initial)lanes BndashE nanoparticles with increasing incubation period (05 115 and 2 days) lane F free ODN

NP2 formulation displayed appreciable stability during thestudy period

762 Serum Stability Stability of nanoparticles formulationsMCHODNNP2 and free ODN were observed in presence ofserum to investigate the behavior in physiological conditionGel electrophoresis results revealed that up to 2nd day ofincubation period at 37∘C nanoparticles remained stable(Figure 8)

8 Cytotoxicity Assay

Cytotoxicity assay was conducted to evaluate the effect ofpolymer concentration on cell viability MTT assay was per-formed to determine the cytotoxicity ofMCH in two differentcell lines that is Raw 2647 cells and Hela cells over a widerange of polymer concentration MCH showed acceptablecell viability in both cell lines at lower concentration MCHexhibited near about 884 cell viability at 200120583gmL in Raw2647 cells The maximum cell viability 1045 and 1002was observed at 10120583gmL with MCH in Raw 2647 and Helacells respectively It might be concluded that the cells growthis appreciable even at higher concentration of 200120583gmLwithMCH(Figure 9) Chitosan is reported to bear low toxicity [67123] although few reports reveal a dose-dependent toxicityof chitosan at high doses in vitro [54] In earlier reportssignificantly higher cell viability of chitosan-treated cells was

0

20

40

60

80

100

120

0 (untreated cell) 10 30 100 200

Cel

l via

bilit

y (

cont

rol)

Hela cellsRaw 2647 cells

Polymer concentration (120583gmL)

Figure 9 Cell Viability studies of MCH at different polymerconcentration in Raw 2647 and Hela cells (119899 = 3)

observed compared to PEI [98 124] In the present studythe cell viability further increased after mannosylation ofchitosan The cell viability of MCH (sim850 of control) wassignificantly higher than that of CH (sim602 of control) ata concentration of 100120583gmL in the Raw 2647 cell linesdata not shown As reported earlier cationic polymers withhigh charge density have strong cell lytic and toxic propertiesand also a reduction of charge density resulted in reducedcell toxicity or increased cell viability [125] Lower toxicityof MCH may be attributed to steric hindrance and thecharge shielding effects offered by mannose moiety Recentevidence suggests that the introduction of sugar into thepolycations affects electrostatic interactions between anioniccell surfaces and cationic polymer This leads to reduction ofmembrane damaging effects which is a typical characteristicof polycationic molecules [126] The results from presentstudy are in accordance with the research work related tothe polymeric nanoparticulate carriers for delivery of genein earlier reports [62 93 127 128] The results further inceptthat modified chitosan being noncytotoxic biocompatibleand safe vector might play a role of compatible gene deliverysystem Therefore nanosized MCHs are expected to besuitable carrier for gene therapy because of their favorableand acceptable physicochemical properties largely leading totheir low cytotoxicity

To further evaluate the efficiency of mannosylated sys-tems the influence of variousODNconcentrations associatedwith MCHODN NPs on the cell viability of Raw 2647macrophages cells was investigated using MTT assay

The results are displayed in Figure 10 Raw 2647 cellsincubated with ODN formulations equivalent to 1 to 10120583gODN displayed variations It was observed that more than90 cells were viable at 1120583g ODN concentration in case ofMCHODN NPs Sign of cytotoxicity was minimum in caseofMCHODNNPs whenODNconcentration increased up to10 120583g More than 75 cells were viable at all the tested ODNconcentrations with MCH

Hashimoto et al [95] studied the transfer of gene byDNAmannosylated chitosan complexes into mouse peri-toneal macrophages and it was reported that on increasingthe DNA concentration from 1 to 10 120583g the cell viability


0

20

40

60

80

100

120

0 1 5 10Equivalent ODN content

Cel

l via

bilit

y (

)

Figure 10 Cell viability study of MCHODN NPs with differentODN concentration in Raw 2647 cells after 24 h (119899 = 3)

of mannosylated chitosanDNA complex was unchanged Itmight be inferred that MCHODN NPs show significantlyhigher cell viability at all ODN concentrations (Figure 10)The cytotoxicity of pDNAPEI complexes was reported tobe higher [52 123] as compared to that of pDNAchitosancomplexes [63 129] The mannosylation of chitosan furtherdecreases the cytotoxicity and might significantly increasethe transfection efficiencyThe low cytotoxicity ofMCHODNNPs might be due to its hydrophilic surface It could behypothesized that the hydrophilic surface of MCHODNNPs suppresses the formation of aggregation followed bynonspecific adhesion to the cell surface

81 In Vitro Transfection Assay with Mannosylated ChitosanNanoparticles In order to evaluate the in vitro gene transfercapability of mannosylated chitosan nanoparticles the trans-fection assay was carried out at different 119873119875 ratio of MCHto ODN in Raw 2647 cells and Hela cells Figure 11 suggeststhat transfection efficiency is dependent on charge ratioof MCHODN There is significant increase in transfectionefficiency of MCHODN NPs in Raw 2647 cells from 2878plusmn 12 ng106 cells to 7318 plusmn 42 ng106 cells on increasing119873119875 ratio of MCH and ODN from 1 1 to 3 1 whereason further increasing the 119873119875 ratio to 5 1 a decrease intransfection efficiency to 6308plusmn 36 ng106 cells was observedand remained almost constant at ratio of 7 1 Maximumtransfection efficiency was recorded at optimum119873119875 ratio of3 1 Further MCHODNNPs displayed approximately 3-foldhigher transfection efficiency to that of free ODN

This might be due to the poor cellular uptake of ODNin free form because of its negative charge and hydrophilicnature [11] The possible reason is that naked ODN getsdegraded rapidly in presence of cellular enzymes unlike theone complexed to mannosylated chitosan [14] It is expectedthat ODNs complexed to mannosylated chitosan get protec-tive shielding against enzymatic degradationTheMCHODNNPs presents high density of mannose for interaction withcell surface Raw 2647 cells that express moderate mannosereceptors might present higher opportunity for transfectionof mannosylated system Kim et al [62] reported the utilityof mannose receptor based endocytosis as they explored

0102030405060708090

Naked ODN 1 3 5 7NP ratio


OD

N (n

g10

6ce

lls)

Figure 11 Transfection efficiency of MCHODN nanoparticles atdifferent119873119875 ratio in Raw 2647 cells and Hela Cells (119899 = 3)

mannosylated chitosan nanoparticles for cytokine gene ther-apy Also in this context Jiang et al [127] explored man-nosylated chitosan PEI grafts for DNA delivery exploitingmannose receptor Few other applications of mannosylatedcationic systems with enhanced transfection efficiency arereported [128 130ndash132] Macrophages and dendritic cellshave substantial amount of mannose receptors on the sur-face suggesting that MCH-mediated gene delivery may beinduced by receptor-mediated endocytosis via surface-boundmannose receptors The results substantiate the findings thatmannosylated system is more effective for gene expression inRaw 2647 cells than nonmannosylated systems

The results from transfection assay at various 119873119875ratios using galactosylated chitosan for gene delivery arein accordance with the present results for mannosylatedcarriers [54 67] Literature cites that for effective expressionof gene vehicle must protect the enzymatic degradationof gene [133] The transfection efficiency of naked ODNMCHODN NPs and lipofectinODN complex in Raw 2647and Hela cells was also compared in order to demonstratethe effect of mannose on receptor mediated gene transferThe differences in transfection efficiency in two cell linesare clearly observed as Raw 2647 cell lines displayed signif-icantly higher transfection at each 119873119875 ratio as comparedto Hela cells in which mannose receptors are not present(Figure 11) Figure 12 demonstrates that transfection effi-ciency of MCHODN NP2 is much higher than naked ODNand similar to that of lipofectin At optimum 119873119875 ratio of3 1 MCHODN NP2 displayed 253-fold higher transfectionin Raw 2647 cells than Hela cells It indicates that themannose ligand onMCHODNNPs played an important rolein mannose receptors recognition and enhanced transfectionin Raw 2647 cell There is no difference observed in case ofMCHODN NPs and naked ODN in regard to transfection inHela cells which lacks mannose receptor on cell membranesurface However transfection efficiency of lipofectinODNcomplexes was found to be much higher as compared toMCHODN NPs Kawakami and coworkers [72 83] earlieremphasized that the mannose receptor-mediated pathway


0102030405060708090

100

Free ODN MCHODN NP2 LipofectinODNFormulations

Raw 2647 cellsHela cells

OD

N (n

g10

6ce

lls)

Figure 12 Comparative study of transfection efficiency in Raw2647 cells and Hela cells with free ODN and MCHODN NP2 andat119873119875 ratio of 3 1 (119899 = 3) Transfection efficiency using Lipofectin2000 was set as a positive control

0102030405060708090

0 10 20 50

Mannose concentration (mM)

LipofectinODN

NakedODN

OD

N (n

g10

6ce

lls)

Figure 13 Competition assay of MCHODNNP2 (119873119875 = 3) in Raw2647 cells with different concentration of mannose

is more effective for gene expression in macrophages thanthe nonspecific pathway [134 135] These results suggest thatthe MCHODN NPs have the capability to transfect the celllines with mannose selectively Thus MCHODN NPs are anefficient carrier for delivery of free ODN

811 Competition Assay For initial studies Raw 2647macrophages cells expressing moderate mannose receptorswere used and it was found that the enhanced cellular uptakeof the MCHODN NPs is due to the mannose mediatedendocytosis The transfection activity of MCHODN NP2prepared at a charge ratio of 3 1 was investigated in thepresence of various amounts of mannose as a competitor forthe nanoparticles and results are given in Figure 13 show therelationship between the transfection efficiency of ODN andMCHODN NP2 (119873119875 = 3) and the mannose inhibitionconcentration

The transfection efficiency of MCHODN NP2 decreaseswhen the concentration of mannose increases and transfec-tion efficiencyuptake ofMCHODNNP2was inhibited about50 at 20mM concentration of mannose

9242

7236

4396

10812

0

20

40

60

80

100

Formulations

Free

OD

N+

LPS

CHO

DN

NP2

+LP

S

MCH

OD

N N

P2RD

+LP

S

MCH

OD

NN

P2+

LPS

TNF-120572

cont

ent (

o

f con

trol)

Figure 14 TNF-120572 expression profile of transfected Raw 2647 cellswith free ODN and MCHODN NP2 after stimulation of cells with100 ngmL LPS (119899 = 3) Control cells treated with PBS + LP

Our results clearly indicate that MCH-mediated ODNdelivery was mannose receptor-dependent because mannosecould block the binding in a concentration-dependent man-ner In other words the availability of mannose receptorsis essential for entry of MCHODN NPs into macrophagesthrough a receptor mediated delivery system Our results arein accordance with the earlier reported work with mannosy-lated cationic polymer for DNA delivery [62 93 128 136]

82 Cellular Uptake Cellular uptake of FITC-ODNalone against MCHODN NPs was studied by fluorescentmicroscopy and confocal laser scanning microscopyfollowing the incubation of Raw 2647 cells with free ODNand nanoparticles formulations for 4 h at 37∘C Resultsshown in Figures 16(a) and 16(b) clearly exhibit an increaseduptake of MCHODN NP2 compared to the FITC-ODNalone Increased fluorescence in the cells displayed highercell uptake of MCHODN NP2 as compared to free ODN asfurther confirmed by CLSM image (Figure 16(c))

9 Antisense Activity

To investigate the inhibitory activity of ODN on LPS(lipopolysaccharide) stimulated TNF-120572 release two differentsequences of ODN namely TJU-2755 (complementary ratTNF-120572 mRNA) and TJU-2755-RD (Random) were used Inorder to determine the potential inhibitory effect of oligonu-cleotides on LPS stimulated TNF-120572 release free ODN andMCHODN NPs formulations were added to the cells simul-taneously with LPS Concentration of TNF-120572 after exposureto ODN either in free form or in NPs formulations measuredusing ELISA method displayed results as shown in Figure 14The oligonucleotide (TJU-2755-ODN) was designed to targetrat TNF-120572mRNA LPS was used to stimulate macrophage forsecretion of cytokines including TNF-120572 Efficient delivery of


(a) (b)

Figure 15 (a) TEM photomicrograph magnification (times80000) (b) SEM photomicrograph of MCHODN NP2

(a) Free ODN (b) MCHODN NP2

(c) MCHODN NP2

Figure 16 Fluorescentmicroscopic images of Raw 2647 cells treated with (a) Free ODN and (b)MCHODNNP2 (c) Confocal laser scanningmicroscopic image of Raw 2647 cells treated with MCHODN NP2

ODN intomacrophage is proposed to suppress the expressionof TNF-120572 [137] The concentration of TNF-120572 after LPStreatment was found to be 10432 plusmn 76 ngmL As shownin the data free ODN bear weak inhibitory effect on TNF-120572 expression while MCH NPs could markedly promoteODN transfer into cells which might block TNF-120572 geneexpression After exposure to various formulations the TNF-120572 concentration was reduced to 9242 and 4396 (ofcontrol) with free ODN and MCHODNNP2 respectivelyTransfection effect of MCH NPs as ODN carrier was higherthan free ODN

To evaluate the inhibitory effect of TJU 2755 ODN onrat TNF-120572 production a control random scrambled sequenceof TJU 2755 was used with MCH NPs formulation Results

shown in Figure 14 indicate that MCHODN NP2 containingTJU 2755 ODN (complementary sequences of rat TNF-120572 mRNA) displayed significant reduction (approx 55 ofcontrol) in rat TNF-120572 production induced by LPS whereasMCHODN NP2 containing TJU2755 RD sequence showedno inhibitory effects on TNF-120572 production The resultssuggest that suppression of TNF-120572 production is dependenton the ODN sequence

10 Conclusion

In the present work mannosylated lowmolecular water solu-ble chitosan was successfully prepared and found as a suitabletargeting gene delivery carrier to macrophages It displayed


good capacity to form nanoconstructs with oligonucleotideand showed suitable physiochemical properties for genedelivery systemThese mannosylated nanoconstructs had nocytotoxicity and exhibit much higher gene transfer efficiencyon Raw 2647 cells TJU 2755 antisense oligonucleotidedisplayed antisense activity by reducing the LPS inducedserum TNF-120572 in 2647 cells Mannosylated chitosan displayspotential regarding safe and efficient gene targeting to themacrophages

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank AIIMS New Delhi forproviding SEM and TEM facility and BMHRC Bhopal forcarrying out cell lines studies and Confocal microscopy

References

[1] I Tamm B Dorken and G Hartmann ldquoAntisense therapy inoncology new hope for an old ideardquo The Lancet vol 358 no9280 pp 489ndash497 2001

[2] S T Crooke and B LebleuAntisense Research and ApplicationsCRC Press Boca Raton Fla USA 1993

[3] C A Stein andY C Cheng ldquoAntisense oligonucleotides as ther-apeutic agentsmdashis the bullet really magicalrdquo Science vol 261no 5124 pp 1004ndash1012 1993

[4] S Akhtar Delivery Strategies for Antisense OligonucleotideTherapeutics CRC Press Boca Raton Fla USA 1995

[5] S T Crooke ldquoMolecular mechanisms of action of antisensedrugsrdquo Biochimica et Biophysica ActamdashGene Structure andExpression vol 1489 no 1 pp 31ndash43 1999

[6] R A Stull and F C Szoka Jr ldquoAntigene ribozyme and aptamernucleic acid drugs progress and prospectsrdquo PharmaceuticalResearch vol 12 no 4 pp 465ndash483 1995

[7] K R Blake A Murakami and P S Miller ldquoInhibition of rabbitglobin mRNA translation by sequence-specific oligodeoxyri-bonucleotidesrdquo Biochemistry vol 24 no 22 pp 6132ndash61381985

[8] K R Blake AMurakami S A Spitz et al ldquoHybridization arrestof globin synthesis in rabbit reticulocyte lysates and cells byoligodeoxyribonucleoside methylphosphonatesrdquo Biochemistryvol 24 no 22 pp 6139ndash6145 1985

[9] M E Gleave and B P Monia ldquoAntisense therapy for cancerrdquoNature Reviews Cancer vol 5 no 6 pp 468ndash479 2005

[10] K J Lewis W J Irwin and S Akhtar ldquoBiodegradable poly(l-lactic acid) matrices for the sustained delivery of antisenseoligonucleotidesrdquo Journal of Controlled Release vol 37 no 1-2pp 173ndash183 1995

[11] S Akhtar and R L Juliano ldquoCellular uptake and intracellularfate of antisense oligonucleotidesrdquo Trends in Cell Biology vol 2no 5 pp 139ndash144 1992

[12] F Shi and D Hoekstra ldquoEffective intracellular delivery ofoligonucleotides in order to make sense of antisenserdquo Journalof Controlled Release vol 97 no 2 pp 189ndash209 2004

[13] CM Liu D P Liu andC C Liang ldquoOligonucleotide-mediatedgene repair at DNA level the potential applications for genetherapyrdquo Journal of Molecular Medicine vol 80 no 10 pp 620ndash628 2002

[14] S Akhtar ldquoAntisense technology selection and delivery ofoptimally acting antisense oligonucleotidesrdquo Journal of DrugTargeting vol 5 no 4 pp 225ndash234 1998

[15] R L Juliano S Alahari H Yoo R Kole andM Cho ldquoAntisensepharmacodynamics critical issues in the transport and deliveryof antisense oligonucleotidesrdquo Pharmaceutical Research vol 16no 4 pp 494ndash502 1999

[16] E Wagner M Cotten and K Zatloukal ldquoReceptor-mediatedgene delivery with synthetic virus-like particlesrdquo in Targetingof Drugs 5 Strategies for Oligonucleotide and Gene Delivery inTherapy G M Gregoriadis and B McCormack Eds pp 67ndash77 Plenum Press London UK 1995

[17] A Harada H Togawa and K Kataoka ldquoPhysicochemicalproperties and nuclease resistance of antisense-oligodeoxy-nucleotides entrapped in the core of polyion complex micellescomposed of poly(ethylene glycol)-poly(L-Lysine) blockcopolymersrdquo European Journal of Pharmaceutical Sciences vol13 no 1 pp 35ndash42 2001

[18] P Couvreur and C Malvy Pharmaceutical Aspects of Oligo-nuceotides vol 1 Taylor and Francis London UK 2000

[19] G Zon ldquoOligonucleotide analogues as potential chemothera-peuticmdashagentsrdquo Pharmaceutical Research vol 5 no 9 pp 539ndash549 1988

[20] NKashihara YMaeshima andHMakino ldquoAntisense oligonu-cleotidesrdquo Experimental Nephrology vol 6 no 1 pp 84ndash881998

[21] S Akhtar S Basu EWickstrom andR L Juliano ldquoInteractionsof antisense DNA oligonucleotide analogs with phospholipidmembranes (liposomes)rdquoNucleic Acids Research vol 19 no 20pp 5551ndash5559 1991

[22] E Wickstrom ldquoNuclease resistant nucleic acid therapeuticsrdquo inDelivery Strategies for Antisense Oligonucleotide Therapeutics SAkhtar Ed pp 85ndash104 CRC Press Boca Raton Fla USA 1995

[23] C A Stein ldquoPhosphorothioate antisense oligodeoxynucleotides questions of specificityrdquo Trends in Biotechnology vol 14no 5 pp 147ndash149 1996

[24] D J Glover H J Lipps and D A Jans ldquoTowards safe non-viral therapeutic gene expression in humansrdquo Nature ReviewsGenetics vol 6 no 4 pp 299ndash310 2005

[25] D D Lasic and N S Templeton ldquoLiposomes in gene therapyrdquoAdvanced Drug Delivery Reviews vol 20 no 2-3 pp 221ndash2661996

[26] A P Rolland ldquoFrom genes to gene medicines Recent advancesin nonviral gene deliveryrdquo Critical Reviews in Therapeutic DrugCarrier Systems vol 15 no 2 pp 143ndash198 1998

[27] M Nishikawa and L Huang ldquoNonviral vectors in the newmillennium delivery barriers in gene transferrdquo Human GeneTherapy vol 12 no 8 pp 861ndash870 2001

[28] H P Zobel J Kreuter D Werner C R Noe G Kumel andA Zimmer ldquoCationic polyhexylcyanoacrylate nanoparticles ascarriers for antisense oligonucleotidesrdquo Antisense and NucleicAcid Drug Development vol 7 no 5 pp 483ndash493 1997

[29] A Zimmer ldquoAntisense oligonucleotide deliverywith polyhexyl-cyanoacrylate nanoparticles as carriersrdquoMethods vol 18 no 3pp 286ndash295 1999


[30] E Fattal C Vauthier I Aynie et al ldquoBiodegradable polyalkyl-cyanoacrylate nanoparticles for the delivery of oligonu-cleotidesrdquo Journal of Controlled Release vol 53 no 1ndash3 pp 137ndash143 1998

[31] M G Ferreiro L Tillman G Hardee and R BodmeierldquoCharacterization of complexes of an antisense oligonucleotidewith protamine and poly-L-lysine saltsrdquo Journal of ControlledRelease vol 73 no 2-3 pp 381ndash390 2001

[32] M Junghans J Kreuter and A Zimmer ldquoPhosphodiester andphosphorothioate oligonucleotide condensation and prepara-tion of antisense nanoparticlesrdquo Biochimica et Biophysica Actavol 1544 no 1-2 pp 177ndash188 2001

[33] H P ZobelM Junghans VMaienschein et al ldquoEnhanced anti-sense efficacy of oligonucleotides adsorbed to monomethylam-inoethylmethacrylate methylmethacrylate copolymer nanopar-ticlesrdquo European Journal of Pharmaceutics and Biopharmaceu-tics vol 49 no 3 pp 203ndash210 2000

[34] M Berton E Allemann C A Stein and R Gurny ldquoHighlyloaded nanoparticulate carrier using an hydrophobic antisenseoligonucleotide complexrdquo European Journal of PharmaceuticalSciences vol 9 no 2 pp 163ndash170 1999

[35] C Chavany T LeDoan P Couvreur F Puisieux andCHeleneldquoPolyalkylcyanoacrylate nanoparticles as polymeric carriers forantisense oligonucleotidesrdquo Pharmaceutical Research vol 9 no4 pp 441ndash449 1992

[36] C Chavany T Saison-Behmoaras T le Doan F Puisieux PCouvreur and C Helene ldquoAdsorption of oligonucleotides ontopolyisohexylcyanoacrylate nanoparticles protects them againstnucleases and increases their cellular uptakerdquo PharmaceuticalResearch vol 11 no 9 pp 1370ndash1378 1994

[37] S Akhtar and K J Lewis ldquoAntisense oligonucleotide deliveryto cultured macrophages is improved by incorporation intosustained-release biodegradable polymer microspheresrdquo Inter-national Journal of Pharmaceutics vol 151 no 1 pp 57ndash67 1997

[38] S T Crooke ldquoDelivery of oligonucleotides and polynu-cleotidesrdquo Journal of Drug Targeting vol 3 no 3 pp 185ndash1901995

[39] R DeLong K Stephenson T Loftus et al ldquoCharacterization ofcomplexes of oligonucleotides with polyamidoamine starburstdendrimers and effects on intracellular deliveryrdquo Journal ofPharmaceutical Sciences vol 86 no 6 pp 762ndash764 1997

[40] J A Hughes A I Aronsohn A V Avrutskaya and R LJuliano ldquoEvaluation of adjuvants that enhance the effectivenessof antisense oligodeoxynucleotidesrdquo Pharmaceutical Researchvol 13 no 3 pp 404ndash410 1996

[41] A M Gewirtz D L Sokol and M Z Ratajczak ldquoNucleic acidtherapeutics state of the art and future prospectsrdquo Blood vol92 no 3 pp 712ndash736 1998

[42] M Junghans J Kreuter and A Zimmer ldquoAntisense deliv-ery using protamine-oligonucleotide particlesrdquo Nucleic AcidsResearch vol 28 no 10 pp E45ndashE47 2000

[43] M Berton P Turelli D Trono C Stein E Allemann and RGurny ldquoInhibition of HIV-1 in cell culture by oligonucleotide-loaded nanoparticlesrdquo Pharmaceutical Research vol 18 no 8pp 1096ndash1101 2001

[44] A Arnedo S Espuelas and J M Irache ldquoAlbumin nanopar-ticles as carriers for a phosphodiester oligonucleotiderdquo Inter-national Journal of Pharmaceutics vol 244 no 1-2 pp 59ndash722002

[45] C Emile D Bazile F Herman C Helene and M VeillardldquoEncapsulation of oligonucleotides in stealth MePEG-PLA

50

nanoparticles by complexation with structured oligopeptidesrdquoDrug Delivery vol 3 no 3 pp 187ndash195 1996

[46] H FritzMMaier and E Bayer ldquoCationic polystyrene nanopar-ticles preparation and characterization of a model drug carriersystem for antisense oligonucleotidesrdquo Journal of Colloid andInterface Science vol 195 no 2 pp 272ndash288 1997

[47] I Aynie C Vauthier H Chacun E Fattal and P CouvreurldquoSpongelike alginate nanoparticles as a new potential systemfor the delivery of antisense oligonucleotidesrdquo Antisense andNucleic Acid Drug Development vol 9 no 3 pp 301ndash312 1999

[48] G Schwab C Chavany I Duroux et al ldquoAntisense oligonu-cleotides adsorbed to polyalkylcyanoacrylate nanoparticlesspecifically inhibit mutated Ha-ras-mediated cell proliferationand tumorigenicity in nude micerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 91 no22 pp 10460ndash10464 1994

[49] G Lambert E Fattal A Brehier J Feger and P CouvreurldquoEffect of polyisobutylcyanoacrylate nanoparticles and Lipo-fectin loaded with oligonucleotides on cell viability and PKC120572neosynthesis inHepG2 cellsrdquo Biochimie vol 80 no 12 pp 969ndash976 1998

[50] A Arnedo J M Irache M Merodio and M S E MillanldquoAlbumin nanoparticles improved the stability nuclear accu-mulation and anticytomegaloviral activity of a phosphodiesteroligonucleotiderdquo Journal of Controlled Release vol 94 no 1 pp217ndash227 2004

[51] K A Janes P Calvo andM J Alonso ldquoPolysaccharide colloidalparticles as delivery systems for macromoleculesrdquo AdvancedDrug Delivery Reviews vol 47 no 1 pp 83ndash97 2001

[52] MKoping-Hoggard I Tubulekas HGuan et al ldquoChitosan as anonviral gene delivery system Structure-property relationshipsand characteristics compared with polyethylenimine in vitroand after lung administration in vivordquo GeneTherapy vol 8 no14 pp 1108ndash1121 2001

[53] K A Howard U L Rahbek X Liu et al ldquoRNA interferencein Vitro and in Vivo using a novel chitosansiRNA nanoparticlesystemrdquoMolecular Therapy vol 14 no 4 pp 476ndash484 2006

[54] S Gao J Chen L Dong Z Ding Y Yang and J ZhangldquoTargeting delivery of oligonucleotide and plasmid DNA tohepatocyte via galactosylated chitosan vectorrdquoEuropean Journalof Pharmaceutics and Biopharmaceutics vol 60 no 3 pp 327ndash334 2005

[55] L Illum ldquoChitosan and its use as a pharmaceutical excipientrdquoPharmaceutical Research vol 15 no 9 pp 1326ndash1331 1998

[56] F C MacLaughlin R J Mumper J Wang et al ldquoChitosan anddepolymerized chitosan oligomers as condensing carriers for invivo plasmid deliveryrdquo Journal of Controlled Release vol 56 no1ndash3 pp 259ndash272 1998

[57] M Lee S Chun W Choi et al ldquoThe use of chitosan as acondensing agent to enhance emulsion-mediated gene transferrdquoBiomaterials vol 26 no 14 pp 2147ndash2156 2005

[58] X Z Shu andK J Zhu ldquoThe influence ofmultivalent phosphatestructure on the properties of ionically cross-linked chitosanfilms for controlled drug releaserdquo European Journal of Pharma-ceutics and Biopharmaceutics vol 54 no 2 pp 235ndash243 2002

[59] S C W Richardson H V J Kolbe and R Duncan ldquoPotentialof low molecular mass chitosan as a DNA delivery systembiocompatibility body distribution and ability to complex andprotect DNArdquo International Journal of Pharmaceutics vol 178no 2 pp 231ndash243 1999

[60] P Erbacher S Zou T Bettinger A M Steffan and J S RemyldquoChitosan-based vectorDNA complexes for gene delivery


biophysical characteristics and transfection abilityrdquoPharmaceu-tical Research vol 15 no 9 pp 1332ndash1339 1998

[61] I-K Park H-L Jiang S-E Cook et al ldquoGalactosylated chi-tosan (GC)-graft-poly(vinyl pyrrolidone) (PVP) as hepatocyte-targeting DNA carrier in vitro transfectionrdquo Archives of Phar-macal Research vol 27 no 12 pp 1284ndash1289 2004

[62] T H Kim H Jin H W Kim M Cho and C S Cho ldquoMan-nosylated chitosan nanoparticle-based cytokine gene therapysuppressed cancer growth in BALBc mice bearing CT-26carcinoma cellsrdquoMolecular CancerTherapeutics vol 5 no 7 pp1723ndash1732 2006

[63] M Thanou B I Florea M Geldof H E Junginger andG Borchard ldquoQuaternized chitosan oligomers as novel genedelivery vectors in epithelial cell linesrdquo Biomaterials vol 23 no1 pp 153ndash159 2002

[64] F LiW G Liu and K D Yao ldquoPreparation of oxidized glucose-crosslinkedN-alkylated chitosanmembrane and in vitro studiesof pH-sensitive drug delivery behaviourrdquo Biomaterials vol 23no 2 pp 343ndash347 2002

[65] Y H Kim S H Gihm C R Park K Y Lee T W Kimand I C Kwon ldquoStructural characteristics of size-controlledself-aggregates of deoxycholic acid-modified chitosan and theirapplication as a DNA delivery carrierrdquo Bioconjugate Chemistryvol 12 no 6 pp 932ndash938 2001

[66] A Sato S Kawakami M Yamada F Yamashita and MHashida ldquoEnhanced gene transfection in macrophages usingmannosylated cationic liposome-polyethylenimine-plasmidDNA complexesrdquo Journal of Drug Targeting vol 9 no 3 pp201ndash207 2001

[67] M Lee J-W Nah Y Kwon J J Koh K S Ko and S WKim ldquoWater-soluble and low molecular weight chitosan-basedplasmid DNA deliveryrdquo Pharmaceutical Research vol 18 no 4pp 427ndash431 2001

[68] S Gao J Chen X Xu et al ldquoGalactosylated low molecularweight chitosan as DNA carrier for hepatocyte-targetingrdquoInternational Journal of Pharmaceutics vol 255 no 1-2 pp 57ndash68 2003

[69] Z Cui and R J Mumper ldquoChitosan-based nanoparticles fortopical genetic immunizationrdquo Journal of Controlled Releasevol 75 no 3 pp 409ndash419 2001

[70] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1ndash3 pp 81ndash96 2001

[71] G Y Wu and C H Wu ldquoReceptor-mediated gene delivery andexpression in vivordquoThe Journal of Biological Chemistry vol 263no 29 pp 14621ndash14624 1988

[72] S Kawakami F Yamashita M Nishikawa Y Takakura and MHashida ldquoAsialoglycoprotein receptor-mediated gene transferusing novel galactosylated cationic liposomesrdquo Biochemical andBiophysical Research Communications vol 252 no 1 pp 78ndash831998

[73] S Kawakami S Fumoto M Nishikawa F Yamashita andM Hashida ldquoIn vivo gene delivery to the liver using novelgalactosylated cationic liposomesrdquo Pharmaceutical Researchvol 17 no 3 pp 306ndash313 2000

[74] M Nishikawa M Yamauchi K Morimoto E Ishida YTakakura and M Hashida ldquoHepatocyte-targeted in vivo geneexpression by intravenous injection of plasmidDNAcomplexedwith synthetic multi-functional gene delivery systemrdquo GeneTherapy vol 7 no 7 pp 548ndash555 2000

[75] MHashida S TakemuraMNishikawa and Y Takakura ldquoTar-geted delivery of plasmid DNA complexed with galactosylated

poly(l- lysine)rdquo Journal of Controlled Release vol 53 no 1ndash3pp 301ndash310 1998

[76] R I Mahato S Takemura K Akamatsu M Nishikawa YTakakura and M Hashida ldquoPhysicochemical and dispositioncharacteristics of antisense oligonucleotides complexed withglycosylated poly(L-lysine)rdquo Biochemical Pharmacology vol 53no 6 pp 887ndash895 1997

[77] T Hara Y Aramaki S Takada K Koike and S TsuchiyaldquoReceptor-mediated transfer of pSV2CAT DNA to a humanhepatoblastoma cell line HepG2 using asialofetuin-labeledcationic liposomesrdquo Gene vol 159 no 2 pp 167ndash174 1995

[78] T Hara Y Aramaki S Takada K Koike and S TsuchiyaldquoReceptor-mediated transfer of pSV2CAT DNA to mouse livercells using asialofetuin-labeled liposomesrdquoGeneTherapy vol 2no 10 pp 784ndash789 1995

[79] T Hara H Kuwasawa Y Aramaki et al ldquoEffects offusogenic and DNA-binding amphiphilic compounds onthe receptor-mediated gene transfer into hepatic cells byasialofetuinmdashlabeled liposomesrdquo Biochimica et BiophysicaActamdashBiomembranes vol 1278 no 1 pp 51ndash58 1996

[80] J S Remy A Kichler V Mordvinov F Schuber and JP Behr ldquoTargeted gene transfer into hepatoma cells withlipopolyamine-condensed DNA particles presenting galactoseligands A stage toward artificial virusesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 92 no 5 pp 1744ndash1748 1995

[81] T Ferkol J C Perales F Mularo and RW Hanson ldquoReceptor-mediated gene transfer into macrophagesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 93 no 1 pp 101ndash105 1996

[82] P Erbacher M T Bousser J Raimond M Monsigny PMidoux and A C Roche ldquoGene transfer by DNAglycosylatedpolylysine complexes into human blood monocyte-derivedmacrophagesrdquo Human Gene Therapy vol 7 no 6 pp 721ndash7291996

[83] S Kawakami A Sato M Nishikawa F Yamashita andM Hashida ldquoMannose receptor-mediated gene transfer intomacrophages using novel mannosylated cationic liposomesrdquoGene Therapy vol 7 no 4 pp 292ndash299 2000

[84] S Kawakami A Sato M Yamada F Yamashita and MHashida ldquoThe effect of lipid composition on receptor-mediatedin vivo gene transfection using mannosylated cationic lipo-somes in micerdquo STP Pharma Sciences vol 11 no 1 pp 117ndash1202001

[85] M Nishikawa S Takemura F Yamashita et al ldquoPharmacoki-netics and in vivo gene transfer of plasmid DNA complexedwith mannosylated poly (L-lysine) in micerdquo Journal of DrugTargeting vol 8 no 1 pp 29ndash38 2000

[86] Y H Choi F Liu J S Park and S W Kim ldquoLactose-poly(ethylene glycol)-grafted poly-L-lysine as hepatoma cell-targeted gene carrierrdquo Bioconjugate Chemistry vol 9 no 6 pp708ndash718 1998

[87] E Wagner M Zenke M Cotten H Beug and M L BirnstielldquoTransferrin-polycation conjugates as carriers for DNA uptakeinto cellsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 87 no 9 pp 3410ndash3414 1990

[88] R Kircheis A Kichler G Wallner et al ldquoCoupling of cell-binding ligands to polyethylenimine for targeted gene deliveryrdquoGene Therapy vol 4 no 5 pp 409ndash418 1997

[89] J Chen S Gamou A Takayanagi and N Shimizu ldquoA novelgene delivery system using EGF receptor-mediated endocyto-sisrdquo FEBS Letters vol 338 no 2 pp 167ndash169 1994


[90] CMVarga T JWickham andDA Lauffenburger ldquoReceptor-mediated targeting of gene delivery vectors insights frommolecular mechanisms for improved vehicle designrdquo Biotech-nology and Bioengineering vol 70 no 6 pp 593ndash605 2000

[91] Y Takakura R I Mahato M Nishikawa and M HashidaldquoControl of pharmacokinetic profiles of drugmdashmacromoleculeconjugatesrdquo Advanced Drug Delivery Reviews vol 19 no 3 pp377ndash399 1996

[92] P Stahl P H Schlesinger E Sigardson J S Rodman andY C Lee ldquoReceptor-mediated pinocytosis of mannose glyco-conjugates by macrophages characterization and evidence forreceptor recyclingrdquo Cell vol 19 no 1 pp 207ndash215 1980

[93] J Kuiper A Brouwer D L Knook and T J C van BerkelldquoKupffer and sinusoidal endothelial cellsrdquo in The Liver BiologyandPathobiology IMArias J L BoyerN FaustoW B JakobyD A Schachter and D A Shafritz Eds pp 791ndash818 RavenPress New York NY USA 3rd edition 1994

[94] W I Weis M E Taylor and K Drickamer ldquoThe C-type lectinsuperfamily in the immune systemrdquo Immunological Reviewsvol 163 pp 19ndash34 1998

[95] M Hashimoto M Morimoto H Saimoto et al ldquoGene transferby DNAmannosylated chitosan complexes into mouse peri-toneal macrophagesrdquo Biotechnology Letters vol 28 no 11 pp815ndash821 2006

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

[97] M Monsigny C Petit and A C Roche ldquoColorimetricdetermination of neutral sugars by a resorcinol sulfuric acidmicromethodrdquo Analytical Biochemistry vol 175 no 2 pp 525ndash530 1988

[98] L Jiang F Qian X He et al ldquoNovel chitosan derivativenanoparticles enhance the immunogenicity of a DNA vaccineencoding hepatitis B virus core antigen inmicerdquo Journal of GeneMedicine vol 9 no 4 pp 253ndash264 2007

[99] M Huang C W Fong E Khor and L Y Lim ldquoTransfectionefficiency of chitosan vectors effect of polymer molecularweight and degree of deacetylationrdquo Journal of ControlledRelease vol 106 no 3 pp 391ndash406 2005

[100] Y K Park Y H Park B A Shin et al ldquoGalactosylated chitosan-graft-dextran as hepatocyte-targeting DNA carrierrdquo Journal ofControlled Release vol 69 no 1 pp 97ndash108 2000

[101] S Sundaram L K Lee and C M Roth ldquoInterplay ofpolyethyleneimine molecular weight and oligonucleotide back-bone chemistry in the dynamics of antisense activityrdquo NucleicAcids Research vol 35 no 13 pp 4396ndash4408 2007

[102] G D Gray and EWickstrom ldquoRapidmeasurement ofmodifiedoligonucleotide levels in plasma samples with a fluorophorespecific for single-stranded DNArdquo Antisense and Nucleic AcidDrug Development vol 7 no 3 pp 133ndash140 1997

[103] T H Kim I K Park J W Nah Y J Choi and C S CholdquoGalactosylated chitosanDNA nanoparticles prepared usingwater-soluble chitosan as a gene carrierrdquo Biomaterials vol 25no 17 pp 3783ndash3792 2004

[104] D Sgouras and R Duncan ldquoMethods for the evaluation ofbiocompatibility of soluble synthetic polymers which havepotential for biomedical use 1mdashuse of the tetrazolium-basedcolorimetric assay (MTT) as a preliminary screen for evaluationof in vitro cytotoxicityrdquo Journal ofMaterials ScienceMaterials inMedicine vol 1 no 2 pp 61ndash68 1990

[105] X Pan L Chen S Liu X Yang J Gao and R J Lee ldquoAnti-tumor activity of G3139 lipid nanoparticles (LNPs)rdquo MolecularPharmaceutics vol 6 no 1 pp 211ndash220 2009

[106] MYamauchiH Kusano E Saito et al ldquoImproved formulationsof antisense oligodeoxynucleotides using wrapped liposomesrdquoJournal of Controlled Release vol 114 no 2 pp 268ndash275 2006

[107] S Kim E Kim J Hou et al ldquoOpsonized erythrocyte ghostsfor liver-targeted delivery of antisense oligodeoxynucleotidesrdquoBiomaterials vol 30 no 5 pp 959ndash967 2009

[108] A Salvati L Ciani S Ristori G Martini A Masi and AArcangeli ldquoPhysico-chemical characterization and transfectionefficacy of cationic liposomes containing the pEGFP plasmidrdquoBiophysical Chemistry vol 121 no 1 pp 21ndash29 2006

[109] L Dong S Xia Y Luo H Diao J Zhang and J Chen ldquoTar-geting delivery oligonucleotide into macrophages by cationicpolysaccharide from Bletilla striata successfully inhibited theexpression of TNF-120572rdquo Journal of Controlled Release vol 134 no3 pp 214ndash220 2009

[110] J Nah and M Jang ldquoSpectroscopic characterization and prepa-ration of lowmolecular water-soluble chitosan with free-aminegroup by novel methodrdquo Journal of Polymer Science A PolymerChemistry vol 40 no 21 pp 3796ndash3803 2002

[111] D G Kim C Choi Y I Jeong et al ldquoAll-trans retinoicacid-associated low molecular weight water-soluble chitosannanoparticles based on ion complexrdquoMacromolecular Researchvol 14 no 1 pp 66ndash72 2006

[112] M C P De Lima S Simoes P Pires H Faneca and NDuzgunes ldquoCationic lipid-DNA complexes in gene deliveryfrom biophysics to biological applicationsrdquo Advanced DrugDelivery Reviews vol 47 no 2-3 pp 277ndash294 2001

[113] M Thomas and A M Klibanov ldquoNon-viral gene therapypolycation-mediated DNA deliveryrdquo Applied Microbiology andBiotechnology vol 62 no 1 pp 27ndash34 2003

[114] L W Seymour ldquoPassive tumor targeting of soluble macro-molecules and drug conjugatesrdquo Critical Reviews inTherapeuticDrug Carrier Systems vol 9 no 2 pp 135ndash187 1992

[115] S Prabha W Zhou J Panyam and V Labhasetwar ldquoSize-dependency of nanoparticle-mediated gene transfection stud-ies with fractionated nanoparticlesrdquo International Journal ofPharmaceutics vol 244 no 1-2 pp 105ndash115 2002

[116] K Kunath A von Harpe D Fischer and T Kissel ldquoGalactose-PEI-DNA complexes for targeted gene delivery degree ofsubstitution affects complex size and transfection efficiencyrdquoJournal of Controlled Release vol 88 no 1 pp 159ndash172 2003

[117] H Petersen P M Fechner A L Martin et al ldquoPolyethyl-enimine-graft-poly(ethylene glycol) copolymers influence ofcopolymer block structure on DNA complexation and biolog-ical activities as gene delivery systemrdquo Bioconjugate Chemistryvol 13 no 4 pp 845ndash854 2002

[118] R I Mahato Y Takakura and M Hashida ldquoNonviral vectorsfor in vivo gene delivery physicochemical and pharmacokineticconsiderationsrdquo Critical Reviews in Therapeutic Drug CarrierSystems vol 14 no 2 pp 133ndash172 1997

[119] R I Mahato K Kawabata T Nomura Y Takakura and MHashida ldquoPhysicochemical and pharmacokinetic characteris-tics of plasmid DNAcationic liposome complexesrdquo Journal ofPharmaceutical Sciences vol 84 no 11 pp 1267ndash1271 1995

[120] J Kim B Kim A Maruyama T Akaike and S W Kim ldquoA newnon-viral DNA delivery vector the terplex systemrdquo Journal ofControlled Release vol 53 no 1ndash3 pp 175ndash182 1998


[121] M E Barry D Pinto-Gonzalez F M Orson G J McKenzie GR Petry and M A Barry ldquoRole of endogenous endonucleasesand tissue site in transfection and CpG-mediated immuneactivation after naked DNA injectionrdquo Human Gene Therapyvol 10 no 15 pp 2461ndash2480 1999

[122] I K Park T H Kim Y H Park et al ldquoGalactosylated chitosan-graft-poly(ethylene glycol) as hepatocyte-targeting DNA car-rierrdquo Journal of Controlled Release vol 76 no 3 pp 349ndash3622001

[123] S M Moghimi P Symonds J C Murray A C Hunter GDebska and A Szewczyk ldquoA two-stage poly(ethylenimine)-mediated cytotoxicity implications for gene transfertherapyrdquoMolecular Therapy vol 11 no 6 pp 990ndash995 2005

[124] Q-Q Zhao J-L Chen M Han W-Q Liang Y Tabata andJ-Q Gao ldquoCombination of poly(ethylenimine) and chitosaninduces high gene transfection efficiency and low cytotoxicityrdquoJournal of Bioscience and Bioengineering vol 105 no 1 pp 65ndash68 2008

[125] B Carreno-Gomez and R Duncan ldquoEvaluation Of the biolog-ical properties of soluble chitosan and chitosan microspheresrdquoInternational Journal of Pharmaceutics vol 148 no 2 pp 231ndash240 1997

[126] K Kunath A von Harpe D Fischer et al ldquoLow-molecular-weight polyethylenimine as a non-viral vector for DNA deliv-ery comparison of physicochemical properties transfectionefficiency and in vivo distribution with high-molecular-weightpolyethyleniminerdquo Journal of Controlled Release vol 89 no 1pp 113ndash125 2003

[127] H Jiang Y Kim R Arote et al ldquoMannosylated chitosan-graft-polyethylenimine as a gene carrier for Raw 2647 cell targetingrdquoInternational Journal of Pharmaceutics vol 375 no 1-2 pp 133ndash139 2009

[128] I Y Park I Y Kim M K Yoo Y J Choi M Cho and CS Cho ldquoMannosylated polyethylenimine coupled mesoporoussilica nanoparticles for receptor-mediated gene deliveryrdquo Inter-national Journal of Pharmaceutics vol 359 no 1-2 pp 280ndash2872008

[129] KCorsi F Chellat L Yahia and J C Fernandes ldquoMesenchymalstem cells MG63 and HEK293 transfection using chitosan-DNA nanoparticlesrdquo Biomaterials vol 24 no 7 pp 1255ndash12642003

[130] X Zhou B Liu X Yu et al ldquoControlled release of PEIDNAcomplexes from mannose-bearing chitosan microspheres as apotent delivery system to enhance immune response to HBVDNA vaccinerdquo Journal of Controlled Release vol 121 no 3 pp200ndash207 2007

[131] H Jiang M L Kang J Quan et al ldquoThe potential of manno-sylated chitosan microspheres to target macrophage mannosereceptors in an adjuvant-delivery system for intranasal immu-nizationrdquo Biomaterials vol 29 no 12 pp 1931ndash1939 2008

[132] S S Diebold M Kursa E Wagner M Cotten and M ZenkeldquoMannose polyethylenimine conjugates for targetedDNAdeliv-ery into dendritic cellsrdquoThe Journal of Biological Chemistry vol274 no 27 pp 19087ndash19094 1999

[133] R PMi OH Ki K H In et al ldquoDegradable polyethylenimine-alt-poly(ethylene glycol) copolymers as novel gene carriersrdquoJournal of Controlled Release vol 105 no 3 pp 367ndash380 2005

[134] Y Lu S Kawakami F Yamashita and M Hashida ldquoDevel-opment of an antigen-presenting cell-targeted DNA vaccineagainst melanoma by mannosylated liposomesrdquo Biomaterialsvol 28 no 21 pp 3255ndash3262 2007

[135] Y Hattori S Suzuki S Kawakami F Yamashita and MHashida ldquoThe role of dioleoylphosphatidylethanolamine(DOPE) in targeted gene delivery with mannosylated cationicliposomes via intravenous routerdquo Journal of Controlled Releasevol 108 no 2-3 pp 484ndash495 2005

[136] Y Rojanasakul D N Weissman X Shi V Castranova J KH Ma and W Liang ldquoAntisense inhibition of silica-inducedtumor necrosis factor in alveolar macrophagesrdquo The Journal ofBiological Chemistry vol 272 no 7 pp 3910ndash3914 1997

[137] M F Taylor J D Paulauskis D D Weller and L KobzikldquoComparison of efficacy of antisense oligomers directed towardTNF-120572 in helper T and macrophage cell linesrdquo Cytokine vol 9no 9 pp 672ndash681 1997

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


For therapeutic application antisense technology promisesgreater advantages over drugs currently on the market byoffering new types of drugs that are easy to design andhave a very high molecular target selectivity and efficacyinhibiting a specific gene expression and an expected lowtoxicity due to metabolism to natural nucleotide componentsby the endogenous systems [5 10] Vitravene is only ODNbased therapeutic (antisense ODN) approved by FDA andseveral others are in Phase 3 clinical trials However ODNstherapeutics have been associated with multiple obstaclesthat is poor physiological stability and cellular uptake [1112] inadequate intracellular ODNs concentration [13 14]inability to target specific cell [14 15] lack of tissue specificity[16] and nuclease degradation [14 17] Thus intracellulardelivery and therapeutic applications in diseases of ODN-based therapeutics have been compromised Although vari-ous chemicalmodifications ofODNs can be used to overcomethese problems [18] they offer other limitations such aslow binding affinity nonsequences specific biological effectstoxicity and acute homeostatic changes after in vivo adminis-tration [19ndash23] Therefore development of effective deliverysystems that are capable of protecting and efficiently deliverODNs intracellularly in target cells becomes essential toexploit the promising applications offered by successful ODNdelivery Among the gene delivery vectors nonviral vectorsare preferred due to its ease of synthesis low immunogenicityand unrestricted gene materials size in addition to potentialbenefits in terms of safety [24ndash27]

Nanoparticles (NPs) were introduced as nonviral genevectors to solve issues related to ODNs delivery [28ndash36]Other carriers such as microspheres matrices complexeswith polycations and starburst dendrimers have beenrecently investigated [10 37ndash40] The major advantage ofusing nanocarriers resides in the possibility of conjugatingligands to them as to direct them to a desired site for localizeddelivery in cells tissue or organ [41] ODNs in complexstate with nanocarriers avail protection against nucleasedegradation [38 42ndash44] Further their cell uptake could beincreased as carrier-ODNs complexes are taken up throughactive endocytosis process

Recently biodegradable nanoparticles have been studiedas potential inert and biocompatible carriers for geneticmaterials for example polylactide polyethylene glycol (PLA-PEG) cationic polystyrene spongelike alginate polyalkyl-cyanoacrylate poly isohexylcyanoacrylate or poly isobutyl-cyanoacrylate and albumin chitosan nanoparticles [36 45ndash51]

Among the large number of cationic polymers describedchitosan is shown to be an effective vector because of con-densing and delivered DNA siRNA in vitro and in vivo [5253] and ODN in vitro [54] It is a natural positively chargedpolymer that can be utilized for preparation ofnanopar-ticulate carriers which represents a novel strategy for thesafe and efficient delivery of gene It has been extensivelyexamined for its potential in the development of controlledrelease drug delivery formulation due to its unique poly-meric cationic characteristic gel forming and film formingproperties [55 56] Chitosan has beneficial qualities such aslow toxicity low immunogenicity excellent biodegradability

and biocompatibility [57 58] It is a suitable candidate forgene delivery system due to its ability to spontaneously forminterpolyelectrolyte stable complexes with genetic material(ODNs or DNA) as a result of cooperative electrostaticinteractions between the positive amino groups of chitosanand the negative phosphate groups ofDNAODN [54 56 59]

Consequently several groups have conducted studiesusing chitosanDNA nanoparticles including use ofgalactosylated chitosan [60] galactosylated chitosan-graft-poly(vinylpyrrolidone) (PVP) [61] mannosylatedchitosanDNA nanoparticles [62] trimethylated chitosanoligomers [63] N-dodecylated chitosan [64] deoxycholicacid modified chitosan [65] galactosylated chitosanODNvector [54] or ligand attached chitosans for targeting cellmembrane receptors [66] However chitosan was difficultto solubilize in water and was dissolved in acidic solutionLow molecular weight water soluble chitosan has beenemployed which is highly water soluble and forms complexwith plasmid DNAODN at physiological pH [54 67 68]They can display high transfection efficiency along withthe higher plasmid DNAODN loading protection againstthe nuclease degradation and being less toxic [54 69 70]Ligands are molecular extension that pave fate of deliverysystem for active targeting and increase overall therapeuticpotential Thus the receptor mediated gene targeting is apromising approach to obtain cell-selective gene transfectionA number of receptor mediated gene delivery systems havebeen developed to introduce the foreign DNAODN intospecific cell types Ligands currently being investigatedinclude galactose [71ndash80] mannose [66 81ndash85] lactose[86] transferrin [87 88] and epidermal growth factor [89]Active targeting using receptor mediated interaction hasbeen effective in gene delivery [90]

Among the various receptors asialoglycoprotein recep-tors and mannose receptors are the most promising for genetargeting because they exhibit high affinity and are rapidlyinternalized [91] Wide variety of macrophages (includingKupffer cells) express mannose specific membrane receptorswhich internalize glycoprotein bearing mannose residue viaclathrin-coated vesicles to allow a delivery into the endoso-mal system [91ndash94]

In gene delivery systems the mannosylation of cationicpolymers such as mannosylated polylysine and PEI wereused to achieve the delivery of gene to macrophages [8285] Mannosylated chitosan for plasmid DNA has also beenreported earlier for macrophage targeting [95]

Although chitosan has been studies for more than adecade as a gene vector for DNA ODN and siRNA [54 96]so for to our knowledge there is no study that has beencarried out to investigate the use of mannosylated chitosannanoparticles to determine ODN in vitro In the presentstudy we formulate mannosylated LMW-water soluble chi-tosan formulations by self-assembled method characterizedfor gel retardation assay particle size particles shape andparticles surface morphology and zeta potential complexingcapacity protection ability and MCHODN NPs stabilityCytotoxicity studies transfection efficiency and antisenseassay were also assessed for developed nanoparticles formu-lations



















2







2






Cell Viability () = (119873119894

119873119888

) times 100 or


sampleOD590

control) times 100

(1)

where 119873119894and 119873



590sample


























3






O

CH2OH CH2OHCH2OH

OH

NH2

CH3

OO OH

O O

NHOC

CH2OH

OOH OH

OH OHHOH H

H

O N C S

OO

OH O

NH

SC

HN H

O

OCH2OH

OHHH

H

CH2OH

OH OH OO

CH2OH

CH3

NH

OC

OO

NH2

Chitosan

+

Stirring



M

K M

X

25∘C24hKminusX


10000

0003500 3000 2500 2000 1500 1000 500

(cmminus1)

3439

32506

23556 21920

19042

16579

16009

16409

15642

15390 14301

14000

13550

12625

12364

11922

10710

10230

9621 9039

8718

8266

7656

7364

6766 6042

5586

5069

4827

T (

)


80 70 60 50 40 30 20 10

(ppm)




10000

0003500 3000 2500 2000 1500 1000 500

(cmminus1)

30847

29215

23724

17312

16514

16261

15621

15234

14174 13

212

12834

11844

11122

10204 9548

8908

8085

7684

7204

6990

6800

6301

6071

5831 55365274 4548

T (

)


80 70 60 50 40 30 20 10

(ppm)






A FEDCB










A FEDCB

(a)

A FEDCB

(b)












033

035

025 05 1 15 2Time (days)


Turb

idity

at340

nm


A FEDCB






0

20

40

60

80

100

120


Cel

l via

bilit

y (

cont

rol)









0

20

40

60

80

100

120


Cel

l via

bilit

y (

)





0102030405060708090



OD

N (n

g10

6ce

lls)





0102030405060708090

100



OD

N (n

g10

6ce

lls)


0102030405060708090

0 10 20 50


LipofectinODN

NakedODN

OD

N (n

g10

6ce

lls)





9242

7236

4396

10812

0

20

40

60

80

100

Formulations

Free

OD

N+

LPS

CHO

DN

NP2

+LP

S

MCH

OD

N N

P2RD

+LP

S

MCH

OD

NN

P2+

LPS

TNF-120572

cont

ent (

o

f con

trol)







(a) (b)



(c) MCHODN NP2





10 Conclusion






Acknowledgments


References















































50










































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















2







2






Cell Viability () = (119873119894

119873119888

) times 100 or


sampleOD590

control) times 100

(1)

where 119873119894and 119873



590sample


























3






O

CH2OH CH2OHCH2OH

OH

NH2

CH3

OO OH

O O

NHOC

CH2OH

OOH OH

OH OHHOH H

H

O N C S

OO

OH O

NH

SC

HN H

O

OCH2OH

OHHH

H

CH2OH

OH OH OO

CH2OH

CH3

NH

OC

OO

NH2

Chitosan

+

Stirring



M

K M

X

25∘C24hKminusX


10000

0003500 3000 2500 2000 1500 1000 500

(cmminus1)

3439

32506

23556 21920

19042

16579

16009

16409

15642

15390 14301

14000

13550

12625

12364

11922

10710

10230

9621 9039

8718

8266

7656

7364

6766 6042

5586

5069

4827

T (

)


80 70 60 50 40 30 20 10

(ppm)




10000

0003500 3000 2500 2000 1500 1000 500

(cmminus1)

30847

29215

23724

17312

16514

16261

15621

15234

14174 13

212

12834

11844

11122

10204 9548

8908

8085

7684

7204

6990

6800

6301

6071

5831 55365274 4548

T (

)


80 70 60 50 40 30 20 10

(ppm)






A FEDCB










A FEDCB

(a)

A FEDCB

(b)












033

035

025 05 1 15 2Time (days)


Turb

idity

at340

nm


A FEDCB






0

20

40

60

80

100

120


Cel

l via

bilit

y (

cont

rol)









0

20

40

60

80

100

120


Cel

l via

bilit

y (

)





0102030405060708090



OD

N (n

g10

6ce

lls)





0102030405060708090

100



OD

N (n

g10

6ce

lls)


0102030405060708090

0 10 20 50


LipofectinODN

NakedODN

OD

N (n

g10

6ce

lls)





9242

7236

4396

10812

0

20

40

60

80

100

Formulations

Free

OD

N+

LPS

CHO

DN

NP2

+LP

S

MCH

OD

N N

P2RD

+LP

S

MCH

OD

NN

P2+

LPS

TNF-120572

cont

ent (

o

f con

trol)







(a) (b)



(c) MCHODN NP2





10 Conclusion






Acknowledgments


References















































50










































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















3






O

CH2OH CH2OHCH2OH

OH

NH2

CH3

OO OH

O O

NHOC

CH2OH

OOH OH

OH OHHOH H

H

O N C S

OO

OH O

NH

SC

HN H

O

OCH2OH

OHHH

H

CH2OH

OH OH OO

CH2OH

CH3

NH

OC

OO

NH2

Chitosan

+

Stirring



M

K M

X

25∘C24hKminusX


10000

0003500 3000 2500 2000 1500 1000 500

(cmminus1)

3439

32506

23556 21920

19042

16579

16009

16409

15642

15390 14301

14000

13550

12625

12364

11922

10710

10230

9621 9039

8718

8266

7656

7364

6766 6042

5586

5069

4827

T (

)


80 70 60 50 40 30 20 10

(ppm)




10000

0003500 3000 2500 2000 1500 1000 500

(cmminus1)

30847

29215

23724

17312

16514

16261

15621

15234

14174 13

212

12834

11844

11122

10204 9548

8908

8085

7684

7204

6990

6800

6301

6071

5831 55365274 4548

T (

)


80 70 60 50 40 30 20 10

(ppm)






A FEDCB










A FEDCB

(a)

A FEDCB

(b)












033

035

025 05 1 15 2Time (days)


Turb

idity

at340

nm


A FEDCB






0

20

40

60

80

100

120


Cel

l via

bilit

y (

cont

rol)









0

20

40

60

80

100

120


Cel

l via

bilit

y (

)





0102030405060708090



OD

N (n

g10

6ce

lls)





0102030405060708090

100



OD

N (n

g10

6ce

lls)


0102030405060708090

0 10 20 50


LipofectinODN

NakedODN

OD

N (n

g10

6ce

lls)





9242

7236

4396

10812

0

20

40

60

80

100

Formulations

Free

OD

N+

LPS

CHO

DN

NP2

+LP

S

MCH

OD

N N

P2RD

+LP

S

MCH

OD

NN

P2+

LPS

TNF-120572

cont

ent (

o

f con

trol)







(a) (b)



(c) MCHODN NP2





10 Conclusion






Acknowledgments


References















































50










































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


O

CH2OH CH2OHCH2OH

OH

NH2

CH3

OO OH

O O

NHOC

CH2OH

OOH OH

OH OHHOH H

H

O N C S

OO

OH O

NH

SC

HN H

O

OCH2OH

OHHH

H

CH2OH

OH OH OO

CH2OH

CH3

NH

OC

OO

NH2

Chitosan

+

Stirring



M

K M

X

25∘C24hKminusX


10000

0003500 3000 2500 2000 1500 1000 500

(cmminus1)

3439

32506

23556 21920

19042

16579

16009

16409

15642

15390 14301

14000

13550

12625

12364

11922

10710

10230

9621 9039

8718

8266

7656

7364

6766 6042

5586

5069

4827

T (

)


80 70 60 50 40 30 20 10

(ppm)




10000

0003500 3000 2500 2000 1500 1000 500

(cmminus1)

30847

29215

23724

17312

16514

16261

15621

15234

14174 13

212

12834

11844

11122

10204 9548

8908

8085

7684

7204

6990

6800

6301

6071

5831 55365274 4548

T (

)


80 70 60 50 40 30 20 10

(ppm)






A FEDCB










A FEDCB

(a)

A FEDCB

(b)












033

035

025 05 1 15 2Time (days)


Turb

idity

at340

nm


A FEDCB






0

20

40

60

80

100

120


Cel

l via

bilit

y (

cont

rol)









0

20

40

60

80

100

120


Cel

l via

bilit

y (

)





0102030405060708090



OD

N (n

g10

6ce

lls)





0102030405060708090

100



OD

N (n

g10

6ce

lls)


0102030405060708090

0 10 20 50


LipofectinODN

NakedODN

OD

N (n

g10

6ce

lls)





9242

7236

4396

10812

0

20

40

60

80

100

Formulations

Free

OD

N+

LPS

CHO

DN

NP2

+LP

S

MCH

OD

N N

P2RD

+LP

S

MCH

OD

NN

P2+

LPS

TNF-120572

cont

ent (

o

f con

trol)







(a) (b)



(c) MCHODN NP2





10 Conclusion






Acknowledgments


References















































50










































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




A FEDCB










A FEDCB

(a)

A FEDCB

(b)












033

035

025 05 1 15 2Time (days)


Turb

idity

at340

nm


A FEDCB






0

20

40

60

80

100

120


Cel

l via

bilit

y (

cont

rol)









0

20

40

60

80

100

120


Cel

l via

bilit

y (

)





0102030405060708090



OD

N (n

g10

6ce

lls)





0102030405060708090

100



OD

N (n

g10

6ce

lls)


0102030405060708090

0 10 20 50


LipofectinODN

NakedODN

OD

N (n

g10

6ce

lls)





9242

7236

4396

10812

0

20

40

60

80

100

Formulations

Free

OD

N+

LPS

CHO

DN

NP2

+LP

S

MCH

OD

N N

P2RD

+LP

S

MCH

OD

NN

P2+

LPS

TNF-120572

cont

ent (

o

f con

trol)







(a) (b)



(c) MCHODN NP2





10 Conclusion






Acknowledgments


References















































50










































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


A FEDCB

(a)

A FEDCB

(b)












033

035

025 05 1 15 2Time (days)


Turb

idity

at340

nm


A FEDCB






0

20

40

60

80

100

120


Cel

l via

bilit

y (

cont

rol)









0

20

40

60

80

100

120


Cel

l via

bilit

y (

)





0102030405060708090



OD

N (n

g10

6ce

lls)





0102030405060708090

100



OD

N (n

g10

6ce

lls)


0102030405060708090

0 10 20 50


LipofectinODN

NakedODN

OD

N (n

g10

6ce

lls)





9242

7236

4396

10812

0

20

40

60

80

100

Formulations

Free

OD

N+

LPS

CHO

DN

NP2

+LP

S

MCH

OD

N N

P2RD

+LP

S

MCH

OD

NN

P2+

LPS

TNF-120572

cont

ent (

o

f con

trol)







(a) (b)



(c) MCHODN NP2





10 Conclusion






Acknowledgments


References















































50










































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


033

035

025 05 1 15 2Time (days)


Turb

idity

at340

nm


A FEDCB






0

20

40

60

80

100

120


Cel

l via

bilit

y (

cont

rol)









0

20

40

60

80

100

120


Cel

l via

bilit

y (

)





0102030405060708090



OD

N (n

g10

6ce

lls)





0102030405060708090

100



OD

N (n

g10

6ce

lls)


0102030405060708090

0 10 20 50


LipofectinODN

NakedODN

OD

N (n

g10

6ce

lls)





9242

7236

4396

10812

0

20

40

60

80

100

Formulations

Free

OD

N+

LPS

CHO

DN

NP2

+LP

S

MCH

OD

N N

P2RD

+LP

S

MCH

OD

NN

P2+

LPS

TNF-120572

cont

ent (

o

f con

trol)







(a) (b)



(c) MCHODN NP2





10 Conclusion






Acknowledgments


References















































50










































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

20

40

60

80

100

120


Cel

l via

bilit

y (

)





0102030405060708090



OD

N (n

g10

6ce

lls)





0102030405060708090

100



OD

N (n

g10

6ce

lls)


0102030405060708090

0 10 20 50


LipofectinODN

NakedODN

OD

N (n

g10

6ce

lls)





9242

7236

4396

10812

0

20

40

60

80

100

Formulations

Free

OD

N+

LPS

CHO

DN

NP2

+LP

S

MCH

OD

N N

P2RD

+LP

S

MCH

OD

NN

P2+

LPS

TNF-120572

cont

ent (

o

f con

trol)







(a) (b)



(c) MCHODN NP2





10 Conclusion






Acknowledgments


References















































50










































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0102030405060708090

100



OD

N (n

g10

6ce

lls)


0102030405060708090

0 10 20 50


LipofectinODN

NakedODN

OD

N (n

g10

6ce

lls)





9242

7236

4396

10812

0

20

40

60

80

100

Formulations

Free

OD

N+

LPS

CHO

DN

NP2

+LP

S

MCH

OD

N N

P2RD

+LP

S

MCH

OD

NN

P2+

LPS

TNF-120572

cont

ent (

o

f con

trol)







(a) (b)



(c) MCHODN NP2





10 Conclusion






Acknowledgments


References















































50










































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)



(c) MCHODN NP2





10 Conclusion






Acknowledgments


References















































50










































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgments


References















































50










































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















50










































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Mannosylated Chitosan Nanoparticles for...

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Transcript of Research Article Mannosylated Chitosan Nanoparticles for...