Preparation and Characterization of Chitosan Nanoparticles-Doped ...

Research ArticlePreparation and Characterization ofChitosan Nanoparticles-Doped Cellulose Filmswith Antimicrobial Property

Ain Nadirah Binti Romainor Suk Fun Chin Suh Cem Pang and Lesley Maurice Bilung

Faculty of Resource Science and Technology Universiti Malaysia Sarawak 94300 Kota Samarahan Sarawak Malaysia

Correspondence should be addressed to Suk Fun Chin sukfunchingmailcom

Received 15 May 2014 Revised 12 August 2014 Accepted 15 August 2014 Published 26 August 2014

Academic Editor Yanbao Zhao

Copyright copy 2014 Ain Nadirah Binti Romainor et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Cellulose films with antimicrobial property were prepared by incorporation of chitosan nanoparticles as antimicrobial agents intothe cellulose films The antimicrobial property of these chitosan nanoparticles-doped cellulose films against Escherichia coli (Ecoli) was evaluated via diffusion assay method minimum inhibitory concentration (MIC) method and minimum bactericidalconcentration (MBC) method The effects of antimicrobial agent amount size-related property (nanoparticles and bulk chitosan)and crosslinking by citric acid on antimicrobial activity of cellulose filmswere studied It was observed that the antimicrobial activitywas enhanced when chitosan nanoparticles were used as compared to when bulk chitosan was used A maximum E coli inhibitionof 85 was achieved with only 5 (vv) doping of chitosan nanoparticles into the cellulose films Crosslinking of the cellulose filmswith citric acid was observed to have resulted in 50 reduction of water absorbency and a slight increase of E coli inhibition by 3for chitosan nanoparticles-doped cellulose films

1 Introduction

Most microbes are harmful and can cause numerous diseaseinfections such as diarrhea respiratory illness whoopingcough and fever [1] Noble metals (silver copper andzinc) and natural products (essential oil biopolymer andorganic acid) are among the antimicrobial agents available forprevention of microbial infection [2 3] Antimicrobial filmswere required to prevent microbial growth in food for foodpackaging industry wound dressing in medical devices andclothing in textile industry and footwear industry [4 5]

Chitosan was commonly used as an antimicrobial agentand blended with other polymer films to produce antimi-crobial films Some examples are cellulosechitosan [6]starchchitosan [7] starchchitosanlauric acid [8] guargumchitosan [9] polyethylene oxide (PEO)chitosan [10]and glucomannanchitosannisin [11] Chitosan inhibitedand suppressed microbial activities through their electro-static charge interaction between positive charges on poly-cationic chitosan molecules (amino groups) with negative

charges on microbial surface [12] This interaction causeddisruption on the microbial cells which then changed theirmetabolism and led to cell death [13 14] However chitosanwas not used in nanoparticulate form The small size ofchitosan nanoparticles rendered them with unique physic-ochemical properties such as large surface area (providingmore cationic sites) and high reactivity and thus couldpotentially enhance the charge interaction on the microbialsurface and lead to more superior antimicrobial effect [15]Some researchers have incorporated chitosan nanoparticlesinto starch and hydroxypropyl methyl cellulose (HPMC)films to prepare antimicrobial films However their workshave focused on the effect of chitosan nanoparticles dopingon the film barrier and their mechanical properties Theyconcluded that the improvement of antimicrobial films prop-erties was attributed to the good interaction between chitosannanoparticles and polymeric-based films [16 17] Howeverit is also useful to investigate the effectiveness of chitosannanoparticles-doped antimicrobial films against microbialactivity

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2014 Article ID 710459 10 pageshttpdxdoiorg1011552014710459

2 Journal of Nanomaterials

Cellulose is a favourable polymeric material for prepara-tion of antimicrobial films due to their abundant availability(most abundant biopolymers) biodegradability low toxicityrenewability and low cost in nature [18] This work focusedon the preparation of chitosan nanoparticles-doped celluloseantimicrobial films and evaluation of their antimicrobialactivity via diffusion assay minimum inhibitory concentra-tion (MIC) andminimumbactericidal concentration (MBC)analysis The effects of chitosan nanoparticles size-relatedproperty (bulky chitosan and chitosan nanoparticles) and theamount of chitosan nanoparticles doping and crosslinking ofcitric acid on the efficacy of cellulose antimicrobial films wereinvestigated against E coli

2 Materials and Methods

21 Materials Chitosan powder with molecular weight of100ndash300 kDa was purchased from Acros Organics (NewJersey USA) Fibrous cellulose powder CF11 was pur-chased from Whatman Ltd (Maidstone England) Sodiumtripolyphosphate (TPP) of technical grade 85 was sup-plied by Sigma-Aldrich (St Louis USA) Acetic acid usedwas from HmbG Chemicals (Hamburg Germany) whilecitric acid and sodium hydroxide (NaOH) were providedby Merck (Darmstadt Germany) Sodium hypophosphatemonohydrate crystal was purchased from J T Baker (China)Thiourea and urea were supplied from Merck (HohenbrunnGermany) The cultivationassay medium for antimicrobialactivities was Muller-Hinton Agar (MHA) purchased fromOxoid (Hampshire UK) Luria broth (Millerrsquos LB broth) forEscherichia coli (E coli) for antibacterial activities testing wassupplied by Conda Pronadisa (Spain) Analytical grade of D-glucose anhydrous was supplied by Fisher Scientific (UK)Ultrapure water (UPW) (182M1015840Ω) from Water PurifyingSystem (ELGA Model Ultra Genetic) was used throughoutthe experiment

22 Preparation of Chitosan Nanoparticles Chitosan nano-particles were prepared by using ionic gelation method asreported by Muhammed Rafeeq et al [19] 03 (wv) ofchitosan was dissolved in 2 (vv) of acetic acid to formchitosan solution Sodium tripolyphosphate (TPP) (1 (wv))was used as an ionic cross linker Chitosan nanoparticles wereobtained upon the addition of 1mL of TPP into 10mL ofchitosan solution under sonication at room temperature for 1hour

23 Preparation of Cellulose Films Cellulose solution wasprepared by dissolution of cellulose powder in NaOH thiourea urea (NTU) (8 65 8 wv ()) solvent systemThemixture was frozen at minus21∘C for 12 hours and thawed in orderto obtain homogeneous cellulose solution [20] Cellulose filmwas prepared by casting cellulose solution (5 (vv)) intopetri dish and then it was dried in oven at 60∘C for at least 2hours until the solution dried and transparent cellulose filmwas formed The cellulose film was rinsed with UPW severaltimes to remove excess NTU salt and then dried at room

temperature for 24 hours Then the film was carefully peeledfrom the petri dish

24 Preparation of Chitosan Nanoparticles- and Chitosan-Doped Cellulose Films Chitosan nanoparticles-doped cellu-lose or chitosan-doped cellulose films solutions were pre-pared by adding various amounts (01 05 1 5 10 and30 (vv)) of chitosan nanoparticles or chitosan solutioninto cellulose solution The mixtures were then magneticallystirred for 30 minutes transferred into petri dish and driedin oven at 60∘C to obtain cellulose film Subsequently thedried film was rinsed with UPW before drying at roomtemperature

25 Preparation of Cross-Linked Chitosan Nanoparticles-Doped Cellulose Films Cellulose solution doped with 5(vv) of chitosan nanoparticles was used for crosslinkingwith citric acid Sodium hypophosphate monohydrate wasadded to themixture of citric acid and chitosan nanoparticlessolution as catalyst and the mixture was magnetically stirredand heated at 80ndash90∘C for 4 hours to allow crosslinkingreaction to occur The solution was then spread evenly intopetri dish and dried in oven at 60∘C to allow the formationof film Finally the film was washed with UPW and dried atroom temperature

26 Characterization of Cellulose Films

261 Scanning Electron Microscopy (SEM) Analysis Themorphology of the samples was observed using a scanningelectron microscope (SEM) (JEOL JSM-6390 LA) The sam-ples were coated with a layer of platinum prior to SEManalysis

262 Fourier Transformed Infrared Spectroscopy (FTIR) Anal-ysis FTIR spectra of samples were obtained from KBrsample pellets within the range of 400ndash4000 cmminus1 on a FTIRspectroscopy (Thermo Scientific Nicole iS10)

263 Water Absorbency Analysis Water absorbency of thesamples was characterized according to the method reportedby Liu et al [21] The films were cut into 15 cm times 30 cmpieces and dialysed for 24 hours for complete removal ofexcessive salt from the filmThen the films were dried (60∘C)and weighed until constant weight (119882

1) was achieved The

dried films were immersed in UPW water for 24 hoursFinally the films were taken out wiped with filter paper andwere weighed until constant weight (119882

2) was achievedWater

absorbency was calculated based on the following

Water absorbency = 1198822 minus11988211198821

times 100 (1)

where1198822is the weight of films after immersion and119882

1is the

weight of films before immersion

27 Antimicrobial Studies The antimicrobial activity of cel-lulose antimicrobial films was investigated against the growth

Journal of Nanomaterials 3

of E coli Diffusion assay minimum inhibitory concentration(MIC) and minimum bactericidal concentration (MBC)methods were used to assess the antimicrobial activity byfollowing the standardmethods fromNational Committee onClinical Laboratory Standard (NCCLS) protocol [22 23]

271 Diffusion Assay The bacteria were cultured in MillerrsquosLuria broth (Millerrsquos LB broth) followed by incubation inincubator shaker for 24 hours Sufficient inoculums wereadded into the new test tube and the suspension turbidity wasadjusted equivalently to 05 McFarland standard (containingapproximately sim432 times 107 CFUmL of bacteria) 20mL ofbacterial suspension was uniformly spread on the sterile petridishes of Muller-Hinton Agar (MHA) using sterile cottonswab and pieces of antimicrobial films were placed on thebacterial culture The plates were sealed and incubated at37∘C for 24 hours After the incubation period clear zonesof inhibitions were observed [22]

272 Minimum Inhibitory Concentration (MIC) Twofoldserial dilution series of samples were prepared volumetricallyforMIC test 1mL ofMillerrsquos LB broth solutions was prepared

in 10 test tubes and the first test tubes were mixed with1mL of sample Then 1mL aliquot of the mixed solution inthe first test tube was transferred into the second test tubeThe same process was repeated until the tenth test tube Theserial dilutions prepared were labelled as 10minus1 to 10minus10 (vv)solution concentration respectively Finally 1mL of E colisuspensionwere added into the resultant serial dilution seriesand incubated in incubator shaker at 37∘C for 24 hours

273 Minimum Bactericidal Concentration (MBC) 20120583L ofmixture from serial dilution test tubes with no signs of tur-bidity was transferred and spread on theMuller-Hinton Agar(MHA) plates The MBC point was determined as the lowestconcentration in serial dilution series that shows no coloniesgrowth after 24 hours incubation at 37∘C The concentrationof samples in serial dilution series concentration solutionwas calculated based on glucose standard curve plottedequation [23 24] Percentage of colonies reduction frombacteriostatic effect was determined based on the bacterialcolonies calculation using haemocytometer by the followingequation

Percentage of inhibition () =Numbers of colonies in control plates minus Numbers of colonies in assays

Numbers of colonies in control platestimes 100 (2)

28 Assessment of Antimicrobial Activity

281 Biophotometer Biophotometer (model Eppendorf Bio-Photometer Plus) was used to determine the glucose concen-tration in the samples for the development of glucose stan-dard curve plotted equation The absorbance was measuredat 485 nm wavelength

282 Haemocytometer The number of viable E coli cells(bacterial colonies) was calculated using a haemocytometer(Hirschmann Laborgerate) The sample suspension was cov-ered by the glass slide on haemocytometer and placed undermicroscope (Motic BA 210) for cell counting

3 Results and Discussion

31 Surface Morphology Homogeneous transparent andflexible films were obtained from cellulose doped with var-ious amount of chitosan or chitosan nanoparticles SEMmicrograph of undoped cellulose film is shown in Figure 1(a)It can be observed that the surface of the cellulose filmwas smooth and homogeneous After the addition of 01(vv) of chitosan into the cellulose film the surface ofthe film became coarse as depicted in Figure 1(b) Whenchitosan content was increased to 10 (vv) the films tendto become denser and rougher as shown in Figure 1(c)Chitosan nanoparticles with mean particles diameter of216 nm were incorporated into cellulose film (Figure 1(d))

The surface of chitosan nanoparticles-doped cellulose film at01 (vv) became rougher and studded with dense granule-like structure as depicted in Figure 1(e) The film exhibiteddenser structure as the amount of incorporated chitosannanoparticles increased to 5 (vv) as shown in Figure 1(f)The surface of chitosan nanoparticles-doped cellulose filmbecame coarse and slightly cavernous after crosslinking withcitric acid (Figure 1(g)) This might be due to the presenceof crosslinking networks between chitosan nanoparticles-doped cellulose films with citric acid [25]

32 FTIR Analysis FTIR spectra of cellulose film chi-tosan chitosan-doped cellulose film chitosan nanoparticles-doped cellulose film and citric acid cross-linked chitosannanoparticles-doped cellulose film were shown in Figures2(a) 2(b) 2(c) 2(d) and 2(e) respectively As shown inFigures 2(a) and 2(b) cellulose and chitosan shared thesimilar functional group of hydroxyl (OH) stretching vibra-tion alkane CndashH stretching vibration and CndashO stretchingvibration from polysaccharide polymers The OH peaks canbe assigned as 3415 and 3422 cmminus1 alkane CndashH stretchingvibration can be assigned as 2896 and 1418 cmminus1 and 2891and 1421 cmminus1 and CndashO stretching vibration from polysac-charide can be assigned as 1157 and 891 cmminus1 and 1156 and1097 cmminus1 for cellulose and chitosan as in Figures 2(a) and2(b) respectively [26ndash28] In contrast peak absorption at1633 cmminus1 in Figure 2(a) was attributed to the OH bendingof cellulose absorbed water molecules [26 27] The finger


(a) (b)

(c) (d)

(e) (f)

(g)

Figure 1 SEM micrograph of (a) cellulose film cellulose film doped with (b) 01 (vv) chitosan and (c) 10 (vv) chitosan (d) chitosannanoparticles cellulose film doped with (e) 01 (vv) chitosan nanoparticles and (f) 5 (vv) chitosan nanoparticles and (g) citric acidcross-linked chitosan nanoparticles-doped cellulose film

print peak absorption of chitosan (amide II andNndashHbendingvibration) appeared at the 1650 and 1595 cmminus1 respectively[29]

After doping with chitosan and chitosan nanoparticlesOH groups of cellulose were shifted to 3422 and 3409 cmminus1

accordingly as revealed in Figures 2(c) and 2(d) respectivelyThis was attributed to the presence of OH stretching fromchitosan and chitosan nanoparticles functional groups inthe cellulose films [30 31] Furthermore the strong peakabsorption of OH bending bound of water in cellulose


Tran

smitt

ance

()

4000 3500 3000 2500 2000 1500 1000 500

(c)

Wavelength (cmminus1)

(d)

(e)

3415

28961633 1418

1157

891

34222891

1650

1595

1421

11561079

34452881 1651

15951418

11561066

894

3409

29001634

1560

14131153

900

3403

29051727 1639

1418 1168

896

(a)

(b)

Figure 2 FTIR spectra of (a) pure cellulose (b) pure chitosan(c) chitosan-doped cellulose film (d) nanoparticulate chitosan-doped cellulose film and (e) citric acid cross-linked nanoparticulatechitosan-doped cellulose film

molecules (1633 cmminus1) was observed to reduce and shiftedto 1651 and 1634 cmminus1 as shown in Figures 2(c) and 2(d)respectively The corresponding peaks were suggested to bethe overlapping peak and interaction betweenOHbending ofwater from cellulose and chitosan and chitosan nanoparticlesmolecules [21 32] The alkane CndashH stretching vibration ofchitosan and chitosan nanoparticles-doped cellulose filmswas assigned at 2881 and 1418 cmminus1 in Figure 2(c) and 2900and 1413 cmminus1 in Figure 2(d) The amide II (NndashH of amidelinkage) bonding was noticed to appear at the peak of 1595and 1560 cmminus1 in Figures 2(c) and 2(d) respectively andwas absent in Figure 2(a) thus this further confirmed thatchitosan and chitosan nanoparticles were incorporated intothe cellulose antimicrobial films

The peak at 1153 cmminus1 in Figure 2(d) indicated the over-lapping peak of CndashO stretching in polysaccharide and for-mation of chitosan nanoparticles due to the interaction ofammonium ion and phosphate ion in chitosan nanoparticlemolecules [29 33] It was observed that the incorporationof chitosan and chitosan nanoparticles into cellulose filmswas not deteriorating the polysaccharide characteristic ofthe antimicrobial films This can be proven by the presenceof finger print of carbohydrate (CndashO stretching) regionregistered at 1156 1066 and 894 cmminus1 in Figure 2(c) and 1153and 900 cmminus1 in Figure 2(d) There are no changes or newpeak was observed in the spectrum of chitosan and chitosannanoparticles-doped cellulose films indicating that chitosanor chitosannanoparticleswere physically doped into cellulosefilms [21]

The result showed the formation of new peaks at1727 cmminus1 in citric acid cross-linked chitosan nanoparticles-doped cellulose film as presented in Figure 2(e) The bond

was produced from the crosslinking reaction between car-boxylic groups (COOH groups) in citric acid with celluloseand chitosan respectively The peak at 1727 cmminus1 was dueto the formation of ester bonding (C=O) resulting from thereaction betweenCOOHgroups of citric acid andOHgroupsof chitosan and cellulose Meanwhile the peak at 1418 cmminus1was attributed to the overlapping peak of CndashH stretching inthe polymer film [29] and CndashN stretching of amide bondingresulting from the interaction between COOH groups ofcitric acid with amino groups (NH

2) from chitosan [34] The

reaction mechanism was shown in Figure 3A shift of the peak from 2900 and 1418 cmminus1 to 2905 and

1418 cmminus1 was observed after chitosan nanoparticles-dopedcellulose film was cross-linked with citric acid (Figure 2(e))This shift was related to the presence of citric acid alkanechains in the film structure [35 36] After the crosslink-ing reaction occurred the polysaccharides glycosidic peakshifted from 900 and 1157 cmminus1 to 891 and 1168 cmminus1 asdepicted in Figures 2(c) and 2(d) respectively [36]

33Water AbsorbencyAnalysis Water sensitivity is one of theimportant criteria for practical application of antimicrobialfilms in various fields [7 9]Thewater absorption of cellulosechitosan nanoparticles-doped cellulose film and citric acidcross-linked chitosan nanoparticles-doped cellulose film isdisplayed in Figure 4 Cellulose film showed the highestwater absorption percentage (4571) followed by chitosannanoparticles-doped cellulose films (2286) The resultsshowed that cellulose film exhibits higher hygroscopicity toabsorb more water inside the film membrane This tendencycould be explained by the interaction between OH groups ofcellulose filmwithwatermolecules [37]The incorporation ofchitosan nanoparticles into cellulose film has made cellulosefilm less water permeable because chitosan nanoparticlescould form hydrogen bond with cellulose molecules thusdecreasing water absorbency Furthermore the nanodimen-sion of chitosan nanoparticles formed a rough and compactfilm structure (as shown in Figure 1(f)) therefore decreasingwater absorbency of cellulose films [17]

After crosslinking with citric acid the water absorbencyof chitosan nanoparticles-doped cellulose film was reducedto 47ndash50 This was due to the formation of ester bondingvia esterification reaction from the carboxylic functionalgroups (COOH) of citric acid and OH functional groupsof cellulose and chitosan polymers Besides the presenceof alkane groups from citric acid molecules also inherentlyaffected the hydrophobicity of the film [8 38]

34 Antimicrobial Assessment

341 Diffusion Assay Figure 5 presented the picture of dif-fusion assay resulting from (Figure 5(a)) pure cellulose film(Figure 5(b)) chitosan-doped cellulose film (Figure 5(c)) chi-tosan nanoparticles-doped cellulose film and (Figure 5(d))citric acid cross-linked chitosan nanoparticles-doped cellu-lose film It was observed that plenty of E coli colonies hadcovered either on the plate or the surface of the film as shownin Figure 5(a) Pure cellulose film did not show any inhibitory


Ester bond

Citric acidO O

OH

+ OHndashcellulose Citric acidndashCndashOndashcellulose

(a)

Ester bond

Citric acid

OO

OH

+ OHndashchitosan Citric acidndashCndashOndashchitosan

(b)

Amide bond

Citric acid

OO

OH

+ H2Nndashchitosan Citric acidndashCndashNndashchitosan

H

(c)

Figure 3 Reaction mechanism of ester bonding formation between citric acid with (a) cellulose (b) chitosan and (c) amide bondingformation between citric acid with chitosan molecules

0

10

20

30

40

50

Cellulose film

Wat

er ab

sorb

ency

()

Nanoparticulatechitosan-doped

cellulose film

Cross-linkednanoparticulatechitosan-doped

cellulose film

Figure 4 Water absorbency of cellulose film nanoparticulatechitosan-doped cellulose film and cross-linked nanoparticulatechitosan-doped cellulose film

effects on E coli due to lack of amino group in their polymerbackboneswhichwas responsible for the antibacterial activity[39 40] Figures 5(b) and 5(c) showed the results of chitosan-doped cellulose films and chitosan nanoparticles-doped cel-lulose films respectively It was observed that there werecolonies growing on the agar plates but not on the surface ofthe film and this phenomenon led to the formation of surfacecontact area of antimicrobial films on the agar plates Suchobservation was due to chitosan and chitosan nanoparticlesbeing less polar which makes them diffuse slowly from thefilms to the agar plates and consequently surface contactarea was formed on the agar plates On the other handthe antimicrobial activity of chitosan nanoparticles-dopedcellulose films was enhanced after crosslinking with citricacid as shown by the appearance of clear zone in Figure 5(d)This was due to the presence of more polar bonds formed in

the cross-linked chitosan nanoparticles-doped cellulose film[41 42]

342 Effect of Chitosan Nanoparticles Doping Tables 1 and 2summarized the quantitative studies of antimicrobial activityof chitosan nanoparticles and chitosan-doped cellulose solu-tions against E coli The antimicrobial activity was attributedto the electrostatic interaction between positive charges(amino group) of chitosan with negative charges of microbialsurface (from the lipopolysaccharide layer of E coli) [4344] The charged interaction broke microbial cell wall anddisturbed their metabolism hence leading to inhibition ofmicrobial proliferation [14 45]

As shown in Tables 1 and 2 antimicrobial activity ofcellulose films was observed to be more effective whenchitosan nanoparticles were incorporated as compared tobulk chitosan The highest inhibition percentage achievedwas 8516 obtained with 5 (vv) of chitosan nanoparticlesdoping Meanwhile the highest inhibition percentage ofchitosan-doped cellulose film achieved was 8148 whichwas obtained with 10 (vv) of chitosan doping The effec-tiveness of the chitosan nanoparticles-doped cellulose filmagainst E coli also was proven by the lower MIC and MBCvalues (1007 and 1304 ppm resp) On the other handMIC and MBC values of chitosan-doped cellulose film wereobserved to bemuch higher whichwere recorded at 1637 and1970 ppm respectively Different from bulky size of chitosannanoparticles system of chitosan offers an advantage of highsurface area to volume ratio which could provide more avail-able charge sites (amino group) formicrobial interaction [46]Due to this reason chitosan nanoparticles-doped cellulosefilm is more effective as an antimicrobial film as comparedto chitosan-doped cellulose film


(a)

Surface contact area

(b)


(c)

Inhibitory zone

(d)

Figure 5 Pictures of diffusion assay of (a) pure cellulose film (b) chitosan-doped cellulose film (c) nanoparticulate chitosan-doped cellulosefilm and (d) cross-linked nanoparticulate chitosan-doped cellulose film

Table 1 Effect of chitosan nanoparticles doped into cellulose solutions on antimicrobial activity of E coli

Antimicrobial analyses Chitosan nanoparticles ( vv)0 01 05 1

MIC values (ppm) mdash 1266 plusmn 037 1193 plusmn 037 1081 plusmn 038

MBC values (ppm) mdash 1495 plusmn 047 1452 plusmn 038 1378 plusmn 037

Percentage of inhibition () mdash 5185 plusmn 080 6296 plusmn 068 7037 plusmn 073

Antimicrobial analyses Chitosan nanoparticles ( vv)5 10 30

MIC values (ppm) 1007 plusmn 037 1304 plusmn 037 1378 plusmn 037

MBC values (ppm) 1304 plusmn 038 1637 plusmn 064 1711 plusmn 037

Percentage of inhibition () 8516 plusmn 054 7771 plusmn 044 7771 plusmn 069

343 Effect of Chitosan and Chitosan Nanoparticles DopingAntimicrobial activity of cellulose films was notably affectedby the doping amount of chitosan or chitosan nanoparticlesas shown in Tables 1 and 2The percentage of E coli inhibitionincreased from 5185 to 8516 and from 4444 to 8148as the chitosan nanoparticles and chitosan doping increasedfrom 01 to 5 (vv) and 01 to 10 (vv) It was believed that

at lower doping amount the electrostatic interaction causedchitosan or chitosan nanoparticles to be tightly absorbedonto the surface of E coli cells through pervasion leadingto the leakage of proteinaceous which then disturbed theirmetabolism (inhibition of mRNA (messenger ribonucleicacid) and protein synthesis when entering their nuclei) andconsequently suppressed the cells activity [47]


Table 2 Effect of chitosan doped into cellulose solutions on antimicrobial activity of E coli

Antimicrobial analyses Chitosan ( vv)0 01 05 1




Antimicrobial analyses Chitosan ( vv)5 10 30




Table 3 Effect of citric acid crosslinking on antimicrobial activityof nanoparticulate chitosan-doped cellulose film against E coli

Antimicrobialanalyses


cellulose

Cross-linkednanoparticulate

chitosan-doped celluloseMIC values (ppm) 1007 plusmn 037 896 plusmn 037

MBC values (ppm) 1304 plusmn 038 1007 plusmn 064

Percentage ofinhibition () 8516 plusmn 058 8822 plusmn 032

The percentage of E coli inhibition decreased after itreached a maximum inhibition percentage at an optimumdoping amount of chitosan nanoparticles or bulk chitosanTables 1 and 2 showed that the percentage of E coli activitywas reduced from 8516 to 7771 and from 8148 to 7778as the chitosan nanoparticles doping increased from 5 to 10(vv) and from 10 to 30 (vv) of chitosan doping Higherdoping amount provided more charge sites (amino groups)and the interaction of charges sites caused the chitosan andchitosan nanoparticles to form cluster and agglomerationConsequently limited charge sites available for attachment ofE coli resulted in reduction of antimicrobial activity [48]

344 Effect of Citric Acid Crosslinking After crosslink-ing with citric acid the antimicrobial activity of chitosannanoparticles-doped cellulose film against E coli was fur-ther investigated and the results were shown in Table 3Cross-linked chitosan nanoparticles-doped cellulose filmgave lower MIC and MBC values (896 and 1007 ppm) andhigher percentage of E coli inhibition (8822) as comparedto films without crosslinking The results suggested thatcrosslinking with citric acid could enhance the antimicrobialactivity due to the synergistic interaction between chitosannanoparticles and citric acid in the films since both of themwere antimicrobial agents [34 49]

4 Conclusions

Antimicrobial cellulose films were successfully prepared byincorporation of chitosan nanoparticles in the cellulosefilms The antimicrobial activity was greatly influenced bythe size-related property of chitosan used (nanoparticles

and bulk chitosan) and also the amount of chitosan or chi-tosan nanoparticles doped into the cellulose films Chitosannanoparticles provided more available charged sites (aminogroup) for interaction with negatively charged bacterial cellsthus having better antimicrobial property Crosslinking withcitric acid enhanced the quality of cellulose antimicrobialfilm by reducing about 50 of the filmrsquos water absorbencyand slightly increased E coli inhibition by 3 Due totheir less hygroscopic and high antibacterial property theresulting cellulose-based films could potentially be used asantimicrobial films in various fields such as in biomedicaltextiles and food packaging

Conflict of Interests

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

Acknowledgment

The authors gratefully acknowledged the financial supportprovided for this work by the COMSTECHIFS (Committeeon Scientific and Technological CooperationInternationalFoundation of Science) under the Grant agreement noF5207-1

References

[1] M F Adegboye O O Babalola and D A Akinpelu ldquoIssuesof resistance of pathogens to antimicrobial agentsrdquo ScientificResearch Essays vol 7 no 41 pp 3468ndash3478 2012

[2] N Luo K Varaprasad G V S Reddy A V Rajulu and J ZhangldquoPreparation and characterization of cellulosecurcumin com-posite filmsrdquo Royal Society of Chemistry vol 2 no 22 pp 8483ndash8488 2012

[3] SNaz S Jabeen FManzoor FAslam andAAli ldquoAntibacterialactivity of Curcuma longa varieties against different strains ofbacteriardquo Pakistan Journal of Botany vol 42 no 1 pp 455ndash4622010

[4] A Pielesz A Machnicka and E Sarna ldquoAntibacterial activ-ity and scanning electron microscopy (SEM) examination ofalginate-based films and wound dressingsrdquo Ecological Chem-istry and Engineering S vol 18 no 2 pp 197ndash210 2011

[5] M C Barros I P Fernandez V Pinto M J Ferreira M FBarreiro and J S Amaral ldquoChitosan as antimicrobial agent for


footwear leather componentsrdquo in Biodegradable Polymers andSustainable Polymers A Jimnez and G E Zairov Eds pp 151ndash162 Nova Science 2011

[6] C-M Shih Y-T Shieh and Y-K Twu ldquoPreparation and char-acterization of cellulosechitosan blend filmsrdquo CarbohydratePolymers vol 78 no 1 pp 169ndash174 2009

[7] Y Zhong X Song and Y Li ldquoAntimicrobial physical andmechanical properties of kudzu starch-chitosan compositefilms as a function of acid solvent typesrdquo Carbohydrate Poly-mers vol 84 no 1 pp 335ndash342 2011

[8] E Salleh I I Muhamad and N Khairuddin ldquoStructuralcharacterization and physical properties of antimicrobial (AM)starch-based filmsrdquoWorld Academy of Science Engineering andTechnology vol 3 no 7 pp 410ndash418 2009

[9] M S Rao S R Kanatt S P Chawla and A Sharma ldquoChitosanand guar gum composite films preparation physical mechan-ical and antimicrobial propertiesrdquo Carbohydrate Polymers vol82 no 4 pp 1243ndash1247 2010

[10] J Li S Zivanovic P M Davidson and K Kit ldquoProductionand characterization of thick thin and ultra-thin chitosanPEOfilmsrdquo Carbohydrate Polymers vol 83 no 2 pp 375ndash382 2011

[11] B Li J F Kennedy J L Peng X Yie and B J Xie ldquoPreparationand performance evaluation of glucomannan-chitosan-nisinternary antimicrobial blend filmrdquo Carbohydrate Polymers vol65 no 4 pp 488ndash494 2006

[12] M A Aziz J D Cabral H J L Brooks S C Moratti andL R Hanton ldquoAntimicrobial properties of a chitosan dextran-based hydrogel for surgical userdquo Antimicrobial Agents andChemotherapy vol 56 no 1 pp 280ndash287 2012

[13] I Leceta P Guerrero I Ibarburu M T Duenas and K de laCaba ldquoCharacterization and antimicrobial analysis of chitosan-based filmsrdquo Journal of Food Engineering vol 116 no 4 pp 889ndash899 2013

[14] A A El-Sharif and M H M Hussain ldquoChitosan-EDTA newcombination is a promising candidate for treatment of bacterialand fungal infectionsrdquo Current Microbiology vol 62 no 3 pp739ndash745 2011

[15] L Zhang D Pornpattananangkul C-M J Hu and C-MHuang ldquoDevelopment of nanoparticles for antimicrobial drugdeliveryrdquo Current Medicinal Chemistry vol 17 no 6 pp 585ndash594 2010

[16] P R Chang R Jian J Yu and X Ma ldquoFabrication andcharacterisation of chitosan nanoparticlesplasticised-starchcompositesrdquo Food Chemistry vol 120 no 3 pp 736ndash740 2010

[17] M R de Moura F A Aouada R J Avena-Bustillos T HMcHugh J M Krochta and L H C Mattoso ldquoImprovedbarrier and mechanical properties of novel hydroxypropylmethylcellulose edible films with chitosantripolyphosphatenanoparticlesrdquo Journal of Food Engineering vol 92 no 4 pp448ndash453 2009

[18] W Z Xu G Gao and J F Kadla ldquoSynthesis of antibacterialcellulose materials using a ldquoclickablerdquo quaternary ammoniumcompoundrdquo Cellulose vol 20 no 3 pp 1187ndash1199 2013

[19] P E Muhammed Rafeeq V Junise R Saraswathi P NKrishnan and C Dilip ldquoDevelopment and characterization ofchitosan nanoparticles loaded with isoniazid for the treatmentof tuberculosisrdquo Research Journal of Pharmaceutical Biologicaland Chemical Sciences vol 1 no 4 pp 383ndash390 2010

[20] S Zhang F-X Li J-Y Yu and Y-L Hsieh ldquoDissolutionbehaviour and solubility of cellulose in NaOH complex solu-tionrdquo Carbohydrate Polymers vol 81 no 3 pp 668ndash674 2010

[21] F Liu B Qin L He and R Song ldquoNovel starchchitosanblending membrane antibacterial permeable and mechanicalpropertiesrdquo Carbohydrate Polymers vol 78 no 1 pp 146ndash1502009

[22] J H Ortez ldquoDisk diffusion testingrdquo inManual of AntimicrobialSusceptibility Testing M B Coyle Ed pp 39ndash52 AmericanSociety for Microbiology 2005

[23] I D Rankin ldquoMIC testingrdquo inManual of Antimicrobial Suscep-tibility Testing M B Coyle Ed pp 53ndash62 American Societyfor Microbiology 2005

[24] M Dubois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[25] M Zhai L Zhao F Yoshii and T Kume ldquoStudy on antibacterialstarchchitosan blend film formed under the action of irradia-tionrdquo Carbohydrate Polymers vol 57 no 1 pp 83ndash88 2004

[26] C Adina F Florinela T Abdelmoumen and S CarmenldquoApplication of FTIR spectroscopy for a rapid determination ofsome hydrolytic enzymes activity on sea buckthorn substraterdquoRomanian Biotechnological Letters vol 15 no 6 pp 5738ndash57442010

[27] S Y Oh D I Yoo Y Shin and G Seo ldquoFTIR analysis ofcellulose treated with sodium hydroxide and carbon dioxiderdquoCarbohydrate Research vol 340 no 3 pp 417ndash428 2005

[28] L L Fernandes C X Resende D S Tavares G A Soares L OCastro and J M Granjeiro ldquoCytocompatibility of chitosan andcollagen-chitosan scaffolds for tissue engineeringrdquo Polimerosvol 21 no 1 pp 1ndash6 2011

[29] G Cardenas and S P Miranda ldquoFTIR and TGA studies ofchitosan composite filmsrdquo Journal of the Chilean ChemicalSociety vol 49 no 4 pp 291ndash295 2004

[30] E P de AzevedoAldehyde-functionalized chitosan and cellulosechitosan composites application as drug carriers and vascularbypass grafts [PhD thesis] University of Iowa 2011

[31] H M Fahmy and M M G Fouda ldquoCrosslinking of alginicacidchitosanmatrices using polycarboxylic acids and their uti-lization for sodium diclofenac releaserdquo Carbohydrate Polymersvol 73 no 4 pp 606ndash611 2008

[32] NAMohamed andMM Fahmy ldquoSynthesis and antimicrobialactivity of some novel cross-linked chitosan hydrogelsrdquo Inter-national Journal of Molecular Sciences vol 13 no 9 pp 11194ndash11209 2012

[33] S F Hosseini M Zandi M Rezaei and F FarahmandghavildquoTwo-step method for encapsulation of oregano essential oilin chitosan nanoparticles preparation characterization and invitro release studyrdquo Carbohydrate Polymers vol 95 no 1 pp50ndash56 2013

[34] K F El-Tahlawy M A El-Bendary A G Elhendawy and SM Hudson ldquoThe antimicrobial activity of cotton fabrics treatedwith different crosslinking agents and chitosanrdquo CarbohydratePolymers vol 60 no 4 pp 421ndash430 2005

[35] O Kuzmina T Heinze and D Wawro ldquoBlending of celluloseand chitosan in alkyl imidazolium ionic liquidsrdquo ISRN PolymerScience vol 2012 Article ID 251950 9 pages 2012

[36] H Kono and S Fujita ldquoBiodegradable superabsorbent hydro-gels derived from cellulose by esterification crosslinking with1234-butanetetracarboxylic dianhydriderdquo Carbohydrate Poly-mers vol 87 no 4 pp 2582ndash2588 2012


[37] E V R Almeida E Frollini A Castellan and V ComaldquoChitosan sisal cellulose and biocomposite chitosansisal cel-lulose films prepared from thioureaNaOH aqueous solutionrdquoCarbohydrate Polymers vol 80 no 3 pp 655ndash664 2010

[38] X Qiu S Tao X Ren and S Hu ldquoModified cellulose films withcontrolled permeatability and biodegradability by crosslinkingwith toluene diisocyanate under homogeneous conditionsrdquoCarbohydrate Polymers vol 88 no 4 pp 1272ndash1280 2012

[39] N Reddy and Y Yang ldquoCitric acid cross-linking of starch filmsrdquoFood Chemistry vol 118 no 3 pp 702ndash711 2010

[40] J Yang J Cai Y Hu D Li and Y Du ldquoPreparation characteri-zation and antimicrobial activity of 6-amino-6-deoxychitosanrdquoCarbohydrate Polymers vol 87 no 1 pp 202ndash209 2012

[41] S Janjic M Kostic V Vucinic et al ldquoBiologically active fibersbased on chitosan-coated lyocell fibersrdquoCarbohydrate Polymersvol 78 no 2 pp 240ndash246 2009

[42] S H Moussa A A Tayel A A Al-Hassan and A FaroukldquoTetrazoliumformazan test as an efficientmethod to determinefungal chitosan antimicrobial activityrdquo Journal of Mycology vol2013 Article ID 753692 7 pages 2013

[43] L Jiang Comparison of disk diffusion agar dilution and brothmicrodilution for antimicrobial susceptibility testing of five chi-tosans [MS thesis] FujianAgricultural and ForestryUniversityFuzhou China

[44] Y Tao L-H Qian and J Xie ldquoEffect of chitosan on membranepermeability and cell morphology of Pseudomonas aeruginosaand Staphyloccocus aureusrdquo Carbohydrate Polymers vol 86 no2 pp 969ndash974 2011

[45] M M S G de Carvalho T C M Stamford E P dos SantosP Tenorio and F Sampaio ldquoChitosan as an oral antimicrobialagentrdquo in Science against Microbial Pathogens CommunicatingCurrent Research and Technological Advances A Mendez-VilasEd Formatex Research Centre 2011

[46] A M Abdelgawad S M Hudson and O J Rojas ldquoAntimi-crobial wound dressing nanofiber mats from multicomponent(chitosansilver-NPspolyvinyl alcohol) systemsrdquoCarbohydratePolymers vol 100 pp 166ndash178 2014

[47] L Qi Z Xu X Jiang C Hu and X Zou ldquoPreparation andantibacterial activity of chitosan nanoparticlesrdquo CarbohydrateResearch vol 339 no 16 pp 2693ndash2700 2004

[48] R C Goy D de Britto and O B G Assis ldquoA review of theantimicrobial activity of chitosanrdquo Polimeros vol 19 no 3 pp241ndash247 2009

[49] A Allende J McEvoy Y Tao and Y Luo ldquoAntimicrobialeffect of acidified sodium chlorite sodium chlorite sodiumhypochlorite and citric acid on Escherichia coli O157H7 andnatural microflora of fresh-cut cilantrordquo Food Control vol 20no 3 pp 230ndash234 2009

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


Cellulose is a favourable polymeric material for prepara-tion of antimicrobial films due to their abundant availability(most abundant biopolymers) biodegradability low toxicityrenewability and low cost in nature [18] This work focusedon the preparation of chitosan nanoparticles-doped celluloseantimicrobial films and evaluation of their antimicrobialactivity via diffusion assay minimum inhibitory concentra-tion (MIC) andminimumbactericidal concentration (MBC)analysis The effects of chitosan nanoparticles size-relatedproperty (bulky chitosan and chitosan nanoparticles) and theamount of chitosan nanoparticles doping and crosslinking ofcitric acid on the efficacy of cellulose antimicrobial films wereinvestigated against E coli

2 Materials and Methods

21 Materials Chitosan powder with molecular weight of100ndash300 kDa was purchased from Acros Organics (NewJersey USA) Fibrous cellulose powder CF11 was pur-chased from Whatman Ltd (Maidstone England) Sodiumtripolyphosphate (TPP) of technical grade 85 was sup-plied by Sigma-Aldrich (St Louis USA) Acetic acid usedwas from HmbG Chemicals (Hamburg Germany) whilecitric acid and sodium hydroxide (NaOH) were providedby Merck (Darmstadt Germany) Sodium hypophosphatemonohydrate crystal was purchased from J T Baker (China)Thiourea and urea were supplied from Merck (HohenbrunnGermany) The cultivationassay medium for antimicrobialactivities was Muller-Hinton Agar (MHA) purchased fromOxoid (Hampshire UK) Luria broth (Millerrsquos LB broth) forEscherichia coli (E coli) for antibacterial activities testing wassupplied by Conda Pronadisa (Spain) Analytical grade of D-glucose anhydrous was supplied by Fisher Scientific (UK)Ultrapure water (UPW) (182M1015840Ω) from Water PurifyingSystem (ELGA Model Ultra Genetic) was used throughoutthe experiment

22 Preparation of Chitosan Nanoparticles Chitosan nano-particles were prepared by using ionic gelation method asreported by Muhammed Rafeeq et al [19] 03 (wv) ofchitosan was dissolved in 2 (vv) of acetic acid to formchitosan solution Sodium tripolyphosphate (TPP) (1 (wv))was used as an ionic cross linker Chitosan nanoparticles wereobtained upon the addition of 1mL of TPP into 10mL ofchitosan solution under sonication at room temperature for 1hour

23 Preparation of Cellulose Films Cellulose solution wasprepared by dissolution of cellulose powder in NaOH thiourea urea (NTU) (8 65 8 wv ()) solvent systemThemixture was frozen at minus21∘C for 12 hours and thawed in orderto obtain homogeneous cellulose solution [20] Cellulose filmwas prepared by casting cellulose solution (5 (vv)) intopetri dish and then it was dried in oven at 60∘C for at least 2hours until the solution dried and transparent cellulose filmwas formed The cellulose film was rinsed with UPW severaltimes to remove excess NTU salt and then dried at room

temperature for 24 hours Then the film was carefully peeledfrom the petri dish

24 Preparation of Chitosan Nanoparticles- and Chitosan-Doped Cellulose Films Chitosan nanoparticles-doped cellu-lose or chitosan-doped cellulose films solutions were pre-pared by adding various amounts (01 05 1 5 10 and30 (vv)) of chitosan nanoparticles or chitosan solutioninto cellulose solution The mixtures were then magneticallystirred for 30 minutes transferred into petri dish and driedin oven at 60∘C to obtain cellulose film Subsequently thedried film was rinsed with UPW before drying at roomtemperature

25 Preparation of Cross-Linked Chitosan Nanoparticles-Doped Cellulose Films Cellulose solution doped with 5(vv) of chitosan nanoparticles was used for crosslinkingwith citric acid Sodium hypophosphate monohydrate wasadded to themixture of citric acid and chitosan nanoparticlessolution as catalyst and the mixture was magnetically stirredand heated at 80ndash90∘C for 4 hours to allow crosslinkingreaction to occur The solution was then spread evenly intopetri dish and dried in oven at 60∘C to allow the formationof film Finally the film was washed with UPW and dried atroom temperature

26 Characterization of Cellulose Films

261 Scanning Electron Microscopy (SEM) Analysis Themorphology of the samples was observed using a scanningelectron microscope (SEM) (JEOL JSM-6390 LA) The sam-ples were coated with a layer of platinum prior to SEManalysis

262 Fourier Transformed Infrared Spectroscopy (FTIR) Anal-ysis FTIR spectra of samples were obtained from KBrsample pellets within the range of 400ndash4000 cmminus1 on a FTIRspectroscopy (Thermo Scientific Nicole iS10)

263 Water Absorbency Analysis Water absorbency of thesamples was characterized according to the method reportedby Liu et al [21] The films were cut into 15 cm times 30 cmpieces and dialysed for 24 hours for complete removal ofexcessive salt from the filmThen the films were dried (60∘C)and weighed until constant weight (119882

1) was achieved The

dried films were immersed in UPW water for 24 hoursFinally the films were taken out wiped with filter paper andwere weighed until constant weight (119882

2) was achievedWater

absorbency was calculated based on the following

Water absorbency = 1198822 minus11988211198821

times 100 (1)

where1198822is the weight of films after immersion and119882

1is the

weight of films before immersion

27 Antimicrobial Studies The antimicrobial activity of cel-lulose antimicrobial films was investigated against the growth

















(a) (b)

(c) (d)

(e) (f)

(g)






Tran

smitt

ance

()

4000 3500 3000 2500 2000 1500 1000 500

(c)


(d)

(e)

3415

28961633 1418

1157

891

34222891

1650

1595

1421

11561079

34452881 1651

15951418

11561066

894

3409

29001634

1560

14131153

900

3403

29051727 1639

1418 1168

896

(a)

(b)














Ester bond

Citric acidO O

OH


(a)

Ester bond

Citric acid

OO

OH


(b)

Amide bond

Citric acid

OO

OH


H

(c)


0

10

20

30

40

50

Cellulose film

Wat

er ab

sorb

ency

()


cellulose film


cellulose film







(a)


(b)


(c)

Inhibitory zone

(d)


























cellulose







4 Conclusions





Acknowledgment


References




























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



















(a) (b)

(c) (d)

(e) (f)

(g)






Tran

smitt

ance

()

4000 3500 3000 2500 2000 1500 1000 500

(c)


(d)

(e)

3415

28961633 1418

1157

891

34222891

1650

1595

1421

11561079

34452881 1651

15951418

11561066

894

3409

29001634

1560

14131153

900

3403

29051727 1639

1418 1168

896

(a)

(b)














Ester bond

Citric acidO O

OH


(a)

Ester bond

Citric acid

OO

OH


(b)

Amide bond

Citric acid

OO

OH


H

(c)


0

10

20

30

40

50

Cellulose film

Wat

er ab

sorb

ency

()


cellulose film


cellulose film







(a)


(b)


(c)

Inhibitory zone

(d)


























cellulose







4 Conclusions





Acknowledgment


References




























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Tran

smitt

ance

()

4000 3500 3000 2500 2000 1500 1000 500

(c)


(d)

(e)

3415

28961633 1418

1157

891

34222891

1650

1595

1421

11561079

34452881 1651

15951418

11561066

894

3409

29001634

1560

14131153

900

3403

29051727 1639

1418 1168

896

(a)

(b)














Ester bond

Citric acidO O

OH


(a)

Ester bond

Citric acid

OO

OH


(b)

Amide bond

Citric acid

OO

OH


H

(c)


0

10

20

30

40

50

Cellulose film

Wat

er ab

sorb

ency

()


cellulose film


cellulose film







(a)


(b)


(c)

Inhibitory zone

(d)


























cellulose







4 Conclusions





Acknowledgment


References




























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Ester bond

Citric acidO O

OH


(a)

Ester bond

Citric acid

OO

OH


(b)

Amide bond

Citric acid

OO

OH


H

(c)


0

10

20

30

40

50

Cellulose film

Wat

er ab

sorb

ency

()


cellulose film


cellulose film







(a)


(b)


(c)

Inhibitory zone

(d)


























cellulose







4 Conclusions





Acknowledgment


References




























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a)


(b)


(c)

Inhibitory zone

(d)


























cellulose







4 Conclusions





Acknowledgment


References




























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials
















cellulose







4 Conclusions





Acknowledgment


References




























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials

























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Preparation and Characterization of Chitosan Nanoparticles-Doped ...

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