Research Article PLGA/Nano-ZnO Composite Particles for Use in ...downloads.hindawi.com/journals/jnm/2016/9425289.pdf · Biomedical Applications: Preparation, Characterization, and

Research ArticlePLGANano-ZnO Composite Particles for Use inBiomedical Applications Preparation Characterizationand Antimicrobial Activity

Ana StankoviT1 Meltem Sezen2 Marina MilenkoviT3 Sonja KaišareviT4

Nebojša AndriT4 and Magdalena StevanoviT1

1 Institute of Technical Sciences of the Serbian Academy of Sciences and Arts Belgrade Serbia2Sabanci University Nanotechnology Research and Application Center (SUNUM) Orhanli Tuzla Istanbul Turkey3Departments of Microbiology and Immunology University of Belgrade Faculty of Pharmacy Belgrade Serbia4Department of Biology and Ecology University of Novi Sad Faculty of Sciences Novi Sad Serbia

Correspondence should be addressed to Magdalena Stevanovic magdalenastevanovicitnsanuacrs

Received 25 July 2016 Revised 8 November 2016 Accepted 21 November 2016

Academic Editor R Torrecillas

Copyright copy 2016 Ana Stankovic et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Copolymer poly (DL-lactide-co-glycolide) (PLGA) is extensively investigated for various biomedical applications such as controlleddrug delivery or carriers in the tissue engineering In addition zinc oxide (ZnO) is widely used in biomedicine especially formaterials like dental composites as a constituent of creams for the treatment of a variety of skin irritations to enhance theantibacterial activity of differentmedicaments and so onUniform spherical ZnOnanoparticles (nano-ZnO) have been synthesizedvia microwave synthesis method In addition to obtaining nano-ZnO a further aim was to examine their immobilization inthe PLGA polymer matrix (PLGAnano-ZnO) and this was done by a simple physicochemical solventnonsolvent method Thesamples were characterized by X-ray diffraction scanning electron microscopy laser diffraction particle size analyzer differentialthermal analysis and thermal gravimetric analysis The synthesized PLGAnano-ZnO particles are spherical uniform and withdiameters below 1 120583m The influence of the different solvents and the drying methods during the synthesis was investigated tooThe biocompatibility of the samples is discussed in terms of in vitro toxicity on human hepatoma HepG2 cells by application ofMTT assay and the antimicrobial activity was evaluated by brothmicrodilutionmethod against different groups of microorganisms(Gram-positive bacteria Gram-negative bacteria and yeast Candida albicans)

1 Introduction

Recently significant effort has been dedicated to developmicro- and nanoparticles for drug delivery since they offeran appropriate means to distribute the therapeutically activedrug molecule only to the site of action without affectinghealthy organs and tissues [1ndash3] Many different polymersboth synthetic and natural have been employed in makingbiocompatible and biodegradable nanoparticles Polyestersmicro- and nanoparticles are used for the controlled deliveryof different classes of active substances such as anticanceragents growth factors antibiotics antimicrobial agentsmetal and metal oxide nanoparticles [4ndash8] Polyester poly(DL-lactide-co-glycolide) (PLGA) is a copolymer approved

by Food and Drug Administration (FDA) for medical andpharmaceutical applications Physicochemical properties ofnanoparticles which include small size and very large specificsurface area significantly improve their bioavailability andminimize drug toxicity Inorganic antibacterial nanoparticlesespecially offer some unique benefits in overcoming majorproblems related to treatment process such as reducingdosage of antibacterial agent minimizing side effects over-coming bacterial resistance and lowering overall cost of thefabrication process [7 8]

ZnO represents a multifunctional material with a wideand direct band gap energy of 337 eV Besides its extraor-dinary optical catalytic semiconducting and piezoelec-tric properties ZnO is a promising material for a variety

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2016 Article ID 9425289 10 pageshttpdxdoiorg10115520169425289

2 Journal of Nanomaterials

of biological applications because of its chemical stabilityantimicrobial activity high biocompatibility and nontoxicityto human cells [9 10] Also zinc is a mineral elementessential to human health and used in the form of ZnO inthe daily supplement for zinc As a consequence of previouslymentioned properties ZnO nanoparticles can be used forcellular imaging [9 10] biodetection [11] drug delivery [1213] and various kinds of therapy [14 15] ZnO has a widerange of different nanostructures already used in numerouscommercial products such as sunscreen products differenttypes of sensors industrial coatings and antibacterial agents[16] Particularly ZnO nanoparticles show a wide range ofantibacterial activities towards Gram-positive and Gram-negative bacteria including a major food borne pathogensattributed to the generation of reactive oxygen species (ROS)on this oxide surfaces In recent years much attention hasbeen paid to preparation of organicnanosized inorganic-particles composite materials for a various types of appli-cations especially in medicine and pharmacy [7 17] Forexample inorganic core-polymer shell hybrid microsphereshave attracted attention because the materials can revealspecial characteristics such as improved strength shapeand chemical resistance [18] Above all the properties ofnanocomposites are greatly influenced by both the dispersingdegree of nanoparticles in the base polymers and the interfa-cial adhesion between the inorganic and organic components[19] Although nanocomposites can be prepared by simplymixing of nanoparticles with base polymers the dispersingdegree of nanoparticles and the interfacial linkage were obvi-ously insufficient to obtain desirable material properties [20]

In previous studies ZnO was incorporated in variety ofdifferent polymers such as poly (methyl methacrylate) [2122] polystyrene [23 24] polyamide [25] polyacrylonitrile[26] and polyurethane [27 28] and the results have shownthat the prepared polymerZnO composites presented betterperformances compared to the polymers alone All of thesecomposite materials were prepared in the forms of filmsscaffolds fibers or grafts [29 30] For the preparation of thesepolymerZnO composites different methods were appliedsuch as dissolution [31] microemulsion polymerization [24]sonication [32] or spin coating method [33]

Our study thus reports on obtaining innovative PLGAnanocomposite spheres with immobilized nano-ZnO(PLGAnano-ZnO) which represent an important system inthe field of medicine pharmacy and controlled drug deliveryand in particular to prevent infections The samples werecharacterized by X-ray diffraction (XRD) scanning electronmicroscopy (SEM) laser diffraction particle size analyzer(PSA) differential thermal analysis (DTA) and thermalgravimetric analysis (TGA) which demonstrated the suc-cessful immobilization of ZnO nanoparticles within PLGApolymer matrix Controlling the morphology of micro- andnanoparticles is essential for exploiting their properties fortheir utilization in different technologies Therefore in thisstudy we have examined influence of different solvents as wellasmethod of drying on themorphology of PLGAnano-ZnOFurther in order to evaluate the in vitro cytotoxic potential ofas prepared ZnO as well as PLGAnano-ZnO we used a testsystem with human hepatoma cells HepG2 This was done

by the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide (MTT) assay The antimicrobial activities of ZnOas well as PLGAnano-ZnO were evaluated by broth microd-ilution method against the Gram-positive bacteria Staph-ylococcus aureus (ATCC 25923) Staphylococcus epidermidis(ATCC 12228) and Bacillus subtilis (ATCC 6633) and theGram-negative bacteria Escherichia coli (ATCC 25922)Klebsiella pneumoniae (ATCC 13883) Salmonella abony(NTCT ndash 6017) and Pseudomonas aeruginosa (ATCC 27853)and the yeast Candida albicans (ATCC 24433)

2 Materials and Methods

21 Materials Poly (DL-lactide-co-glycolide) (PLGA)was obtained from Durect Lactel Adsorbable PolymersInternational and had lactide to glycolide ratio of 50 50Molecular weight of polymer was 40000ndash50000 gmolPolyvinylpyrrolidone (PVP) zinc acetate dihydrate(Zn(CH3COO)2sdot2H2O) and sodium hydroxide (NaOH)were obtained fromMerck Chemicals Ltd (Merck Germany)All chemicals and solvents were of reagent grade

22 Methods

221 Synthesis of ZnO Nanoparticles ZnO nanoparticleswere prepared via microwave assisted synthesis methodas previously described but with modifications[34] Starting chemicals were zinc acetate dihydrate(Zn(CH3COO)2sdot2H2O) and sodium hydroxide (NaOH) Inthe synthesis procedure 175M solution of NaOH was slowlyadded into a 007mol solution of Zn(CH3COO)2 2H2Oat permanent temperature of 60∘C and under continuousstirring at 1000 rpm The white precipitate was formedindicating enactment of a chemical reactionThe as preparedsuspension was treated in a microwave field through 5mintime period Strength of the applied microwave field was150W Final reaction product was cooled to the roomtemperature and then centrifuged at 8000 rpm for 10minto obtain white precipitate Synthesized ZnO powder waswashed out several times with absolute ethanol and deionizedwater and dried at 60∘C for couple hours

222 Synthesis of PLGANano-ZnO Particles with Acetone asSolvent and Ethanol as Nonsolvent PLGAnano-ZnO parti-cles were synthesized by physicochemical solventnonsolventmethod Immobilization of ZnO nanoparticles into PLGApolymer matrix was performed using a homogenizationprocess of water and organic phase (Figure 1) Firstly 200mgof PLGA was dissolved in 10mL of acetone This solutionwas constantly mixed on a magnetic stirrer for about 15minand at room temperature Thereafter 1mL of the ZnOdispersion in acetone (055ww) was added dropwise into aPLGAacetone solution while constantly being homogenizedat 1200 rpm for 30minThis was followed by precipitation byaddition of 15mL ethanol with the solution obtained beingpoured very slowly into 40mL aqueous 005 (ww) PVPsolution with continuous stirring at 1200 rpm PVP was usedas a stabilizer in order to prevent particles agglomeration

Journal of Nanomaterials 3

ZnO

PLGA

PLGA

ZnO

Particle PLGAnano-ZnO

Figure 1 Scheme for preparation of PLGAnano-ZnO particles

Finally the prepared PLGAnano-ZnO dispersion wasdecanted and left overnight to dry at room temperature

223 Synthesis of PLGANano-ZnO Particles with EthylAcetate as Solvent and Ethanol as Nonsolvent In order toexamine the influence of different solvents on morphologyof PLGAnano-ZnO particles ethyl acetate (C4H8O2) wasused as solvent in the experiment instead of acetone PLGAcommercial granules (200mg) were dissolved in 10mL ethylacetate over approximately 15min at room temperature Allother reaction parameters were caped the same as in theexperiment described above

224 Synthesis of PLGANano-ZnO Particles Using Freeze-Drying in the Synthesis Freeze-drying or lyophilisation isa method used to transform solutions of formulations intosolids of sufficient stability for distribution and storageExamination of the influence of freeze-drying on morphol-ogy of PLGAnano-ZnO particles was also part of this workAll steps in the synthesis were the same as in the experimentsdescribed above except that the PLGAnano-ZnO particleswere freeze-dried After the freezing method of lyophilisa-tion was utilized at minus55∘C and the pressure of 03mbar Maindrying was performed for 85 h and final drying for 30minusing Freeze Dryer Christ Alpha 1-2LD plus

225 Morphology Studies To define the microstructure ofZnO particles field-emission scanning electron microscopy(FE-SEM) analysis was carried out on a Carl Zeiss SUPRA35 VP instrument The size distribution of the synthesizedsamples was measured using the laser diffraction particle sizeanalyzer (Mastersizer 2000 Malvern Instruments Ltd) Thesize measurement range of this instrument is from 20 nmup to 2mm Prior to measuring the sample was treatedfor 15min in an ultrasonic bath The morphology of thesynthesized PLGAnano-ZnO particles was examined usingscanning electron microscopy (SEM) on JEOL JIB-4601 FMulti Beam Platform

226 XRD Analysis For identification of the phase com-position (phase analysis) of the synthesized powders X-raydiffraction (XRD) method was applied with a Philips PW1050 diffractometer with Cu-K12057212 radiation The measure-ments were carried out in the 2120579 range of 10∘ to 70∘ with ascanning step width of 005∘ and 2 s per step

227 Thermal Analysis Differential thermal analysis (DTA)and thermal gravimetric analysis (TGA) measurements werecarried out using a SETSYS Evolution TGA-DTADSCinstrument (SETERAM Instrumentation France) on pow-dered sample PLGAnano-ZnO of approximately 15mgAssigned temperature range was from room temperature to1200∘C The data were baseline corrected by carrying out ablank run and subtracting this from the original data

228 Cytotoxicity Study The samples were evaluated fortheir in vitro cytotoxicity towards human hepatoma HepG2cells by application of MTT assay This is a colorimetricassay based on the measurement of conversion of the yel-low thiazolyl blue tetrazolium bromide (MTT) to a purpleformazan derivative by mitochondrial dehydrogenase inviable cells The HepG2 cells were cultured in MinimumEssential Medium Eagle (MEM Sigma) supplemented with10 fetal bovine serum (FBS Gibco) sodium bicarbonate(15 gL) sodium pyruvate (011 gL) HEPES buffer (20mM)streptomycin (01 gL) and penicillin (100 000 IUL) at 37∘Cin a humidified atmosphere containing 5 CO2 For theexperiments the cells were seeded in 96-well plates at thedensity 25 000 cellswell in 02mL of MEM 10 FBS andincubated for 24 h to attach Then the medium was removedand the cells were treated by samplesrsquo dilutions at the con-centrations 0000001 000001 00001 0001 and 001wvfor 24 h Two independent experiments were conducted andin each experiment cells were treated in triplicates withcontrol (nontreated) wells included in each plate In eachexperiment solvent controls were also tested in solventconcentrations corresponding to the ones in the samplestreatments Following incubation medium was removedand cells were incubated for 3 h with 005mg01mLwellMTT dissolved in serum-free MEM Formazan salts weredissolved in 01mLwell of 004M HCl in isopropanol andlight absorption was measured using a plate reader (ThermoLabsystems) on 540 nm with reference wavelength 690 nmCell viabilitywas expressed as a percentage of the correspond-ing control value (100) The data were analyzed by one-wayANOVA followed byDunnettrsquosMultiple Comparison Test(119875 le 005) using the GraphPad Prism 5 software

229 Antimicrobial Activity Theantimicrobial activity of theZnO and PLGAnano-ZnO particles was investigated againstGram-positive bacteria Staphylococcus aureus (ATCC 25923)Staphylococcus epidermidis (ATCC 12228) and Bacillus sub-tilis (ATCC6633) and theGram-negative bacteriaEscherichiacoli (ATCC 25922) Klebsiella pneumoniae (ATCC 13883)Salmonella abony (NTCT ndash 6017) and Pseudomonas aerugi-nosa (ATCC 27853) and the yeast Candida albicans (ATCC


100nm

d(01) 0034120583m d(05) 0063120583m d(09) 0118 120583m

(a)

0

2

4

6

8

10

12

14

Num

ber (

)

01 1 10 100001Particle size (120583m)

d(01) 0034120583m

d(05) 0063120583m

d(09) 0118 120583m

(b)

Figure 2 (a) Representative FE-SEM image of ZnO particles (bar 100 nm) (b) Particle size distribution profile for ZnO particles

24433) In order to determine minimum inhibitory concen-trations (MICs) of the prepared samples a broth microdilu-tion method was used MIC represents the lowest concentra-tion of a compound at which the microorganism does notdemonstrate visible growth MICs were determined accord-ing to Clinical and Laboratory Standards Institute (CLSI2007) [35] Tests were performed in Muller Hinton broth forthe bacterial strains and in Sabouraud dextrose broth for Calbicans Overnight broth cultures were prepared for eachstrain and the final concentration in each well was adjustedto 2 times 106CFUmL The tested samples were dissolved in 1dimethylsulfoxide and diluted to the desired concentrations(ranging from 625 to 1000 120583gmL) in fresh Muller-Hintonbroth for bacteria and Sabouraud broth for C albicans Inthe tests 005 triphenyl tetrazolium chloride (TTC AldrichChemical Company Inc USA) was also added to the culturemedium as a growth indicator TTC is a redox indicatorused for differentiation between metabolically active andnonactive cells The colorless compound is enzymaticallyreduced to red 135-triphenylformazan by cell dehydroge-nases indicating metabolic activity (red color of the mediuminmicrotiter plate well) As a positive control of growth wellscontaining only the microorganisms in the broth were usedBacteria growth was determined after 24 h while growth ofCandida albicans was determined after 48 h of incubationat 37∘C All of the MIC determinations were performed induplicate and two positive growth controls were included

3 Results and Discussion

Even though the morphology of micro- and nanoparticles isof great importance it is generally not well characterized andpractically very hard to control Nevertheless this is of primeimportance Morphological characteristics of the obtainedZnO particles are revealed by FE-SEM and PSA Figure 2(a)shows nanostructured ZnO powder composed of uniformspherical particles with a narrow particle size distribution

and average diameter of approximately 63 nm (Figure 2(b))From the presented FE-SEM image (Figure 2(a)) it canbe seen that obtained ZnO powder is composed of welldispersed nanoparticles Particles size distribution of theZnOnanopowder is presented in Figure 2(b) Designations 1198891011988950 and 11988990 means that 10 50 and 90 of total number ofthe measured particles have diameter less than 34 nm 63 nmand 118 nm respectively

SEM images of PLGAnano-ZnO particles prepared byphysicochemical solventnonsolvent method with acetoneas solvent and dried at room temperature are presented inFigure 3(A) SEM analysis showed that PLGAnano-ZnOparticles have uniform spherical morphology with a narrowparticle size distribution and smooth surfaces Diametersof the particles are of about 200 nm In contrast to welldefined morphology of PLGAnano-ZnO particles preparedwith acetone PLGAnano-ZnOparticles preparedwith ethylacetate as solvent and dried at room temperature are veryagglomerated with no clearly defined forms and dimensions(Figure 3(B)) Only small number of spherical micron sizeparticles with a broad particle size distribution and a smoothsurface can be seen in the sample presented in Figure 3(B)PLGAnano-ZnO particles prepared by physicochemical sol-ventnonsolvent method with acetone as solvent and freeze-dried are presented in Figure 3(C) From these images it canbe seen that sample consisted of particles which are withsmall sizes comparable with particlersquos sizes of the samplealso obtained with acetone but dried at room temperatureHowever particles are agglomerated Obviously this processgenerates various stresses during freezing and desiccationsteps The most likely reason why particles are so agglomer-ated (Figure 3(C)) is that we did not use any cryoprotectantin the synthesis

Further we have characterized ZnO and PLGAnano-ZnO particles prepared by physicochemical solventnonsolventmethodwith acetone as solvent and dried at roomtemperature In order to determine crystal structure and


Figure 3 (A) Representative SEM images of PLGAnano-ZnO particles prepared with acetone and dried at room temperature (bars 05 120583m1 120583m and 5120583m) (B) Representative SEM images of PLGAnano-ZnO particles prepared with ethyl acetate and dried at room temperature(bars 2 120583m 5 120583m and 5 120583m) (C) Representative SEM images of PLGAnano-ZnO particles prepared with acetone and freeze-dried (bars05 120583m 2 120583m and 5120583m)

phase composition of PLGAnano-ZnO XRD method wasapplied As it is shown in Figure 4 characteristic diffractionpeaks corresponding to a pure hexagonal wurtzite phase ofthe crystalline ZnO nanostructured powder are obtainedaccording to the JCPDS card number 36-145 PLGA asan amorphous compound did not show any crystallinediffraction peaks in the presented spectra Broad and verylow intensity peak in the 2120579 range of 10 to 25∘ was observedindicating its amorphous structure

Figure 5(a) shows differential thermal analysis DTA(green) and relative mass loss TGA (black) curves corre-sponding to synthesized nanostructured ZnO powder TheTGA curve represents the total weight loss of approximately5 wt due to the elimination of the residual hydroxyl groupsThe presence of the reaction precursor Zn(CH3COO)2 in thefinal product can be distinguished by the three characteristicendothermic processes that take place during heating to350∘C The first process which corresponds to dehydrationof zinc acetate dihydrate occurs at 1198791 asymp 100

∘C the secondrepresents process ofmelting of anhydrous salt at1198792 asymp 250

∘Cand the third evaporation of zinc species 1198793 asymp 320

∘C Theendothermic peak at 1198794 asymp 450

∘C can be attributed to crystal-lization process of the ZnO hexagonal crystal structure [36]

10311

0

102

101

002

100

PLGAnano-ZnO

ZnO0

200

400

600

800

1000

1200

1400

1600

Inte

nsity

(au

)

20 30 40 50 60 70102120579 (∘)

Figure 4 XRD patterns of as prepared ZnO nanoparticles andPLGAnano-ZnO powder

DTA-TGA curves (blue and red resp) of preparedPLGAnano-ZnO powder are displayed in Figure 5(b) It canbe clearly seen that the TG curve demonstrates major weight


ZnOTGADTA

100 200 300 400 500 600 700 800 900 100011000Temperature (∘C)

949596979899

100101102103104105

Mas

s los

s (

)

T4T3

T2

T1

minus09

minus08

minus07

minus06

minus05

minus04

minus03

minus02

minus01

00

Hea

t flow

(Exo

-Up)

(a)

PLGAZnOTGADTA

100 200 300 400 500 600 700 800 900 100011000Temperature (∘C)

T2

T1 minus4

minus2

0

2

4

6

8

Hea

t flow

(Exo

-Up)

0102030405060708090

100

Mas

s los

s (

)

T1998400

T2998400

(b)

Figure 5 DTA-TGA curves of (a) ZnO nanoparticles and (b) PLGAnano-ZnO particles prepared by physicochemical solventnonsolventmethod with acetone as solvent and dried at room temperature

ZnO

Concentration ( wv)

PLGAnano-ZnO

Concentration ( wv)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

lowastlowast

00100010000100000100000010 00100010000100000100000010

Figure 6 Viability of HepG2 cells treated with ZnO nanoparticles or PLGAnano-ZnO for 24 hThe data are presented as mean values of sixreplicates from two independent experiments plusmnSEM lowastlowast statistically significant difference compared to the control (0wv) 119875 le 001

loss of approximately 95wt in the considered temperaturerange The weight loss of PLGAnano-ZnO attributed tothermal decomposition of polymer occurs in two phases(11987911015840 asymp 398

∘Cmdash91 25 and 11987921015840 asymp 545∘Cmdash3 30) DTA

curve reveals an endothermic process at about 50∘C whichcan be assigned to the relaxation peak that follows the glasstransition of the polymer The exothermic peaks around1198791 asymp 350 1198792 asymp 400 and 1198793 asymp 500

∘C are related to thethermal decomposition of the polymer The decompositionprocess characterized by an endothermic peak has started atapproximately 119879 asymp 320∘C [37]

For the cytotoxicity study ZnO nanoparticles andPLGAnano-ZnO particles prepared with acetone as solventand ethanol as nonsolvent were used In vitro cytotoxicityof ZnO nanoparticles and PLGAnano-ZnO was determinedwith MTT assay after exposure of HepG2 cells to 0000001

000001 00001 0001 and 001 vv of ZnO nanoparticlesor of PLGAnano-ZnO prepared with acetone for 24 h Atthe applied exposure conditions ZnO nanoparticles as well asPLGAnano-ZnO did not affect the viability of HepG2 cells(Figure 6)The cell viability data for the vehicle control are notshown in Figure 6 as they were almost identical as negativecontrol

One of the aims of our study was to examine antimicro-bial activity of ZnO and PLGAnano-ZnO against differentgroups of microorganisms (Gram-positive bacteria Gram-negative bacteria and yeast Candida albicans) In order todetermine minimum inhibitory concentrations (MICs) ofthe tested samples we performed broth microdilution testaccording to CLSI (2007) which is standard method [38]Minimal inhibitory concentrations of the samples were deter-mined against Gram-positive bacteria Staphylococcus aureus


ZnOPLGAnano-ZnO

00

02

04

06

08

10

Min

imum

inhi

bito

ry co

ncen

trat

ions

(mg

mL)

Bacil

lus s

ubtil

is

Esch

erich

ia co

li

Salm

onell

a ab

ony

Cand

ida

albi

cans

Pseu

dom

onas

aeru

gino

sa

Stap

hylo

cocc

usep

ider

mid

is

Kleb

siella

pneu

mon

iae

Stap

hylo

cocc

usau

reus

Figure 7 Minimal inhibitory concentrations of ZnO and PLGAnano-ZnO nanoparticles determined using a broth microdilutionassay against Gram-positive bacteria Staphylococcus aureus (ATCC25923) Staphylococcus epidermidis (ATCC 12228) and Bacillus sub-tilis (ATCC 6633) and the Gram-negative bacteria Klebsiella pneu-moniae (ATCC 13883) Escherichia coli (ATCC 25922) Salmonellaabony (NTCT ndash 6017) and Pseudomonas aeruginosa (ATCC 27853)and the yeast Candida albicans (ATCC 24433)

(ATCC 25923) Staphylococcus epidermidis (ATCC 12228)and Bacillus subtilis (ATCC 6633) and the Gram-negativebacteria Klebsiella pneumoniae (ATCC 13883) Escherichiacoli (ATCC 25922) Salmonella abony (NTCT ndash 6017) andPseudomonas aeruginosa (ATCC 27853) and the yeast Can-dida albicans (ATCC 24433) (Figure 7) Generally antimicro-bial activity of nanoparticles has received significant interestworldwide since many microorganisms exist in the rangefrom few of nanometers to tens of micrometers One of theexplanation why particles such as ZnO display antimicrobialactivities is due to increased specific surface area as a resultof the reduction of the particle size which leads to enhancedparticle surface reactivity Also reactive oxygen species suchas hydrogen peroxide hydroxyl radicals and peroxide havebeen important bactericidal and bacteriostatic factor forseveral mechanisms involving cell wall damage becauseof zinc oxide localized interaction enhanced membranepermeability internalization of nanoparticles due to loss ofproton motive force and so forth [39] Results of this study(Figure 7) provided the evidence of a considerable antibac-terial activity of ZnO as well as PLGAnano-ZnO againstall of the tested strains The samples exhibited bacteriostaticeffect Some standard antimicrobial drugs (tetracyclines andchloramphenicol) are also mainly bacteriostatic but regard-less of this fact they are used in clinical practice In thiscase ZnO exhibited stronger activity against Gram-positivebacteriaBacillus subtilis (ATCC 6633) and theGram-negative

bacteria Klebsiella pneumoniae (ATCC 13883) Escherichiacoli (ATCC 25922) and Pseudomonas aeruginosa (ATCC27853) compared to the PLGAnano-ZnO As it is alreadymentioned above the inorganic core-polymer shell hybridmicrospheres have attracted attention because the materialscan reveal special characteristics such as improved strengthshape and chemical resistance So far very few studies reportsuccess to preserve antibacterial activity when blended withpolymer [25 40] Here even if there is obviously the differ-ence between MICs of ZnO and PLGAnano-ZnO particlesof PLGAnano-ZnO also showed antimicrobial propertiesagainst eight different microbial strains From the literature itis known in some cases that improved antimicrobial activityof ZnO can be attributed to surface defects on its abrasivesurface texture [39] On the other hand PLGAnano-ZnOparticles exhibited more pronounced antimicrobial activityagainst yeast Candida albicans In the literature differentmechanisms for the microorganism and material surfaceinteractions have been reported such as electrostatic andhydrophobic forces van der Waals forces and adhesins suchas lectins [41] Adhesion is an essential step for microorgan-ism to colonize material surfaces Previous studies describedthat the adhesion should be higher on hydrophobic thanon hydrophilic material [41] However the strength of theinteraction between microorganism and material dependsnot only on the material properties but also on the microor-ganism surface structure itself [42] The differences observedfor the minimum inhibitory concentrations of ZnO andPLGAnano-ZnO when they were evaluated versus Bacillussubtilis Klebsiella pneumoniae Escherichia coli Salmonellaabony and Pseudomonas aeruginosa can be also explainedby differences in their cell envelopes Most Gram-positivecell walls contain considerable amounts of teichoic andteichuronic acids which may account for up to 50 of thedry weight of the wall In addition some Gram-positivewalls may contain polysaccharide molecules Gram-negativecell walls contain three components that lie outside ofthe peptidoglycan layer (lipoprotein outer membrane andlipopolysaccharide)The permeability of the outermembranevaries widely from one Gram-negative species to another[43 44] In accordance with this we observed strongerbacteriostatic activity of tested samples against Klebsiellapneumoniae Escherichia coli and Pseudomonas aeruginosathan against Salmonella abony Although it would be goodthat the MIC concentrations are tested also in cytotoxicityassay to make sure that these compounds could be usedas antimicrobial without any risk unfortunately it was notpossible due to the limits of the experimental setup andgreat solvent concentrations that could mask the compoundrsquoseffect However since not a single indication of cytotoxicitywas observed for PLGAnano-ZnO in 001wv concentra-tion we believe it is unlikely that 005ndash006wv wouldinduce significant cytotoxic response Our assumption canalso be supported by literature data where for example nocytotoxic effects of zinc containing glasses were observed atconcentration of 05wv while MIC concentrations were3-4 times lower [45] All of the tested bacterial strainsmay be highly resistant to traditional antibiotic therapies[46 47] However the antimicrobial mechanism is very


complex and it is not easy to fully understand the exactmechanismof cellmembrane damage involvedwith particlesWhen immobilized in a polymer matrix amount of fillerinteractions between filler and polymer matrix and particledispersion within polymeric particles may all influence theantimicrobial activity of the material All of this as well askinetic release of ZnO from the PLGA polymer matrix willbe the subject of our next study Also the subject of ourfuture research will be to determine minimum bactericidalconcentration (MBC) and influence of different externalfactors of antibacterial activity of ZnO and PLGAnano-ZnO against selected bacterial strains (laboratory and clinicalisolates)

4 Conclusion

Uniform spherical ZnO particles with average diameterof 63 nm were synthesized and then successfully immobi-lized within a PLGA polymer matrix (PLGAnano-ZnO)This was confirmed by X-ray diffraction scanning electronmicroscopy laser diffraction differential thermal analysisand thermal gravimetric analysis Influence of different sol-vents as well as different drying methods on morphology ofPLGAnano-ZnO particles was evaluatedThe best morphol-ogy was obtained in the case of PLGAnano-ZnO particlesprepared by physicochemical solventnonsolvent methodwith acetone as solvent and dried at room temperatureThesePLGAnano-ZnO particles are spherical in shape uniformand with diameters below 1120583m The MTT assay data indi-cate good biocompatibility of these ZnO and PLGAnano-ZnO Both types of particles ZnO and PLGAnano-ZnOexhibited antimicrobial inhibition activity against Gram-positive bacteria Staphylococcus aureus Staphylococcus epi-dermidis andBacillus subtilis and theGram-negative bacteriaEscherichia coliKlebsiella pneumoniae Salmonella abony andPseudomonas aeruginosa and the yeast Candida albicansOur data suggest that PLGAnano-ZnO nanoparticles arepromising candidates for applications in biomedical fieldsince it is expected that such particles will demonstrate acombination of desirable properties in one device that iscontrolled drug-delivery function and prevention or elimi-nation of possible infections

Competing Interests

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

Acknowledgments

TheMinistry of Education Science and Technological Devel-opment of the Republic of Serbia supported this work finan-cially through the project Grant no III45004 The authorswould like to express gratitude to Dr Miodrag J Lukicfor particle size distribution measurements to MSc NenadFilipovic for the technical assistance during the experimentsand to MSc Ljljana Veselinovic for performing the XRDmeasurements This work is also supported by the COST

Action CA15114 Anti-MIcrobial Coating Innovations to pre-vent infectious diseases (AMICI)

References

[1] L S Nair and C T Laurencin ldquoBiodegradable polymers asbiomaterialsrdquo Progress in Polymer Science vol 32 no 8-9 pp762ndash798 2007

[2] A Kumari S K Yadav and S C Yadav ldquoBiodegradablepolymeric nanoparticles based drug delivery systemsrdquo Colloidsand Surfaces B Biointerfaces vol 75 no 1 pp 1ndash18 2010

[3] P L Lam and R Gambari ldquoAdvanced progress of microencap-sulation technologies in vivo and in vitro models for studyingoral and transdermal drug deliveriesrdquo Journal of ControlledRelease vol 178 no 1 pp 25ndash45 2014

[4] H K Makadia and S J Siegel ldquoPoly Lactic-co-Glycolic Acid(PLGA) as biodegradable controlled drug delivery carrierrdquoPolymers vol 3 no 3 pp 1377ndash1397 2011

[5] F Danhier E Ansorena J M Silva R Coco A Le Bretonand V Preat ldquoPLGA-based nanoparticles an overview ofbiomedical applicationsrdquo Journal of Controlled Release vol 161no 2 pp 505ndash522 2012

[6] T K Dash and V B Konkimalla ldquoPoly-120598-caprolactone basedformulations for drug delivery and tissue engineering a reviewrdquoJournal of Controlled Release vol 158 pp 15ndash33 2012

[7] M M Stevanovic S D Skapin I Bracko et al ldquoPoly(lactide-co-glycolide)silver nanoparticles synthesis characterizationantimicrobial activity cytotoxicity assessment and ROS-inducing potentialrdquo Polymer vol 53 no 14 pp 2818ndash28282012

[8] M Stevanovic N Filipovic J Djurdjevic M Lukic MMilenkovic and A Boccaccini ldquo45S5Bioglass-based scaf-folds coated with selenium nanoparticles or with poly(lactide-co-glycolide)selenium particles processing evaluation andantibacterial activityrdquoColloids and Surfaces B Biointerfaces vol132 pp 208ndash215 2015

[9] H Hong J Shi Y Yang et al ldquoCancer-targeted optical imagingwith fluorescent zinc oxide nanowiresrdquoNano Letters vol 11 no9 pp 3744ndash3750 2011

[10] H-J Zhang H-M Xiong Q-G Ren Y-Y Xia and J-LKong ldquoZnOsilica corendashshell nanoparticles with remarkableluminescence and stability in cell imagingrdquo Journal of MaterialsChemistry vol 22 no 26 pp 13159ndash13165 2012

[11] A A Ansari A Kaushik P R Solanki and B D MalhotraldquoNanostructured zinc oxide platform for mycotoxin detectionrdquoBioelectrochemistry vol 77 no 2 pp 75ndash81 2010

[12] K C Barick S Nigam andD Bahadur ldquoNanoscale assembly ofmesoporous ZnO a potential drug carrierrdquo Journal of MaterialsChemistry vol 20 no 31 pp 6446ndash6452 2010

[13] F Muhammad M Guo Y Guo et al ldquoAcid degradable ZnOquantum dots as a platform for targeted delivery of an anti-cancer drugrdquo Journal of Materials Chemistry vol 21 no 35 pp13406ndash13412 2011

[14] H Zhang B Chen H Jiang C Wang H Wang and X WangldquoA strategy for ZnO nanorod mediated multi-mode cancertreatmentrdquo Biomaterials vol 32 no 7 pp 1906ndash1914 2011

[15] D Guo C Wu H Jiang Q Li X Wang and B ChenldquoSynergistic cytotoxic effect of different sized ZnO nanopar-ticles and daunorubicin against leukemia cancer cells underUV irradiationrdquo Journal of Photochemistry and Photobiology BBiology vol 93 no 3 pp 119ndash126 2008


[16] A Stankovic S Dimitrijevic and D Uskokovic ldquoInfluence ofsize scale and morphology on antibacterial properties of ZnOpowders hydrothemally synthesized using different surfacestabilizing agentsrdquo Colloids and Surfaces B Biointerfaces vol102 pp 21ndash28 2013

[17] M Stevanovic I Bracko MMilenkovic et al ldquoMultifunctionalPLGA particles containing poly(l-glutamic acid)-capped silvernanoparticles and ascorbic acid with simultaneous antioxida-tive and prolonged antimicrobial activityrdquo Acta Biomaterialiavol 10 no 1 pp 151ndash162 2014

[18] E Tang and S Dong ldquoPreparation of styrene polymerZnOnanocomposite latex via miniemulsion polymerization and itsantibacterial propertyrdquoColloid and Polymer Science vol 287 no9 pp 1025ndash1032 2009

[19] D-G Yu and J H An ldquoPreparation and characterization of tita-niumdioxide core and polymer shell hybrid composite particlesprepared by two-step dispersion polymerizationrdquo Polymer vol45 no 14 pp 4761ndash4768 2004

[20] M Xiong L Wu S Zhou and B You ldquoPreparation and char-acterization of acrylic latexnano-SiO2 compositesrdquo PolymerInternational vol 51 no 8 pp 693ndash698 2002

[21] Y Zhang X Wang Y Liu S Song and D Liu ldquoHighly trans-parent bulk PMMAZnO nanocomposites with bright visibleluminescence and efficient UV-shielding capabilityrdquo Journal ofMaterials Chemistry vol 22 no 24 pp 11971ndash11977 2012

[22] G Gedda H N Abdelhamid M S Khan and H-F WuldquoZnO nanoparticle-modified polymethyl methacrylate-assisteddispersive liquid-liquid microextraction coupled withMALDI-MS for rapid pathogenic bacteria analysisrdquo RSC Advances vol4 no 86 pp 45973ndash45983 2014

[23] J Zhou Y Gu P Fei et al ldquoFlexible piezotronic strain sensorrdquoNano Letters vol 8 no 9 pp 3035ndash3040 2008

[24] P Liu ldquoFacile preparation of monodispersed coreshell zincoxidepolystyrene (ZnOPS) nanoparticles via soaplessseeded microemulsion polymerizationrdquo Colloids and SurfacesA Physicochemical and Engineering Aspects vol 291 no 1ndash3pp 155ndash161 2006

[25] G Droval I Aranberri L German et al ldquoThermal andrheological characterization of antibacterial nanocompositespoly(amide) 6 and low-density poly(ethylene) filled with zincoxiderdquo Journal ofThermoplastic CompositeMaterials vol 27 no2 pp 268ndash284 2014

[26] Y-Y Chen C-C Kuo B-Y Chen P-C Chiu and P-CTsai ldquoMultifunctional polyacrylonitrile-ZnOAg electrospunnanofiber membranes with various ZnO morphologies forphotocatalytic UV-shielding and antibacterial applicationsrdquoJournal of Polymer Science Part B Polymer Physics vol 53 no4 pp 262ndash269 2015

[27] T Amna M S Hassan F A Sheikh et al ldquoZinc oxide-dopedpoly(urethane) spider web nanofibrous scaffold via one-stepelectrospinning a novel matrix for tissue engineeringrdquo AppliedMicrobiology and Biotechnology vol 97 no 4 pp 1725ndash17342013

[28] S Awad H Chen G Chen et al ldquoFree volumes glass transi-tions and cross-links in zinc oxidewaterborne polyurethanenanocompositesrdquoMacromolecules vol 44 no 1 pp 29ndash38 2011

[29] A Kołodziejczak-Radzimska and T Jesionowski ldquoZinc oxidemdashfrom synthesis to application a reviewrdquoMaterials vol 7 no 4pp 2833ndash2881 2014

[30] Y-S Park K-N Kim K-M Kim et al ldquoFeasibility of three-dimensional macroporous scaffold using calcium phosphate

glass and polyurethane spongerdquo Journal of Materials Sciencevol 41 no 13 pp 4357ndash4364 2006

[31] T Kos A Anzlovar D Pahovnik E Zagar Z C Orel andM Zigon ldquoZinc-containing block copolymer as a precursorfor the in situ formation of nano ZnO and PMMAZnOnanocompositesrdquo Macromolecules vol 46 no 17 pp 6942ndash6948 2013

[32] J T Seil and T J Webster ldquoReduced Staphylococcus aureusproliferation and biofilm formation on zinc oxide nanoparticlePVC composite surfacesrdquo Acta Biomaterialia vol 7 no 6 pp2579ndash2584 2011

[33] Q Peng Y-C Tseng S B Darling and J W Elam ldquoA routeto nanoscopic materials via sequential infiltration synthesis onblock copolymer templatesrdquo ACS Nano vol 5 no 6 pp 4600ndash4606 2011

[34] S Markovic V Rajic A Stankovic et al ldquoEffect of PEOmolecular weight on sunlight induced photocatalytic activity ofZnOPEO compositesrdquo Solar Energy vol 127 pp 124ndash135 2016

[35] Clinical and Laboratory Standards Institute (CLSI) ldquoPerfor-mance standards for antimicrobial susceptibility testing 17thinformational supplementrdquo CLSI Document M100-S17 CLSIWayne Pa USA 2007

[36] M S Tokumoto S H Pulcinelli C V Santilli and V BrioisldquoCatalysis and temperature dependence on the formation ofZnO nanoparticles and of zinc acetate derivatives prepared bythe sol-gel routerdquo Journal of Physical Chemistry B vol 107 no2 pp 568ndash574 2003

[37] R M Mainardes M P D Gremiao and R C Evange-lista ldquoThermoanalytical study of praziquantel-loaded PLGAnanoparticlesrdquo Revista Brasileira de Ciencias Farmaceuti-casBrazilian Journal of Pharmaceutical Sciences vol 42 no 4pp 523ndash530 2006

[38] G Brađan A Pevec I Turel et al ldquoSynthesis characterizationDFT calculations and antimicrobial activity of pentagonal-bipyramidal Zn(II) and Cd(II) complexes with 26-diacetylpyridine-bis(trimethylammoniumacetohydrazone)rdquoJournal of Coordination Chemistry vol 69 no 18 pp 2754ndash2765 2016

[39] S Amna M Shahrom S Azman et al ldquoReview on zinc oxidenanoparticles antibacterial activity and toxicity mechanismrdquoNano-Micro Letters vol 7 no 3 pp 219ndash242 2015

[40] J H Li R Y Hong M Y Li H Z Li Y Zheng and JDing ldquoEffects of ZnO nanoparticles on the mechanical andantibacterial properties of polyurethane coatingsrdquo Progress inOrganic Coatings vol 64 no 4 pp 504ndash509 2009

[41] A Al-Ahmad M Wiedmann-Al-Ahmad C Carvalho et alldquoBacterial andCandida albicans adhesion on rapid prototyping-produced 3D-scaffolds manufactured as bone replacementmaterialsrdquo Journal of Biomedical Materials ResearchmdashPart Avol 87 no 4 pp 933ndash943 2008

[42] Y H An and R J Friedman ldquoConcise review of mechanisms ofbacterial adhesion to biomaterial surfacesrdquo Journal of Biomedi-cal Materials Research vol 43 no 3 pp 338ndash348 1998

[43] G F Brooks K C Carroll J S Butel S A Morse and T AMietzner Jawetz Melnick amp Adelbergrsquos Medical Microbiologychapter 2 McGraw-Hill 26th edition 2013

[44] P R Murray K S Rosenthal and M A Pfaller BacterialClassification Structure and Replication Medical MicrobiologyElsevier 7th edition 2013

[45] L Esteban-Tejeda C Prado B Cabal J Sanz R Torrecillasand J S Moya ldquoAntibacterial and antifungal activity of ZnO


containing glassesrdquo PLoS ONE vol 10 no 7 Article IDe0132709 2015

[46] M Stevanovic V Uskokovic M Filipovic S D Skapin and DUskokovic ldquoComposite PLGAAgNpPGAAscH nanosphereswith combined osteoinductive antioxidative and antimicrobialactivitiesrdquo ACS Applied Materials and Interfaces vol 5 no 18pp 9034ndash9042 2013

[47] E Oldfield and X Feng ldquoResistance-resistant antibioticsrdquoTrends in Pharmacological Sciences vol 35 no 12 pp 664ndash6742014

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


of biological applications because of its chemical stabilityantimicrobial activity high biocompatibility and nontoxicityto human cells [9 10] Also zinc is a mineral elementessential to human health and used in the form of ZnO inthe daily supplement for zinc As a consequence of previouslymentioned properties ZnO nanoparticles can be used forcellular imaging [9 10] biodetection [11] drug delivery [1213] and various kinds of therapy [14 15] ZnO has a widerange of different nanostructures already used in numerouscommercial products such as sunscreen products differenttypes of sensors industrial coatings and antibacterial agents[16] Particularly ZnO nanoparticles show a wide range ofantibacterial activities towards Gram-positive and Gram-negative bacteria including a major food borne pathogensattributed to the generation of reactive oxygen species (ROS)on this oxide surfaces In recent years much attention hasbeen paid to preparation of organicnanosized inorganic-particles composite materials for a various types of appli-cations especially in medicine and pharmacy [7 17] Forexample inorganic core-polymer shell hybrid microsphereshave attracted attention because the materials can revealspecial characteristics such as improved strength shapeand chemical resistance [18] Above all the properties ofnanocomposites are greatly influenced by both the dispersingdegree of nanoparticles in the base polymers and the interfa-cial adhesion between the inorganic and organic components[19] Although nanocomposites can be prepared by simplymixing of nanoparticles with base polymers the dispersingdegree of nanoparticles and the interfacial linkage were obvi-ously insufficient to obtain desirable material properties [20]

In previous studies ZnO was incorporated in variety ofdifferent polymers such as poly (methyl methacrylate) [2122] polystyrene [23 24] polyamide [25] polyacrylonitrile[26] and polyurethane [27 28] and the results have shownthat the prepared polymerZnO composites presented betterperformances compared to the polymers alone All of thesecomposite materials were prepared in the forms of filmsscaffolds fibers or grafts [29 30] For the preparation of thesepolymerZnO composites different methods were appliedsuch as dissolution [31] microemulsion polymerization [24]sonication [32] or spin coating method [33]

Our study thus reports on obtaining innovative PLGAnanocomposite spheres with immobilized nano-ZnO(PLGAnano-ZnO) which represent an important system inthe field of medicine pharmacy and controlled drug deliveryand in particular to prevent infections The samples werecharacterized by X-ray diffraction (XRD) scanning electronmicroscopy (SEM) laser diffraction particle size analyzer(PSA) differential thermal analysis (DTA) and thermalgravimetric analysis (TGA) which demonstrated the suc-cessful immobilization of ZnO nanoparticles within PLGApolymer matrix Controlling the morphology of micro- andnanoparticles is essential for exploiting their properties fortheir utilization in different technologies Therefore in thisstudy we have examined influence of different solvents as wellasmethod of drying on themorphology of PLGAnano-ZnOFurther in order to evaluate the in vitro cytotoxic potential ofas prepared ZnO as well as PLGAnano-ZnO we used a testsystem with human hepatoma cells HepG2 This was done

by the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide (MTT) assay The antimicrobial activities of ZnOas well as PLGAnano-ZnO were evaluated by broth microd-ilution method against the Gram-positive bacteria Staph-ylococcus aureus (ATCC 25923) Staphylococcus epidermidis(ATCC 12228) and Bacillus subtilis (ATCC 6633) and theGram-negative bacteria Escherichia coli (ATCC 25922)Klebsiella pneumoniae (ATCC 13883) Salmonella abony(NTCT ndash 6017) and Pseudomonas aeruginosa (ATCC 27853)and the yeast Candida albicans (ATCC 24433)

2 Materials and Methods

21 Materials Poly (DL-lactide-co-glycolide) (PLGA)was obtained from Durect Lactel Adsorbable PolymersInternational and had lactide to glycolide ratio of 50 50Molecular weight of polymer was 40000ndash50000 gmolPolyvinylpyrrolidone (PVP) zinc acetate dihydrate(Zn(CH3COO)2sdot2H2O) and sodium hydroxide (NaOH)were obtained fromMerck Chemicals Ltd (Merck Germany)All chemicals and solvents were of reagent grade

22 Methods

221 Synthesis of ZnO Nanoparticles ZnO nanoparticleswere prepared via microwave assisted synthesis methodas previously described but with modifications[34] Starting chemicals were zinc acetate dihydrate(Zn(CH3COO)2sdot2H2O) and sodium hydroxide (NaOH) Inthe synthesis procedure 175M solution of NaOH was slowlyadded into a 007mol solution of Zn(CH3COO)2 2H2Oat permanent temperature of 60∘C and under continuousstirring at 1000 rpm The white precipitate was formedindicating enactment of a chemical reactionThe as preparedsuspension was treated in a microwave field through 5mintime period Strength of the applied microwave field was150W Final reaction product was cooled to the roomtemperature and then centrifuged at 8000 rpm for 10minto obtain white precipitate Synthesized ZnO powder waswashed out several times with absolute ethanol and deionizedwater and dried at 60∘C for couple hours

222 Synthesis of PLGANano-ZnO Particles with Acetone asSolvent and Ethanol as Nonsolvent PLGAnano-ZnO parti-cles were synthesized by physicochemical solventnonsolventmethod Immobilization of ZnO nanoparticles into PLGApolymer matrix was performed using a homogenizationprocess of water and organic phase (Figure 1) Firstly 200mgof PLGA was dissolved in 10mL of acetone This solutionwas constantly mixed on a magnetic stirrer for about 15minand at room temperature Thereafter 1mL of the ZnOdispersion in acetone (055ww) was added dropwise into aPLGAacetone solution while constantly being homogenizedat 1200 rpm for 30minThis was followed by precipitation byaddition of 15mL ethanol with the solution obtained beingpoured very slowly into 40mL aqueous 005 (ww) PVPsolution with continuous stirring at 1200 rpm PVP was usedas a stabilizer in order to prevent particles agglomeration


ZnO

PLGA

PLGA

ZnO












100nm

d(01) 0034120583m d(05) 0063120583m d(09) 0118 120583m

(a)

0

2

4

6

8

10

12

14

Num

ber (

)

01 1 10 100001Particle size (120583m)

d(01) 0034120583m

d(05) 0063120583m

d(09) 0118 120583m

(b)
















10311

0

102

101

002

100

PLGAnano-ZnO

ZnO0

200

400

600

800

1000

1200

1400

1600

Inte

nsity

(au

)

20 30 40 50 60 70102120579 (∘)




ZnOTGADTA

100 200 300 400 500 600 700 800 900 100011000Temperature (∘C)

949596979899

100101102103104105

Mas

s los

s (

)

T4T3

T2

T1

minus09

minus08

minus07

minus06

minus05

minus04

minus03

minus02

minus01

00

Hea

t flow

(Exo

-Up)

(a)

PLGAZnOTGADTA

100 200 300 400 500 600 700 800 900 100011000Temperature (∘C)

T2

T1 minus4

minus2

0

2

4

6

8

Hea

t flow

(Exo

-Up)

0102030405060708090

100

Mas

s los

s (

)

T1998400

T2998400

(b)


ZnO

Concentration ( wv)

PLGAnano-ZnO

Concentration ( wv)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

lowastlowast

00100010000100000100000010 00100010000100000100000010










ZnOPLGAnano-ZnO

00

02

04

06

08

10

Min

imum

inhi

bito

ry co

ncen

trat

ions

(mg

mL)

Bacil

lus s

ubtil

is

Esch

erich

ia co

li

Salm

onell

a ab

ony

Cand

ida

albi

cans

Pseu

dom

onas

aeru

gino

sa

Stap

hylo

cocc

usep

ider

mid

is

Kleb

siella

pneu

mon

iae

Stap

hylo

cocc

usau

reus






4 Conclusion


Competing Interests


Acknowledgments



References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




ZnO

PLGA

PLGA

ZnO












100nm

d(01) 0034120583m d(05) 0063120583m d(09) 0118 120583m

(a)

0

2

4

6

8

10

12

14

Num

ber (

)

01 1 10 100001Particle size (120583m)

d(01) 0034120583m

d(05) 0063120583m

d(09) 0118 120583m

(b)
















10311

0

102

101

002

100

PLGAnano-ZnO

ZnO0

200

400

600

800

1000

1200

1400

1600

Inte

nsity

(au

)

20 30 40 50 60 70102120579 (∘)




ZnOTGADTA

100 200 300 400 500 600 700 800 900 100011000Temperature (∘C)

949596979899

100101102103104105

Mas

s los

s (

)

T4T3

T2

T1

minus09

minus08

minus07

minus06

minus05

minus04

minus03

minus02

minus01

00

Hea

t flow

(Exo

-Up)

(a)

PLGAZnOTGADTA

100 200 300 400 500 600 700 800 900 100011000Temperature (∘C)

T2

T1 minus4

minus2

0

2

4

6

8

Hea

t flow

(Exo

-Up)

0102030405060708090

100

Mas

s los

s (

)

T1998400

T2998400

(b)


ZnO

Concentration ( wv)

PLGAnano-ZnO

Concentration ( wv)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

lowastlowast

00100010000100000100000010 00100010000100000100000010










ZnOPLGAnano-ZnO

00

02

04

06

08

10

Min

imum

inhi

bito

ry co

ncen

trat

ions

(mg

mL)

Bacil

lus s

ubtil

is

Esch

erich

ia co

li

Salm

onell

a ab

ony

Cand

ida

albi

cans

Pseu

dom

onas

aeru

gino

sa

Stap

hylo

cocc

usep

ider

mid

is

Kleb

siella

pneu

mon

iae

Stap

hylo

cocc

usau

reus






4 Conclusion


Competing Interests


Acknowledgments



References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




100nm

d(01) 0034120583m d(05) 0063120583m d(09) 0118 120583m

(a)

0

2

4

6

8

10

12

14

Num

ber (

)

01 1 10 100001Particle size (120583m)

d(01) 0034120583m

d(05) 0063120583m

d(09) 0118 120583m

(b)
















10311

0

102

101

002

100

PLGAnano-ZnO

ZnO0

200

400

600

800

1000

1200

1400

1600

Inte

nsity

(au

)

20 30 40 50 60 70102120579 (∘)




ZnOTGADTA

100 200 300 400 500 600 700 800 900 100011000Temperature (∘C)

949596979899

100101102103104105

Mas

s los

s (

)

T4T3

T2

T1

minus09

minus08

minus07

minus06

minus05

minus04

minus03

minus02

minus01

00

Hea

t flow

(Exo

-Up)

(a)

PLGAZnOTGADTA

100 200 300 400 500 600 700 800 900 100011000Temperature (∘C)

T2

T1 minus4

minus2

0

2

4

6

8

Hea

t flow

(Exo

-Up)

0102030405060708090

100

Mas

s los

s (

)

T1998400

T2998400

(b)


ZnO

Concentration ( wv)

PLGAnano-ZnO

Concentration ( wv)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

lowastlowast

00100010000100000100000010 00100010000100000100000010










ZnOPLGAnano-ZnO

00

02

04

06

08

10

Min

imum

inhi

bito

ry co

ncen

trat

ions

(mg

mL)

Bacil

lus s

ubtil

is

Esch

erich

ia co

li

Salm

onell

a ab

ony

Cand

ida

albi

cans

Pseu

dom

onas

aeru

gino

sa

Stap

hylo

cocc

usep

ider

mid

is

Kleb

siella

pneu

mon

iae

Stap

hylo

cocc

usau

reus






4 Conclusion


Competing Interests


Acknowledgments



References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials











10311

0

102

101

002

100

PLGAnano-ZnO

ZnO0

200

400

600

800

1000

1200

1400

1600

Inte

nsity

(au

)

20 30 40 50 60 70102120579 (∘)




ZnOTGADTA

100 200 300 400 500 600 700 800 900 100011000Temperature (∘C)

949596979899

100101102103104105

Mas

s los

s (

)

T4T3

T2

T1

minus09

minus08

minus07

minus06

minus05

minus04

minus03

minus02

minus01

00

Hea

t flow

(Exo

-Up)

(a)

PLGAZnOTGADTA

100 200 300 400 500 600 700 800 900 100011000Temperature (∘C)

T2

T1 minus4

minus2

0

2

4

6

8

Hea

t flow

(Exo

-Up)

0102030405060708090

100

Mas

s los

s (

)

T1998400

T2998400

(b)


ZnO

Concentration ( wv)

PLGAnano-ZnO

Concentration ( wv)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

lowastlowast

00100010000100000100000010 00100010000100000100000010










ZnOPLGAnano-ZnO

00

02

04

06

08

10

Min

imum

inhi

bito

ry co

ncen

trat

ions

(mg

mL)

Bacil

lus s

ubtil

is

Esch

erich

ia co

li

Salm

onell

a ab

ony

Cand

ida

albi

cans

Pseu

dom

onas

aeru

gino

sa

Stap

hylo

cocc

usep

ider

mid

is

Kleb

siella

pneu

mon

iae

Stap

hylo

cocc

usau

reus






4 Conclusion


Competing Interests


Acknowledgments



References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




ZnOTGADTA

100 200 300 400 500 600 700 800 900 100011000Temperature (∘C)

949596979899

100101102103104105

Mas

s los

s (

)

T4T3

T2

T1

minus09

minus08

minus07

minus06

minus05

minus04

minus03

minus02

minus01

00

Hea

t flow

(Exo

-Up)

(a)

PLGAZnOTGADTA

100 200 300 400 500 600 700 800 900 100011000Temperature (∘C)

T2

T1 minus4

minus2

0

2

4

6

8

Hea

t flow

(Exo

-Up)

0102030405060708090

100

Mas

s los

s (

)

T1998400

T2998400

(b)


ZnO

Concentration ( wv)

PLGAnano-ZnO

Concentration ( wv)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

lowastlowast

00100010000100000100000010 00100010000100000100000010










ZnOPLGAnano-ZnO

00

02

04

06

08

10

Min

imum

inhi

bito

ry co

ncen

trat

ions

(mg

mL)

Bacil

lus s

ubtil

is

Esch

erich

ia co

li

Salm

onell

a ab

ony

Cand

ida

albi

cans

Pseu

dom

onas

aeru

gino

sa

Stap

hylo

cocc

usep

ider

mid

is

Kleb

siella

pneu

mon

iae

Stap

hylo

cocc

usau

reus






4 Conclusion


Competing Interests


Acknowledgments



References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




ZnOPLGAnano-ZnO

00

02

04

06

08

10

Min

imum

inhi

bito

ry co

ncen

trat

ions

(mg

mL)

Bacil

lus s

ubtil

is

Esch

erich

ia co

li

Salm

onell

a ab

ony

Cand

ida

albi

cans

Pseu

dom

onas

aeru

gino

sa

Stap

hylo

cocc

usep

ider

mid

is

Kleb

siella

pneu

mon

iae

Stap

hylo

cocc

usau

reus






4 Conclusion


Competing Interests


Acknowledgments



References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





4 Conclusion


Competing Interests


Acknowledgments



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














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



Documents

Research Article PLGA/Nano-ZnO Composite Particles for Use in ...downloads.hindawi.com/journals/jnm/2016/9425289.pdf · Biomedical Applications: Preparation, Characterization, and