10
Research Article Antimicrobial Properties of Microparticles Based on Carmellose Cross-Linked by Cu 2+ Ions Martina Kejdušová, 1 Jakub VyslouDil, 1 Katelina Kubová, 1 Vladimír Celer, 2 Magdaléna Krásna, 2 Alena Pechová, 3 Vjra VyskoIilová, 3 and Vratislav Koš2ál 4 1 Department of Pharmaceutics, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palack´ eho Tˇ ıda 1/3, 612 42 Brno, Czech Republic 2 Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, Palack´ eho 1/3, 612 42 Brno, Czech Republic 3 Department of Biochemistry and Biophysics, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Palack´ eho 1/3, 612 42 Brno, Czech Republic 4 Tescan, Libuˇ sina Tˇ ıda 863/21, 623 00 Brno-Kohoutovice, Czech Republic Correspondence should be addressed to Kateˇ rina Kubov´ a; [email protected] Received 16 September 2014; Accepted 14 November 2014 Academic Editor: Kin Tam Copyright © 2015 Martina Kejduˇ sov´ a et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Carmellose (CMC) is frequently used due to its high biocompatibility, biodegradability, and low immunogenicity for development of site-specific or controlled release drug delivery systems. In this experimental work, CMC dispersions in two different concentrations (1% and 2%) cross-linked by copper (II) ions (0.5, 1, 1.5, or 2.0M CuCl 2 ) were used to prepare microspheres with antimicrobial activity against Escherichia coli and Candida albicans, both frequently occurring pathogens which cause vaginal infections. e microparticles were prepared by an ionotropic gelation technique which offers the unique possibility to entrap divalent copper ions in a CMC structure and thus ensure their antibacterial activity. Prepared CMC microspheres exhibited sufficient sphericity. Both equivalent diameter and copper content were influenced by CMC concentration, and the molarity of copper (II) solution affected only the copper content results. Selected samples exhibited stable but pH-responsive behaviour in environments which corresponded with natural (pH 4.5) and inflamed (pH 6.0) vaginal conditions. All the tested samples exhibited proven substantial antimicrobial activity against both Gram-negative bacteria Escherichia coli and yeast Candida albicans. Unexpectedly, a crucial parameter for microsphere antimicrobial activity was not found in the copper content but in the swelling capacity of the microparticles and in the degree of CMC surface shrinking. 1. Introduction Carmellose (CMC) is a water-soluble anionic polysaccharide and semisynthetic derivative of cellulose [1] which has no harmful effects on human health. Its chains are linear (1 → 4)-linked glucopyranose units. In addition, it contains a hydrophobic polysaccharide backbone and many hydrophilic carboxyl groups and, as a result, exhibits amphiphilic charac- teristics. CMC is used in a number of applications throughout the food, cosmetic, textile, paper, and ceramic industries as a viscosity modifier, thickener, emulsion stabilizer, and water retention and adhesive agent [2]. It also has tremendous potential for use in pharmaceutical products including site- specific or controlled release drug delivery carrier matrices thanks to its high biocompatibility, biodegradability [3], and low immunogenicity [4]. In spite of the numerous positive properties of CMC, it does not have any antimicrobial proper- ties as it lacks antimicrobial functional groups [5]. To bestow CMC with antimicrobial properties, different possibilities have been proposed, such as (i) adding different antimicrobial agents, for example, potassium sorbate [6] or silver nitrate [7], (ii) graſting CMC with different antimicrobial substances, for example, guanidine hydrochloride [8], (iii) combining CMC with other polymers that exhibit antimicrobial properties, Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 790720, 9 pages http://dx.doi.org/10.1155/2015/790720

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Research ArticleAntimicrobial Properties of Microparticles Based onCarmellose Cross-Linked by Cu2+ Ions

Martina Kejdušovaacute1 Jakub VyslouDil1 Katelina Kubovaacute1 Vladimiacuter Celer2

Magdaleacutena Kraacutesna2 Alena Pechovaacute3 Vjra VyskoIilovaacute3 and Vratislav Koš2aacutel4

1Department of Pharmaceutics Faculty of Pharmacy University of Veterinary and Pharmaceutical Sciences BrnoPalackeho Trıda 13 612 42 Brno Czech Republic2Department of Infectious Diseases and Microbiology Faculty of Veterinary Medicine University of Veterinary andPharmaceutical Sciences Brno Palackeho 13 612 42 Brno Czech Republic3Department of Biochemistry and Biophysics Faculty of Veterinary Hygiene and Ecology University of Veterinary andPharmaceutical Sciences Brno Palackeho 13 612 42 Brno Czech Republic4Tescan Libusina Trıda 86321 623 00 Brno-Kohoutovice Czech Republic

Correspondence should be addressed to Katerina Kubova kubovakvfucz

Received 16 September 2014 Accepted 14 November 2014

Academic Editor Kin Tam

Copyright copy 2015 Martina Kejdusova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Carmellose (CMC) is frequently used due to its high biocompatibility biodegradability and low immunogenicity for development ofsite-specific or controlled release drug delivery systems In this experimental work CMCdispersions in two different concentrations(1 and 2) cross-linked by copper (II) ions (05 1 15 or 20M CuCl

2) were used to prepare microspheres with antimicrobial

activity against Escherichia coli and Candida albicans both frequently occurring pathogens which cause vaginal infections Themicroparticles were prepared by an ionotropic gelation technique which offers the unique possibility to entrap divalent copperions in a CMC structure and thus ensure their antibacterial activity Prepared CMC microspheres exhibited sufficient sphericityBoth equivalent diameter and copper content were influenced by CMC concentration and the molarity of copper (II) solutionaffected only the copper content results Selected samples exhibited stable but pH-responsive behaviour in environments whichcorresponded with natural (pH 45) and inflamed (pH 60) vaginal conditions All the tested samples exhibited proven substantialantimicrobial activity against both Gram-negative bacteria Escherichia coli and yeast Candida albicans Unexpectedly a crucialparameter for microsphere antimicrobial activity was not found in the copper content but in the swelling capacity of themicroparticles and in the degree of CMC surface shrinking

1 Introduction

Carmellose (CMC) is a water-soluble anionic polysaccharideand semisynthetic derivative of cellulose [1] which has noharmful effects on human health Its chains are linear 120573(1 rarr4)-linked glucopyranose units In addition it contains ahydrophobic polysaccharide backbone andmany hydrophiliccarboxyl groups and as a result exhibits amphiphilic charac-teristics CMC is used in a number of applications throughoutthe food cosmetic textile paper and ceramic industries as aviscosity modifier thickener emulsion stabilizer and waterretention and adhesive agent [2] It also has tremendous

potential for use in pharmaceutical products including site-specific or controlled release drug delivery carrier matricesthanks to its high biocompatibility biodegradability [3] andlow immunogenicity [4] In spite of the numerous positiveproperties ofCMC it does not have any antimicrobial proper-ties as it lacks antimicrobial functional groups [5] To bestowCMC with antimicrobial properties different possibilitieshave been proposed such as (i) adding different antimicrobialagents for example potassium sorbate [6] or silver nitrate [7](ii) grafting CMCwith different antimicrobial substances forexample guanidine hydrochloride [8] (iii) combining CMCwith other polymers that exhibit antimicrobial properties

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 790720 9 pageshttpdxdoiorg1011552015790720

2 BioMed Research International

for example carboxymethyl chitosan [9 10] or (iv) preparingnanoparticles of certainmetals for example with silver [7] orcopper [11] and incorporating them into the CMC structure

Copper is one of the most abundant trace elements foundin the human body It is an essential nutrient involved incatalyzing biochemical reactions However excessive copperlevels can be toxic mainly because they cause oxidativedamage to the body Copper can change its redox status byaccepting and donating electrons shifting between cuprous(Cu+) and cupric (Cu2+) forms and therefore participatein reactions that generate superoxide radicals and hydrogenperoxide which are major reactive oxygen species in thebody That is why a systemic usage of copper is limitedin the human body with exception of rheumatoid arthritistreatment [12ndash14] New options for copper utilization canbe found in its local effect via vaginal application of dosageforms based on copper ions for their strong antibacterialand spermicidal andor spermiostatic effect The vaginaproduces fluid at a rate of 3-4 g4 h [15] which can reducethe potent local toxic effect of the copper compounds Mucusefficiently traps foreign particles and particulates by bothsteric and adhesive mechanisms facilitating rapid clearance[16]Daunter used copper ethylenediaminetetraacetic acidL-ascorbic acid as a fertilization-preventing agent that can bedelivered via nonbiodegradable polyurethane or polyvinylacetate vaginal discs [17]

An aqueous CMC solution can undergo a sol-gel trans-formation in the presence of cross-linking cations and thusallow for the creation of solid gel microparticlesThismethodis known as an ionotropic gelation technique and is basedon the impact of physical (electrostatic) forces and polyelec-trolyte complexation with the presence of polyvalent ions[18] In general divalent cations (eg Ba2+ Sr2+ Cd2+ Co2+Ca2+ Mn2+ Ni2+ Pb2+ and Zn2+) are suitable cross-linkingagents for this method [19] Thus ionotropic gelation offersa unique possibility to incorporate divalent copper ions intothe CMC structure and form microspheres with potentialantimicrobial properties

The aim of the presented research was to prepare Cu2+cross-linkedCMCmicrospheres using external ionic gelationand assess their antimicrobial activity against Gram-negativeEscherichia coli and Candida albicans yeast both frequentlyoccurring pathogens responsible for vaginal infections

2 Materials and Methods

21 Materials Blanose-carmellose (CMC) with a mediumviscosity grade (1500ndash3100mPasdots for 2 in water) and a DSof 12 was used as the polymer carrier (Ashland CovingtonUSA) copper (II) chloride was used as a cross-linking agent(Sigma Aldrich St Louis USA) and HNO

3(65 vv) and

H2O2(30 vv) used for the determination of copper content

were purchased from Analytika (Prague Czech Republic)Calibration solutions were prepared using a dilution of1000mgL stock copper reference solvent (Analytika PragueCzech Republic) Deionised water with a resistivity of 18MΩwas used for all necessary dilution

Table 1 Variables during preparation of microparticles samples

Sample CMCconcentration ()

Molarity of CuCl2(moldm3)

MP-1 05 1 05MP-1 10 1 10MP-1 15 1 15MP-1 20 1 20MP-2 05 2 05MP-2 10 2 10MP-2 15 2 15MP-2 20 2 20

22 Methods

221 Microparticles Preparation Copper cross-linked CMCmicroparticles were prepared by a method of external ionicgelation CMC dispersions (1 and 2) were prepared bydispersing 1 g and 2 g of CMC in purified water respectivelyThe dispersions were heated to 80∘C to increase the rate ofswellingThey were then homogenized using anUltra-Turrax(T25 basic IKA-Werke Staufen Germany) at 13000 rpm for5minThe volume of each dispersion was ultimately adjustedto 100mL with purified water The resulting dispersions werethen extruded through a needle with an internal diameter of04mm at a dropping rate of 20mLmin into 50mL of 0510 15 or 20M CuCl

2aqueous solution respectively The

distance between the edge of the needle and the surface of thesolution was adjusted to 50 cm Microparticle formation wasinstantaneous and the particles were left in the cross-linkingsolution for 1 hour to harden while being gently mixed Theresulting beads were subsequently washed three times withpurified water and dried at 25∘C in a cabinet drier (HORO ndash048B Dr Hofmann GmbH Ostfildern Germany) for 24 hrsbefore testing Prepared samples were named in accordancewith the type and values of the altered variables (CMCconcentration and molarity of the cross-linking medium)Samples characteristics are shown in Table 1

222 Viscosity Measurement To investigate the influence ofthe viscosity on the characteristics of the prepared micropar-ticles rheological measurements of CMC dispersions wereperformed Dispersions (10 and 20 wt) were preparedand then homogenized with Ultra-Turrax (T25 BASIC IKA-Werke GmbH amp Co KG Staufen Germany) at 16000 rpmDynamic viscosity was measured by a Brookfield DV-II+Pro rotary viscometer (Brookfield Engineering Middle-boro USA) and Rheocalc software (Brookfield EngineeringMiddleboro USA) at 37∘C and 200 rpm A small sampleadapter was employed for this step Each sample was mea-sured three times and the results were expressed as meanvalues with standard deviations (SD)

223 Scanning Electron Microscopy In order to observemicroparticle morphology and surface topography scanningelectron microscopy (SEM) was employedThe samples were

BioMed Research International 3

mounted directly onto the SEM sample holder using double-sided sticking tape and then coated with a 10 nm thick layerof Au Images were taken using theMIRA3 scanning electronmicroscope (Tescan Brno CzechRepublic) at an acceleratingvoltage of 50 kV

224 Optical Microscope Analysis Particle size of the coppermicroparticles was measured using a NIKON SMZ 1500stereoscopic microscope (Nikon Tokyo Japan) equippedwith a 72AUC02USB camera (The Imaging Source Bre-men Germany) Microparticles were visualized under times15magnification The obtained images of 200 randomly chosenmicroparticles were stored and subsequently processed usingthe NIS-Elements AR 40 computer software (Nikon TokyoJapan)

Equivalent diameter (ED) and sphericity factor (SF)were calculated from the measured values and expressed asarithmetic mean plusmn standard deviation

225 Copper Content in Microparticles The copper contentin the prepared microparticles was determined by atomicabsorption To digest the microparticles 6mL of concen-trated nitric acid (65 vv) and 2mL of hydrogen peroxide(30 vv) were added to 10mg of every sample and placedin a TFM digestion vessel The vessels were closed andplaced into the segment and the content was mineralizedusing an Ethos SEL Microwave Labstation (Milestone Italy)at 220∘C for 35min applying a maximal power of 1000WThe microwave programme was started by steadily increas-ing the temperature over 15min followed by holding thetemperature for an additional 20min After cooling eachresulting solution was transferred to 50mL glass flasks andfilled to the mark with deionized water Samples were dilutedto 1 19 with deionized water prior to further analysis Coppercontent was measured using air-acetylene flame atomizationin a contrAA 700 atomic absorption spectrometer (AnalytikJena Germany) All samples were measured in triplicate andthe obtained values were processed by Aspect CS softwareversion 21

226 Swelling Capacity To determine swelling capacity apreviously reported method was improved [20] The testwas performed in a medium that properly simulates vaginalconditions 100mg of each sample was put into fine meshbaskets and immersed in a pH 45 medium (natural vaginalenvironmentmdash680 g of potassium dihydrogen phosphate Rin 10000mL of water R) and a pH 60 medium (infectedvaginal environmentmdash68 g of sodiumdihydrogen phosphateR in 10000mLofwater R pH adjustmentwith strong sodiumhydroxide solution R) The baskets were pulled out at timeintervals of 5 10 15 30 and 45min and 1 2 3 4 5 and 6hours after the first immersion properly dried and weighedSwelling capacity was calculated using the following equation[21]

119878SW = (119882119905minus1198820

1198820

) times 100 () (1)

119878SW is swelling capacity expressed as a percentage of weightaddition 119882

119905is the weight of a sample at the relevant time

interval and 1198820represents the initial weight of the sample

For each batch the measurement was performed three timesand results were expressed as mean values with standarddeviations

227 Antimicrobial Activity Bacterial Strains andMIC Deter-mination E coli (CCM4517) wasmaintained in a blood agarTo determine minimal inhibition concentration (MIC) anovernight culture of E coli cells was suspended in a fresh LB(Luria broth) medium and grown to OD

60005 at 37∘C at a

shaking speed of 250 rpmCandida albicans (CCM 8186) was maintained in an

YNB (Yeast Nitrogen Base with ammonium sulfate) mediumat 37∘C For MIC determination an overnight culture wassuspended in a fresh YNB medium and grown to OD

60005

at 37∘C at a shaking speed of 250 rpmA suitable amount of CMCmicroparticles was suspended

in the growth media (LB medium for E coli YNB for C albi-cans) to prepare a 10 suspension and incubated for 60minat room temperature to release the copper into solutionUndissolved particles were then separated by centrifugationand discarded and atomic absorption was used to determinecopper concentration Twofold dilutions of copper suspen-sion were prepared in appropriate mediums in a microtiterplate (100 120583Lwell in triplicate) Then 10e6 cells (E coli orC albicans) were added to each well of the microtiter plateand incubated at 37∘C Tomonitor cell growth optical densitywas measured spectrophotometrically (OD

600) after 20 hrs of

incubation The mean of the three wells was calculated toevaluate antimicrobial activity Wells containing the E colior C albicans without copper inhibition were included intriplicate in all tested plates as controls The MIC value wasexpressed as the concentration of copper in a well showing atleast a fourfold reduction of OD

600absorbance compared to

each subsequent well

3 Results and Discussion

The copper cross-linked carmellosemicroparticles were eval-uated for particle size sphericity factor and copper content(Table 2) The equivalent diameter of the prepared particlesranged from 7381 plusmn 303 to 1078 plusmn 124 120583m It seems thatparticle size did not depend on the concentration of theCu2+ hardening solution However it was observed that itdid increase with increasing CMC concentration which canbe attributed to the increased viscosity [22 23] Viscosity ofthe 1 CMC dispersion was found to be 162 plusmn 021mPasdotsand that of the 2 CMC dispersion 76667 plusmn 047mPasdotsA polymer dispersion with higher viscosity is more difficultto form into smaller droplets [24] thus these dispersionsyielded larger particles Another influence on the particle sizeof microspheres could be seen in the degree of CMC chainshrinkage Generally particle size decreases inversely withthe degree of shrinkage It has been well documented thatthe degree of shrinkage is typically higher for beads preparedfrom a dispersion with lower polymer concentration [25 26]

4 BioMed Research International

(a) (b)

(c) (d)

Figure 1 SEM photographs of surface topography of CMCmicroparticles (a) MP-1 05 (magnification 4330x) (b) surface detail of MP-1 05(magnification 9890x) (c) MP-2 05 (magnification 4330x) and (d) surface detail of MP-2 05 (magnification 9890x)

0

50

100

150

200

250

300

350

400

0 5 10 15 20

Swel

ling

capa

city

()

Time (hrs)MP-1 05 (SD max 2411) MP-1 10 (SD max 1253)MP-2 05 (SD max 1410) MP-2 10 (SD max 630)

Figure 2 Degree of swelling in Cu2+ cross-linked microparticles atpH 45

A comparison of the microparticle surfaces (MP-1 05 ED =7757120583m versus MP-2 05 ED = 10169) in Figure 1 confirmsthese findings From these SEMphotographs it is evident thatthe degree of CMC shrinkage was significantly higher for thesmaller 1 CMCmicrospheres

0

1000

2000

3000

4000

5000

6000

0 5 10 15 20

Swel

ling

capa

city

()

Time (hrs)

MP-1 05 (SD max 13837) MP-1 10 (SD max 2476)MP-2 05 (SD max 7730) MP-2 10 (SD max 4300)

Figure 3 Degree of swelling in Cu2+ cross-linked microparticles atpH 60

PreparedCMCmicrospheres exhibited sufficient spheric-ity ranging from 0850 plusmn 0067 to 0934 plusmn 0038 Previousstudies indicate that particles with this parameter value above08 are considered to have good sphericity [27] It can beobserved from the results that the sphericity was not clearly

BioMed Research International 5

(a) (b)

(c) (d)

Figure 4 Optical microscope images of CMC particles after 20-hour swelling capacity test in pH 60 buffer bars correspond to 1000120583m (a)MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

Table 2 Microparticle characteristics equivalent diameter sphericity factor and copper content

Sample ED (120583m) SD (120583m) SF SD Copper content (gkg) SD (gkg)MP-1 05 7757 302 0906 0044 1013 065MP-1 10 8885 584 0934 0038 1436 231MP-1 15 7381 303 0887 0056 1523 019MP-1 20 7986 879 0855 0059 1730 215MP-2 05 10169 228 0891 0040 1436 026MP-2 10 10780 124 0886 0071 1647 040MP-2 15 9221 576 0850 0067 1876 132MP-2 20 9336 386 0874 0057 2003 103

influenced by increasing the concentration of the hardeningsolution but it was noticed that hardening solutions withlower molarity of CuCl

2(05 and 10) yielded microspheres

with higher sphericity values On the other hand particlesphericity was lower in samples prepared with a higherpolymer concentration with the exception of samples MP-1 20 and MP-2 20 which were prepared with 2M of CuCl

2

hardening solution Previously reported results confirmedthat increasing the concentration of CMC solutions lin-early increases the solution viscosity [28] and sphericity ofparticles Our results generally coincide with findings thatoverly viscous polymer solutions (2 in our case) form lessspherical tail-shaped particles [29] and particles with a roughsurface [30]

Table 2 also shows the results of atomic absorptionanalysis for copper content Copper content in the preparedmicroparticles ranged from 1013 plusmn 065 to 2003 plusmn 103 gkgIt was observed that the content significantly increased with

increases in hardening solution concentration as well aspolymer concentration

The samples of microspheres with the highest sphericity(MP-1 05 MP-1 10 MP-2 05 and MP-2 10) which is onethe most important criteria for the preparation of particlesystems were then investigated for swelling capacity in phos-phate buffers with pH 45 [31] and pH 60 [32] respectivelyto simulate natural and inflamed vaginal conditions and topredict their behaviour on vaginal mucosa for a 20-hourtime interval (20-hour interval represents an estimated timeof therapy) The obtained results can be seen in Figure 2for pH 45 and in Figure 3 for pH 60 Figure 4 showsimages of microparticles after a 20-hour swelling capacitytest in a pH 60 buffer Generally it was observed that theswelling capacity of all samples gradually increased duringthe test Selected samples exhibited pH-responsive behaviourwhich differed based on the tested environments swellingwas substantially higher at pH 60 [33] It is obvious from

6 BioMed Research International

00

01

02

03

04

05

06

07

08

09

0369 0184 0092 0046 0023 0012

A

c (mgmL)

(a)

00

01

02

03

04

05

06

07

08

09

3871 1936 0968 0484 0242 0121

A

c (mgmL)

(b)

00

01

02

03

04

05

06

07

08

09

0707 0353 0177 0088 0044 0022

A

c (mgmL)

20hrs

(c)

00

01

02

03

04

05

06

07

08

09

4148 2074 1037 0519 0259 0130

20hrs

A

c (mgmL)

(d)

Figure 5 MIC results of CMCmicroparticles against Escherichia coli (a) MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

Figures 2 and 3 that sample MP-1 05 prepared with a less-concentrated hardening solution (05M) and a lower CMCconcentration (1) had the highest swelling capacity at bothpH values (3662 at pH 45 after 20 hrs 52963 at pH60 after 20 hrs) These values of formulation variables ledto the creation of favourable conditions for water uptakeinto the microsphere matrix resulting in the creation of anamorphous gel structure at pH 60 (see Figure 4(a)) Also theenormous loss of the blue colour in comparison with sampleMP-2 10 (Figure 4(d)) can be noticed probably related tohigh water uptake and higher release of the copper ionswhich are responsible for the blue colour This is uniquelyin accordance with previously published data saying thatincreased polymer or hardening solution concentrations cansignificantly reduce water uptake due to the increased densityof the polymer network [34] At pH 60 sample MP-2 05followed with swelling capacity of 24360 in 20 hrs At pH45 however its swelling was comparable with samples cross-linked with 1M CuCl

2 which exhibited a lower swelling

capacity ranging from 1372 to 1607 after 20 hrs at pH 45and 4330ndash5391 at pH 60 (results for pH 60 are clearlyevident in Figures 4(b) and 4(d)) No influence on swellingcapacity as a result of particle size was observed

The mechanism for the antibacterial activity of Cu+ions is based on their energetically easier movement acrossa lipid bilayer and uptake by the cell generating reactiveoxygen species leading to lipid peroxidation and proteinoxidation [35] The excess of copper causes a decrease in themembrane integrity of a microorganism which causes theparticular cell to leak nutritional elements like potassiumand glutamate which lead to desiccation and ultimatelycell death [36] The antimicrobial activity of selected CMCmicroparticles cross-linked by Cu2+ was evaluated by MICtest using Gram-negative Escherichia coli and Candida albi-cans yeast (see Figures 5 and 6) The test expresses theminimum concentration of antimicrobial agent that inhibitsthe visible growth of microorganisms [37] The test wastriplicated For E coli MIC values after 20 hrs of incubationwere 0184 0484 0353 and 0519mgmL for samples MP-1 05 MP-1 10 MP-2 05 and MP-2 10 respectively MICvalues for C albicans were higher for the same samples0740 1639 0598 and 1087mgmL respectively HigherMIC values for C albicans in comparison with the valuesfor E coli can be explained by the higher resistance of theyeast to the copperrsquos antibacterial effect Weissman et alconfirm this reporting on the isolation of two genes involved

BioMed Research International 7

00

01

02

03

04

05

06

07

08

09

10

0740 0370 0185 0093 0046 0023

A

c (mgmL)

(a)

00

01

02

03

04

05

06

07

08

09

10

3277 1639 0819 0410 0205 0102

A

c (mgmL)

(b)

0001020304050607080910

0598 0299 0150 0075 0037 0019

20hrs

A

c (mgmL)

(c)

0001020304050607080910

2173 1087 0543 0272 0136 0068

A

20hrs

c (mgmL)

(d)

Figure 6 MIC results of CMC microparticles against Candida albicans (a) MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

in copper detoxification in C albicans metallothioneinCaCUP1 and a copper-transporting P-type ATPase CaCRP1[38] These two genes account for C albicansrsquos resistanceto copper and consequently the vast differences in ourresults

The obtained MIC values prove substantial antimicrobialactivity against both microorganisms tested in this study andare in good agreementwith copperMIC values against strainsof E coli and C albicans described by different authors inother studies Martınez Medina et al reported MIC valuesfor Cu(II) complexes of 0375mgmL and gt15mgmL for Ecoli and C albicans respectively [39] Copper nanoparticlesprepared by Zain et al (200 nm) showed MIC values of0433mgL for E coli [40] and those prepared by Rupareliaet al (9 nm) showed MIC values of 0280mgmL for E coli[41]

Despite the lower content of copper in microspheresthe samples prepared using the less-concentrated CuCl

2

(05M) solutionmdashMP-1 05 and MP-2 05mdashexhibited lowerMIC values and so better antibacterial activity against bothtested pathogens in comparison with samples cross-linked in

the more potent 1M CuCl2solutionThese results confirmed

the great influence of the microparticle swellability on MICvalues Both samples exhibited high swelling capacity at pH60 (see Figure 3) and thus good conditions for the releaseof copper ions from particles (pH of LB and YNB mediumwas also close to 60) Against E coli sample MP-1 05 wasfound to be the most effective as it probably exhibited fastercopper releaseThis could be a result of the higher cumulationof copper ions on more rougher surface (see Figures 1(a)and 1(b)) and also smaller particle size (see Table 2) and thuslarger surface area [42] These characteristics could promotefaster swelling leading to the creation of an amorphousstructure during the 20-hour interval (Figure 4(a)) Againstthe more resistant C albicans however the most effectivesample was MP-2 05 exhibiting probably more uniformcopper release due to the higher CMC concentraction (2)more uniform copper distribution in the microspheres andlarger particle size (see Table 2) This theory is also sup-ported by the fact that the MP-2 05 microparticles main-tained their shape-specific structure after 20 hrs of swelling(Figure 4(c))

8 BioMed Research International

4 Conclusion

In this experimental work CMC dispersions of two differentconcentrations (1 and 2) were cross-linked by copper (II)ions (05 1 15 or 20MCuCl

2) to prepare microspheres with

antimicrobial activity against frequently occurring vaginalpathogens Escherichia coli and Candida albicans All testedsamples exhibited stable but pH responsive behaviour inenvironments corresponding with natural and inflamed vagi-nal conditions and proved substantial antimicrobial activityagainst both pathogens The most effective samples werethose hardened in a less-concentrated CuCl

2(05M) solu-

tion Unexpectedly a crucial parameter for microsphereantimicrobial activity was not found in the copper contentbut in the swelling capacity of the microparticles and in thedegree of CMC surface shrinkingThe sample prepared usinga 1 CMC dispersion cross-linked by 05M CuCl

2seemed to

be the most suitable for potential vaginal use not only due toits antibacterial activity but also due to its gradual change to anonspecific-shaped gel which is preferable when consideringvaginal dosage forms

Conflict of Interests

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

References

[1] L Fan M Peng Z Zhou et al ldquoModification of carboxymethylcellulose Grafted with collagen peptide and its antioxidantaktivityrdquo Carbohydrate Polymers vol 112 pp 32ndash38 2014

[2] N HaleemM Arsah M Shahid and AM Tahir ldquoSynthesis ofcarboxymethyl cellulose fromwaste of cotton ginning industryrdquoCarbohydrate Polymers vol 113 pp 249ndash255 2014

[3] J-F Su Z Huang X-Y Yuan X-YWang andM Li ldquoStructureand properties of carboxymethyl cellulosesoy protein isolateblend edible films crosslinked by Maillard reactionsrdquo Carbohy-drate Polymers vol 79 no 1 pp 145ndash153 2010

[4] H Kono ldquoCharacterization and properties of carboxymethylcellulose hydrogels crosslinked by polyethylene glycolrdquo Carbo-hydrate Polymers vol 106 no 1 pp 84ndash93 2014

[5] E A Hassn M L Hassan N C Moorefield and GR Newkome ldquoNew supramolecular metallo-terpyridine car-boxymethyl cellulose derivativeswith antimicrobial propertiesrdquoCarbohydrate Polymers vol 116 pp 2ndash8 2014

[6] S Sayanjali B Ghanbarzadeh and S Ghiassifar ldquoEvaluation ofantimicrobial and physical properties of edible film based oncarboxymethyl cellulose containing potassium sorbate on somemycotoxigenic Aspergillus species in fresh pistachiosrdquo LWTmdashFood Science and Technology vol 44 no 4 pp 1133ndash1138 2011

[7] A A Hebeish M H El-Rafie F A Abdel-Mohdy E S Abdel-Halim and H E Emam ldquoCarboxymethyl cellulose for greensynthesis and stabilization of silver nanoparticlesrdquo Carbohy-drate Polymers vol 82 no 3 pp 933ndash941 2010

[8] K Liu Y Xu X Lin et al ldquoSynergistic effects of guanidine-grafted CMC on enhancing antimicrobial activity and drystrength of paperrdquo Carbohydrate Polymers vol 110 pp 382ndash3872014

[9] L Upadhyaya J Singh V Agarwal and R P Tewari ldquoBiomed-ical applications of carboxymethyl chitosansrdquo CarbohydratePolymers vol 91 no 1 pp 452ndash466 2013

[10] A-H Yu H-Y Hsieh J-C Pang et al ldquoActive films fromwater-soluble chitosancellulose composites incorporating releasablecaffeic acid for inhibition of lipid oxidation in fish oil emul-sionsrdquo Food Hydrocolloids vol 32 no 1 pp 9ndash19 2013

[11] T Zhong G S Oporto J Jaczynski A T Tesfai and JArmstrong ldquoAntimicrobial properties of the hybrid coppernanoparticles-carboxymethyl celluloserdquo Wood and Fiber Sci-ence vol 45 no 2 pp 215ndash222 2013

[12] G E Jackson P M May and D R Williams ldquoMetal-ligandcomplexes involved in rheumatoid arthritis-I justificationsfor copper administrationrdquo Journal of Inorganic and NuclearChemistry vol 40 no 6 pp 1189ndash1194 1978

[13] O A El-Gammal E A Elmorsy and Y E Sherif ldquoEvaluationof the anti-inflammatory and analgesic effects of Cu(II) andZn(II) complexes derived from 2-(naphthalen-1-yloxy)-119873

1015840

-(1-(pyridin-2-1) ethylidene) acetohydraziderdquo Spectrochimica ActaPart A Molecular and Biomolecular Spectroscopy vol 120 pp332ndash339 2014

[14] S Odisitse and G E Jackson ldquoIn vitro and in vivo studiesof NN1015840-bis[2(2-pyridyl)-methyl]pyridine-26-dicarboxamide-copper(II) and rheumatoid arthritisrdquo Polyhedron vol 27 no 1pp 453ndash464 2008

[15] A Hussain and F Ahsan ldquoThe vagina as a route for systemicdrug deliveryrdquo Journal of Controlled Release vol 103 no 2 pp301ndash313 2005

[16] L M Ensign T E Hoen K Maisel R A Cone and J S HanesldquoEnhanced vaginal drug delivery through the use of hypotonicformulations that induce fluid uptakerdquo Biomaterials vol 34 no28 pp 6922ndash6929 2013

[17] B B Saxena M Singh R M Gospin C C Chu and W JLedger ldquoEfficacy of nonhormonal vaginal contraceptives from ahydrogel delivery systemrdquo Contraception vol 70 no 3 pp 213ndash219 2004

[18] M L Gonzalez-Rodrıguez M A Holgado C Sanchez-Lafuente A M Rabasco and A Fini ldquoAlginatechitosan par-ticulate systems for sodium diclofenac releaserdquo InternationalJournal of Pharmaceutics vol 232 no 1-2 pp 225ndash234 2002

[19] C H Goh P W S Heng and L W Chan ldquoAlginates as auseful natural polymer for microencapsulation and therapeuticapplicationsrdquo Carbohydrate Polymers vol 88 no 1 pp 1ndash122012

[20] W-C Lin D-G Yu and M-C Yang ldquopH-sensitive poly-electrolyte complex gel microspheres composed of chitosansodium tripolyphosphatedextran sulfate swelling kinetics anddrug delivery propertiesrdquo Colloids and Surfaces B Biointerfacesvol 44 no 2-3 pp 143ndash151 2005

[21] K G H Desai and H J Park ldquoPreparation and characteriza-tion of drug-loaded chitosan-tripolyphosphatemicrospheres byspray dryingrdquo Drug Development Research vol 64 no 2 pp114ndash128 2005

[22] K M Rao B Mallikarjuna K K Rao M N Prabhakar K CRao and M C Subha ldquoPreparation and characterization of pHsensitive poly(vinyl alcohol)sodium carboxymethyl celluloseIPN microspheres for in vitro release studies of an anti-cancerdrugrdquo Polymer Bulletin vol 68 no 7 pp 1905ndash1919 2012

[23] M A Saleem Y D Murali M D Naheed P Jaydeep andM Dhaval ldquoPrepapation and evaluation of valsartan loadedhydrogel beadsrdquo International Research Journal of Pharmacyvol 3 no 6 pp 80ndash85 2012

BioMed Research International 9

[24] Y Y Yang J P Wan T S Chung P K Pallathadka S Ng andJ Heller ldquoPOE-PEG-POE triblock copolymeric microspherescontaining protein I Preparation and characterizationrdquo Journalof Controlled Release vol 75 no 1-2 pp 115ndash128 2001

[25] H Brandenberger and F Widmer ldquoA new multinozzle encap-sulationimmobilisation system to produce uniform beads ofalginaterdquo Journal of Biotechnology vol 63 no 1 pp 73ndash80 1998

[26] E-S Chan B-B Lee P Ravindra and D Poncelet ldquoPredictionmodels for shape and size of ca-alginate macrobeads producedthrough extrusion-dripping methodrdquo Journal of Colloid andInterface Science vol 338 no 1 pp 63ndash72 2009

[27] P B Deasy and M F L Law ldquoUse of extrusion-spheronizationto develop an improved oral dosage form of indomethacinrdquoInternational Journal of Pharmaceutics vol 148 no 2 pp 201ndash209 1997

[28] X H Yang and W L Zhu ldquoViscosity properties of sodiumcarboxymethylcellulose solutionsrdquo Cellulose vol 14 no 5 pp409ndash417 2007

[29] E-S Chan ldquoPreparation of Ca-alginate beads containing highoil content influence of process variables on encapsulationefficiency and bead propertiesrdquo Carbohydrate Polymers vol 84no 4 pp 1267ndash1275 2011

[30] M S Kim S J Park B K Gu and C-H Kim ldquoIonicallycrosslinked alginate-carboxymethyl cellulose beads for thedelivery of protein therapeuticsrdquo Applied Surface Science vol262 pp 28ndash33 2012

[31] C Valenta ldquoThe use of mucoadhesive polymers in vaginaldeliveryrdquo Advanced Drug Delivery Reviews vol 57 no 11 pp1692ndash1712 2005

[32] S F Borges J G L Silva and P C M Teixeira ldquoSurvival andbiofilm formation of Listeria monocytogenes in simulated vagi-nal fluid influence of pH and strain originrdquo FEMS Immunologyand Medical Microbiology vol 62 no 3 pp 315ndash320 2011

[33] R Barbucci A Magnani and M Consumi ldquoSwelling behav-ior of carboxymethylcellulose hydrogels in relation to cross-linking pH and charge densityrdquo Macromolecules vol 33 no20 pp 7475ndash7480 2000

[34] J Berger M Reist J M Mayer O Felt N A Peppas and RGurny ldquoStructure and interactions in covalently and ionicallycrosslinked chitosan hydrogels for biomedical applicationsrdquoEuropean Journal of Pharmaceutics and Biopharmaceutics vol57 no 1 pp 19ndash34 2004

[35] U Bogdanovic V Lazic V Vodnik M Budimir Z Markovicand S Dimitrijevic ldquoCopper nanoparticles with high antimi-crobial activityrdquoMaterials Letters vol 128 pp 75ndash78 2014

[36] J Konieczny and Z Rdzawski ldquoAntibacterial properties of cop-per and its alloysrdquoArchives ofMaterials Science and Engineeringvol 56 no 2 pp 53ndash60 2012

[37] S M Magana P Quintana D H Aguilar et al ldquoAntibacterialactivity of montmorillonites modified with silverrdquo Journal ofMolecular Catalysis A Chemical vol 281 no 1-2 pp 192ndash1992008

[38] Z Weissman I Berdicevsky B-Z Cavari and D KornitzerldquoThe high copper tolerance ofCandida albicans is mediated by aP-type ATPaserdquo Proceedings of the National Academy of Sciencesof the United States of America vol 97 no 7 pp 3520ndash35252000

[39] J J Martınez Medina M S Islas L L Lopez Tevez E GFerrer N B Okulik and P A M Williams ldquoCopper(II) com-plexes with cyanoguanidine and o-phenanthroline theoreticalstudies in vitro antimicrobial activity and alkaline phosphatase

inhibitory effectrdquo Journal of Molecular Structure vol 1058 no1 pp 298ndash307 2014

[40] N M Zain A G F Stapley and G Shama ldquoGreen synthesis ofsilver and copper nanoparticles using ascorbic acid and chitosanfor antimicrobial applicationsrdquo Carbohydrate Polymers vol 112pp 195ndash202 2014

[41] J P Ruparelia A K Chatterjee S P Duttagupta and SMukherji ldquoStrain specificity in antimicrobial activity of silverand copper nanoparticlesrdquo Acta Biomaterialia vol 4 no 3 pp707ndash716 2008

[42] J Zhao H J Feng H Q Tang and J H Zheng ldquoBactericidaland corrosive properties of silver implanted TiN thin filmscoated on AISI317 stainless steelrdquo Surface and Coatings Technol-ogy vol 201 no 9ndash11 pp 5676ndash5679 2007

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MEDIATORSINFLAMMATION

of

Page 2: Research Article Antimicrobial Properties of ...downloads.hindawi.com/journals/bmri/2015/790720.pdf · Research Article Antimicrobial Properties of Microparticles Based on ... ect

2 BioMed Research International

for example carboxymethyl chitosan [9 10] or (iv) preparingnanoparticles of certainmetals for example with silver [7] orcopper [11] and incorporating them into the CMC structure

Copper is one of the most abundant trace elements foundin the human body It is an essential nutrient involved incatalyzing biochemical reactions However excessive copperlevels can be toxic mainly because they cause oxidativedamage to the body Copper can change its redox status byaccepting and donating electrons shifting between cuprous(Cu+) and cupric (Cu2+) forms and therefore participatein reactions that generate superoxide radicals and hydrogenperoxide which are major reactive oxygen species in thebody That is why a systemic usage of copper is limitedin the human body with exception of rheumatoid arthritistreatment [12ndash14] New options for copper utilization canbe found in its local effect via vaginal application of dosageforms based on copper ions for their strong antibacterialand spermicidal andor spermiostatic effect The vaginaproduces fluid at a rate of 3-4 g4 h [15] which can reducethe potent local toxic effect of the copper compounds Mucusefficiently traps foreign particles and particulates by bothsteric and adhesive mechanisms facilitating rapid clearance[16]Daunter used copper ethylenediaminetetraacetic acidL-ascorbic acid as a fertilization-preventing agent that can bedelivered via nonbiodegradable polyurethane or polyvinylacetate vaginal discs [17]

An aqueous CMC solution can undergo a sol-gel trans-formation in the presence of cross-linking cations and thusallow for the creation of solid gel microparticlesThismethodis known as an ionotropic gelation technique and is basedon the impact of physical (electrostatic) forces and polyelec-trolyte complexation with the presence of polyvalent ions[18] In general divalent cations (eg Ba2+ Sr2+ Cd2+ Co2+Ca2+ Mn2+ Ni2+ Pb2+ and Zn2+) are suitable cross-linkingagents for this method [19] Thus ionotropic gelation offersa unique possibility to incorporate divalent copper ions intothe CMC structure and form microspheres with potentialantimicrobial properties

The aim of the presented research was to prepare Cu2+cross-linkedCMCmicrospheres using external ionic gelationand assess their antimicrobial activity against Gram-negativeEscherichia coli and Candida albicans yeast both frequentlyoccurring pathogens responsible for vaginal infections

2 Materials and Methods

21 Materials Blanose-carmellose (CMC) with a mediumviscosity grade (1500ndash3100mPasdots for 2 in water) and a DSof 12 was used as the polymer carrier (Ashland CovingtonUSA) copper (II) chloride was used as a cross-linking agent(Sigma Aldrich St Louis USA) and HNO

3(65 vv) and

H2O2(30 vv) used for the determination of copper content

were purchased from Analytika (Prague Czech Republic)Calibration solutions were prepared using a dilution of1000mgL stock copper reference solvent (Analytika PragueCzech Republic) Deionised water with a resistivity of 18MΩwas used for all necessary dilution

Table 1 Variables during preparation of microparticles samples

Sample CMCconcentration ()

Molarity of CuCl2(moldm3)

MP-1 05 1 05MP-1 10 1 10MP-1 15 1 15MP-1 20 1 20MP-2 05 2 05MP-2 10 2 10MP-2 15 2 15MP-2 20 2 20

22 Methods

221 Microparticles Preparation Copper cross-linked CMCmicroparticles were prepared by a method of external ionicgelation CMC dispersions (1 and 2) were prepared bydispersing 1 g and 2 g of CMC in purified water respectivelyThe dispersions were heated to 80∘C to increase the rate ofswellingThey were then homogenized using anUltra-Turrax(T25 basic IKA-Werke Staufen Germany) at 13000 rpm for5minThe volume of each dispersion was ultimately adjustedto 100mL with purified water The resulting dispersions werethen extruded through a needle with an internal diameter of04mm at a dropping rate of 20mLmin into 50mL of 0510 15 or 20M CuCl

2aqueous solution respectively The

distance between the edge of the needle and the surface of thesolution was adjusted to 50 cm Microparticle formation wasinstantaneous and the particles were left in the cross-linkingsolution for 1 hour to harden while being gently mixed Theresulting beads were subsequently washed three times withpurified water and dried at 25∘C in a cabinet drier (HORO ndash048B Dr Hofmann GmbH Ostfildern Germany) for 24 hrsbefore testing Prepared samples were named in accordancewith the type and values of the altered variables (CMCconcentration and molarity of the cross-linking medium)Samples characteristics are shown in Table 1

222 Viscosity Measurement To investigate the influence ofthe viscosity on the characteristics of the prepared micropar-ticles rheological measurements of CMC dispersions wereperformed Dispersions (10 and 20 wt) were preparedand then homogenized with Ultra-Turrax (T25 BASIC IKA-Werke GmbH amp Co KG Staufen Germany) at 16000 rpmDynamic viscosity was measured by a Brookfield DV-II+Pro rotary viscometer (Brookfield Engineering Middle-boro USA) and Rheocalc software (Brookfield EngineeringMiddleboro USA) at 37∘C and 200 rpm A small sampleadapter was employed for this step Each sample was mea-sured three times and the results were expressed as meanvalues with standard deviations (SD)

223 Scanning Electron Microscopy In order to observemicroparticle morphology and surface topography scanningelectron microscopy (SEM) was employedThe samples were

BioMed Research International 3

mounted directly onto the SEM sample holder using double-sided sticking tape and then coated with a 10 nm thick layerof Au Images were taken using theMIRA3 scanning electronmicroscope (Tescan Brno CzechRepublic) at an acceleratingvoltage of 50 kV

224 Optical Microscope Analysis Particle size of the coppermicroparticles was measured using a NIKON SMZ 1500stereoscopic microscope (Nikon Tokyo Japan) equippedwith a 72AUC02USB camera (The Imaging Source Bre-men Germany) Microparticles were visualized under times15magnification The obtained images of 200 randomly chosenmicroparticles were stored and subsequently processed usingthe NIS-Elements AR 40 computer software (Nikon TokyoJapan)

Equivalent diameter (ED) and sphericity factor (SF)were calculated from the measured values and expressed asarithmetic mean plusmn standard deviation

225 Copper Content in Microparticles The copper contentin the prepared microparticles was determined by atomicabsorption To digest the microparticles 6mL of concen-trated nitric acid (65 vv) and 2mL of hydrogen peroxide(30 vv) were added to 10mg of every sample and placedin a TFM digestion vessel The vessels were closed andplaced into the segment and the content was mineralizedusing an Ethos SEL Microwave Labstation (Milestone Italy)at 220∘C for 35min applying a maximal power of 1000WThe microwave programme was started by steadily increas-ing the temperature over 15min followed by holding thetemperature for an additional 20min After cooling eachresulting solution was transferred to 50mL glass flasks andfilled to the mark with deionized water Samples were dilutedto 1 19 with deionized water prior to further analysis Coppercontent was measured using air-acetylene flame atomizationin a contrAA 700 atomic absorption spectrometer (AnalytikJena Germany) All samples were measured in triplicate andthe obtained values were processed by Aspect CS softwareversion 21

226 Swelling Capacity To determine swelling capacity apreviously reported method was improved [20] The testwas performed in a medium that properly simulates vaginalconditions 100mg of each sample was put into fine meshbaskets and immersed in a pH 45 medium (natural vaginalenvironmentmdash680 g of potassium dihydrogen phosphate Rin 10000mL of water R) and a pH 60 medium (infectedvaginal environmentmdash68 g of sodiumdihydrogen phosphateR in 10000mLofwater R pH adjustmentwith strong sodiumhydroxide solution R) The baskets were pulled out at timeintervals of 5 10 15 30 and 45min and 1 2 3 4 5 and 6hours after the first immersion properly dried and weighedSwelling capacity was calculated using the following equation[21]

119878SW = (119882119905minus1198820

1198820

) times 100 () (1)

119878SW is swelling capacity expressed as a percentage of weightaddition 119882

119905is the weight of a sample at the relevant time

interval and 1198820represents the initial weight of the sample

For each batch the measurement was performed three timesand results were expressed as mean values with standarddeviations

227 Antimicrobial Activity Bacterial Strains andMIC Deter-mination E coli (CCM4517) wasmaintained in a blood agarTo determine minimal inhibition concentration (MIC) anovernight culture of E coli cells was suspended in a fresh LB(Luria broth) medium and grown to OD

60005 at 37∘C at a

shaking speed of 250 rpmCandida albicans (CCM 8186) was maintained in an

YNB (Yeast Nitrogen Base with ammonium sulfate) mediumat 37∘C For MIC determination an overnight culture wassuspended in a fresh YNB medium and grown to OD

60005

at 37∘C at a shaking speed of 250 rpmA suitable amount of CMCmicroparticles was suspended

in the growth media (LB medium for E coli YNB for C albi-cans) to prepare a 10 suspension and incubated for 60minat room temperature to release the copper into solutionUndissolved particles were then separated by centrifugationand discarded and atomic absorption was used to determinecopper concentration Twofold dilutions of copper suspen-sion were prepared in appropriate mediums in a microtiterplate (100 120583Lwell in triplicate) Then 10e6 cells (E coli orC albicans) were added to each well of the microtiter plateand incubated at 37∘C Tomonitor cell growth optical densitywas measured spectrophotometrically (OD

600) after 20 hrs of

incubation The mean of the three wells was calculated toevaluate antimicrobial activity Wells containing the E colior C albicans without copper inhibition were included intriplicate in all tested plates as controls The MIC value wasexpressed as the concentration of copper in a well showing atleast a fourfold reduction of OD

600absorbance compared to

each subsequent well

3 Results and Discussion

The copper cross-linked carmellosemicroparticles were eval-uated for particle size sphericity factor and copper content(Table 2) The equivalent diameter of the prepared particlesranged from 7381 plusmn 303 to 1078 plusmn 124 120583m It seems thatparticle size did not depend on the concentration of theCu2+ hardening solution However it was observed that itdid increase with increasing CMC concentration which canbe attributed to the increased viscosity [22 23] Viscosity ofthe 1 CMC dispersion was found to be 162 plusmn 021mPasdotsand that of the 2 CMC dispersion 76667 plusmn 047mPasdotsA polymer dispersion with higher viscosity is more difficultto form into smaller droplets [24] thus these dispersionsyielded larger particles Another influence on the particle sizeof microspheres could be seen in the degree of CMC chainshrinkage Generally particle size decreases inversely withthe degree of shrinkage It has been well documented thatthe degree of shrinkage is typically higher for beads preparedfrom a dispersion with lower polymer concentration [25 26]

4 BioMed Research International

(a) (b)

(c) (d)

Figure 1 SEM photographs of surface topography of CMCmicroparticles (a) MP-1 05 (magnification 4330x) (b) surface detail of MP-1 05(magnification 9890x) (c) MP-2 05 (magnification 4330x) and (d) surface detail of MP-2 05 (magnification 9890x)

0

50

100

150

200

250

300

350

400

0 5 10 15 20

Swel

ling

capa

city

()

Time (hrs)MP-1 05 (SD max 2411) MP-1 10 (SD max 1253)MP-2 05 (SD max 1410) MP-2 10 (SD max 630)

Figure 2 Degree of swelling in Cu2+ cross-linked microparticles atpH 45

A comparison of the microparticle surfaces (MP-1 05 ED =7757120583m versus MP-2 05 ED = 10169) in Figure 1 confirmsthese findings From these SEMphotographs it is evident thatthe degree of CMC shrinkage was significantly higher for thesmaller 1 CMCmicrospheres

0

1000

2000

3000

4000

5000

6000

0 5 10 15 20

Swel

ling

capa

city

()

Time (hrs)

MP-1 05 (SD max 13837) MP-1 10 (SD max 2476)MP-2 05 (SD max 7730) MP-2 10 (SD max 4300)

Figure 3 Degree of swelling in Cu2+ cross-linked microparticles atpH 60

PreparedCMCmicrospheres exhibited sufficient spheric-ity ranging from 0850 plusmn 0067 to 0934 plusmn 0038 Previousstudies indicate that particles with this parameter value above08 are considered to have good sphericity [27] It can beobserved from the results that the sphericity was not clearly

BioMed Research International 5

(a) (b)

(c) (d)

Figure 4 Optical microscope images of CMC particles after 20-hour swelling capacity test in pH 60 buffer bars correspond to 1000120583m (a)MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

Table 2 Microparticle characteristics equivalent diameter sphericity factor and copper content

Sample ED (120583m) SD (120583m) SF SD Copper content (gkg) SD (gkg)MP-1 05 7757 302 0906 0044 1013 065MP-1 10 8885 584 0934 0038 1436 231MP-1 15 7381 303 0887 0056 1523 019MP-1 20 7986 879 0855 0059 1730 215MP-2 05 10169 228 0891 0040 1436 026MP-2 10 10780 124 0886 0071 1647 040MP-2 15 9221 576 0850 0067 1876 132MP-2 20 9336 386 0874 0057 2003 103

influenced by increasing the concentration of the hardeningsolution but it was noticed that hardening solutions withlower molarity of CuCl

2(05 and 10) yielded microspheres

with higher sphericity values On the other hand particlesphericity was lower in samples prepared with a higherpolymer concentration with the exception of samples MP-1 20 and MP-2 20 which were prepared with 2M of CuCl

2

hardening solution Previously reported results confirmedthat increasing the concentration of CMC solutions lin-early increases the solution viscosity [28] and sphericity ofparticles Our results generally coincide with findings thatoverly viscous polymer solutions (2 in our case) form lessspherical tail-shaped particles [29] and particles with a roughsurface [30]

Table 2 also shows the results of atomic absorptionanalysis for copper content Copper content in the preparedmicroparticles ranged from 1013 plusmn 065 to 2003 plusmn 103 gkgIt was observed that the content significantly increased with

increases in hardening solution concentration as well aspolymer concentration

The samples of microspheres with the highest sphericity(MP-1 05 MP-1 10 MP-2 05 and MP-2 10) which is onethe most important criteria for the preparation of particlesystems were then investigated for swelling capacity in phos-phate buffers with pH 45 [31] and pH 60 [32] respectivelyto simulate natural and inflamed vaginal conditions and topredict their behaviour on vaginal mucosa for a 20-hourtime interval (20-hour interval represents an estimated timeof therapy) The obtained results can be seen in Figure 2for pH 45 and in Figure 3 for pH 60 Figure 4 showsimages of microparticles after a 20-hour swelling capacitytest in a pH 60 buffer Generally it was observed that theswelling capacity of all samples gradually increased duringthe test Selected samples exhibited pH-responsive behaviourwhich differed based on the tested environments swellingwas substantially higher at pH 60 [33] It is obvious from

6 BioMed Research International

00

01

02

03

04

05

06

07

08

09

0369 0184 0092 0046 0023 0012

A

c (mgmL)

(a)

00

01

02

03

04

05

06

07

08

09

3871 1936 0968 0484 0242 0121

A

c (mgmL)

(b)

00

01

02

03

04

05

06

07

08

09

0707 0353 0177 0088 0044 0022

A

c (mgmL)

20hrs

(c)

00

01

02

03

04

05

06

07

08

09

4148 2074 1037 0519 0259 0130

20hrs

A

c (mgmL)

(d)

Figure 5 MIC results of CMCmicroparticles against Escherichia coli (a) MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

Figures 2 and 3 that sample MP-1 05 prepared with a less-concentrated hardening solution (05M) and a lower CMCconcentration (1) had the highest swelling capacity at bothpH values (3662 at pH 45 after 20 hrs 52963 at pH60 after 20 hrs) These values of formulation variables ledto the creation of favourable conditions for water uptakeinto the microsphere matrix resulting in the creation of anamorphous gel structure at pH 60 (see Figure 4(a)) Also theenormous loss of the blue colour in comparison with sampleMP-2 10 (Figure 4(d)) can be noticed probably related tohigh water uptake and higher release of the copper ionswhich are responsible for the blue colour This is uniquelyin accordance with previously published data saying thatincreased polymer or hardening solution concentrations cansignificantly reduce water uptake due to the increased densityof the polymer network [34] At pH 60 sample MP-2 05followed with swelling capacity of 24360 in 20 hrs At pH45 however its swelling was comparable with samples cross-linked with 1M CuCl

2 which exhibited a lower swelling

capacity ranging from 1372 to 1607 after 20 hrs at pH 45and 4330ndash5391 at pH 60 (results for pH 60 are clearlyevident in Figures 4(b) and 4(d)) No influence on swellingcapacity as a result of particle size was observed

The mechanism for the antibacterial activity of Cu+ions is based on their energetically easier movement acrossa lipid bilayer and uptake by the cell generating reactiveoxygen species leading to lipid peroxidation and proteinoxidation [35] The excess of copper causes a decrease in themembrane integrity of a microorganism which causes theparticular cell to leak nutritional elements like potassiumand glutamate which lead to desiccation and ultimatelycell death [36] The antimicrobial activity of selected CMCmicroparticles cross-linked by Cu2+ was evaluated by MICtest using Gram-negative Escherichia coli and Candida albi-cans yeast (see Figures 5 and 6) The test expresses theminimum concentration of antimicrobial agent that inhibitsthe visible growth of microorganisms [37] The test wastriplicated For E coli MIC values after 20 hrs of incubationwere 0184 0484 0353 and 0519mgmL for samples MP-1 05 MP-1 10 MP-2 05 and MP-2 10 respectively MICvalues for C albicans were higher for the same samples0740 1639 0598 and 1087mgmL respectively HigherMIC values for C albicans in comparison with the valuesfor E coli can be explained by the higher resistance of theyeast to the copperrsquos antibacterial effect Weissman et alconfirm this reporting on the isolation of two genes involved

BioMed Research International 7

00

01

02

03

04

05

06

07

08

09

10

0740 0370 0185 0093 0046 0023

A

c (mgmL)

(a)

00

01

02

03

04

05

06

07

08

09

10

3277 1639 0819 0410 0205 0102

A

c (mgmL)

(b)

0001020304050607080910

0598 0299 0150 0075 0037 0019

20hrs

A

c (mgmL)

(c)

0001020304050607080910

2173 1087 0543 0272 0136 0068

A

20hrs

c (mgmL)

(d)

Figure 6 MIC results of CMC microparticles against Candida albicans (a) MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

in copper detoxification in C albicans metallothioneinCaCUP1 and a copper-transporting P-type ATPase CaCRP1[38] These two genes account for C albicansrsquos resistanceto copper and consequently the vast differences in ourresults

The obtained MIC values prove substantial antimicrobialactivity against both microorganisms tested in this study andare in good agreementwith copperMIC values against strainsof E coli and C albicans described by different authors inother studies Martınez Medina et al reported MIC valuesfor Cu(II) complexes of 0375mgmL and gt15mgmL for Ecoli and C albicans respectively [39] Copper nanoparticlesprepared by Zain et al (200 nm) showed MIC values of0433mgL for E coli [40] and those prepared by Rupareliaet al (9 nm) showed MIC values of 0280mgmL for E coli[41]

Despite the lower content of copper in microspheresthe samples prepared using the less-concentrated CuCl

2

(05M) solutionmdashMP-1 05 and MP-2 05mdashexhibited lowerMIC values and so better antibacterial activity against bothtested pathogens in comparison with samples cross-linked in

the more potent 1M CuCl2solutionThese results confirmed

the great influence of the microparticle swellability on MICvalues Both samples exhibited high swelling capacity at pH60 (see Figure 3) and thus good conditions for the releaseof copper ions from particles (pH of LB and YNB mediumwas also close to 60) Against E coli sample MP-1 05 wasfound to be the most effective as it probably exhibited fastercopper releaseThis could be a result of the higher cumulationof copper ions on more rougher surface (see Figures 1(a)and 1(b)) and also smaller particle size (see Table 2) and thuslarger surface area [42] These characteristics could promotefaster swelling leading to the creation of an amorphousstructure during the 20-hour interval (Figure 4(a)) Againstthe more resistant C albicans however the most effectivesample was MP-2 05 exhibiting probably more uniformcopper release due to the higher CMC concentraction (2)more uniform copper distribution in the microspheres andlarger particle size (see Table 2) This theory is also sup-ported by the fact that the MP-2 05 microparticles main-tained their shape-specific structure after 20 hrs of swelling(Figure 4(c))

8 BioMed Research International

4 Conclusion

In this experimental work CMC dispersions of two differentconcentrations (1 and 2) were cross-linked by copper (II)ions (05 1 15 or 20MCuCl

2) to prepare microspheres with

antimicrobial activity against frequently occurring vaginalpathogens Escherichia coli and Candida albicans All testedsamples exhibited stable but pH responsive behaviour inenvironments corresponding with natural and inflamed vagi-nal conditions and proved substantial antimicrobial activityagainst both pathogens The most effective samples werethose hardened in a less-concentrated CuCl

2(05M) solu-

tion Unexpectedly a crucial parameter for microsphereantimicrobial activity was not found in the copper contentbut in the swelling capacity of the microparticles and in thedegree of CMC surface shrinkingThe sample prepared usinga 1 CMC dispersion cross-linked by 05M CuCl

2seemed to

be the most suitable for potential vaginal use not only due toits antibacterial activity but also due to its gradual change to anonspecific-shaped gel which is preferable when consideringvaginal dosage forms

Conflict of Interests

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

References

[1] L Fan M Peng Z Zhou et al ldquoModification of carboxymethylcellulose Grafted with collagen peptide and its antioxidantaktivityrdquo Carbohydrate Polymers vol 112 pp 32ndash38 2014

[2] N HaleemM Arsah M Shahid and AM Tahir ldquoSynthesis ofcarboxymethyl cellulose fromwaste of cotton ginning industryrdquoCarbohydrate Polymers vol 113 pp 249ndash255 2014

[3] J-F Su Z Huang X-Y Yuan X-YWang andM Li ldquoStructureand properties of carboxymethyl cellulosesoy protein isolateblend edible films crosslinked by Maillard reactionsrdquo Carbohy-drate Polymers vol 79 no 1 pp 145ndash153 2010

[4] H Kono ldquoCharacterization and properties of carboxymethylcellulose hydrogels crosslinked by polyethylene glycolrdquo Carbo-hydrate Polymers vol 106 no 1 pp 84ndash93 2014

[5] E A Hassn M L Hassan N C Moorefield and GR Newkome ldquoNew supramolecular metallo-terpyridine car-boxymethyl cellulose derivativeswith antimicrobial propertiesrdquoCarbohydrate Polymers vol 116 pp 2ndash8 2014

[6] S Sayanjali B Ghanbarzadeh and S Ghiassifar ldquoEvaluation ofantimicrobial and physical properties of edible film based oncarboxymethyl cellulose containing potassium sorbate on somemycotoxigenic Aspergillus species in fresh pistachiosrdquo LWTmdashFood Science and Technology vol 44 no 4 pp 1133ndash1138 2011

[7] A A Hebeish M H El-Rafie F A Abdel-Mohdy E S Abdel-Halim and H E Emam ldquoCarboxymethyl cellulose for greensynthesis and stabilization of silver nanoparticlesrdquo Carbohy-drate Polymers vol 82 no 3 pp 933ndash941 2010

[8] K Liu Y Xu X Lin et al ldquoSynergistic effects of guanidine-grafted CMC on enhancing antimicrobial activity and drystrength of paperrdquo Carbohydrate Polymers vol 110 pp 382ndash3872014

[9] L Upadhyaya J Singh V Agarwal and R P Tewari ldquoBiomed-ical applications of carboxymethyl chitosansrdquo CarbohydratePolymers vol 91 no 1 pp 452ndash466 2013

[10] A-H Yu H-Y Hsieh J-C Pang et al ldquoActive films fromwater-soluble chitosancellulose composites incorporating releasablecaffeic acid for inhibition of lipid oxidation in fish oil emul-sionsrdquo Food Hydrocolloids vol 32 no 1 pp 9ndash19 2013

[11] T Zhong G S Oporto J Jaczynski A T Tesfai and JArmstrong ldquoAntimicrobial properties of the hybrid coppernanoparticles-carboxymethyl celluloserdquo Wood and Fiber Sci-ence vol 45 no 2 pp 215ndash222 2013

[12] G E Jackson P M May and D R Williams ldquoMetal-ligandcomplexes involved in rheumatoid arthritis-I justificationsfor copper administrationrdquo Journal of Inorganic and NuclearChemistry vol 40 no 6 pp 1189ndash1194 1978

[13] O A El-Gammal E A Elmorsy and Y E Sherif ldquoEvaluationof the anti-inflammatory and analgesic effects of Cu(II) andZn(II) complexes derived from 2-(naphthalen-1-yloxy)-119873

1015840

-(1-(pyridin-2-1) ethylidene) acetohydraziderdquo Spectrochimica ActaPart A Molecular and Biomolecular Spectroscopy vol 120 pp332ndash339 2014

[14] S Odisitse and G E Jackson ldquoIn vitro and in vivo studiesof NN1015840-bis[2(2-pyridyl)-methyl]pyridine-26-dicarboxamide-copper(II) and rheumatoid arthritisrdquo Polyhedron vol 27 no 1pp 453ndash464 2008

[15] A Hussain and F Ahsan ldquoThe vagina as a route for systemicdrug deliveryrdquo Journal of Controlled Release vol 103 no 2 pp301ndash313 2005

[16] L M Ensign T E Hoen K Maisel R A Cone and J S HanesldquoEnhanced vaginal drug delivery through the use of hypotonicformulations that induce fluid uptakerdquo Biomaterials vol 34 no28 pp 6922ndash6929 2013

[17] B B Saxena M Singh R M Gospin C C Chu and W JLedger ldquoEfficacy of nonhormonal vaginal contraceptives from ahydrogel delivery systemrdquo Contraception vol 70 no 3 pp 213ndash219 2004

[18] M L Gonzalez-Rodrıguez M A Holgado C Sanchez-Lafuente A M Rabasco and A Fini ldquoAlginatechitosan par-ticulate systems for sodium diclofenac releaserdquo InternationalJournal of Pharmaceutics vol 232 no 1-2 pp 225ndash234 2002

[19] C H Goh P W S Heng and L W Chan ldquoAlginates as auseful natural polymer for microencapsulation and therapeuticapplicationsrdquo Carbohydrate Polymers vol 88 no 1 pp 1ndash122012

[20] W-C Lin D-G Yu and M-C Yang ldquopH-sensitive poly-electrolyte complex gel microspheres composed of chitosansodium tripolyphosphatedextran sulfate swelling kinetics anddrug delivery propertiesrdquo Colloids and Surfaces B Biointerfacesvol 44 no 2-3 pp 143ndash151 2005

[21] K G H Desai and H J Park ldquoPreparation and characteriza-tion of drug-loaded chitosan-tripolyphosphatemicrospheres byspray dryingrdquo Drug Development Research vol 64 no 2 pp114ndash128 2005

[22] K M Rao B Mallikarjuna K K Rao M N Prabhakar K CRao and M C Subha ldquoPreparation and characterization of pHsensitive poly(vinyl alcohol)sodium carboxymethyl celluloseIPN microspheres for in vitro release studies of an anti-cancerdrugrdquo Polymer Bulletin vol 68 no 7 pp 1905ndash1919 2012

[23] M A Saleem Y D Murali M D Naheed P Jaydeep andM Dhaval ldquoPrepapation and evaluation of valsartan loadedhydrogel beadsrdquo International Research Journal of Pharmacyvol 3 no 6 pp 80ndash85 2012

BioMed Research International 9

[24] Y Y Yang J P Wan T S Chung P K Pallathadka S Ng andJ Heller ldquoPOE-PEG-POE triblock copolymeric microspherescontaining protein I Preparation and characterizationrdquo Journalof Controlled Release vol 75 no 1-2 pp 115ndash128 2001

[25] H Brandenberger and F Widmer ldquoA new multinozzle encap-sulationimmobilisation system to produce uniform beads ofalginaterdquo Journal of Biotechnology vol 63 no 1 pp 73ndash80 1998

[26] E-S Chan B-B Lee P Ravindra and D Poncelet ldquoPredictionmodels for shape and size of ca-alginate macrobeads producedthrough extrusion-dripping methodrdquo Journal of Colloid andInterface Science vol 338 no 1 pp 63ndash72 2009

[27] P B Deasy and M F L Law ldquoUse of extrusion-spheronizationto develop an improved oral dosage form of indomethacinrdquoInternational Journal of Pharmaceutics vol 148 no 2 pp 201ndash209 1997

[28] X H Yang and W L Zhu ldquoViscosity properties of sodiumcarboxymethylcellulose solutionsrdquo Cellulose vol 14 no 5 pp409ndash417 2007

[29] E-S Chan ldquoPreparation of Ca-alginate beads containing highoil content influence of process variables on encapsulationefficiency and bead propertiesrdquo Carbohydrate Polymers vol 84no 4 pp 1267ndash1275 2011

[30] M S Kim S J Park B K Gu and C-H Kim ldquoIonicallycrosslinked alginate-carboxymethyl cellulose beads for thedelivery of protein therapeuticsrdquo Applied Surface Science vol262 pp 28ndash33 2012

[31] C Valenta ldquoThe use of mucoadhesive polymers in vaginaldeliveryrdquo Advanced Drug Delivery Reviews vol 57 no 11 pp1692ndash1712 2005

[32] S F Borges J G L Silva and P C M Teixeira ldquoSurvival andbiofilm formation of Listeria monocytogenes in simulated vagi-nal fluid influence of pH and strain originrdquo FEMS Immunologyand Medical Microbiology vol 62 no 3 pp 315ndash320 2011

[33] R Barbucci A Magnani and M Consumi ldquoSwelling behav-ior of carboxymethylcellulose hydrogels in relation to cross-linking pH and charge densityrdquo Macromolecules vol 33 no20 pp 7475ndash7480 2000

[34] J Berger M Reist J M Mayer O Felt N A Peppas and RGurny ldquoStructure and interactions in covalently and ionicallycrosslinked chitosan hydrogels for biomedical applicationsrdquoEuropean Journal of Pharmaceutics and Biopharmaceutics vol57 no 1 pp 19ndash34 2004

[35] U Bogdanovic V Lazic V Vodnik M Budimir Z Markovicand S Dimitrijevic ldquoCopper nanoparticles with high antimi-crobial activityrdquoMaterials Letters vol 128 pp 75ndash78 2014

[36] J Konieczny and Z Rdzawski ldquoAntibacterial properties of cop-per and its alloysrdquoArchives ofMaterials Science and Engineeringvol 56 no 2 pp 53ndash60 2012

[37] S M Magana P Quintana D H Aguilar et al ldquoAntibacterialactivity of montmorillonites modified with silverrdquo Journal ofMolecular Catalysis A Chemical vol 281 no 1-2 pp 192ndash1992008

[38] Z Weissman I Berdicevsky B-Z Cavari and D KornitzerldquoThe high copper tolerance ofCandida albicans is mediated by aP-type ATPaserdquo Proceedings of the National Academy of Sciencesof the United States of America vol 97 no 7 pp 3520ndash35252000

[39] J J Martınez Medina M S Islas L L Lopez Tevez E GFerrer N B Okulik and P A M Williams ldquoCopper(II) com-plexes with cyanoguanidine and o-phenanthroline theoreticalstudies in vitro antimicrobial activity and alkaline phosphatase

inhibitory effectrdquo Journal of Molecular Structure vol 1058 no1 pp 298ndash307 2014

[40] N M Zain A G F Stapley and G Shama ldquoGreen synthesis ofsilver and copper nanoparticles using ascorbic acid and chitosanfor antimicrobial applicationsrdquo Carbohydrate Polymers vol 112pp 195ndash202 2014

[41] J P Ruparelia A K Chatterjee S P Duttagupta and SMukherji ldquoStrain specificity in antimicrobial activity of silverand copper nanoparticlesrdquo Acta Biomaterialia vol 4 no 3 pp707ndash716 2008

[42] J Zhao H J Feng H Q Tang and J H Zheng ldquoBactericidaland corrosive properties of silver implanted TiN thin filmscoated on AISI317 stainless steelrdquo Surface and Coatings Technol-ogy vol 201 no 9ndash11 pp 5676ndash5679 2007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

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MEDIATORSINFLAMMATION

of

Page 3: Research Article Antimicrobial Properties of ...downloads.hindawi.com/journals/bmri/2015/790720.pdf · Research Article Antimicrobial Properties of Microparticles Based on ... ect

BioMed Research International 3

mounted directly onto the SEM sample holder using double-sided sticking tape and then coated with a 10 nm thick layerof Au Images were taken using theMIRA3 scanning electronmicroscope (Tescan Brno CzechRepublic) at an acceleratingvoltage of 50 kV

224 Optical Microscope Analysis Particle size of the coppermicroparticles was measured using a NIKON SMZ 1500stereoscopic microscope (Nikon Tokyo Japan) equippedwith a 72AUC02USB camera (The Imaging Source Bre-men Germany) Microparticles were visualized under times15magnification The obtained images of 200 randomly chosenmicroparticles were stored and subsequently processed usingthe NIS-Elements AR 40 computer software (Nikon TokyoJapan)

Equivalent diameter (ED) and sphericity factor (SF)were calculated from the measured values and expressed asarithmetic mean plusmn standard deviation

225 Copper Content in Microparticles The copper contentin the prepared microparticles was determined by atomicabsorption To digest the microparticles 6mL of concen-trated nitric acid (65 vv) and 2mL of hydrogen peroxide(30 vv) were added to 10mg of every sample and placedin a TFM digestion vessel The vessels were closed andplaced into the segment and the content was mineralizedusing an Ethos SEL Microwave Labstation (Milestone Italy)at 220∘C for 35min applying a maximal power of 1000WThe microwave programme was started by steadily increas-ing the temperature over 15min followed by holding thetemperature for an additional 20min After cooling eachresulting solution was transferred to 50mL glass flasks andfilled to the mark with deionized water Samples were dilutedto 1 19 with deionized water prior to further analysis Coppercontent was measured using air-acetylene flame atomizationin a contrAA 700 atomic absorption spectrometer (AnalytikJena Germany) All samples were measured in triplicate andthe obtained values were processed by Aspect CS softwareversion 21

226 Swelling Capacity To determine swelling capacity apreviously reported method was improved [20] The testwas performed in a medium that properly simulates vaginalconditions 100mg of each sample was put into fine meshbaskets and immersed in a pH 45 medium (natural vaginalenvironmentmdash680 g of potassium dihydrogen phosphate Rin 10000mL of water R) and a pH 60 medium (infectedvaginal environmentmdash68 g of sodiumdihydrogen phosphateR in 10000mLofwater R pH adjustmentwith strong sodiumhydroxide solution R) The baskets were pulled out at timeintervals of 5 10 15 30 and 45min and 1 2 3 4 5 and 6hours after the first immersion properly dried and weighedSwelling capacity was calculated using the following equation[21]

119878SW = (119882119905minus1198820

1198820

) times 100 () (1)

119878SW is swelling capacity expressed as a percentage of weightaddition 119882

119905is the weight of a sample at the relevant time

interval and 1198820represents the initial weight of the sample

For each batch the measurement was performed three timesand results were expressed as mean values with standarddeviations

227 Antimicrobial Activity Bacterial Strains andMIC Deter-mination E coli (CCM4517) wasmaintained in a blood agarTo determine minimal inhibition concentration (MIC) anovernight culture of E coli cells was suspended in a fresh LB(Luria broth) medium and grown to OD

60005 at 37∘C at a

shaking speed of 250 rpmCandida albicans (CCM 8186) was maintained in an

YNB (Yeast Nitrogen Base with ammonium sulfate) mediumat 37∘C For MIC determination an overnight culture wassuspended in a fresh YNB medium and grown to OD

60005

at 37∘C at a shaking speed of 250 rpmA suitable amount of CMCmicroparticles was suspended

in the growth media (LB medium for E coli YNB for C albi-cans) to prepare a 10 suspension and incubated for 60minat room temperature to release the copper into solutionUndissolved particles were then separated by centrifugationand discarded and atomic absorption was used to determinecopper concentration Twofold dilutions of copper suspen-sion were prepared in appropriate mediums in a microtiterplate (100 120583Lwell in triplicate) Then 10e6 cells (E coli orC albicans) were added to each well of the microtiter plateand incubated at 37∘C Tomonitor cell growth optical densitywas measured spectrophotometrically (OD

600) after 20 hrs of

incubation The mean of the three wells was calculated toevaluate antimicrobial activity Wells containing the E colior C albicans without copper inhibition were included intriplicate in all tested plates as controls The MIC value wasexpressed as the concentration of copper in a well showing atleast a fourfold reduction of OD

600absorbance compared to

each subsequent well

3 Results and Discussion

The copper cross-linked carmellosemicroparticles were eval-uated for particle size sphericity factor and copper content(Table 2) The equivalent diameter of the prepared particlesranged from 7381 plusmn 303 to 1078 plusmn 124 120583m It seems thatparticle size did not depend on the concentration of theCu2+ hardening solution However it was observed that itdid increase with increasing CMC concentration which canbe attributed to the increased viscosity [22 23] Viscosity ofthe 1 CMC dispersion was found to be 162 plusmn 021mPasdotsand that of the 2 CMC dispersion 76667 plusmn 047mPasdotsA polymer dispersion with higher viscosity is more difficultto form into smaller droplets [24] thus these dispersionsyielded larger particles Another influence on the particle sizeof microspheres could be seen in the degree of CMC chainshrinkage Generally particle size decreases inversely withthe degree of shrinkage It has been well documented thatthe degree of shrinkage is typically higher for beads preparedfrom a dispersion with lower polymer concentration [25 26]

4 BioMed Research International

(a) (b)

(c) (d)

Figure 1 SEM photographs of surface topography of CMCmicroparticles (a) MP-1 05 (magnification 4330x) (b) surface detail of MP-1 05(magnification 9890x) (c) MP-2 05 (magnification 4330x) and (d) surface detail of MP-2 05 (magnification 9890x)

0

50

100

150

200

250

300

350

400

0 5 10 15 20

Swel

ling

capa

city

()

Time (hrs)MP-1 05 (SD max 2411) MP-1 10 (SD max 1253)MP-2 05 (SD max 1410) MP-2 10 (SD max 630)

Figure 2 Degree of swelling in Cu2+ cross-linked microparticles atpH 45

A comparison of the microparticle surfaces (MP-1 05 ED =7757120583m versus MP-2 05 ED = 10169) in Figure 1 confirmsthese findings From these SEMphotographs it is evident thatthe degree of CMC shrinkage was significantly higher for thesmaller 1 CMCmicrospheres

0

1000

2000

3000

4000

5000

6000

0 5 10 15 20

Swel

ling

capa

city

()

Time (hrs)

MP-1 05 (SD max 13837) MP-1 10 (SD max 2476)MP-2 05 (SD max 7730) MP-2 10 (SD max 4300)

Figure 3 Degree of swelling in Cu2+ cross-linked microparticles atpH 60

PreparedCMCmicrospheres exhibited sufficient spheric-ity ranging from 0850 plusmn 0067 to 0934 plusmn 0038 Previousstudies indicate that particles with this parameter value above08 are considered to have good sphericity [27] It can beobserved from the results that the sphericity was not clearly

BioMed Research International 5

(a) (b)

(c) (d)

Figure 4 Optical microscope images of CMC particles after 20-hour swelling capacity test in pH 60 buffer bars correspond to 1000120583m (a)MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

Table 2 Microparticle characteristics equivalent diameter sphericity factor and copper content

Sample ED (120583m) SD (120583m) SF SD Copper content (gkg) SD (gkg)MP-1 05 7757 302 0906 0044 1013 065MP-1 10 8885 584 0934 0038 1436 231MP-1 15 7381 303 0887 0056 1523 019MP-1 20 7986 879 0855 0059 1730 215MP-2 05 10169 228 0891 0040 1436 026MP-2 10 10780 124 0886 0071 1647 040MP-2 15 9221 576 0850 0067 1876 132MP-2 20 9336 386 0874 0057 2003 103

influenced by increasing the concentration of the hardeningsolution but it was noticed that hardening solutions withlower molarity of CuCl

2(05 and 10) yielded microspheres

with higher sphericity values On the other hand particlesphericity was lower in samples prepared with a higherpolymer concentration with the exception of samples MP-1 20 and MP-2 20 which were prepared with 2M of CuCl

2

hardening solution Previously reported results confirmedthat increasing the concentration of CMC solutions lin-early increases the solution viscosity [28] and sphericity ofparticles Our results generally coincide with findings thatoverly viscous polymer solutions (2 in our case) form lessspherical tail-shaped particles [29] and particles with a roughsurface [30]

Table 2 also shows the results of atomic absorptionanalysis for copper content Copper content in the preparedmicroparticles ranged from 1013 plusmn 065 to 2003 plusmn 103 gkgIt was observed that the content significantly increased with

increases in hardening solution concentration as well aspolymer concentration

The samples of microspheres with the highest sphericity(MP-1 05 MP-1 10 MP-2 05 and MP-2 10) which is onethe most important criteria for the preparation of particlesystems were then investigated for swelling capacity in phos-phate buffers with pH 45 [31] and pH 60 [32] respectivelyto simulate natural and inflamed vaginal conditions and topredict their behaviour on vaginal mucosa for a 20-hourtime interval (20-hour interval represents an estimated timeof therapy) The obtained results can be seen in Figure 2for pH 45 and in Figure 3 for pH 60 Figure 4 showsimages of microparticles after a 20-hour swelling capacitytest in a pH 60 buffer Generally it was observed that theswelling capacity of all samples gradually increased duringthe test Selected samples exhibited pH-responsive behaviourwhich differed based on the tested environments swellingwas substantially higher at pH 60 [33] It is obvious from

6 BioMed Research International

00

01

02

03

04

05

06

07

08

09

0369 0184 0092 0046 0023 0012

A

c (mgmL)

(a)

00

01

02

03

04

05

06

07

08

09

3871 1936 0968 0484 0242 0121

A

c (mgmL)

(b)

00

01

02

03

04

05

06

07

08

09

0707 0353 0177 0088 0044 0022

A

c (mgmL)

20hrs

(c)

00

01

02

03

04

05

06

07

08

09

4148 2074 1037 0519 0259 0130

20hrs

A

c (mgmL)

(d)

Figure 5 MIC results of CMCmicroparticles against Escherichia coli (a) MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

Figures 2 and 3 that sample MP-1 05 prepared with a less-concentrated hardening solution (05M) and a lower CMCconcentration (1) had the highest swelling capacity at bothpH values (3662 at pH 45 after 20 hrs 52963 at pH60 after 20 hrs) These values of formulation variables ledto the creation of favourable conditions for water uptakeinto the microsphere matrix resulting in the creation of anamorphous gel structure at pH 60 (see Figure 4(a)) Also theenormous loss of the blue colour in comparison with sampleMP-2 10 (Figure 4(d)) can be noticed probably related tohigh water uptake and higher release of the copper ionswhich are responsible for the blue colour This is uniquelyin accordance with previously published data saying thatincreased polymer or hardening solution concentrations cansignificantly reduce water uptake due to the increased densityof the polymer network [34] At pH 60 sample MP-2 05followed with swelling capacity of 24360 in 20 hrs At pH45 however its swelling was comparable with samples cross-linked with 1M CuCl

2 which exhibited a lower swelling

capacity ranging from 1372 to 1607 after 20 hrs at pH 45and 4330ndash5391 at pH 60 (results for pH 60 are clearlyevident in Figures 4(b) and 4(d)) No influence on swellingcapacity as a result of particle size was observed

The mechanism for the antibacterial activity of Cu+ions is based on their energetically easier movement acrossa lipid bilayer and uptake by the cell generating reactiveoxygen species leading to lipid peroxidation and proteinoxidation [35] The excess of copper causes a decrease in themembrane integrity of a microorganism which causes theparticular cell to leak nutritional elements like potassiumand glutamate which lead to desiccation and ultimatelycell death [36] The antimicrobial activity of selected CMCmicroparticles cross-linked by Cu2+ was evaluated by MICtest using Gram-negative Escherichia coli and Candida albi-cans yeast (see Figures 5 and 6) The test expresses theminimum concentration of antimicrobial agent that inhibitsthe visible growth of microorganisms [37] The test wastriplicated For E coli MIC values after 20 hrs of incubationwere 0184 0484 0353 and 0519mgmL for samples MP-1 05 MP-1 10 MP-2 05 and MP-2 10 respectively MICvalues for C albicans were higher for the same samples0740 1639 0598 and 1087mgmL respectively HigherMIC values for C albicans in comparison with the valuesfor E coli can be explained by the higher resistance of theyeast to the copperrsquos antibacterial effect Weissman et alconfirm this reporting on the isolation of two genes involved

BioMed Research International 7

00

01

02

03

04

05

06

07

08

09

10

0740 0370 0185 0093 0046 0023

A

c (mgmL)

(a)

00

01

02

03

04

05

06

07

08

09

10

3277 1639 0819 0410 0205 0102

A

c (mgmL)

(b)

0001020304050607080910

0598 0299 0150 0075 0037 0019

20hrs

A

c (mgmL)

(c)

0001020304050607080910

2173 1087 0543 0272 0136 0068

A

20hrs

c (mgmL)

(d)

Figure 6 MIC results of CMC microparticles against Candida albicans (a) MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

in copper detoxification in C albicans metallothioneinCaCUP1 and a copper-transporting P-type ATPase CaCRP1[38] These two genes account for C albicansrsquos resistanceto copper and consequently the vast differences in ourresults

The obtained MIC values prove substantial antimicrobialactivity against both microorganisms tested in this study andare in good agreementwith copperMIC values against strainsof E coli and C albicans described by different authors inother studies Martınez Medina et al reported MIC valuesfor Cu(II) complexes of 0375mgmL and gt15mgmL for Ecoli and C albicans respectively [39] Copper nanoparticlesprepared by Zain et al (200 nm) showed MIC values of0433mgL for E coli [40] and those prepared by Rupareliaet al (9 nm) showed MIC values of 0280mgmL for E coli[41]

Despite the lower content of copper in microspheresthe samples prepared using the less-concentrated CuCl

2

(05M) solutionmdashMP-1 05 and MP-2 05mdashexhibited lowerMIC values and so better antibacterial activity against bothtested pathogens in comparison with samples cross-linked in

the more potent 1M CuCl2solutionThese results confirmed

the great influence of the microparticle swellability on MICvalues Both samples exhibited high swelling capacity at pH60 (see Figure 3) and thus good conditions for the releaseof copper ions from particles (pH of LB and YNB mediumwas also close to 60) Against E coli sample MP-1 05 wasfound to be the most effective as it probably exhibited fastercopper releaseThis could be a result of the higher cumulationof copper ions on more rougher surface (see Figures 1(a)and 1(b)) and also smaller particle size (see Table 2) and thuslarger surface area [42] These characteristics could promotefaster swelling leading to the creation of an amorphousstructure during the 20-hour interval (Figure 4(a)) Againstthe more resistant C albicans however the most effectivesample was MP-2 05 exhibiting probably more uniformcopper release due to the higher CMC concentraction (2)more uniform copper distribution in the microspheres andlarger particle size (see Table 2) This theory is also sup-ported by the fact that the MP-2 05 microparticles main-tained their shape-specific structure after 20 hrs of swelling(Figure 4(c))

8 BioMed Research International

4 Conclusion

In this experimental work CMC dispersions of two differentconcentrations (1 and 2) were cross-linked by copper (II)ions (05 1 15 or 20MCuCl

2) to prepare microspheres with

antimicrobial activity against frequently occurring vaginalpathogens Escherichia coli and Candida albicans All testedsamples exhibited stable but pH responsive behaviour inenvironments corresponding with natural and inflamed vagi-nal conditions and proved substantial antimicrobial activityagainst both pathogens The most effective samples werethose hardened in a less-concentrated CuCl

2(05M) solu-

tion Unexpectedly a crucial parameter for microsphereantimicrobial activity was not found in the copper contentbut in the swelling capacity of the microparticles and in thedegree of CMC surface shrinkingThe sample prepared usinga 1 CMC dispersion cross-linked by 05M CuCl

2seemed to

be the most suitable for potential vaginal use not only due toits antibacterial activity but also due to its gradual change to anonspecific-shaped gel which is preferable when consideringvaginal dosage forms

Conflict of Interests

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

References

[1] L Fan M Peng Z Zhou et al ldquoModification of carboxymethylcellulose Grafted with collagen peptide and its antioxidantaktivityrdquo Carbohydrate Polymers vol 112 pp 32ndash38 2014

[2] N HaleemM Arsah M Shahid and AM Tahir ldquoSynthesis ofcarboxymethyl cellulose fromwaste of cotton ginning industryrdquoCarbohydrate Polymers vol 113 pp 249ndash255 2014

[3] J-F Su Z Huang X-Y Yuan X-YWang andM Li ldquoStructureand properties of carboxymethyl cellulosesoy protein isolateblend edible films crosslinked by Maillard reactionsrdquo Carbohy-drate Polymers vol 79 no 1 pp 145ndash153 2010

[4] H Kono ldquoCharacterization and properties of carboxymethylcellulose hydrogels crosslinked by polyethylene glycolrdquo Carbo-hydrate Polymers vol 106 no 1 pp 84ndash93 2014

[5] E A Hassn M L Hassan N C Moorefield and GR Newkome ldquoNew supramolecular metallo-terpyridine car-boxymethyl cellulose derivativeswith antimicrobial propertiesrdquoCarbohydrate Polymers vol 116 pp 2ndash8 2014

[6] S Sayanjali B Ghanbarzadeh and S Ghiassifar ldquoEvaluation ofantimicrobial and physical properties of edible film based oncarboxymethyl cellulose containing potassium sorbate on somemycotoxigenic Aspergillus species in fresh pistachiosrdquo LWTmdashFood Science and Technology vol 44 no 4 pp 1133ndash1138 2011

[7] A A Hebeish M H El-Rafie F A Abdel-Mohdy E S Abdel-Halim and H E Emam ldquoCarboxymethyl cellulose for greensynthesis and stabilization of silver nanoparticlesrdquo Carbohy-drate Polymers vol 82 no 3 pp 933ndash941 2010

[8] K Liu Y Xu X Lin et al ldquoSynergistic effects of guanidine-grafted CMC on enhancing antimicrobial activity and drystrength of paperrdquo Carbohydrate Polymers vol 110 pp 382ndash3872014

[9] L Upadhyaya J Singh V Agarwal and R P Tewari ldquoBiomed-ical applications of carboxymethyl chitosansrdquo CarbohydratePolymers vol 91 no 1 pp 452ndash466 2013

[10] A-H Yu H-Y Hsieh J-C Pang et al ldquoActive films fromwater-soluble chitosancellulose composites incorporating releasablecaffeic acid for inhibition of lipid oxidation in fish oil emul-sionsrdquo Food Hydrocolloids vol 32 no 1 pp 9ndash19 2013

[11] T Zhong G S Oporto J Jaczynski A T Tesfai and JArmstrong ldquoAntimicrobial properties of the hybrid coppernanoparticles-carboxymethyl celluloserdquo Wood and Fiber Sci-ence vol 45 no 2 pp 215ndash222 2013

[12] G E Jackson P M May and D R Williams ldquoMetal-ligandcomplexes involved in rheumatoid arthritis-I justificationsfor copper administrationrdquo Journal of Inorganic and NuclearChemistry vol 40 no 6 pp 1189ndash1194 1978

[13] O A El-Gammal E A Elmorsy and Y E Sherif ldquoEvaluationof the anti-inflammatory and analgesic effects of Cu(II) andZn(II) complexes derived from 2-(naphthalen-1-yloxy)-119873

1015840

-(1-(pyridin-2-1) ethylidene) acetohydraziderdquo Spectrochimica ActaPart A Molecular and Biomolecular Spectroscopy vol 120 pp332ndash339 2014

[14] S Odisitse and G E Jackson ldquoIn vitro and in vivo studiesof NN1015840-bis[2(2-pyridyl)-methyl]pyridine-26-dicarboxamide-copper(II) and rheumatoid arthritisrdquo Polyhedron vol 27 no 1pp 453ndash464 2008

[15] A Hussain and F Ahsan ldquoThe vagina as a route for systemicdrug deliveryrdquo Journal of Controlled Release vol 103 no 2 pp301ndash313 2005

[16] L M Ensign T E Hoen K Maisel R A Cone and J S HanesldquoEnhanced vaginal drug delivery through the use of hypotonicformulations that induce fluid uptakerdquo Biomaterials vol 34 no28 pp 6922ndash6929 2013

[17] B B Saxena M Singh R M Gospin C C Chu and W JLedger ldquoEfficacy of nonhormonal vaginal contraceptives from ahydrogel delivery systemrdquo Contraception vol 70 no 3 pp 213ndash219 2004

[18] M L Gonzalez-Rodrıguez M A Holgado C Sanchez-Lafuente A M Rabasco and A Fini ldquoAlginatechitosan par-ticulate systems for sodium diclofenac releaserdquo InternationalJournal of Pharmaceutics vol 232 no 1-2 pp 225ndash234 2002

[19] C H Goh P W S Heng and L W Chan ldquoAlginates as auseful natural polymer for microencapsulation and therapeuticapplicationsrdquo Carbohydrate Polymers vol 88 no 1 pp 1ndash122012

[20] W-C Lin D-G Yu and M-C Yang ldquopH-sensitive poly-electrolyte complex gel microspheres composed of chitosansodium tripolyphosphatedextran sulfate swelling kinetics anddrug delivery propertiesrdquo Colloids and Surfaces B Biointerfacesvol 44 no 2-3 pp 143ndash151 2005

[21] K G H Desai and H J Park ldquoPreparation and characteriza-tion of drug-loaded chitosan-tripolyphosphatemicrospheres byspray dryingrdquo Drug Development Research vol 64 no 2 pp114ndash128 2005

[22] K M Rao B Mallikarjuna K K Rao M N Prabhakar K CRao and M C Subha ldquoPreparation and characterization of pHsensitive poly(vinyl alcohol)sodium carboxymethyl celluloseIPN microspheres for in vitro release studies of an anti-cancerdrugrdquo Polymer Bulletin vol 68 no 7 pp 1905ndash1919 2012

[23] M A Saleem Y D Murali M D Naheed P Jaydeep andM Dhaval ldquoPrepapation and evaluation of valsartan loadedhydrogel beadsrdquo International Research Journal of Pharmacyvol 3 no 6 pp 80ndash85 2012

BioMed Research International 9

[24] Y Y Yang J P Wan T S Chung P K Pallathadka S Ng andJ Heller ldquoPOE-PEG-POE triblock copolymeric microspherescontaining protein I Preparation and characterizationrdquo Journalof Controlled Release vol 75 no 1-2 pp 115ndash128 2001

[25] H Brandenberger and F Widmer ldquoA new multinozzle encap-sulationimmobilisation system to produce uniform beads ofalginaterdquo Journal of Biotechnology vol 63 no 1 pp 73ndash80 1998

[26] E-S Chan B-B Lee P Ravindra and D Poncelet ldquoPredictionmodels for shape and size of ca-alginate macrobeads producedthrough extrusion-dripping methodrdquo Journal of Colloid andInterface Science vol 338 no 1 pp 63ndash72 2009

[27] P B Deasy and M F L Law ldquoUse of extrusion-spheronizationto develop an improved oral dosage form of indomethacinrdquoInternational Journal of Pharmaceutics vol 148 no 2 pp 201ndash209 1997

[28] X H Yang and W L Zhu ldquoViscosity properties of sodiumcarboxymethylcellulose solutionsrdquo Cellulose vol 14 no 5 pp409ndash417 2007

[29] E-S Chan ldquoPreparation of Ca-alginate beads containing highoil content influence of process variables on encapsulationefficiency and bead propertiesrdquo Carbohydrate Polymers vol 84no 4 pp 1267ndash1275 2011

[30] M S Kim S J Park B K Gu and C-H Kim ldquoIonicallycrosslinked alginate-carboxymethyl cellulose beads for thedelivery of protein therapeuticsrdquo Applied Surface Science vol262 pp 28ndash33 2012

[31] C Valenta ldquoThe use of mucoadhesive polymers in vaginaldeliveryrdquo Advanced Drug Delivery Reviews vol 57 no 11 pp1692ndash1712 2005

[32] S F Borges J G L Silva and P C M Teixeira ldquoSurvival andbiofilm formation of Listeria monocytogenes in simulated vagi-nal fluid influence of pH and strain originrdquo FEMS Immunologyand Medical Microbiology vol 62 no 3 pp 315ndash320 2011

[33] R Barbucci A Magnani and M Consumi ldquoSwelling behav-ior of carboxymethylcellulose hydrogels in relation to cross-linking pH and charge densityrdquo Macromolecules vol 33 no20 pp 7475ndash7480 2000

[34] J Berger M Reist J M Mayer O Felt N A Peppas and RGurny ldquoStructure and interactions in covalently and ionicallycrosslinked chitosan hydrogels for biomedical applicationsrdquoEuropean Journal of Pharmaceutics and Biopharmaceutics vol57 no 1 pp 19ndash34 2004

[35] U Bogdanovic V Lazic V Vodnik M Budimir Z Markovicand S Dimitrijevic ldquoCopper nanoparticles with high antimi-crobial activityrdquoMaterials Letters vol 128 pp 75ndash78 2014

[36] J Konieczny and Z Rdzawski ldquoAntibacterial properties of cop-per and its alloysrdquoArchives ofMaterials Science and Engineeringvol 56 no 2 pp 53ndash60 2012

[37] S M Magana P Quintana D H Aguilar et al ldquoAntibacterialactivity of montmorillonites modified with silverrdquo Journal ofMolecular Catalysis A Chemical vol 281 no 1-2 pp 192ndash1992008

[38] Z Weissman I Berdicevsky B-Z Cavari and D KornitzerldquoThe high copper tolerance ofCandida albicans is mediated by aP-type ATPaserdquo Proceedings of the National Academy of Sciencesof the United States of America vol 97 no 7 pp 3520ndash35252000

[39] J J Martınez Medina M S Islas L L Lopez Tevez E GFerrer N B Okulik and P A M Williams ldquoCopper(II) com-plexes with cyanoguanidine and o-phenanthroline theoreticalstudies in vitro antimicrobial activity and alkaline phosphatase

inhibitory effectrdquo Journal of Molecular Structure vol 1058 no1 pp 298ndash307 2014

[40] N M Zain A G F Stapley and G Shama ldquoGreen synthesis ofsilver and copper nanoparticles using ascorbic acid and chitosanfor antimicrobial applicationsrdquo Carbohydrate Polymers vol 112pp 195ndash202 2014

[41] J P Ruparelia A K Chatterjee S P Duttagupta and SMukherji ldquoStrain specificity in antimicrobial activity of silverand copper nanoparticlesrdquo Acta Biomaterialia vol 4 no 3 pp707ndash716 2008

[42] J Zhao H J Feng H Q Tang and J H Zheng ldquoBactericidaland corrosive properties of silver implanted TiN thin filmscoated on AISI317 stainless steelrdquo Surface and Coatings Technol-ogy vol 201 no 9ndash11 pp 5676ndash5679 2007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Autoimmune Diseases

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Anesthesiology Research and Practice

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Pharmaceutics

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MEDIATORSINFLAMMATION

of

Page 4: Research Article Antimicrobial Properties of ...downloads.hindawi.com/journals/bmri/2015/790720.pdf · Research Article Antimicrobial Properties of Microparticles Based on ... ect

4 BioMed Research International

(a) (b)

(c) (d)

Figure 1 SEM photographs of surface topography of CMCmicroparticles (a) MP-1 05 (magnification 4330x) (b) surface detail of MP-1 05(magnification 9890x) (c) MP-2 05 (magnification 4330x) and (d) surface detail of MP-2 05 (magnification 9890x)

0

50

100

150

200

250

300

350

400

0 5 10 15 20

Swel

ling

capa

city

()

Time (hrs)MP-1 05 (SD max 2411) MP-1 10 (SD max 1253)MP-2 05 (SD max 1410) MP-2 10 (SD max 630)

Figure 2 Degree of swelling in Cu2+ cross-linked microparticles atpH 45

A comparison of the microparticle surfaces (MP-1 05 ED =7757120583m versus MP-2 05 ED = 10169) in Figure 1 confirmsthese findings From these SEMphotographs it is evident thatthe degree of CMC shrinkage was significantly higher for thesmaller 1 CMCmicrospheres

0

1000

2000

3000

4000

5000

6000

0 5 10 15 20

Swel

ling

capa

city

()

Time (hrs)

MP-1 05 (SD max 13837) MP-1 10 (SD max 2476)MP-2 05 (SD max 7730) MP-2 10 (SD max 4300)

Figure 3 Degree of swelling in Cu2+ cross-linked microparticles atpH 60

PreparedCMCmicrospheres exhibited sufficient spheric-ity ranging from 0850 plusmn 0067 to 0934 plusmn 0038 Previousstudies indicate that particles with this parameter value above08 are considered to have good sphericity [27] It can beobserved from the results that the sphericity was not clearly

BioMed Research International 5

(a) (b)

(c) (d)

Figure 4 Optical microscope images of CMC particles after 20-hour swelling capacity test in pH 60 buffer bars correspond to 1000120583m (a)MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

Table 2 Microparticle characteristics equivalent diameter sphericity factor and copper content

Sample ED (120583m) SD (120583m) SF SD Copper content (gkg) SD (gkg)MP-1 05 7757 302 0906 0044 1013 065MP-1 10 8885 584 0934 0038 1436 231MP-1 15 7381 303 0887 0056 1523 019MP-1 20 7986 879 0855 0059 1730 215MP-2 05 10169 228 0891 0040 1436 026MP-2 10 10780 124 0886 0071 1647 040MP-2 15 9221 576 0850 0067 1876 132MP-2 20 9336 386 0874 0057 2003 103

influenced by increasing the concentration of the hardeningsolution but it was noticed that hardening solutions withlower molarity of CuCl

2(05 and 10) yielded microspheres

with higher sphericity values On the other hand particlesphericity was lower in samples prepared with a higherpolymer concentration with the exception of samples MP-1 20 and MP-2 20 which were prepared with 2M of CuCl

2

hardening solution Previously reported results confirmedthat increasing the concentration of CMC solutions lin-early increases the solution viscosity [28] and sphericity ofparticles Our results generally coincide with findings thatoverly viscous polymer solutions (2 in our case) form lessspherical tail-shaped particles [29] and particles with a roughsurface [30]

Table 2 also shows the results of atomic absorptionanalysis for copper content Copper content in the preparedmicroparticles ranged from 1013 plusmn 065 to 2003 plusmn 103 gkgIt was observed that the content significantly increased with

increases in hardening solution concentration as well aspolymer concentration

The samples of microspheres with the highest sphericity(MP-1 05 MP-1 10 MP-2 05 and MP-2 10) which is onethe most important criteria for the preparation of particlesystems were then investigated for swelling capacity in phos-phate buffers with pH 45 [31] and pH 60 [32] respectivelyto simulate natural and inflamed vaginal conditions and topredict their behaviour on vaginal mucosa for a 20-hourtime interval (20-hour interval represents an estimated timeof therapy) The obtained results can be seen in Figure 2for pH 45 and in Figure 3 for pH 60 Figure 4 showsimages of microparticles after a 20-hour swelling capacitytest in a pH 60 buffer Generally it was observed that theswelling capacity of all samples gradually increased duringthe test Selected samples exhibited pH-responsive behaviourwhich differed based on the tested environments swellingwas substantially higher at pH 60 [33] It is obvious from

6 BioMed Research International

00

01

02

03

04

05

06

07

08

09

0369 0184 0092 0046 0023 0012

A

c (mgmL)

(a)

00

01

02

03

04

05

06

07

08

09

3871 1936 0968 0484 0242 0121

A

c (mgmL)

(b)

00

01

02

03

04

05

06

07

08

09

0707 0353 0177 0088 0044 0022

A

c (mgmL)

20hrs

(c)

00

01

02

03

04

05

06

07

08

09

4148 2074 1037 0519 0259 0130

20hrs

A

c (mgmL)

(d)

Figure 5 MIC results of CMCmicroparticles against Escherichia coli (a) MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

Figures 2 and 3 that sample MP-1 05 prepared with a less-concentrated hardening solution (05M) and a lower CMCconcentration (1) had the highest swelling capacity at bothpH values (3662 at pH 45 after 20 hrs 52963 at pH60 after 20 hrs) These values of formulation variables ledto the creation of favourable conditions for water uptakeinto the microsphere matrix resulting in the creation of anamorphous gel structure at pH 60 (see Figure 4(a)) Also theenormous loss of the blue colour in comparison with sampleMP-2 10 (Figure 4(d)) can be noticed probably related tohigh water uptake and higher release of the copper ionswhich are responsible for the blue colour This is uniquelyin accordance with previously published data saying thatincreased polymer or hardening solution concentrations cansignificantly reduce water uptake due to the increased densityof the polymer network [34] At pH 60 sample MP-2 05followed with swelling capacity of 24360 in 20 hrs At pH45 however its swelling was comparable with samples cross-linked with 1M CuCl

2 which exhibited a lower swelling

capacity ranging from 1372 to 1607 after 20 hrs at pH 45and 4330ndash5391 at pH 60 (results for pH 60 are clearlyevident in Figures 4(b) and 4(d)) No influence on swellingcapacity as a result of particle size was observed

The mechanism for the antibacterial activity of Cu+ions is based on their energetically easier movement acrossa lipid bilayer and uptake by the cell generating reactiveoxygen species leading to lipid peroxidation and proteinoxidation [35] The excess of copper causes a decrease in themembrane integrity of a microorganism which causes theparticular cell to leak nutritional elements like potassiumand glutamate which lead to desiccation and ultimatelycell death [36] The antimicrobial activity of selected CMCmicroparticles cross-linked by Cu2+ was evaluated by MICtest using Gram-negative Escherichia coli and Candida albi-cans yeast (see Figures 5 and 6) The test expresses theminimum concentration of antimicrobial agent that inhibitsthe visible growth of microorganisms [37] The test wastriplicated For E coli MIC values after 20 hrs of incubationwere 0184 0484 0353 and 0519mgmL for samples MP-1 05 MP-1 10 MP-2 05 and MP-2 10 respectively MICvalues for C albicans were higher for the same samples0740 1639 0598 and 1087mgmL respectively HigherMIC values for C albicans in comparison with the valuesfor E coli can be explained by the higher resistance of theyeast to the copperrsquos antibacterial effect Weissman et alconfirm this reporting on the isolation of two genes involved

BioMed Research International 7

00

01

02

03

04

05

06

07

08

09

10

0740 0370 0185 0093 0046 0023

A

c (mgmL)

(a)

00

01

02

03

04

05

06

07

08

09

10

3277 1639 0819 0410 0205 0102

A

c (mgmL)

(b)

0001020304050607080910

0598 0299 0150 0075 0037 0019

20hrs

A

c (mgmL)

(c)

0001020304050607080910

2173 1087 0543 0272 0136 0068

A

20hrs

c (mgmL)

(d)

Figure 6 MIC results of CMC microparticles against Candida albicans (a) MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

in copper detoxification in C albicans metallothioneinCaCUP1 and a copper-transporting P-type ATPase CaCRP1[38] These two genes account for C albicansrsquos resistanceto copper and consequently the vast differences in ourresults

The obtained MIC values prove substantial antimicrobialactivity against both microorganisms tested in this study andare in good agreementwith copperMIC values against strainsof E coli and C albicans described by different authors inother studies Martınez Medina et al reported MIC valuesfor Cu(II) complexes of 0375mgmL and gt15mgmL for Ecoli and C albicans respectively [39] Copper nanoparticlesprepared by Zain et al (200 nm) showed MIC values of0433mgL for E coli [40] and those prepared by Rupareliaet al (9 nm) showed MIC values of 0280mgmL for E coli[41]

Despite the lower content of copper in microspheresthe samples prepared using the less-concentrated CuCl

2

(05M) solutionmdashMP-1 05 and MP-2 05mdashexhibited lowerMIC values and so better antibacterial activity against bothtested pathogens in comparison with samples cross-linked in

the more potent 1M CuCl2solutionThese results confirmed

the great influence of the microparticle swellability on MICvalues Both samples exhibited high swelling capacity at pH60 (see Figure 3) and thus good conditions for the releaseof copper ions from particles (pH of LB and YNB mediumwas also close to 60) Against E coli sample MP-1 05 wasfound to be the most effective as it probably exhibited fastercopper releaseThis could be a result of the higher cumulationof copper ions on more rougher surface (see Figures 1(a)and 1(b)) and also smaller particle size (see Table 2) and thuslarger surface area [42] These characteristics could promotefaster swelling leading to the creation of an amorphousstructure during the 20-hour interval (Figure 4(a)) Againstthe more resistant C albicans however the most effectivesample was MP-2 05 exhibiting probably more uniformcopper release due to the higher CMC concentraction (2)more uniform copper distribution in the microspheres andlarger particle size (see Table 2) This theory is also sup-ported by the fact that the MP-2 05 microparticles main-tained their shape-specific structure after 20 hrs of swelling(Figure 4(c))

8 BioMed Research International

4 Conclusion

In this experimental work CMC dispersions of two differentconcentrations (1 and 2) were cross-linked by copper (II)ions (05 1 15 or 20MCuCl

2) to prepare microspheres with

antimicrobial activity against frequently occurring vaginalpathogens Escherichia coli and Candida albicans All testedsamples exhibited stable but pH responsive behaviour inenvironments corresponding with natural and inflamed vagi-nal conditions and proved substantial antimicrobial activityagainst both pathogens The most effective samples werethose hardened in a less-concentrated CuCl

2(05M) solu-

tion Unexpectedly a crucial parameter for microsphereantimicrobial activity was not found in the copper contentbut in the swelling capacity of the microparticles and in thedegree of CMC surface shrinkingThe sample prepared usinga 1 CMC dispersion cross-linked by 05M CuCl

2seemed to

be the most suitable for potential vaginal use not only due toits antibacterial activity but also due to its gradual change to anonspecific-shaped gel which is preferable when consideringvaginal dosage forms

Conflict of Interests

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

References

[1] L Fan M Peng Z Zhou et al ldquoModification of carboxymethylcellulose Grafted with collagen peptide and its antioxidantaktivityrdquo Carbohydrate Polymers vol 112 pp 32ndash38 2014

[2] N HaleemM Arsah M Shahid and AM Tahir ldquoSynthesis ofcarboxymethyl cellulose fromwaste of cotton ginning industryrdquoCarbohydrate Polymers vol 113 pp 249ndash255 2014

[3] J-F Su Z Huang X-Y Yuan X-YWang andM Li ldquoStructureand properties of carboxymethyl cellulosesoy protein isolateblend edible films crosslinked by Maillard reactionsrdquo Carbohy-drate Polymers vol 79 no 1 pp 145ndash153 2010

[4] H Kono ldquoCharacterization and properties of carboxymethylcellulose hydrogels crosslinked by polyethylene glycolrdquo Carbo-hydrate Polymers vol 106 no 1 pp 84ndash93 2014

[5] E A Hassn M L Hassan N C Moorefield and GR Newkome ldquoNew supramolecular metallo-terpyridine car-boxymethyl cellulose derivativeswith antimicrobial propertiesrdquoCarbohydrate Polymers vol 116 pp 2ndash8 2014

[6] S Sayanjali B Ghanbarzadeh and S Ghiassifar ldquoEvaluation ofantimicrobial and physical properties of edible film based oncarboxymethyl cellulose containing potassium sorbate on somemycotoxigenic Aspergillus species in fresh pistachiosrdquo LWTmdashFood Science and Technology vol 44 no 4 pp 1133ndash1138 2011

[7] A A Hebeish M H El-Rafie F A Abdel-Mohdy E S Abdel-Halim and H E Emam ldquoCarboxymethyl cellulose for greensynthesis and stabilization of silver nanoparticlesrdquo Carbohy-drate Polymers vol 82 no 3 pp 933ndash941 2010

[8] K Liu Y Xu X Lin et al ldquoSynergistic effects of guanidine-grafted CMC on enhancing antimicrobial activity and drystrength of paperrdquo Carbohydrate Polymers vol 110 pp 382ndash3872014

[9] L Upadhyaya J Singh V Agarwal and R P Tewari ldquoBiomed-ical applications of carboxymethyl chitosansrdquo CarbohydratePolymers vol 91 no 1 pp 452ndash466 2013

[10] A-H Yu H-Y Hsieh J-C Pang et al ldquoActive films fromwater-soluble chitosancellulose composites incorporating releasablecaffeic acid for inhibition of lipid oxidation in fish oil emul-sionsrdquo Food Hydrocolloids vol 32 no 1 pp 9ndash19 2013

[11] T Zhong G S Oporto J Jaczynski A T Tesfai and JArmstrong ldquoAntimicrobial properties of the hybrid coppernanoparticles-carboxymethyl celluloserdquo Wood and Fiber Sci-ence vol 45 no 2 pp 215ndash222 2013

[12] G E Jackson P M May and D R Williams ldquoMetal-ligandcomplexes involved in rheumatoid arthritis-I justificationsfor copper administrationrdquo Journal of Inorganic and NuclearChemistry vol 40 no 6 pp 1189ndash1194 1978

[13] O A El-Gammal E A Elmorsy and Y E Sherif ldquoEvaluationof the anti-inflammatory and analgesic effects of Cu(II) andZn(II) complexes derived from 2-(naphthalen-1-yloxy)-119873

1015840

-(1-(pyridin-2-1) ethylidene) acetohydraziderdquo Spectrochimica ActaPart A Molecular and Biomolecular Spectroscopy vol 120 pp332ndash339 2014

[14] S Odisitse and G E Jackson ldquoIn vitro and in vivo studiesof NN1015840-bis[2(2-pyridyl)-methyl]pyridine-26-dicarboxamide-copper(II) and rheumatoid arthritisrdquo Polyhedron vol 27 no 1pp 453ndash464 2008

[15] A Hussain and F Ahsan ldquoThe vagina as a route for systemicdrug deliveryrdquo Journal of Controlled Release vol 103 no 2 pp301ndash313 2005

[16] L M Ensign T E Hoen K Maisel R A Cone and J S HanesldquoEnhanced vaginal drug delivery through the use of hypotonicformulations that induce fluid uptakerdquo Biomaterials vol 34 no28 pp 6922ndash6929 2013

[17] B B Saxena M Singh R M Gospin C C Chu and W JLedger ldquoEfficacy of nonhormonal vaginal contraceptives from ahydrogel delivery systemrdquo Contraception vol 70 no 3 pp 213ndash219 2004

[18] M L Gonzalez-Rodrıguez M A Holgado C Sanchez-Lafuente A M Rabasco and A Fini ldquoAlginatechitosan par-ticulate systems for sodium diclofenac releaserdquo InternationalJournal of Pharmaceutics vol 232 no 1-2 pp 225ndash234 2002

[19] C H Goh P W S Heng and L W Chan ldquoAlginates as auseful natural polymer for microencapsulation and therapeuticapplicationsrdquo Carbohydrate Polymers vol 88 no 1 pp 1ndash122012

[20] W-C Lin D-G Yu and M-C Yang ldquopH-sensitive poly-electrolyte complex gel microspheres composed of chitosansodium tripolyphosphatedextran sulfate swelling kinetics anddrug delivery propertiesrdquo Colloids and Surfaces B Biointerfacesvol 44 no 2-3 pp 143ndash151 2005

[21] K G H Desai and H J Park ldquoPreparation and characteriza-tion of drug-loaded chitosan-tripolyphosphatemicrospheres byspray dryingrdquo Drug Development Research vol 64 no 2 pp114ndash128 2005

[22] K M Rao B Mallikarjuna K K Rao M N Prabhakar K CRao and M C Subha ldquoPreparation and characterization of pHsensitive poly(vinyl alcohol)sodium carboxymethyl celluloseIPN microspheres for in vitro release studies of an anti-cancerdrugrdquo Polymer Bulletin vol 68 no 7 pp 1905ndash1919 2012

[23] M A Saleem Y D Murali M D Naheed P Jaydeep andM Dhaval ldquoPrepapation and evaluation of valsartan loadedhydrogel beadsrdquo International Research Journal of Pharmacyvol 3 no 6 pp 80ndash85 2012

BioMed Research International 9

[24] Y Y Yang J P Wan T S Chung P K Pallathadka S Ng andJ Heller ldquoPOE-PEG-POE triblock copolymeric microspherescontaining protein I Preparation and characterizationrdquo Journalof Controlled Release vol 75 no 1-2 pp 115ndash128 2001

[25] H Brandenberger and F Widmer ldquoA new multinozzle encap-sulationimmobilisation system to produce uniform beads ofalginaterdquo Journal of Biotechnology vol 63 no 1 pp 73ndash80 1998

[26] E-S Chan B-B Lee P Ravindra and D Poncelet ldquoPredictionmodels for shape and size of ca-alginate macrobeads producedthrough extrusion-dripping methodrdquo Journal of Colloid andInterface Science vol 338 no 1 pp 63ndash72 2009

[27] P B Deasy and M F L Law ldquoUse of extrusion-spheronizationto develop an improved oral dosage form of indomethacinrdquoInternational Journal of Pharmaceutics vol 148 no 2 pp 201ndash209 1997

[28] X H Yang and W L Zhu ldquoViscosity properties of sodiumcarboxymethylcellulose solutionsrdquo Cellulose vol 14 no 5 pp409ndash417 2007

[29] E-S Chan ldquoPreparation of Ca-alginate beads containing highoil content influence of process variables on encapsulationefficiency and bead propertiesrdquo Carbohydrate Polymers vol 84no 4 pp 1267ndash1275 2011

[30] M S Kim S J Park B K Gu and C-H Kim ldquoIonicallycrosslinked alginate-carboxymethyl cellulose beads for thedelivery of protein therapeuticsrdquo Applied Surface Science vol262 pp 28ndash33 2012

[31] C Valenta ldquoThe use of mucoadhesive polymers in vaginaldeliveryrdquo Advanced Drug Delivery Reviews vol 57 no 11 pp1692ndash1712 2005

[32] S F Borges J G L Silva and P C M Teixeira ldquoSurvival andbiofilm formation of Listeria monocytogenes in simulated vagi-nal fluid influence of pH and strain originrdquo FEMS Immunologyand Medical Microbiology vol 62 no 3 pp 315ndash320 2011

[33] R Barbucci A Magnani and M Consumi ldquoSwelling behav-ior of carboxymethylcellulose hydrogels in relation to cross-linking pH and charge densityrdquo Macromolecules vol 33 no20 pp 7475ndash7480 2000

[34] J Berger M Reist J M Mayer O Felt N A Peppas and RGurny ldquoStructure and interactions in covalently and ionicallycrosslinked chitosan hydrogels for biomedical applicationsrdquoEuropean Journal of Pharmaceutics and Biopharmaceutics vol57 no 1 pp 19ndash34 2004

[35] U Bogdanovic V Lazic V Vodnik M Budimir Z Markovicand S Dimitrijevic ldquoCopper nanoparticles with high antimi-crobial activityrdquoMaterials Letters vol 128 pp 75ndash78 2014

[36] J Konieczny and Z Rdzawski ldquoAntibacterial properties of cop-per and its alloysrdquoArchives ofMaterials Science and Engineeringvol 56 no 2 pp 53ndash60 2012

[37] S M Magana P Quintana D H Aguilar et al ldquoAntibacterialactivity of montmorillonites modified with silverrdquo Journal ofMolecular Catalysis A Chemical vol 281 no 1-2 pp 192ndash1992008

[38] Z Weissman I Berdicevsky B-Z Cavari and D KornitzerldquoThe high copper tolerance ofCandida albicans is mediated by aP-type ATPaserdquo Proceedings of the National Academy of Sciencesof the United States of America vol 97 no 7 pp 3520ndash35252000

[39] J J Martınez Medina M S Islas L L Lopez Tevez E GFerrer N B Okulik and P A M Williams ldquoCopper(II) com-plexes with cyanoguanidine and o-phenanthroline theoreticalstudies in vitro antimicrobial activity and alkaline phosphatase

inhibitory effectrdquo Journal of Molecular Structure vol 1058 no1 pp 298ndash307 2014

[40] N M Zain A G F Stapley and G Shama ldquoGreen synthesis ofsilver and copper nanoparticles using ascorbic acid and chitosanfor antimicrobial applicationsrdquo Carbohydrate Polymers vol 112pp 195ndash202 2014

[41] J P Ruparelia A K Chatterjee S P Duttagupta and SMukherji ldquoStrain specificity in antimicrobial activity of silverand copper nanoparticlesrdquo Acta Biomaterialia vol 4 no 3 pp707ndash716 2008

[42] J Zhao H J Feng H Q Tang and J H Zheng ldquoBactericidaland corrosive properties of silver implanted TiN thin filmscoated on AISI317 stainless steelrdquo Surface and Coatings Technol-ogy vol 201 no 9ndash11 pp 5676ndash5679 2007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 5: Research Article Antimicrobial Properties of ...downloads.hindawi.com/journals/bmri/2015/790720.pdf · Research Article Antimicrobial Properties of Microparticles Based on ... ect

BioMed Research International 5

(a) (b)

(c) (d)

Figure 4 Optical microscope images of CMC particles after 20-hour swelling capacity test in pH 60 buffer bars correspond to 1000120583m (a)MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

Table 2 Microparticle characteristics equivalent diameter sphericity factor and copper content

Sample ED (120583m) SD (120583m) SF SD Copper content (gkg) SD (gkg)MP-1 05 7757 302 0906 0044 1013 065MP-1 10 8885 584 0934 0038 1436 231MP-1 15 7381 303 0887 0056 1523 019MP-1 20 7986 879 0855 0059 1730 215MP-2 05 10169 228 0891 0040 1436 026MP-2 10 10780 124 0886 0071 1647 040MP-2 15 9221 576 0850 0067 1876 132MP-2 20 9336 386 0874 0057 2003 103

influenced by increasing the concentration of the hardeningsolution but it was noticed that hardening solutions withlower molarity of CuCl

2(05 and 10) yielded microspheres

with higher sphericity values On the other hand particlesphericity was lower in samples prepared with a higherpolymer concentration with the exception of samples MP-1 20 and MP-2 20 which were prepared with 2M of CuCl

2

hardening solution Previously reported results confirmedthat increasing the concentration of CMC solutions lin-early increases the solution viscosity [28] and sphericity ofparticles Our results generally coincide with findings thatoverly viscous polymer solutions (2 in our case) form lessspherical tail-shaped particles [29] and particles with a roughsurface [30]

Table 2 also shows the results of atomic absorptionanalysis for copper content Copper content in the preparedmicroparticles ranged from 1013 plusmn 065 to 2003 plusmn 103 gkgIt was observed that the content significantly increased with

increases in hardening solution concentration as well aspolymer concentration

The samples of microspheres with the highest sphericity(MP-1 05 MP-1 10 MP-2 05 and MP-2 10) which is onethe most important criteria for the preparation of particlesystems were then investigated for swelling capacity in phos-phate buffers with pH 45 [31] and pH 60 [32] respectivelyto simulate natural and inflamed vaginal conditions and topredict their behaviour on vaginal mucosa for a 20-hourtime interval (20-hour interval represents an estimated timeof therapy) The obtained results can be seen in Figure 2for pH 45 and in Figure 3 for pH 60 Figure 4 showsimages of microparticles after a 20-hour swelling capacitytest in a pH 60 buffer Generally it was observed that theswelling capacity of all samples gradually increased duringthe test Selected samples exhibited pH-responsive behaviourwhich differed based on the tested environments swellingwas substantially higher at pH 60 [33] It is obvious from

6 BioMed Research International

00

01

02

03

04

05

06

07

08

09

0369 0184 0092 0046 0023 0012

A

c (mgmL)

(a)

00

01

02

03

04

05

06

07

08

09

3871 1936 0968 0484 0242 0121

A

c (mgmL)

(b)

00

01

02

03

04

05

06

07

08

09

0707 0353 0177 0088 0044 0022

A

c (mgmL)

20hrs

(c)

00

01

02

03

04

05

06

07

08

09

4148 2074 1037 0519 0259 0130

20hrs

A

c (mgmL)

(d)

Figure 5 MIC results of CMCmicroparticles against Escherichia coli (a) MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

Figures 2 and 3 that sample MP-1 05 prepared with a less-concentrated hardening solution (05M) and a lower CMCconcentration (1) had the highest swelling capacity at bothpH values (3662 at pH 45 after 20 hrs 52963 at pH60 after 20 hrs) These values of formulation variables ledto the creation of favourable conditions for water uptakeinto the microsphere matrix resulting in the creation of anamorphous gel structure at pH 60 (see Figure 4(a)) Also theenormous loss of the blue colour in comparison with sampleMP-2 10 (Figure 4(d)) can be noticed probably related tohigh water uptake and higher release of the copper ionswhich are responsible for the blue colour This is uniquelyin accordance with previously published data saying thatincreased polymer or hardening solution concentrations cansignificantly reduce water uptake due to the increased densityof the polymer network [34] At pH 60 sample MP-2 05followed with swelling capacity of 24360 in 20 hrs At pH45 however its swelling was comparable with samples cross-linked with 1M CuCl

2 which exhibited a lower swelling

capacity ranging from 1372 to 1607 after 20 hrs at pH 45and 4330ndash5391 at pH 60 (results for pH 60 are clearlyevident in Figures 4(b) and 4(d)) No influence on swellingcapacity as a result of particle size was observed

The mechanism for the antibacterial activity of Cu+ions is based on their energetically easier movement acrossa lipid bilayer and uptake by the cell generating reactiveoxygen species leading to lipid peroxidation and proteinoxidation [35] The excess of copper causes a decrease in themembrane integrity of a microorganism which causes theparticular cell to leak nutritional elements like potassiumand glutamate which lead to desiccation and ultimatelycell death [36] The antimicrobial activity of selected CMCmicroparticles cross-linked by Cu2+ was evaluated by MICtest using Gram-negative Escherichia coli and Candida albi-cans yeast (see Figures 5 and 6) The test expresses theminimum concentration of antimicrobial agent that inhibitsthe visible growth of microorganisms [37] The test wastriplicated For E coli MIC values after 20 hrs of incubationwere 0184 0484 0353 and 0519mgmL for samples MP-1 05 MP-1 10 MP-2 05 and MP-2 10 respectively MICvalues for C albicans were higher for the same samples0740 1639 0598 and 1087mgmL respectively HigherMIC values for C albicans in comparison with the valuesfor E coli can be explained by the higher resistance of theyeast to the copperrsquos antibacterial effect Weissman et alconfirm this reporting on the isolation of two genes involved

BioMed Research International 7

00

01

02

03

04

05

06

07

08

09

10

0740 0370 0185 0093 0046 0023

A

c (mgmL)

(a)

00

01

02

03

04

05

06

07

08

09

10

3277 1639 0819 0410 0205 0102

A

c (mgmL)

(b)

0001020304050607080910

0598 0299 0150 0075 0037 0019

20hrs

A

c (mgmL)

(c)

0001020304050607080910

2173 1087 0543 0272 0136 0068

A

20hrs

c (mgmL)

(d)

Figure 6 MIC results of CMC microparticles against Candida albicans (a) MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

in copper detoxification in C albicans metallothioneinCaCUP1 and a copper-transporting P-type ATPase CaCRP1[38] These two genes account for C albicansrsquos resistanceto copper and consequently the vast differences in ourresults

The obtained MIC values prove substantial antimicrobialactivity against both microorganisms tested in this study andare in good agreementwith copperMIC values against strainsof E coli and C albicans described by different authors inother studies Martınez Medina et al reported MIC valuesfor Cu(II) complexes of 0375mgmL and gt15mgmL for Ecoli and C albicans respectively [39] Copper nanoparticlesprepared by Zain et al (200 nm) showed MIC values of0433mgL for E coli [40] and those prepared by Rupareliaet al (9 nm) showed MIC values of 0280mgmL for E coli[41]

Despite the lower content of copper in microspheresthe samples prepared using the less-concentrated CuCl

2

(05M) solutionmdashMP-1 05 and MP-2 05mdashexhibited lowerMIC values and so better antibacterial activity against bothtested pathogens in comparison with samples cross-linked in

the more potent 1M CuCl2solutionThese results confirmed

the great influence of the microparticle swellability on MICvalues Both samples exhibited high swelling capacity at pH60 (see Figure 3) and thus good conditions for the releaseof copper ions from particles (pH of LB and YNB mediumwas also close to 60) Against E coli sample MP-1 05 wasfound to be the most effective as it probably exhibited fastercopper releaseThis could be a result of the higher cumulationof copper ions on more rougher surface (see Figures 1(a)and 1(b)) and also smaller particle size (see Table 2) and thuslarger surface area [42] These characteristics could promotefaster swelling leading to the creation of an amorphousstructure during the 20-hour interval (Figure 4(a)) Againstthe more resistant C albicans however the most effectivesample was MP-2 05 exhibiting probably more uniformcopper release due to the higher CMC concentraction (2)more uniform copper distribution in the microspheres andlarger particle size (see Table 2) This theory is also sup-ported by the fact that the MP-2 05 microparticles main-tained their shape-specific structure after 20 hrs of swelling(Figure 4(c))

8 BioMed Research International

4 Conclusion

In this experimental work CMC dispersions of two differentconcentrations (1 and 2) were cross-linked by copper (II)ions (05 1 15 or 20MCuCl

2) to prepare microspheres with

antimicrobial activity against frequently occurring vaginalpathogens Escherichia coli and Candida albicans All testedsamples exhibited stable but pH responsive behaviour inenvironments corresponding with natural and inflamed vagi-nal conditions and proved substantial antimicrobial activityagainst both pathogens The most effective samples werethose hardened in a less-concentrated CuCl

2(05M) solu-

tion Unexpectedly a crucial parameter for microsphereantimicrobial activity was not found in the copper contentbut in the swelling capacity of the microparticles and in thedegree of CMC surface shrinkingThe sample prepared usinga 1 CMC dispersion cross-linked by 05M CuCl

2seemed to

be the most suitable for potential vaginal use not only due toits antibacterial activity but also due to its gradual change to anonspecific-shaped gel which is preferable when consideringvaginal dosage forms

Conflict of Interests

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

References

[1] L Fan M Peng Z Zhou et al ldquoModification of carboxymethylcellulose Grafted with collagen peptide and its antioxidantaktivityrdquo Carbohydrate Polymers vol 112 pp 32ndash38 2014

[2] N HaleemM Arsah M Shahid and AM Tahir ldquoSynthesis ofcarboxymethyl cellulose fromwaste of cotton ginning industryrdquoCarbohydrate Polymers vol 113 pp 249ndash255 2014

[3] J-F Su Z Huang X-Y Yuan X-YWang andM Li ldquoStructureand properties of carboxymethyl cellulosesoy protein isolateblend edible films crosslinked by Maillard reactionsrdquo Carbohy-drate Polymers vol 79 no 1 pp 145ndash153 2010

[4] H Kono ldquoCharacterization and properties of carboxymethylcellulose hydrogels crosslinked by polyethylene glycolrdquo Carbo-hydrate Polymers vol 106 no 1 pp 84ndash93 2014

[5] E A Hassn M L Hassan N C Moorefield and GR Newkome ldquoNew supramolecular metallo-terpyridine car-boxymethyl cellulose derivativeswith antimicrobial propertiesrdquoCarbohydrate Polymers vol 116 pp 2ndash8 2014

[6] S Sayanjali B Ghanbarzadeh and S Ghiassifar ldquoEvaluation ofantimicrobial and physical properties of edible film based oncarboxymethyl cellulose containing potassium sorbate on somemycotoxigenic Aspergillus species in fresh pistachiosrdquo LWTmdashFood Science and Technology vol 44 no 4 pp 1133ndash1138 2011

[7] A A Hebeish M H El-Rafie F A Abdel-Mohdy E S Abdel-Halim and H E Emam ldquoCarboxymethyl cellulose for greensynthesis and stabilization of silver nanoparticlesrdquo Carbohy-drate Polymers vol 82 no 3 pp 933ndash941 2010

[8] K Liu Y Xu X Lin et al ldquoSynergistic effects of guanidine-grafted CMC on enhancing antimicrobial activity and drystrength of paperrdquo Carbohydrate Polymers vol 110 pp 382ndash3872014

[9] L Upadhyaya J Singh V Agarwal and R P Tewari ldquoBiomed-ical applications of carboxymethyl chitosansrdquo CarbohydratePolymers vol 91 no 1 pp 452ndash466 2013

[10] A-H Yu H-Y Hsieh J-C Pang et al ldquoActive films fromwater-soluble chitosancellulose composites incorporating releasablecaffeic acid for inhibition of lipid oxidation in fish oil emul-sionsrdquo Food Hydrocolloids vol 32 no 1 pp 9ndash19 2013

[11] T Zhong G S Oporto J Jaczynski A T Tesfai and JArmstrong ldquoAntimicrobial properties of the hybrid coppernanoparticles-carboxymethyl celluloserdquo Wood and Fiber Sci-ence vol 45 no 2 pp 215ndash222 2013

[12] G E Jackson P M May and D R Williams ldquoMetal-ligandcomplexes involved in rheumatoid arthritis-I justificationsfor copper administrationrdquo Journal of Inorganic and NuclearChemistry vol 40 no 6 pp 1189ndash1194 1978

[13] O A El-Gammal E A Elmorsy and Y E Sherif ldquoEvaluationof the anti-inflammatory and analgesic effects of Cu(II) andZn(II) complexes derived from 2-(naphthalen-1-yloxy)-119873

1015840

-(1-(pyridin-2-1) ethylidene) acetohydraziderdquo Spectrochimica ActaPart A Molecular and Biomolecular Spectroscopy vol 120 pp332ndash339 2014

[14] S Odisitse and G E Jackson ldquoIn vitro and in vivo studiesof NN1015840-bis[2(2-pyridyl)-methyl]pyridine-26-dicarboxamide-copper(II) and rheumatoid arthritisrdquo Polyhedron vol 27 no 1pp 453ndash464 2008

[15] A Hussain and F Ahsan ldquoThe vagina as a route for systemicdrug deliveryrdquo Journal of Controlled Release vol 103 no 2 pp301ndash313 2005

[16] L M Ensign T E Hoen K Maisel R A Cone and J S HanesldquoEnhanced vaginal drug delivery through the use of hypotonicformulations that induce fluid uptakerdquo Biomaterials vol 34 no28 pp 6922ndash6929 2013

[17] B B Saxena M Singh R M Gospin C C Chu and W JLedger ldquoEfficacy of nonhormonal vaginal contraceptives from ahydrogel delivery systemrdquo Contraception vol 70 no 3 pp 213ndash219 2004

[18] M L Gonzalez-Rodrıguez M A Holgado C Sanchez-Lafuente A M Rabasco and A Fini ldquoAlginatechitosan par-ticulate systems for sodium diclofenac releaserdquo InternationalJournal of Pharmaceutics vol 232 no 1-2 pp 225ndash234 2002

[19] C H Goh P W S Heng and L W Chan ldquoAlginates as auseful natural polymer for microencapsulation and therapeuticapplicationsrdquo Carbohydrate Polymers vol 88 no 1 pp 1ndash122012

[20] W-C Lin D-G Yu and M-C Yang ldquopH-sensitive poly-electrolyte complex gel microspheres composed of chitosansodium tripolyphosphatedextran sulfate swelling kinetics anddrug delivery propertiesrdquo Colloids and Surfaces B Biointerfacesvol 44 no 2-3 pp 143ndash151 2005

[21] K G H Desai and H J Park ldquoPreparation and characteriza-tion of drug-loaded chitosan-tripolyphosphatemicrospheres byspray dryingrdquo Drug Development Research vol 64 no 2 pp114ndash128 2005

[22] K M Rao B Mallikarjuna K K Rao M N Prabhakar K CRao and M C Subha ldquoPreparation and characterization of pHsensitive poly(vinyl alcohol)sodium carboxymethyl celluloseIPN microspheres for in vitro release studies of an anti-cancerdrugrdquo Polymer Bulletin vol 68 no 7 pp 1905ndash1919 2012

[23] M A Saleem Y D Murali M D Naheed P Jaydeep andM Dhaval ldquoPrepapation and evaluation of valsartan loadedhydrogel beadsrdquo International Research Journal of Pharmacyvol 3 no 6 pp 80ndash85 2012

BioMed Research International 9

[24] Y Y Yang J P Wan T S Chung P K Pallathadka S Ng andJ Heller ldquoPOE-PEG-POE triblock copolymeric microspherescontaining protein I Preparation and characterizationrdquo Journalof Controlled Release vol 75 no 1-2 pp 115ndash128 2001

[25] H Brandenberger and F Widmer ldquoA new multinozzle encap-sulationimmobilisation system to produce uniform beads ofalginaterdquo Journal of Biotechnology vol 63 no 1 pp 73ndash80 1998

[26] E-S Chan B-B Lee P Ravindra and D Poncelet ldquoPredictionmodels for shape and size of ca-alginate macrobeads producedthrough extrusion-dripping methodrdquo Journal of Colloid andInterface Science vol 338 no 1 pp 63ndash72 2009

[27] P B Deasy and M F L Law ldquoUse of extrusion-spheronizationto develop an improved oral dosage form of indomethacinrdquoInternational Journal of Pharmaceutics vol 148 no 2 pp 201ndash209 1997

[28] X H Yang and W L Zhu ldquoViscosity properties of sodiumcarboxymethylcellulose solutionsrdquo Cellulose vol 14 no 5 pp409ndash417 2007

[29] E-S Chan ldquoPreparation of Ca-alginate beads containing highoil content influence of process variables on encapsulationefficiency and bead propertiesrdquo Carbohydrate Polymers vol 84no 4 pp 1267ndash1275 2011

[30] M S Kim S J Park B K Gu and C-H Kim ldquoIonicallycrosslinked alginate-carboxymethyl cellulose beads for thedelivery of protein therapeuticsrdquo Applied Surface Science vol262 pp 28ndash33 2012

[31] C Valenta ldquoThe use of mucoadhesive polymers in vaginaldeliveryrdquo Advanced Drug Delivery Reviews vol 57 no 11 pp1692ndash1712 2005

[32] S F Borges J G L Silva and P C M Teixeira ldquoSurvival andbiofilm formation of Listeria monocytogenes in simulated vagi-nal fluid influence of pH and strain originrdquo FEMS Immunologyand Medical Microbiology vol 62 no 3 pp 315ndash320 2011

[33] R Barbucci A Magnani and M Consumi ldquoSwelling behav-ior of carboxymethylcellulose hydrogels in relation to cross-linking pH and charge densityrdquo Macromolecules vol 33 no20 pp 7475ndash7480 2000

[34] J Berger M Reist J M Mayer O Felt N A Peppas and RGurny ldquoStructure and interactions in covalently and ionicallycrosslinked chitosan hydrogels for biomedical applicationsrdquoEuropean Journal of Pharmaceutics and Biopharmaceutics vol57 no 1 pp 19ndash34 2004

[35] U Bogdanovic V Lazic V Vodnik M Budimir Z Markovicand S Dimitrijevic ldquoCopper nanoparticles with high antimi-crobial activityrdquoMaterials Letters vol 128 pp 75ndash78 2014

[36] J Konieczny and Z Rdzawski ldquoAntibacterial properties of cop-per and its alloysrdquoArchives ofMaterials Science and Engineeringvol 56 no 2 pp 53ndash60 2012

[37] S M Magana P Quintana D H Aguilar et al ldquoAntibacterialactivity of montmorillonites modified with silverrdquo Journal ofMolecular Catalysis A Chemical vol 281 no 1-2 pp 192ndash1992008

[38] Z Weissman I Berdicevsky B-Z Cavari and D KornitzerldquoThe high copper tolerance ofCandida albicans is mediated by aP-type ATPaserdquo Proceedings of the National Academy of Sciencesof the United States of America vol 97 no 7 pp 3520ndash35252000

[39] J J Martınez Medina M S Islas L L Lopez Tevez E GFerrer N B Okulik and P A M Williams ldquoCopper(II) com-plexes with cyanoguanidine and o-phenanthroline theoreticalstudies in vitro antimicrobial activity and alkaline phosphatase

inhibitory effectrdquo Journal of Molecular Structure vol 1058 no1 pp 298ndash307 2014

[40] N M Zain A G F Stapley and G Shama ldquoGreen synthesis ofsilver and copper nanoparticles using ascorbic acid and chitosanfor antimicrobial applicationsrdquo Carbohydrate Polymers vol 112pp 195ndash202 2014

[41] J P Ruparelia A K Chatterjee S P Duttagupta and SMukherji ldquoStrain specificity in antimicrobial activity of silverand copper nanoparticlesrdquo Acta Biomaterialia vol 4 no 3 pp707ndash716 2008

[42] J Zhao H J Feng H Q Tang and J H Zheng ldquoBactericidaland corrosive properties of silver implanted TiN thin filmscoated on AISI317 stainless steelrdquo Surface and Coatings Technol-ogy vol 201 no 9ndash11 pp 5676ndash5679 2007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 6: Research Article Antimicrobial Properties of ...downloads.hindawi.com/journals/bmri/2015/790720.pdf · Research Article Antimicrobial Properties of Microparticles Based on ... ect

6 BioMed Research International

00

01

02

03

04

05

06

07

08

09

0369 0184 0092 0046 0023 0012

A

c (mgmL)

(a)

00

01

02

03

04

05

06

07

08

09

3871 1936 0968 0484 0242 0121

A

c (mgmL)

(b)

00

01

02

03

04

05

06

07

08

09

0707 0353 0177 0088 0044 0022

A

c (mgmL)

20hrs

(c)

00

01

02

03

04

05

06

07

08

09

4148 2074 1037 0519 0259 0130

20hrs

A

c (mgmL)

(d)

Figure 5 MIC results of CMCmicroparticles against Escherichia coli (a) MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

Figures 2 and 3 that sample MP-1 05 prepared with a less-concentrated hardening solution (05M) and a lower CMCconcentration (1) had the highest swelling capacity at bothpH values (3662 at pH 45 after 20 hrs 52963 at pH60 after 20 hrs) These values of formulation variables ledto the creation of favourable conditions for water uptakeinto the microsphere matrix resulting in the creation of anamorphous gel structure at pH 60 (see Figure 4(a)) Also theenormous loss of the blue colour in comparison with sampleMP-2 10 (Figure 4(d)) can be noticed probably related tohigh water uptake and higher release of the copper ionswhich are responsible for the blue colour This is uniquelyin accordance with previously published data saying thatincreased polymer or hardening solution concentrations cansignificantly reduce water uptake due to the increased densityof the polymer network [34] At pH 60 sample MP-2 05followed with swelling capacity of 24360 in 20 hrs At pH45 however its swelling was comparable with samples cross-linked with 1M CuCl

2 which exhibited a lower swelling

capacity ranging from 1372 to 1607 after 20 hrs at pH 45and 4330ndash5391 at pH 60 (results for pH 60 are clearlyevident in Figures 4(b) and 4(d)) No influence on swellingcapacity as a result of particle size was observed

The mechanism for the antibacterial activity of Cu+ions is based on their energetically easier movement acrossa lipid bilayer and uptake by the cell generating reactiveoxygen species leading to lipid peroxidation and proteinoxidation [35] The excess of copper causes a decrease in themembrane integrity of a microorganism which causes theparticular cell to leak nutritional elements like potassiumand glutamate which lead to desiccation and ultimatelycell death [36] The antimicrobial activity of selected CMCmicroparticles cross-linked by Cu2+ was evaluated by MICtest using Gram-negative Escherichia coli and Candida albi-cans yeast (see Figures 5 and 6) The test expresses theminimum concentration of antimicrobial agent that inhibitsthe visible growth of microorganisms [37] The test wastriplicated For E coli MIC values after 20 hrs of incubationwere 0184 0484 0353 and 0519mgmL for samples MP-1 05 MP-1 10 MP-2 05 and MP-2 10 respectively MICvalues for C albicans were higher for the same samples0740 1639 0598 and 1087mgmL respectively HigherMIC values for C albicans in comparison with the valuesfor E coli can be explained by the higher resistance of theyeast to the copperrsquos antibacterial effect Weissman et alconfirm this reporting on the isolation of two genes involved

BioMed Research International 7

00

01

02

03

04

05

06

07

08

09

10

0740 0370 0185 0093 0046 0023

A

c (mgmL)

(a)

00

01

02

03

04

05

06

07

08

09

10

3277 1639 0819 0410 0205 0102

A

c (mgmL)

(b)

0001020304050607080910

0598 0299 0150 0075 0037 0019

20hrs

A

c (mgmL)

(c)

0001020304050607080910

2173 1087 0543 0272 0136 0068

A

20hrs

c (mgmL)

(d)

Figure 6 MIC results of CMC microparticles against Candida albicans (a) MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

in copper detoxification in C albicans metallothioneinCaCUP1 and a copper-transporting P-type ATPase CaCRP1[38] These two genes account for C albicansrsquos resistanceto copper and consequently the vast differences in ourresults

The obtained MIC values prove substantial antimicrobialactivity against both microorganisms tested in this study andare in good agreementwith copperMIC values against strainsof E coli and C albicans described by different authors inother studies Martınez Medina et al reported MIC valuesfor Cu(II) complexes of 0375mgmL and gt15mgmL for Ecoli and C albicans respectively [39] Copper nanoparticlesprepared by Zain et al (200 nm) showed MIC values of0433mgL for E coli [40] and those prepared by Rupareliaet al (9 nm) showed MIC values of 0280mgmL for E coli[41]

Despite the lower content of copper in microspheresthe samples prepared using the less-concentrated CuCl

2

(05M) solutionmdashMP-1 05 and MP-2 05mdashexhibited lowerMIC values and so better antibacterial activity against bothtested pathogens in comparison with samples cross-linked in

the more potent 1M CuCl2solutionThese results confirmed

the great influence of the microparticle swellability on MICvalues Both samples exhibited high swelling capacity at pH60 (see Figure 3) and thus good conditions for the releaseof copper ions from particles (pH of LB and YNB mediumwas also close to 60) Against E coli sample MP-1 05 wasfound to be the most effective as it probably exhibited fastercopper releaseThis could be a result of the higher cumulationof copper ions on more rougher surface (see Figures 1(a)and 1(b)) and also smaller particle size (see Table 2) and thuslarger surface area [42] These characteristics could promotefaster swelling leading to the creation of an amorphousstructure during the 20-hour interval (Figure 4(a)) Againstthe more resistant C albicans however the most effectivesample was MP-2 05 exhibiting probably more uniformcopper release due to the higher CMC concentraction (2)more uniform copper distribution in the microspheres andlarger particle size (see Table 2) This theory is also sup-ported by the fact that the MP-2 05 microparticles main-tained their shape-specific structure after 20 hrs of swelling(Figure 4(c))

8 BioMed Research International

4 Conclusion

In this experimental work CMC dispersions of two differentconcentrations (1 and 2) were cross-linked by copper (II)ions (05 1 15 or 20MCuCl

2) to prepare microspheres with

antimicrobial activity against frequently occurring vaginalpathogens Escherichia coli and Candida albicans All testedsamples exhibited stable but pH responsive behaviour inenvironments corresponding with natural and inflamed vagi-nal conditions and proved substantial antimicrobial activityagainst both pathogens The most effective samples werethose hardened in a less-concentrated CuCl

2(05M) solu-

tion Unexpectedly a crucial parameter for microsphereantimicrobial activity was not found in the copper contentbut in the swelling capacity of the microparticles and in thedegree of CMC surface shrinkingThe sample prepared usinga 1 CMC dispersion cross-linked by 05M CuCl

2seemed to

be the most suitable for potential vaginal use not only due toits antibacterial activity but also due to its gradual change to anonspecific-shaped gel which is preferable when consideringvaginal dosage forms

Conflict of Interests

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

References

[1] L Fan M Peng Z Zhou et al ldquoModification of carboxymethylcellulose Grafted with collagen peptide and its antioxidantaktivityrdquo Carbohydrate Polymers vol 112 pp 32ndash38 2014

[2] N HaleemM Arsah M Shahid and AM Tahir ldquoSynthesis ofcarboxymethyl cellulose fromwaste of cotton ginning industryrdquoCarbohydrate Polymers vol 113 pp 249ndash255 2014

[3] J-F Su Z Huang X-Y Yuan X-YWang andM Li ldquoStructureand properties of carboxymethyl cellulosesoy protein isolateblend edible films crosslinked by Maillard reactionsrdquo Carbohy-drate Polymers vol 79 no 1 pp 145ndash153 2010

[4] H Kono ldquoCharacterization and properties of carboxymethylcellulose hydrogels crosslinked by polyethylene glycolrdquo Carbo-hydrate Polymers vol 106 no 1 pp 84ndash93 2014

[5] E A Hassn M L Hassan N C Moorefield and GR Newkome ldquoNew supramolecular metallo-terpyridine car-boxymethyl cellulose derivativeswith antimicrobial propertiesrdquoCarbohydrate Polymers vol 116 pp 2ndash8 2014

[6] S Sayanjali B Ghanbarzadeh and S Ghiassifar ldquoEvaluation ofantimicrobial and physical properties of edible film based oncarboxymethyl cellulose containing potassium sorbate on somemycotoxigenic Aspergillus species in fresh pistachiosrdquo LWTmdashFood Science and Technology vol 44 no 4 pp 1133ndash1138 2011

[7] A A Hebeish M H El-Rafie F A Abdel-Mohdy E S Abdel-Halim and H E Emam ldquoCarboxymethyl cellulose for greensynthesis and stabilization of silver nanoparticlesrdquo Carbohy-drate Polymers vol 82 no 3 pp 933ndash941 2010

[8] K Liu Y Xu X Lin et al ldquoSynergistic effects of guanidine-grafted CMC on enhancing antimicrobial activity and drystrength of paperrdquo Carbohydrate Polymers vol 110 pp 382ndash3872014

[9] L Upadhyaya J Singh V Agarwal and R P Tewari ldquoBiomed-ical applications of carboxymethyl chitosansrdquo CarbohydratePolymers vol 91 no 1 pp 452ndash466 2013

[10] A-H Yu H-Y Hsieh J-C Pang et al ldquoActive films fromwater-soluble chitosancellulose composites incorporating releasablecaffeic acid for inhibition of lipid oxidation in fish oil emul-sionsrdquo Food Hydrocolloids vol 32 no 1 pp 9ndash19 2013

[11] T Zhong G S Oporto J Jaczynski A T Tesfai and JArmstrong ldquoAntimicrobial properties of the hybrid coppernanoparticles-carboxymethyl celluloserdquo Wood and Fiber Sci-ence vol 45 no 2 pp 215ndash222 2013

[12] G E Jackson P M May and D R Williams ldquoMetal-ligandcomplexes involved in rheumatoid arthritis-I justificationsfor copper administrationrdquo Journal of Inorganic and NuclearChemistry vol 40 no 6 pp 1189ndash1194 1978

[13] O A El-Gammal E A Elmorsy and Y E Sherif ldquoEvaluationof the anti-inflammatory and analgesic effects of Cu(II) andZn(II) complexes derived from 2-(naphthalen-1-yloxy)-119873

1015840

-(1-(pyridin-2-1) ethylidene) acetohydraziderdquo Spectrochimica ActaPart A Molecular and Biomolecular Spectroscopy vol 120 pp332ndash339 2014

[14] S Odisitse and G E Jackson ldquoIn vitro and in vivo studiesof NN1015840-bis[2(2-pyridyl)-methyl]pyridine-26-dicarboxamide-copper(II) and rheumatoid arthritisrdquo Polyhedron vol 27 no 1pp 453ndash464 2008

[15] A Hussain and F Ahsan ldquoThe vagina as a route for systemicdrug deliveryrdquo Journal of Controlled Release vol 103 no 2 pp301ndash313 2005

[16] L M Ensign T E Hoen K Maisel R A Cone and J S HanesldquoEnhanced vaginal drug delivery through the use of hypotonicformulations that induce fluid uptakerdquo Biomaterials vol 34 no28 pp 6922ndash6929 2013

[17] B B Saxena M Singh R M Gospin C C Chu and W JLedger ldquoEfficacy of nonhormonal vaginal contraceptives from ahydrogel delivery systemrdquo Contraception vol 70 no 3 pp 213ndash219 2004

[18] M L Gonzalez-Rodrıguez M A Holgado C Sanchez-Lafuente A M Rabasco and A Fini ldquoAlginatechitosan par-ticulate systems for sodium diclofenac releaserdquo InternationalJournal of Pharmaceutics vol 232 no 1-2 pp 225ndash234 2002

[19] C H Goh P W S Heng and L W Chan ldquoAlginates as auseful natural polymer for microencapsulation and therapeuticapplicationsrdquo Carbohydrate Polymers vol 88 no 1 pp 1ndash122012

[20] W-C Lin D-G Yu and M-C Yang ldquopH-sensitive poly-electrolyte complex gel microspheres composed of chitosansodium tripolyphosphatedextran sulfate swelling kinetics anddrug delivery propertiesrdquo Colloids and Surfaces B Biointerfacesvol 44 no 2-3 pp 143ndash151 2005

[21] K G H Desai and H J Park ldquoPreparation and characteriza-tion of drug-loaded chitosan-tripolyphosphatemicrospheres byspray dryingrdquo Drug Development Research vol 64 no 2 pp114ndash128 2005

[22] K M Rao B Mallikarjuna K K Rao M N Prabhakar K CRao and M C Subha ldquoPreparation and characterization of pHsensitive poly(vinyl alcohol)sodium carboxymethyl celluloseIPN microspheres for in vitro release studies of an anti-cancerdrugrdquo Polymer Bulletin vol 68 no 7 pp 1905ndash1919 2012

[23] M A Saleem Y D Murali M D Naheed P Jaydeep andM Dhaval ldquoPrepapation and evaluation of valsartan loadedhydrogel beadsrdquo International Research Journal of Pharmacyvol 3 no 6 pp 80ndash85 2012

BioMed Research International 9

[24] Y Y Yang J P Wan T S Chung P K Pallathadka S Ng andJ Heller ldquoPOE-PEG-POE triblock copolymeric microspherescontaining protein I Preparation and characterizationrdquo Journalof Controlled Release vol 75 no 1-2 pp 115ndash128 2001

[25] H Brandenberger and F Widmer ldquoA new multinozzle encap-sulationimmobilisation system to produce uniform beads ofalginaterdquo Journal of Biotechnology vol 63 no 1 pp 73ndash80 1998

[26] E-S Chan B-B Lee P Ravindra and D Poncelet ldquoPredictionmodels for shape and size of ca-alginate macrobeads producedthrough extrusion-dripping methodrdquo Journal of Colloid andInterface Science vol 338 no 1 pp 63ndash72 2009

[27] P B Deasy and M F L Law ldquoUse of extrusion-spheronizationto develop an improved oral dosage form of indomethacinrdquoInternational Journal of Pharmaceutics vol 148 no 2 pp 201ndash209 1997

[28] X H Yang and W L Zhu ldquoViscosity properties of sodiumcarboxymethylcellulose solutionsrdquo Cellulose vol 14 no 5 pp409ndash417 2007

[29] E-S Chan ldquoPreparation of Ca-alginate beads containing highoil content influence of process variables on encapsulationefficiency and bead propertiesrdquo Carbohydrate Polymers vol 84no 4 pp 1267ndash1275 2011

[30] M S Kim S J Park B K Gu and C-H Kim ldquoIonicallycrosslinked alginate-carboxymethyl cellulose beads for thedelivery of protein therapeuticsrdquo Applied Surface Science vol262 pp 28ndash33 2012

[31] C Valenta ldquoThe use of mucoadhesive polymers in vaginaldeliveryrdquo Advanced Drug Delivery Reviews vol 57 no 11 pp1692ndash1712 2005

[32] S F Borges J G L Silva and P C M Teixeira ldquoSurvival andbiofilm formation of Listeria monocytogenes in simulated vagi-nal fluid influence of pH and strain originrdquo FEMS Immunologyand Medical Microbiology vol 62 no 3 pp 315ndash320 2011

[33] R Barbucci A Magnani and M Consumi ldquoSwelling behav-ior of carboxymethylcellulose hydrogels in relation to cross-linking pH and charge densityrdquo Macromolecules vol 33 no20 pp 7475ndash7480 2000

[34] J Berger M Reist J M Mayer O Felt N A Peppas and RGurny ldquoStructure and interactions in covalently and ionicallycrosslinked chitosan hydrogels for biomedical applicationsrdquoEuropean Journal of Pharmaceutics and Biopharmaceutics vol57 no 1 pp 19ndash34 2004

[35] U Bogdanovic V Lazic V Vodnik M Budimir Z Markovicand S Dimitrijevic ldquoCopper nanoparticles with high antimi-crobial activityrdquoMaterials Letters vol 128 pp 75ndash78 2014

[36] J Konieczny and Z Rdzawski ldquoAntibacterial properties of cop-per and its alloysrdquoArchives ofMaterials Science and Engineeringvol 56 no 2 pp 53ndash60 2012

[37] S M Magana P Quintana D H Aguilar et al ldquoAntibacterialactivity of montmorillonites modified with silverrdquo Journal ofMolecular Catalysis A Chemical vol 281 no 1-2 pp 192ndash1992008

[38] Z Weissman I Berdicevsky B-Z Cavari and D KornitzerldquoThe high copper tolerance ofCandida albicans is mediated by aP-type ATPaserdquo Proceedings of the National Academy of Sciencesof the United States of America vol 97 no 7 pp 3520ndash35252000

[39] J J Martınez Medina M S Islas L L Lopez Tevez E GFerrer N B Okulik and P A M Williams ldquoCopper(II) com-plexes with cyanoguanidine and o-phenanthroline theoreticalstudies in vitro antimicrobial activity and alkaline phosphatase

inhibitory effectrdquo Journal of Molecular Structure vol 1058 no1 pp 298ndash307 2014

[40] N M Zain A G F Stapley and G Shama ldquoGreen synthesis ofsilver and copper nanoparticles using ascorbic acid and chitosanfor antimicrobial applicationsrdquo Carbohydrate Polymers vol 112pp 195ndash202 2014

[41] J P Ruparelia A K Chatterjee S P Duttagupta and SMukherji ldquoStrain specificity in antimicrobial activity of silverand copper nanoparticlesrdquo Acta Biomaterialia vol 4 no 3 pp707ndash716 2008

[42] J Zhao H J Feng H Q Tang and J H Zheng ldquoBactericidaland corrosive properties of silver implanted TiN thin filmscoated on AISI317 stainless steelrdquo Surface and Coatings Technol-ogy vol 201 no 9ndash11 pp 5676ndash5679 2007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 7: Research Article Antimicrobial Properties of ...downloads.hindawi.com/journals/bmri/2015/790720.pdf · Research Article Antimicrobial Properties of Microparticles Based on ... ect

BioMed Research International 7

00

01

02

03

04

05

06

07

08

09

10

0740 0370 0185 0093 0046 0023

A

c (mgmL)

(a)

00

01

02

03

04

05

06

07

08

09

10

3277 1639 0819 0410 0205 0102

A

c (mgmL)

(b)

0001020304050607080910

0598 0299 0150 0075 0037 0019

20hrs

A

c (mgmL)

(c)

0001020304050607080910

2173 1087 0543 0272 0136 0068

A

20hrs

c (mgmL)

(d)

Figure 6 MIC results of CMC microparticles against Candida albicans (a) MP-1 05 (b) MP-1 10 (c) MP-2 05 and (d) MP-2 10

in copper detoxification in C albicans metallothioneinCaCUP1 and a copper-transporting P-type ATPase CaCRP1[38] These two genes account for C albicansrsquos resistanceto copper and consequently the vast differences in ourresults

The obtained MIC values prove substantial antimicrobialactivity against both microorganisms tested in this study andare in good agreementwith copperMIC values against strainsof E coli and C albicans described by different authors inother studies Martınez Medina et al reported MIC valuesfor Cu(II) complexes of 0375mgmL and gt15mgmL for Ecoli and C albicans respectively [39] Copper nanoparticlesprepared by Zain et al (200 nm) showed MIC values of0433mgL for E coli [40] and those prepared by Rupareliaet al (9 nm) showed MIC values of 0280mgmL for E coli[41]

Despite the lower content of copper in microspheresthe samples prepared using the less-concentrated CuCl

2

(05M) solutionmdashMP-1 05 and MP-2 05mdashexhibited lowerMIC values and so better antibacterial activity against bothtested pathogens in comparison with samples cross-linked in

the more potent 1M CuCl2solutionThese results confirmed

the great influence of the microparticle swellability on MICvalues Both samples exhibited high swelling capacity at pH60 (see Figure 3) and thus good conditions for the releaseof copper ions from particles (pH of LB and YNB mediumwas also close to 60) Against E coli sample MP-1 05 wasfound to be the most effective as it probably exhibited fastercopper releaseThis could be a result of the higher cumulationof copper ions on more rougher surface (see Figures 1(a)and 1(b)) and also smaller particle size (see Table 2) and thuslarger surface area [42] These characteristics could promotefaster swelling leading to the creation of an amorphousstructure during the 20-hour interval (Figure 4(a)) Againstthe more resistant C albicans however the most effectivesample was MP-2 05 exhibiting probably more uniformcopper release due to the higher CMC concentraction (2)more uniform copper distribution in the microspheres andlarger particle size (see Table 2) This theory is also sup-ported by the fact that the MP-2 05 microparticles main-tained their shape-specific structure after 20 hrs of swelling(Figure 4(c))

8 BioMed Research International

4 Conclusion

In this experimental work CMC dispersions of two differentconcentrations (1 and 2) were cross-linked by copper (II)ions (05 1 15 or 20MCuCl

2) to prepare microspheres with

antimicrobial activity against frequently occurring vaginalpathogens Escherichia coli and Candida albicans All testedsamples exhibited stable but pH responsive behaviour inenvironments corresponding with natural and inflamed vagi-nal conditions and proved substantial antimicrobial activityagainst both pathogens The most effective samples werethose hardened in a less-concentrated CuCl

2(05M) solu-

tion Unexpectedly a crucial parameter for microsphereantimicrobial activity was not found in the copper contentbut in the swelling capacity of the microparticles and in thedegree of CMC surface shrinkingThe sample prepared usinga 1 CMC dispersion cross-linked by 05M CuCl

2seemed to

be the most suitable for potential vaginal use not only due toits antibacterial activity but also due to its gradual change to anonspecific-shaped gel which is preferable when consideringvaginal dosage forms

Conflict of Interests

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

References

[1] L Fan M Peng Z Zhou et al ldquoModification of carboxymethylcellulose Grafted with collagen peptide and its antioxidantaktivityrdquo Carbohydrate Polymers vol 112 pp 32ndash38 2014

[2] N HaleemM Arsah M Shahid and AM Tahir ldquoSynthesis ofcarboxymethyl cellulose fromwaste of cotton ginning industryrdquoCarbohydrate Polymers vol 113 pp 249ndash255 2014

[3] J-F Su Z Huang X-Y Yuan X-YWang andM Li ldquoStructureand properties of carboxymethyl cellulosesoy protein isolateblend edible films crosslinked by Maillard reactionsrdquo Carbohy-drate Polymers vol 79 no 1 pp 145ndash153 2010

[4] H Kono ldquoCharacterization and properties of carboxymethylcellulose hydrogels crosslinked by polyethylene glycolrdquo Carbo-hydrate Polymers vol 106 no 1 pp 84ndash93 2014

[5] E A Hassn M L Hassan N C Moorefield and GR Newkome ldquoNew supramolecular metallo-terpyridine car-boxymethyl cellulose derivativeswith antimicrobial propertiesrdquoCarbohydrate Polymers vol 116 pp 2ndash8 2014

[6] S Sayanjali B Ghanbarzadeh and S Ghiassifar ldquoEvaluation ofantimicrobial and physical properties of edible film based oncarboxymethyl cellulose containing potassium sorbate on somemycotoxigenic Aspergillus species in fresh pistachiosrdquo LWTmdashFood Science and Technology vol 44 no 4 pp 1133ndash1138 2011

[7] A A Hebeish M H El-Rafie F A Abdel-Mohdy E S Abdel-Halim and H E Emam ldquoCarboxymethyl cellulose for greensynthesis and stabilization of silver nanoparticlesrdquo Carbohy-drate Polymers vol 82 no 3 pp 933ndash941 2010

[8] K Liu Y Xu X Lin et al ldquoSynergistic effects of guanidine-grafted CMC on enhancing antimicrobial activity and drystrength of paperrdquo Carbohydrate Polymers vol 110 pp 382ndash3872014

[9] L Upadhyaya J Singh V Agarwal and R P Tewari ldquoBiomed-ical applications of carboxymethyl chitosansrdquo CarbohydratePolymers vol 91 no 1 pp 452ndash466 2013

[10] A-H Yu H-Y Hsieh J-C Pang et al ldquoActive films fromwater-soluble chitosancellulose composites incorporating releasablecaffeic acid for inhibition of lipid oxidation in fish oil emul-sionsrdquo Food Hydrocolloids vol 32 no 1 pp 9ndash19 2013

[11] T Zhong G S Oporto J Jaczynski A T Tesfai and JArmstrong ldquoAntimicrobial properties of the hybrid coppernanoparticles-carboxymethyl celluloserdquo Wood and Fiber Sci-ence vol 45 no 2 pp 215ndash222 2013

[12] G E Jackson P M May and D R Williams ldquoMetal-ligandcomplexes involved in rheumatoid arthritis-I justificationsfor copper administrationrdquo Journal of Inorganic and NuclearChemistry vol 40 no 6 pp 1189ndash1194 1978

[13] O A El-Gammal E A Elmorsy and Y E Sherif ldquoEvaluationof the anti-inflammatory and analgesic effects of Cu(II) andZn(II) complexes derived from 2-(naphthalen-1-yloxy)-119873

1015840

-(1-(pyridin-2-1) ethylidene) acetohydraziderdquo Spectrochimica ActaPart A Molecular and Biomolecular Spectroscopy vol 120 pp332ndash339 2014

[14] S Odisitse and G E Jackson ldquoIn vitro and in vivo studiesof NN1015840-bis[2(2-pyridyl)-methyl]pyridine-26-dicarboxamide-copper(II) and rheumatoid arthritisrdquo Polyhedron vol 27 no 1pp 453ndash464 2008

[15] A Hussain and F Ahsan ldquoThe vagina as a route for systemicdrug deliveryrdquo Journal of Controlled Release vol 103 no 2 pp301ndash313 2005

[16] L M Ensign T E Hoen K Maisel R A Cone and J S HanesldquoEnhanced vaginal drug delivery through the use of hypotonicformulations that induce fluid uptakerdquo Biomaterials vol 34 no28 pp 6922ndash6929 2013

[17] B B Saxena M Singh R M Gospin C C Chu and W JLedger ldquoEfficacy of nonhormonal vaginal contraceptives from ahydrogel delivery systemrdquo Contraception vol 70 no 3 pp 213ndash219 2004

[18] M L Gonzalez-Rodrıguez M A Holgado C Sanchez-Lafuente A M Rabasco and A Fini ldquoAlginatechitosan par-ticulate systems for sodium diclofenac releaserdquo InternationalJournal of Pharmaceutics vol 232 no 1-2 pp 225ndash234 2002

[19] C H Goh P W S Heng and L W Chan ldquoAlginates as auseful natural polymer for microencapsulation and therapeuticapplicationsrdquo Carbohydrate Polymers vol 88 no 1 pp 1ndash122012

[20] W-C Lin D-G Yu and M-C Yang ldquopH-sensitive poly-electrolyte complex gel microspheres composed of chitosansodium tripolyphosphatedextran sulfate swelling kinetics anddrug delivery propertiesrdquo Colloids and Surfaces B Biointerfacesvol 44 no 2-3 pp 143ndash151 2005

[21] K G H Desai and H J Park ldquoPreparation and characteriza-tion of drug-loaded chitosan-tripolyphosphatemicrospheres byspray dryingrdquo Drug Development Research vol 64 no 2 pp114ndash128 2005

[22] K M Rao B Mallikarjuna K K Rao M N Prabhakar K CRao and M C Subha ldquoPreparation and characterization of pHsensitive poly(vinyl alcohol)sodium carboxymethyl celluloseIPN microspheres for in vitro release studies of an anti-cancerdrugrdquo Polymer Bulletin vol 68 no 7 pp 1905ndash1919 2012

[23] M A Saleem Y D Murali M D Naheed P Jaydeep andM Dhaval ldquoPrepapation and evaluation of valsartan loadedhydrogel beadsrdquo International Research Journal of Pharmacyvol 3 no 6 pp 80ndash85 2012

BioMed Research International 9

[24] Y Y Yang J P Wan T S Chung P K Pallathadka S Ng andJ Heller ldquoPOE-PEG-POE triblock copolymeric microspherescontaining protein I Preparation and characterizationrdquo Journalof Controlled Release vol 75 no 1-2 pp 115ndash128 2001

[25] H Brandenberger and F Widmer ldquoA new multinozzle encap-sulationimmobilisation system to produce uniform beads ofalginaterdquo Journal of Biotechnology vol 63 no 1 pp 73ndash80 1998

[26] E-S Chan B-B Lee P Ravindra and D Poncelet ldquoPredictionmodels for shape and size of ca-alginate macrobeads producedthrough extrusion-dripping methodrdquo Journal of Colloid andInterface Science vol 338 no 1 pp 63ndash72 2009

[27] P B Deasy and M F L Law ldquoUse of extrusion-spheronizationto develop an improved oral dosage form of indomethacinrdquoInternational Journal of Pharmaceutics vol 148 no 2 pp 201ndash209 1997

[28] X H Yang and W L Zhu ldquoViscosity properties of sodiumcarboxymethylcellulose solutionsrdquo Cellulose vol 14 no 5 pp409ndash417 2007

[29] E-S Chan ldquoPreparation of Ca-alginate beads containing highoil content influence of process variables on encapsulationefficiency and bead propertiesrdquo Carbohydrate Polymers vol 84no 4 pp 1267ndash1275 2011

[30] M S Kim S J Park B K Gu and C-H Kim ldquoIonicallycrosslinked alginate-carboxymethyl cellulose beads for thedelivery of protein therapeuticsrdquo Applied Surface Science vol262 pp 28ndash33 2012

[31] C Valenta ldquoThe use of mucoadhesive polymers in vaginaldeliveryrdquo Advanced Drug Delivery Reviews vol 57 no 11 pp1692ndash1712 2005

[32] S F Borges J G L Silva and P C M Teixeira ldquoSurvival andbiofilm formation of Listeria monocytogenes in simulated vagi-nal fluid influence of pH and strain originrdquo FEMS Immunologyand Medical Microbiology vol 62 no 3 pp 315ndash320 2011

[33] R Barbucci A Magnani and M Consumi ldquoSwelling behav-ior of carboxymethylcellulose hydrogels in relation to cross-linking pH and charge densityrdquo Macromolecules vol 33 no20 pp 7475ndash7480 2000

[34] J Berger M Reist J M Mayer O Felt N A Peppas and RGurny ldquoStructure and interactions in covalently and ionicallycrosslinked chitosan hydrogels for biomedical applicationsrdquoEuropean Journal of Pharmaceutics and Biopharmaceutics vol57 no 1 pp 19ndash34 2004

[35] U Bogdanovic V Lazic V Vodnik M Budimir Z Markovicand S Dimitrijevic ldquoCopper nanoparticles with high antimi-crobial activityrdquoMaterials Letters vol 128 pp 75ndash78 2014

[36] J Konieczny and Z Rdzawski ldquoAntibacterial properties of cop-per and its alloysrdquoArchives ofMaterials Science and Engineeringvol 56 no 2 pp 53ndash60 2012

[37] S M Magana P Quintana D H Aguilar et al ldquoAntibacterialactivity of montmorillonites modified with silverrdquo Journal ofMolecular Catalysis A Chemical vol 281 no 1-2 pp 192ndash1992008

[38] Z Weissman I Berdicevsky B-Z Cavari and D KornitzerldquoThe high copper tolerance ofCandida albicans is mediated by aP-type ATPaserdquo Proceedings of the National Academy of Sciencesof the United States of America vol 97 no 7 pp 3520ndash35252000

[39] J J Martınez Medina M S Islas L L Lopez Tevez E GFerrer N B Okulik and P A M Williams ldquoCopper(II) com-plexes with cyanoguanidine and o-phenanthroline theoreticalstudies in vitro antimicrobial activity and alkaline phosphatase

inhibitory effectrdquo Journal of Molecular Structure vol 1058 no1 pp 298ndash307 2014

[40] N M Zain A G F Stapley and G Shama ldquoGreen synthesis ofsilver and copper nanoparticles using ascorbic acid and chitosanfor antimicrobial applicationsrdquo Carbohydrate Polymers vol 112pp 195ndash202 2014

[41] J P Ruparelia A K Chatterjee S P Duttagupta and SMukherji ldquoStrain specificity in antimicrobial activity of silverand copper nanoparticlesrdquo Acta Biomaterialia vol 4 no 3 pp707ndash716 2008

[42] J Zhao H J Feng H Q Tang and J H Zheng ldquoBactericidaland corrosive properties of silver implanted TiN thin filmscoated on AISI317 stainless steelrdquo Surface and Coatings Technol-ogy vol 201 no 9ndash11 pp 5676ndash5679 2007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 8: Research Article Antimicrobial Properties of ...downloads.hindawi.com/journals/bmri/2015/790720.pdf · Research Article Antimicrobial Properties of Microparticles Based on ... ect

8 BioMed Research International

4 Conclusion

In this experimental work CMC dispersions of two differentconcentrations (1 and 2) were cross-linked by copper (II)ions (05 1 15 or 20MCuCl

2) to prepare microspheres with

antimicrobial activity against frequently occurring vaginalpathogens Escherichia coli and Candida albicans All testedsamples exhibited stable but pH responsive behaviour inenvironments corresponding with natural and inflamed vagi-nal conditions and proved substantial antimicrobial activityagainst both pathogens The most effective samples werethose hardened in a less-concentrated CuCl

2(05M) solu-

tion Unexpectedly a crucial parameter for microsphereantimicrobial activity was not found in the copper contentbut in the swelling capacity of the microparticles and in thedegree of CMC surface shrinkingThe sample prepared usinga 1 CMC dispersion cross-linked by 05M CuCl

2seemed to

be the most suitable for potential vaginal use not only due toits antibacterial activity but also due to its gradual change to anonspecific-shaped gel which is preferable when consideringvaginal dosage forms

Conflict of Interests

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

References

[1] L Fan M Peng Z Zhou et al ldquoModification of carboxymethylcellulose Grafted with collagen peptide and its antioxidantaktivityrdquo Carbohydrate Polymers vol 112 pp 32ndash38 2014

[2] N HaleemM Arsah M Shahid and AM Tahir ldquoSynthesis ofcarboxymethyl cellulose fromwaste of cotton ginning industryrdquoCarbohydrate Polymers vol 113 pp 249ndash255 2014

[3] J-F Su Z Huang X-Y Yuan X-YWang andM Li ldquoStructureand properties of carboxymethyl cellulosesoy protein isolateblend edible films crosslinked by Maillard reactionsrdquo Carbohy-drate Polymers vol 79 no 1 pp 145ndash153 2010

[4] H Kono ldquoCharacterization and properties of carboxymethylcellulose hydrogels crosslinked by polyethylene glycolrdquo Carbo-hydrate Polymers vol 106 no 1 pp 84ndash93 2014

[5] E A Hassn M L Hassan N C Moorefield and GR Newkome ldquoNew supramolecular metallo-terpyridine car-boxymethyl cellulose derivativeswith antimicrobial propertiesrdquoCarbohydrate Polymers vol 116 pp 2ndash8 2014

[6] S Sayanjali B Ghanbarzadeh and S Ghiassifar ldquoEvaluation ofantimicrobial and physical properties of edible film based oncarboxymethyl cellulose containing potassium sorbate on somemycotoxigenic Aspergillus species in fresh pistachiosrdquo LWTmdashFood Science and Technology vol 44 no 4 pp 1133ndash1138 2011

[7] A A Hebeish M H El-Rafie F A Abdel-Mohdy E S Abdel-Halim and H E Emam ldquoCarboxymethyl cellulose for greensynthesis and stabilization of silver nanoparticlesrdquo Carbohy-drate Polymers vol 82 no 3 pp 933ndash941 2010

[8] K Liu Y Xu X Lin et al ldquoSynergistic effects of guanidine-grafted CMC on enhancing antimicrobial activity and drystrength of paperrdquo Carbohydrate Polymers vol 110 pp 382ndash3872014

[9] L Upadhyaya J Singh V Agarwal and R P Tewari ldquoBiomed-ical applications of carboxymethyl chitosansrdquo CarbohydratePolymers vol 91 no 1 pp 452ndash466 2013

[10] A-H Yu H-Y Hsieh J-C Pang et al ldquoActive films fromwater-soluble chitosancellulose composites incorporating releasablecaffeic acid for inhibition of lipid oxidation in fish oil emul-sionsrdquo Food Hydrocolloids vol 32 no 1 pp 9ndash19 2013

[11] T Zhong G S Oporto J Jaczynski A T Tesfai and JArmstrong ldquoAntimicrobial properties of the hybrid coppernanoparticles-carboxymethyl celluloserdquo Wood and Fiber Sci-ence vol 45 no 2 pp 215ndash222 2013

[12] G E Jackson P M May and D R Williams ldquoMetal-ligandcomplexes involved in rheumatoid arthritis-I justificationsfor copper administrationrdquo Journal of Inorganic and NuclearChemistry vol 40 no 6 pp 1189ndash1194 1978

[13] O A El-Gammal E A Elmorsy and Y E Sherif ldquoEvaluationof the anti-inflammatory and analgesic effects of Cu(II) andZn(II) complexes derived from 2-(naphthalen-1-yloxy)-119873

1015840

-(1-(pyridin-2-1) ethylidene) acetohydraziderdquo Spectrochimica ActaPart A Molecular and Biomolecular Spectroscopy vol 120 pp332ndash339 2014

[14] S Odisitse and G E Jackson ldquoIn vitro and in vivo studiesof NN1015840-bis[2(2-pyridyl)-methyl]pyridine-26-dicarboxamide-copper(II) and rheumatoid arthritisrdquo Polyhedron vol 27 no 1pp 453ndash464 2008

[15] A Hussain and F Ahsan ldquoThe vagina as a route for systemicdrug deliveryrdquo Journal of Controlled Release vol 103 no 2 pp301ndash313 2005

[16] L M Ensign T E Hoen K Maisel R A Cone and J S HanesldquoEnhanced vaginal drug delivery through the use of hypotonicformulations that induce fluid uptakerdquo Biomaterials vol 34 no28 pp 6922ndash6929 2013

[17] B B Saxena M Singh R M Gospin C C Chu and W JLedger ldquoEfficacy of nonhormonal vaginal contraceptives from ahydrogel delivery systemrdquo Contraception vol 70 no 3 pp 213ndash219 2004

[18] M L Gonzalez-Rodrıguez M A Holgado C Sanchez-Lafuente A M Rabasco and A Fini ldquoAlginatechitosan par-ticulate systems for sodium diclofenac releaserdquo InternationalJournal of Pharmaceutics vol 232 no 1-2 pp 225ndash234 2002

[19] C H Goh P W S Heng and L W Chan ldquoAlginates as auseful natural polymer for microencapsulation and therapeuticapplicationsrdquo Carbohydrate Polymers vol 88 no 1 pp 1ndash122012

[20] W-C Lin D-G Yu and M-C Yang ldquopH-sensitive poly-electrolyte complex gel microspheres composed of chitosansodium tripolyphosphatedextran sulfate swelling kinetics anddrug delivery propertiesrdquo Colloids and Surfaces B Biointerfacesvol 44 no 2-3 pp 143ndash151 2005

[21] K G H Desai and H J Park ldquoPreparation and characteriza-tion of drug-loaded chitosan-tripolyphosphatemicrospheres byspray dryingrdquo Drug Development Research vol 64 no 2 pp114ndash128 2005

[22] K M Rao B Mallikarjuna K K Rao M N Prabhakar K CRao and M C Subha ldquoPreparation and characterization of pHsensitive poly(vinyl alcohol)sodium carboxymethyl celluloseIPN microspheres for in vitro release studies of an anti-cancerdrugrdquo Polymer Bulletin vol 68 no 7 pp 1905ndash1919 2012

[23] M A Saleem Y D Murali M D Naheed P Jaydeep andM Dhaval ldquoPrepapation and evaluation of valsartan loadedhydrogel beadsrdquo International Research Journal of Pharmacyvol 3 no 6 pp 80ndash85 2012

BioMed Research International 9

[24] Y Y Yang J P Wan T S Chung P K Pallathadka S Ng andJ Heller ldquoPOE-PEG-POE triblock copolymeric microspherescontaining protein I Preparation and characterizationrdquo Journalof Controlled Release vol 75 no 1-2 pp 115ndash128 2001

[25] H Brandenberger and F Widmer ldquoA new multinozzle encap-sulationimmobilisation system to produce uniform beads ofalginaterdquo Journal of Biotechnology vol 63 no 1 pp 73ndash80 1998

[26] E-S Chan B-B Lee P Ravindra and D Poncelet ldquoPredictionmodels for shape and size of ca-alginate macrobeads producedthrough extrusion-dripping methodrdquo Journal of Colloid andInterface Science vol 338 no 1 pp 63ndash72 2009

[27] P B Deasy and M F L Law ldquoUse of extrusion-spheronizationto develop an improved oral dosage form of indomethacinrdquoInternational Journal of Pharmaceutics vol 148 no 2 pp 201ndash209 1997

[28] X H Yang and W L Zhu ldquoViscosity properties of sodiumcarboxymethylcellulose solutionsrdquo Cellulose vol 14 no 5 pp409ndash417 2007

[29] E-S Chan ldquoPreparation of Ca-alginate beads containing highoil content influence of process variables on encapsulationefficiency and bead propertiesrdquo Carbohydrate Polymers vol 84no 4 pp 1267ndash1275 2011

[30] M S Kim S J Park B K Gu and C-H Kim ldquoIonicallycrosslinked alginate-carboxymethyl cellulose beads for thedelivery of protein therapeuticsrdquo Applied Surface Science vol262 pp 28ndash33 2012

[31] C Valenta ldquoThe use of mucoadhesive polymers in vaginaldeliveryrdquo Advanced Drug Delivery Reviews vol 57 no 11 pp1692ndash1712 2005

[32] S F Borges J G L Silva and P C M Teixeira ldquoSurvival andbiofilm formation of Listeria monocytogenes in simulated vagi-nal fluid influence of pH and strain originrdquo FEMS Immunologyand Medical Microbiology vol 62 no 3 pp 315ndash320 2011

[33] R Barbucci A Magnani and M Consumi ldquoSwelling behav-ior of carboxymethylcellulose hydrogels in relation to cross-linking pH and charge densityrdquo Macromolecules vol 33 no20 pp 7475ndash7480 2000

[34] J Berger M Reist J M Mayer O Felt N A Peppas and RGurny ldquoStructure and interactions in covalently and ionicallycrosslinked chitosan hydrogels for biomedical applicationsrdquoEuropean Journal of Pharmaceutics and Biopharmaceutics vol57 no 1 pp 19ndash34 2004

[35] U Bogdanovic V Lazic V Vodnik M Budimir Z Markovicand S Dimitrijevic ldquoCopper nanoparticles with high antimi-crobial activityrdquoMaterials Letters vol 128 pp 75ndash78 2014

[36] J Konieczny and Z Rdzawski ldquoAntibacterial properties of cop-per and its alloysrdquoArchives ofMaterials Science and Engineeringvol 56 no 2 pp 53ndash60 2012

[37] S M Magana P Quintana D H Aguilar et al ldquoAntibacterialactivity of montmorillonites modified with silverrdquo Journal ofMolecular Catalysis A Chemical vol 281 no 1-2 pp 192ndash1992008

[38] Z Weissman I Berdicevsky B-Z Cavari and D KornitzerldquoThe high copper tolerance ofCandida albicans is mediated by aP-type ATPaserdquo Proceedings of the National Academy of Sciencesof the United States of America vol 97 no 7 pp 3520ndash35252000

[39] J J Martınez Medina M S Islas L L Lopez Tevez E GFerrer N B Okulik and P A M Williams ldquoCopper(II) com-plexes with cyanoguanidine and o-phenanthroline theoreticalstudies in vitro antimicrobial activity and alkaline phosphatase

inhibitory effectrdquo Journal of Molecular Structure vol 1058 no1 pp 298ndash307 2014

[40] N M Zain A G F Stapley and G Shama ldquoGreen synthesis ofsilver and copper nanoparticles using ascorbic acid and chitosanfor antimicrobial applicationsrdquo Carbohydrate Polymers vol 112pp 195ndash202 2014

[41] J P Ruparelia A K Chatterjee S P Duttagupta and SMukherji ldquoStrain specificity in antimicrobial activity of silverand copper nanoparticlesrdquo Acta Biomaterialia vol 4 no 3 pp707ndash716 2008

[42] J Zhao H J Feng H Q Tang and J H Zheng ldquoBactericidaland corrosive properties of silver implanted TiN thin filmscoated on AISI317 stainless steelrdquo Surface and Coatings Technol-ogy vol 201 no 9ndash11 pp 5676ndash5679 2007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 9: Research Article Antimicrobial Properties of ...downloads.hindawi.com/journals/bmri/2015/790720.pdf · Research Article Antimicrobial Properties of Microparticles Based on ... ect

BioMed Research International 9

[24] Y Y Yang J P Wan T S Chung P K Pallathadka S Ng andJ Heller ldquoPOE-PEG-POE triblock copolymeric microspherescontaining protein I Preparation and characterizationrdquo Journalof Controlled Release vol 75 no 1-2 pp 115ndash128 2001

[25] H Brandenberger and F Widmer ldquoA new multinozzle encap-sulationimmobilisation system to produce uniform beads ofalginaterdquo Journal of Biotechnology vol 63 no 1 pp 73ndash80 1998

[26] E-S Chan B-B Lee P Ravindra and D Poncelet ldquoPredictionmodels for shape and size of ca-alginate macrobeads producedthrough extrusion-dripping methodrdquo Journal of Colloid andInterface Science vol 338 no 1 pp 63ndash72 2009

[27] P B Deasy and M F L Law ldquoUse of extrusion-spheronizationto develop an improved oral dosage form of indomethacinrdquoInternational Journal of Pharmaceutics vol 148 no 2 pp 201ndash209 1997

[28] X H Yang and W L Zhu ldquoViscosity properties of sodiumcarboxymethylcellulose solutionsrdquo Cellulose vol 14 no 5 pp409ndash417 2007

[29] E-S Chan ldquoPreparation of Ca-alginate beads containing highoil content influence of process variables on encapsulationefficiency and bead propertiesrdquo Carbohydrate Polymers vol 84no 4 pp 1267ndash1275 2011

[30] M S Kim S J Park B K Gu and C-H Kim ldquoIonicallycrosslinked alginate-carboxymethyl cellulose beads for thedelivery of protein therapeuticsrdquo Applied Surface Science vol262 pp 28ndash33 2012

[31] C Valenta ldquoThe use of mucoadhesive polymers in vaginaldeliveryrdquo Advanced Drug Delivery Reviews vol 57 no 11 pp1692ndash1712 2005

[32] S F Borges J G L Silva and P C M Teixeira ldquoSurvival andbiofilm formation of Listeria monocytogenes in simulated vagi-nal fluid influence of pH and strain originrdquo FEMS Immunologyand Medical Microbiology vol 62 no 3 pp 315ndash320 2011

[33] R Barbucci A Magnani and M Consumi ldquoSwelling behav-ior of carboxymethylcellulose hydrogels in relation to cross-linking pH and charge densityrdquo Macromolecules vol 33 no20 pp 7475ndash7480 2000

[34] J Berger M Reist J M Mayer O Felt N A Peppas and RGurny ldquoStructure and interactions in covalently and ionicallycrosslinked chitosan hydrogels for biomedical applicationsrdquoEuropean Journal of Pharmaceutics and Biopharmaceutics vol57 no 1 pp 19ndash34 2004

[35] U Bogdanovic V Lazic V Vodnik M Budimir Z Markovicand S Dimitrijevic ldquoCopper nanoparticles with high antimi-crobial activityrdquoMaterials Letters vol 128 pp 75ndash78 2014

[36] J Konieczny and Z Rdzawski ldquoAntibacterial properties of cop-per and its alloysrdquoArchives ofMaterials Science and Engineeringvol 56 no 2 pp 53ndash60 2012

[37] S M Magana P Quintana D H Aguilar et al ldquoAntibacterialactivity of montmorillonites modified with silverrdquo Journal ofMolecular Catalysis A Chemical vol 281 no 1-2 pp 192ndash1992008

[38] Z Weissman I Berdicevsky B-Z Cavari and D KornitzerldquoThe high copper tolerance ofCandida albicans is mediated by aP-type ATPaserdquo Proceedings of the National Academy of Sciencesof the United States of America vol 97 no 7 pp 3520ndash35252000

[39] J J Martınez Medina M S Islas L L Lopez Tevez E GFerrer N B Okulik and P A M Williams ldquoCopper(II) com-plexes with cyanoguanidine and o-phenanthroline theoreticalstudies in vitro antimicrobial activity and alkaline phosphatase

inhibitory effectrdquo Journal of Molecular Structure vol 1058 no1 pp 298ndash307 2014

[40] N M Zain A G F Stapley and G Shama ldquoGreen synthesis ofsilver and copper nanoparticles using ascorbic acid and chitosanfor antimicrobial applicationsrdquo Carbohydrate Polymers vol 112pp 195ndash202 2014

[41] J P Ruparelia A K Chatterjee S P Duttagupta and SMukherji ldquoStrain specificity in antimicrobial activity of silverand copper nanoparticlesrdquo Acta Biomaterialia vol 4 no 3 pp707ndash716 2008

[42] J Zhao H J Feng H Q Tang and J H Zheng ldquoBactericidaland corrosive properties of silver implanted TiN thin filmscoated on AISI317 stainless steelrdquo Surface and Coatings Technol-ogy vol 201 no 9ndash11 pp 5676ndash5679 2007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 10: Research Article Antimicrobial Properties of ...downloads.hindawi.com/journals/bmri/2015/790720.pdf · Research Article Antimicrobial Properties of Microparticles Based on ... ect

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of