CHARACTERIZATION AND IN VITRO EVALUATION OF THE BEHAVIOUR OF 6-CARBOXYCELLULOSE/AG(I) IN THE PRESENCE OF MICROORGANISMS

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1 CELLULOSE CHEMISTRY AND TECHNOLOGY CHARACTERIZATION AND IN VITRO EVALUATION OF THE BEHAVIOUR OF 6-CARBOXYCELLULOSE/AG(I) IN THE PRESENCE OF MICROORGANISMS LENKA HUSÁKOVÁ, IVA URBANOVÁ, VLASTA KULIŠOVÁ, * JAN MUSELÍK, ** VÁCLAV LOCHAŘ, JIŘÍ BRUS, *** JANA VESELÁ-LAŇAROVÁ, JARMILA VYTŘASOVÁ, ANDREA KALENDOVÁ, PAVEL HAVELKA, **** PETRA MOŤKOVÁ, JITKA ŠRÁMKOVÁ and LUDVÍK BENEŠ ***** Faculty of Chmical Tchnology, Univrsity of Pardubic, Studntska 573, Pardubic, CZ , Czch Rpublic * Hxion Spcialty Chmicals, Inc., Tovární 2093, CZ Sokolov, Czch Rpublic ** Faculty of Pharmacy, Univrsity of Vtrinary and Pharmacutical Scincs Brno, Palackéhotř. 1/3, CZ Brno, Czch Rpublic *** Institut of Macromolcular Chmistry of th Acadmy of Scinc of th Czch Rpublic, Hyrovsky sq. 2, CZ , Pragu 6, Czch Rpublic **** VUOS, Inc., Rybitví 296, CZ , Pardubic, Czch Rpublic ***** Joint Laboratory of Solid Stat Chmistry of th Institut of Macromolcular Chmistry of th Acadmy of Scincs, v.v.i., and Univrsity of Pardubic, Studntska 84, CZ , Pardubic, Czch Rpublic Corrsponding author: LnkaHusáková,Lnka.Husakova@upc.cz Rcivd Fbruary 13, 2014 A nw typ of an antimicrobial matrial basd on silvr salt (chlorid) adsorbd onto 6-carboxycllulos (OC/Ag(I)) was charactrizd using solid-stat carbon-13 cross-polarization magic angl spinning nuclar magntic rsonanc ( 13 C CP/MAS NMR) spctroscopy, flam atomic absorption spctromtry (FAAS), X-ray diffraction (XRD) and siz xclusion chromatography (SEC). In vitro invstigations of th bhaviour of th matrial wr carrid out in buffrd nutrint mdia (ph 7.0 or 5.4) in th prsnc of svral human pathogns, such as Asprgillus fumigatus, Candida albicans, Psudomonas aruginosa and Staphylococcus pidrmidis at 37 C within a priod of on wk. Th concntration of Ag rlasd into th liquid nutrint mdia was masurd by FAAS. Furthr, th composition of th rsulting liquid and solid phas was charactrizd by diffrnt chromatographic mthods, Fourir transform infrard spctroscopy (FTIR), FAAS, and inductivly coupld plasma orthogonal acclration tim-of-flight mass spctromtry (ICP-oa-TOF-MS) and scanning lctron microscopy (SEM). Th rsults showd that non of th invstigatd microorganisms hav shown significant dgradation potntial of OC structur. Only a partial rlas of Ag was obsrvd, compard to th initial sampls, rgardlss of th prsnc of th microorganism strain and both invstigatd ph valus. Othr noticabl changs obsrvd in th molcular structur of OC wr du to th incrasing nutralization dgr upon cationization by th ions prsnt in th nutrint mdia. Kywords: oxidizd cllulos, silvr salt of oxidizd cllulos, dgradation, antimicrobial matrials, analysis of polymr, silvr INTRODUCTION Silvr plays an important rol in promoting wound haling and in prvnting infction of a wound 1-6 and thus, silvr-containing drssings ar wll-stablishd in th mdical industry as saf and ffctiv antimicrobial agnts. 5,7-11 Many typs of ths drssings ar nowadays commrcially availabl for this purpos. 10,11 Ths matrials ar mainly dsignd: (i) to rlas Ag Cllulos Chm. Tchnol., 49 (3-4), (2015)

2 LENKA HUSÁKOVÁ t al. into th wound bd for antimicrobial action (Acticoat 7, Smith & Nphw), (ii) to absorb wound fluid and xudats containing infctious organisms into th drssing fabric, whr th silvr xrts its antibactrial action (Aquacl Ag, ConvaTc and Actisorb Silvr, Johnson & Johnson), (iii) both to absorb wound xudats and to rlas Ag into th wound bd (Contrt Foam, Coloplast) or (iv) to rlas Ag sulphadiazin which librats, in th prsnc of wound fluid, Ag and sulphadiazin acting against silvrrsistant bactria (Urgotul Ag, Urgo). 8,10,11 Numrous approachs for th dvlopmnt of ths matrials wr usd by individual producrs 10,11 rlating to th usag of diffrnt typs of drssing matrials (polythyln, polystr, sodium carboxymthylcllulos, nylon, tc.) and silvr coating (nanocrystallin silvr, silvr in an ionic form, silvr sulphadiazin, tc.). Th prsnt rsarch prformd by VUOS, Synthsia Inc., th Czch Rpublic, is focusd on th dvlopmnt of ths typs of silvr-rlasing agnts basd on a combination of Ag salts and oxidizd cllulos (OC/Ag(I)). Ths matrials ar dsignd to rlas Ag ions to xrt a sustaind antimicrobial ffct of oxidizd rgnratd cllulos (OC), prviously dmonstratd by svral authors and attributd to th significant acidity of OC solutions, 14 and to tak othr advantags of OC, such as its bio absorbability or hmostatic action. 16 Th concntration of silvr in commrcially availabl silvr-containing drssings of diffrnt typs varis considrably and diffrnt factors lik th distribution of silvr in a drssing, its chmical and physical form and drssing structur influnc th amount of silvr rlasd from th drssing and th fficacy of silvr as an antimicrobial agnt. 11 An improvd antimicrobial activity of OC/Ag(I) towards th diffrnt tstd bactria whn compard to unmodifid OC sampls was prviously rportd. 12 Howvr, th rduction of th silvr ion accompanid by changs of surroundings to brown and/or black colors in contact with som bactria and additionally th changs in sampl composition prviously rportd 12 ndd to b rsolvd. Information about th bhaviour of OC/Ag(I) during th in vitro studis in th prsnc of svral human pathogns has not bn rportd to this dat, similarly as th information about th matrial structur. This is du to th fact that th 292 charactrization of th original matrial with Ag contnt of only about 1% (w/w), and of th rsulting complx sampl of a high molcular wight obtaind aftr th in vitro dgradation study is still an analytical challng. Th objctiv of this study was thus to valuat th in vitro dgradation of a novl typ of antimicrobial matrial basd on a silvr salt of 6-carboxycllulos (OC/Ag(I)) causd by th mtabolic action of svral human pathogns undr diffrnt xprimntal conditions, as wll as to charactriz th matrial. EXPERIMENTAL Sampls and sampl prparation Th sampls of OC/Ag(I) wr prpard at VUOS Synthsia Inc. (Pardubic, Czch Rpublic) using th following procdur. Th amount of 100 g of oxycllulos was disprsd in 1 L of 0.205% aquous silvr nitrat. Aftr stirring th mixtur for 12 h in th dark, th sampl was filtrd and placd into 1 L of 0.5% solution of NaCl in 50% thanol for 1 h for photostabilization. Aftr that th sampls wr wrung out and placd into 96% thanol for 1 h in ordr to rmov watr and drid undr th laboratory conditions. Chmical and physical charactristics of both OC and OC/Ag(I) sampls wr givn by ph valu of 1% (w/w) solutions of ths sampls (ph was masurd at 25 C), concntration of fr carboxyl groups ( COOH), carbonyl groups contnt (aldhyd and kton) 17 and ash lvl wr 2.9, 19.3% (w/w), 1.7% (w/w), 0.1% for OC sampls, rspctivly 3.2, 18.1% (w/w), 0.60% (w/w), 1.8% (w/w) for OC/Ag(I). Th concntration of Ag in OC/Ag(I) sampls was 1.2% (w/w). All sampls wr stord in non-transparnt PE sachts in a rfrigrator. Bactrial culturs Two bactrial (Psudomonas aruginosa CCM 1960 and Staphylococcus pidrmidis CCM 4418), on yast (Candida albicans CCM 8180), and on fungal (Asprgillus fumigatus CCM 373) strains wr usd throughout this work. All th strains wr obtaind from th Czch Collction of Microorganisms (CCM, Brno, Czch Rpublic). A suspnsion of bactrial and yast strains was prpard from frsh colonis on blood agar plats (HiMdia, India) (for bactria) and malt agar (HiMdia, India) (for yast) aftr 24 h incubation for all strains. Th concntration of clls was adjustd to 10 8 cfu ml -1 using th 0.5 McFarland standard turbidity scal and srially dilutd to initial inoculum of 10 5 cfu ml -1. A suspnsion of fungal spors was prpard from culturs grown for 7 days on malt agar at 24 C and th dnsity of spors was adjustd to 10 5 spors ml -1.

3 Oxidizd cllulos FT-IR mthod Th FT-IR spctra of KBr pllts wr takn by th Thrmo Scintific Nicolt 6700 spctromtr at room tmpratur. Spctra wr rcordd in an absorbanc mod and thoroughly analyzd for all absorption paks from wavnumbrs 4000 to 800 cm -1. Th rsolution was of 4 cm -l and 64 scans wr collctd. Th rportd spctra of th OC and OC/Ag(I) wr obtaind by subtraction of th spctra of pur KBr from th raw data. Solid stat NMR spctroscopy Solid-stat 13 C CP/MAS (cross polarization/magic angl spinning) NMR spctra wr masurd at ambint tmpratur by a Brukr Avanc 500 spctromtr at MHz with spinning frquncy 11 khz and contact tim 2 ms, i.., undr conditions that allowd quantitativ analysis. Chmical shifts in th 13 C NMR spctra wr rfrrd to th carbonyl lin of glycin (with a signal at ppm from TMS) by sampl rplacmnt. X-ray diffraction (XRD) Powdr X-ray diffraction data (Cu Kα, λ = Å) wr collctd on a D8 Advanc diffractomtr (Brukr AXS, Grmany) with Bragg-Brntano Θ-Θ goniomtr (radius mm) quippd with a scondary bam curvd graphit monochromator and Na(Tl)I scintilation dtctor. Th gnrator was opratd at 40 kv and 30 ma. Th scan was prformd at room tmpratur from 5 to 60 (2 Θ) in 0.02 stp with a counting tim of 10 s pr stp. Chromatographic analyss HPLC mthod Th HP Agilnt 1100 HPLC systm (Agilnt Tchnologis, USA), quippd with a vacuum dgassr, quatrnary pump, autosamplr and vaporativ light scattring dtctor (ELSD), was usd for th chromatographic xprimnts. All sparations wr carrid out on a LiChrosphr Diol 100 column (4 150 mm; 5 µm). Sampls (10 µl) wr lutd by a mthod that was optimizd for th sparation of ithr oligomrs or monomrs of th saccharid units of th original matrials. Oligomrs and monomrs wr lutd using a mobil phas comprisd of 85% actonitril (A) and 15% watr (B) usd with on of th following gradint conditions: 75% (A), 25% (B) until 15 min, 50% (A), 50% (B) until 30 min, 0% (A), 100% (B) until 31 min, 0% (A), 100% (B) until 35 min. Flow rats and column tmpraturs for th sparation of oligosaccharids and monosaccharids wr of 1.0 ml min -1 and 40 C. Siz-xclusion chromatography (SEC) Siz-xclusion chromatography (SEC) masurmnts wr carrid out using th Agilnt 1100 Sris systm quippd with a vacuum dgassr, isocratic pump, manual injctor (100 µl sampling loop), thrmostattd column compartmnt, rfractiv indx (RI) dtctor. Data valuation was prformd with standard ChmStation GPC data analysis softwar (GPC-Addon). Th following paramtrs wr usd in th SEC masurmnts: flow, 1.00 ml min -1 ; mobil phas 0.2 mol L -1 NaNO 3 and 0.01 mol L -1 NaH 2 PO 4 in dmi watr, column tmpratur 25 C, optical unit tmpratur 35 C, columns, PL aquagl OH guard 8 µm (50x7.5 mm), PL aquagl-oh MIX 8 µm (300x7.5 mm), PL aquagl-oh 40 8 µm (300x7.5 mm); injction volum, 20 µl; run tim, 27 min. Sampls wr filtrd through a 0.45 µm nylon syring filtr (Whatman Autovial) prior to th analysis. A st of analytical pullulan polysaccharid standards, for GPC, Mp g mol -1 was usd for calibration purposs. Scanning lctron microscopy Th morphology of th sampls was xamind using a scanning lctron microscop JEOL-JSM 5600 LV manufacturd by Jol, Japan. Th sampls wr mountd on mtal stubs using doubl sidd adhsiv taps. Scanning lctron micrographs of th sampls wr obtaind at an acclration voltag of 15 kv. Th sampls wr prviously sputtr coatd with gold for 30 s, using a Crsingtor plasma sputtr coatr. Microwav digstion In ordr to study th homognity of Ag distribution in OC/Ag(I) sampls, th microwav digstion of th sampls was carrid out in Spdwav TM MWS-3 + (Brghof, Grmany) microwav systm with th maximum total output of th microwav gnrator 1450 W. For this purpos, a mg portion of th powdrd sampl was wighd and placd into a 100-mL prssur rsistant PTFE vssl, whrupon 6 ml of HNO 3 (65%, w/v) was addd. Th sampls wr digstd in 5 stps: (i) 5 min at 80% powr and 160 C (ramp 3 min), (ii) 10 min at 90% powr and 210 C (ramp 10 min), (iii-v) 5 min at 10% powr and 100 C (ramp 1 min). Th rsulting solution was transfrrd into 100 ml volumtric flask and dilutd to approximatly 50 ml with dionizd watr. Aftr that, 5.5 ml of ammonia solution (25%, w/v) was addd to dissolv AgCl and th volum was adjustd with dionizd watr to 100 ml.th solutions wr analysd by F-AAS along with blank sampl containing th sam amount of individual ragnts and prpard in th sam way as th sampls. Bfor th analysis, th sampls wr dilutd with dionisd watr. AAS mthod Th dtrmination of K, Na and Ag was carrid out with an Avanta P doubl bam atomic absorption spctromtr (GBC Scintific Equipmnt Pty. Ltd., 293

4 LENKA HUSÁKOVÁ t al. Australia) quippd with dutrium arc background corrction in th flam mission and absorption mod, rspctivly. Sampl solutions wr introducd to th spctromtr using a standard GBC pnumatic nbulizr. Th hollow cathod lamps obtaind from Photron Pty. (Australia) wr usd throughout. Th wavlngth was st at nm for K, nm for Na, and nm for Ag with a lamp currnt of 4 ma. Spctral rsolution of 0.2 and 0.3 nm was usd for Na, K and Ag, rspctivly. Th gas flam mixtur was formd with 10.0 L min -1 air and 1.5 L min -1 actyln (99.5%, from Lind Gas, Inc., Czch Rpublic). In ordr to valuat th silvr rlas from th OC/Ag(I) sampls, an amount of 0.20 g of powdrd sampl was placd in stril vials in an asptic mannr and Brain Hart Infusion broth (BHI) (HiMdia) (for bactria) or Malt Extract Broth bas (MEB) (HiMdia) (for yast and fungi) was thn addd to ach vial to mak up to a total volum of 10 ml. An aliquot (1 ml) of th bactrial, yast or fungal suspnsion was addd to ach vial containing th sampl. Control broths with and without bactrial, yast or fungal inoculation wr also includd. Th vials wr thn incubatd undr diffrnt xprimntal conditions. Aliquots of 1 ml bactrial broth wr sampld from ach vial at spcific tim intrvals and srial 25 fold dilutions for ach aliquot wr usd, which allowd Ag quantification by th applid analytical tchniqu. Bfor th dtrmination of Na and K contnt in OC and/or OC/Ag(I), th sampls wr rconstitutd in cadoxn. Th concntration of solutions rangd from 0.5 to 10 mg L 1 for Ag and 0-20 mg L -1 for Na and K. A singl calibration graph was constructd for ach lmnt, from which all calculations wr mad in on analytical run. Fiv aquous standards wr usd to obtain th calibration curvs. Fiv rplicats at 3 sconds intgration tim wr usd. All th calibration plots wr linar in th invstigatd concntration rangs with th corrlation cofficints bttr than ICP-TOF-MS ICP-MS masurmnts wr prformd by th Optimass8000 ICP-oa-TOF-MS instrumnt (GBC Scintific Equipmnt Pty. Ltd., Australia). Dtails of th instrumnt and th oprating conditions ar summarizd in an arlir study. 18 Dtrmination of carboxyl contnt Th carboxyl contnt was dtrmind by th wllstablishd volumtric mthod dscribd prviously. 19, 20 Statistical analysis Th calculations and statistical valuation wr carrid out using commrcial softwar Statistica 6.1 (StatSoft., Inc. Tulsa, USA), and Origin 7.0 (OriginLab Corporation, Northampton, MA, USA). 294 RESULTS AND DISCUSSION Charactrization of OC/Ag(I) sampls 13 C CP/MAS NMR 13 C CP/MAS NMR mthod, although lss accssibl to routin laboratoris, was usd for charactrization of th sampls as FTIR mthod, i.. th most frquntly usd tool for obtaining rapid information about oxycllulos structur and physical and chmical changs taking plac with various tratmnts, 20, 21 is unabl to rval changs among th spctra of OC and OC/Ag(I) for th sampls with th contnt of Ag of about 1% (w/w). 13 C CP/MAS NMR spctra of oxidizd cllulos and modifid oxidizd cllulos sampls wr masurd and ar prsntd in Fig. 1. Th solid-stat 13 C CP/MAS spctrum of oxycllulos has bn prviously rportd by svral authors. 19 Thus, analogically, in th spctrum of oxidizd cllulos (Fig. 1a), th 170 ppm pak is assignd to th carboxyl carbon, that is C 6 (COOH) in th oxidizd anhydroglucos unit of th cllulos chain, th pak at 93 ppm is attributd to C 1 of th trminal α-d-glucos unit, th 63 ppm componnt of C 6 (CH2OH) rsonanc, th pak at ppm is assignd to C 1 -carbon of basic cllulos chain, th paks at 87.3 and 82.9 ppm ar du to carbons on th chains locatd in th crystallin (C 4 a) and amorphous intriors (C 4 b), rspctivly, th clustr of paks in th rgion ppm du to C 2, C 3, and C 5 rsonancs, from both amorphous and crystallin part, shows a maximum at 75 ppm, th signal at 14 ppm is du to th CH 3 group. Whn comparing th spctra of OC and OC/Ag(I) (Fig. 1), a diffrnc in th chmical shift for C 6 (COOH) can b sn, most probably du to th chmical influnc by Ag salt. In addition, two signals for th CH 3 group wr obsrvd in th spctra of OC/Ag(I) (Fig. 1b) having th sam ovrall intnsity, probably du to th fact that th modification by Ag silvr salts also impacts th CH 3 group. Anothr notabl fatur in th spctra of modifid oxidizd cllulos, compard to th spctrum of th non-modifid sampl, is th twic highr incras in snsitivity at th 93 ppm pak (C 1 t), showing that th OC/Ag(I) sampl contains shortr chains. AAS mthod Atomic absorption spctromtry was applid both to dtrmin th contnt of silvr and to valuat its distribution in th sampl. Th

5 Oxidizd cllulos homognity of a sampl is an important prrquisit for attaining a dsirabl thraputical ffct and obtaining accurat rsults during th studis on Ag rlas from OC/Ag(I) sampls. A total numbr of 16 OC/Ag(I) with th sam Ag concntration obtaind from diffrnt placs of th original sampls wr analysd in ordr to chck th homognity of ths matrials. Th valus for Ag contnt (man ± S.D.) in OC/Ag(I) wr found to b 1.2 ± 0.1% (w/w). Basd on ths obsrvations, it is vidnt that th analytical valus for silvr contnt will not dpnd strongly on th sampling ara. Th rsults of AAS masurmnts additionally rvald that th total contnt of silvr corrspondd to its halid form and no dtctabl part was prsnt as Ag(0) or as a silvr salt of oxycllulos. As th halid compound was asily solubl in aquous ammonia, it was xpctd that it was most probably AgCl. Howvr, in ordr to prov this assumption X-ray diffraction was furthr mployd. X-ray diffraction A typical X-ray diffraction spctrum obtaind for a powdrd OC/Ag(I) is shown in Fig. 2. All of th diffraction paks markd in Fig. 2 can b attributd to th structur of AgCl for which th lattic paramtrs wr in a good agrmnt with thos of th standard Chlorargyrit, syn in th databas (PDF-4+/ICDD). Thr is no vidnc of additional silvr containing phass. Figur 1: 13 C CP/MAS NMR spctra of (a) oxidizd cllulos and (b) oxidizd cllulos with Ag salt Figur 2: X-ray diffractogram of oxidizd cllulos modifid with Ag salt 295

6 LENKA HUSÁKOVÁ t al. Studis on OC/Ag(I) in vitro bhaviour To valuat th amount of silvr bing rlasd from th OC/Ag(I) sampl and to study th possibl mtabolic vrsatility of svral human pathogns for th dgradation of ths sampls, diffrnt chromatographic mthods, FTIR, AAS, ICP-TOF-MS and SIMS wr applid to th analysis of both nutrint mdia and th rsulting solid sampl. Exprimnts wr carrid out undr similar conditions as thos prviously applid during th valuation of antimicrobial activity of OC salts, 12 including th priod of on wk and th tmpratur of 37 C, both bing optimum for th cultivation of bactria and corrsponding to th conditions found in vivo, sinc th matrial is aimd to b usd as an adhsion barrir to assist natural wound rpair. In ordr to prvnt th photorduction of silvr ions from th OC/Ag(I) sampls, dark conditions wr maintaind throughout th study. AAS dtrmination of Ag Silvr was dtrmind in nutrint mdia containing OC/Ag(I) without and in th prsnc of diffrnt kinds of microorganisms at an incubation tmpratur of 37 C in th dark for a priod of on wk. To find whthr thr wr any diffrncs btwn th rsults dpnding on th factor slctd, th analysis of varianc (ANOVA) was usd. Finally, a total numbr of 20 sampls was analysd and two-way ANOVA was usd for th data tratmnt. Microorganism strain with fiv lvls (no microorganism, A. fumigatus, C. albicans, P. aruginosa, and S. pidrmidis) was slctd as th first factor and tim with two lvls (2 or 7 days) was slctd as th scond factor. Both factors, as wll as th intraction btwn factors, wr found to b insignificant (p = 0.100, 0.631, 0.879) at th significanc lvl of This mans that th silvr concntration in th analysd solutions was not dpndnt on th prsnc of th invstigatd microorganism strain and did not significantly chang during th priod of on wk. In addition, silvr rlas was not influncd by th intraction btwn microorganism strain and tim. Man Ag concntration in th analysd solutions varid from 6.7 to 22.6 mg L -1, which mans that approximatly % of Ag from OC/Ag(I) was rlasd. Ths valus ar significantly highr than thos xpctd basd on th solubility constant of AgCl. This fact may b attributd to th cration of diffrnt complxs 296 lik AgCl - 2, AgCl 2-3 and AgCl 3-4. If th solution contains xcss chlorid du to th prsnc of, say, sodium chlorid, thr is a tndncy toward complx formation. 22 For BHI mdia, th valu of 5 g L -1 of NaCl is dclard by th producr; an important concntration of NaCl to th xprimnts is also introducd via OC/Ag(I) as will b discussd latr. As chlorids may act by forming complxs, thy influnc both th solubility of AgCl and th microbiological ffctivnss of Ag +. 6 In gnral, silvr rlas was not dpndnt on th typ of th cultur mdium (MEB or BHI) with various ph valus of solutions (5.4 and 7.0 for MEB and BHI, rspctivly). Undr th conditions whr no microorganism was prsnt, no significant changs wr indicatd using paird t-tst (p = 0.129) for silvr concntration valus dtrmind in both typs of mdia for a priod of 7 days. Th apparanc of both liquid mdia and OC/Ag(I) did not chang bcaus of th rduction of silvr ions during all of th incubation xprimnts as long as dark conditions wr st vn in th prsnc of bactria, as prviously rportd, 12 which is attributd to th slfprotction mchanism of bactria 23 as silvr in its lmntal form is purly absorbd by mammal and bactrial clls. Visual changs wr obsrvd howvr in th light du to th photorduction of silvr ions. Chromatographic analysis HPLC As shown prviously, 24,25 during th solubilization and dgradation of rgnratd cllulos undr conditions which approximat thos found in vivo, th oxidizd polymr radily undrgos chain shortning to giv oligomrs which, in th prsnc of plasma or srum, ar furthr hydrolyzd to smallr fragmnts, including glucuronic acid and glucos. In ordr to monitor possibl oligosacharids and monosacharids products from OC/Ag(I) dgradation procsss in nutrint mdia, high prformanc liquid chromatography coupld with ELSD dtction was mployd. Fig. 3 shows a comparison of th ELSD-HPLC profils of solutions obtaind aftr incubation of OC/Ag(I) in MEB and BHI in th absnc and prsnc of th invstigatd microorganisms at 37 C for 7 days. It can b sn from Fig. 3 that in th prsnc of th invstigatd microorganisms no significant diffrncs wr obsrvd in th

7 Oxidizd cllulos ara blow 30 min, i.. lution tims of oligosaccharids and monosaccharids, with th xcption of th conditions including C. albicans (s Fig. 3a vs Fig. 3b). In th prsnc of C. albicans, a significant dcras in glucos concntration at rtntion tims of 4 min (Fig. 3b) was obsrvd in comparison with thos obsrvd in nutrint mdium (Fig. 3a). Ths findings may b attributd to th diffrnt inhibition action of OC/Ag(I) obsrvd prviously for bactria, yast and fungi. It was rportd 12 that th minimal inhibitory concntration of th OC/Ag(I) sampl for bactria is lowr than thos for fungi and no inhibition by th OC/Ag(I) sampl was obsrvd for yasts. Basd on ths findings, it may b xpctd that a highr cll count in th solution containing C. albicans will rsult in th consumption of saccharid and thus in th dcras of th signal. Th oligosaccharids and monosaccharids obsrvd in Fig. 3 originat from nutrint mdia and thy ar not products of solubilization and dgradation of OC/Ag(I). Figur 3: ELSD-HPLC profils of (a) MEB and (d) BHI background and thos of OC/Ag(I) partially solubilizd with ths mdia and aftr incubation with (b) C. albicans, (c) A. fumigatis, () P. aruginosa and (f) S. pidrmidis for 7 days Tabl 1 Rsults of SEC analysis for pak molcular wight (Mp, g mol -1 ), wight-avrag molcular wight (Mw, g mol -1 ), numbr-avrag molcular wight (Mn, g mol -1 ), polydisprsity (PDI) and pak aras (P A, %) of OC/Ag(I) for initial sampls and aftr application of diffrnt microorganisms at 37 C for 7 days Pak 1 Pak 2 Microorganism Mp Mw Mn PDI P A Mp Mw Mn PDI P A No A. fumigatus C. albicans P. aruginosa S. pidrmidis SEC In ordr to sparat and charactriz highr molcular fractions, SEC was additionally mployd. Molar mass distributions (MMDs) dtrmind for initial sampls and thos stord at 37 C for 7 days in th absnc and prsnc of diffrnt microorganisms rvald two similar paks in all cass. Th lft pak (Pak 1) was a high molcular pak with an Mp (molcular wight at pak maximum) of about g mol - 1 ; for th right pak (Pak 2), th Mp was of about g mol -1. Th Mp valus wr dtrmind by comparing th lution tim of th paks of th sampl with th lution tims of th pullulan standards. Tabl 1 prsnts a summary of th xprimntal rsults for th OC/Ag(I) with rspct 297

8 LENKA HUSÁKOVÁ t al. to numbr-avrag molcular wight (Mn), wight avrag molcular wight (Mw), and polydisprsity indx (PDI) at diffrnt xprimntal conditions. In addition, pak aras (P A ) for all idntifid compounds wr normalizd so that th total pak ara addd up to 100 and is prsntd in Tabl 1. Aftr that, quantitativ rsults ar shown by xprssing th ara of a givn pak as a prcntag of th sum of th aras of all th paks. As follows from th data in Tabl 1, th valus of Mn, Mw and PDI of both Pak 1 and Pak 2 did not show a significant dcras, Pak 2 bing prdominant in all cass; also, th polydisprsity rmaind quit constant. Th rsults suggst that th tratmnt of OC/Ag(I) with th invstigatd microorganisms undr th st xprimntal conditions did not rsult in significant dgradation of th OC structur. FTIR analysis Figur 4 shows th infrard spctra of OC/Ag(I) and of th sampls aftr th tratmnt with diffrnt microorganisms. Th analysis of th spctra was focusd on th rgion cm -1, which is th most valuabl for unambiguous spctral changs. This rgion contains svral distinct absorption bands at 1736, 1636, 1420, 1160, 1031 and 1063 cm -1. Furthrmor, thr is a numbr of low-intnsity absorption bands at 1383, 1282, 925, 895 and 855 cm -1. Th pak around 1740 cm -1 is du to th strtching of C=O, and th pak at 1636 cm -1 is rlatd to th O H bnding vibration of th watr molculs adsorbd onto OC and also to th carboxylat anion COO -. Th bands around 1420 and 1380 cm -1 ar assignd to COO and/or to O H bnding in COOH and O H in plan dformation, rspctivly. Th 1160 cm -1 band is assignd to th asymmtric vibration of C O C strtching, th band at 1063 cm -1 is attributd to skltal vibrations involving strtching of C O and C C bonds attachd to th glucos rings and th band at 900 cm -1 is attributd to th asymmtric out-of-plan ring strtching in cllulos du to th β-linkag. 20, 21 Th analysis of OC/Ag(I) spctra valuatd bfor and aftr th dgradation xprimnt (Fig. 4) shows that th main spctral changs ar th rdistribution of th absorption intnsity at cm -1 (s Fig. 4). A significant dcras in intnsity was obsrvd for th charactristic carbonyl pak ( COOH) at 1740 cm -1 in all cass, compard with th original OC/Ag(I) sampl, consistnt with an incras in 298 th intnsity of th band of th carboxylat anion at 1636 cm -1 in th products. This ffct was mor pronouncd in BHI mdia at ph 7.0. This fact may b attributd to th cration of sodium and/or potassium salts of OC from nutrint mdia as BHI contains high concntrations of potassium and spcially of sodium ions, in contrast to MEB (s Tabl 2). Lowr absorbanc intnsity ratio for highr substitutd sampls du to th blocking of th carboxyl group by th raction was prviously obsrvd by diffrnt authors 20,21 upon cationization of oxidizd cllulos. Carboxyl contnt In ordr to valuat th nutralization dgr of th OC/Ag(I) sampls bfor and aftr th tratmnts undr diffrnt xprimntal conditions, th rsidual carboxyl contnt has bn dtrmind. Carboxyl contnt rducd from 18.1% for initial OC/Ag(I) to 14.1, 13.6, 11.4 and 12.1% for conditions with A. fumigatus (ph = 5.4), C. albicans (ph = 5.4), S. pidrmidis (ph = 7.0) and P. aruginosa (ph = 7.0), rspctivly. Th dcras of fr carboxyl groups bing mor pronouncd at ph 7 corrsponds wll with th rsults obtaind by th FTIR mthod and confirms th furthr cationization of th OC/Ag(I) sampls by th ions prsnt in th nutrint mdia. ICP-TOF-MS qualitativ analysis and AAS dtrmination of Na and K ICP-TOF-MS spctra of all invstigatd sampls wr valuatd within th rang of amu to idntify th main lmntal composition changs among th original OC/Ag(I) sampl and thos aftr diffrnt xprimntal tratmnt (data not shown hr). Th visual inspction of th spctra rvald th significant diffrncs in Na and K ions concntration. Du to this, th concntration of both K and Na in all invstigatd sampls, i.. in OC/Ag(I) bfor and aftr th tratmnt with diffrnt microorganisms and MEB and BHI mdia, was dtrmind by AAS and th data ar includd in Tabl 2. A significant incras in K concntration from 0.09 mg g -1 for th initial OC/Ag(I) sampl up to mg g -1 for th sampls incubatd undr diffrnt xprimntal conditions (s Tabl 2) was obsrvd. Highr lvls of K in th OC/Ag(I) sampls wr du to its prsnc in both nutrint mdia. Along with an incras of K concntration in th OC/Ag(I) sampls, a dcras was obsrvd rgarding th K contnt in both MEB and BHI

9 Oxidizd cllulos (s Tabl 2). As to th prsnc of Na in th OC/Ag(I) sampl, it can b sn from th data prsntd in Tabl 2 that rlativly high Na contnt was prsnt alrady in th initial OC/Ag(I) sampl. This is du to th us of NaCl for photostabilization during th prparation of OC/Ag(I). Th contnt of Na in OC usd for th prparation of th OC/Ag(I) sampl was lowr than 0.05 µg g -1 (s Tabl 2). During th tratmnt of th OC/Ag(I) sampl in MEB mdia, th concntration of Na in th sampl dcrasd and th contnt of Na in th nutrint mdia incrasd, which may b du to th dissolution of NaCl from OC/Ag(I) into th solutions. In BHI mdia, whr significantly highr concntrations of Na wr prsnt in contrast to MEB (2917 vrsus 117 mg L -1 ), a dcras of th Na concntration was obsrvd accompanid by an incras in Na concntration in th OC/Ag(I) sampls. Basd on th computation of th analyzd data, a significant corrlation of r = btwn th amount of K and th contnt of carboxylic groups was notd. No significant corrlation at th significanc lvl of 0.05 was rmarkd in th cas of Na with r = Figur 4: FTIR spctra of (a) control OC/Ag(I) sampl (rd) and thos submittd to a 7 day incubation with (b) A. fumigatus (MEB, ph = 5.4) (black), (c) C. albicans (MEB, ph = 5.4) (grn), (d) P. aruginosa (BHI, ph = 7.0) (blu) and () S.pidrmidis (BHI, ph = 7.0) (cyan) in th rgion cm -1 Tabl 2 Concntrations a of Na and K dtrmind in original OC b and in OC/Ag(I) and nutrint mdia (MEB, ph = 5.4, and BHI, ph = 7.0) bfor and aftr incubation with diffrnt microorganisms for 7 days K (mg g -1 ) c K (mg L -1 ) d Na (mg g -1 ) c Na (mg L -1 ) d OC/Ag(I) MEB BHI OC/Ag(I) MEB BHI No microorg ± ± ± ± ± ± 53 A.fumigatus 3.3 ± ± ± ± 11 C. albicans 2.4 ± ± ± ± 11 P. aruginosa 3.3 ± ± ± ± 107 S. pidrmidis 3.5 ± ± ± ± 53 a Man ± 2SD (n = 3); b Concntration of K and Na in OC was ± mg g -1, rspctivly < 0.05 µg g -1 ; c Concntration in mg g -1 dtrmind in th original OC/Ag(I) sampl or in OC/Ag(I) analyzd aftr incubation with diffrnt microorganisms in MEB or BHI mdia aftr dissolving th sparatd solid phas in cadoxn. Cadoxn solution containd ± mg L -1 of K and < 0.2 mg L -1 of Na; d Concntration in mg L -1 dtrmind in original MEB and BHI mdia or thos sparatd aftr incubation of OC/Ag(I) with diffrnt microorganisms; Not dtrmind 299

10 LENKA HUSÁKOVÁ t al. Howvr, it may b also xpctd that in both MEB and BHI mdia OC-Na salts wr partially cratd as a significant dcras in fr carboxylic groups was obsrvd within % for diffrnt xprimntal conditions and only th contnt of K is not satisfactory for ths changs. On th othr hand, th imprfct sparation of th solid sampl from th rmaining liquid mdia may also b rsponsibl. SEM SEM photographs of th starting OC/Ag(I) and thos aftr incubation with C. albicans and S. pidrmidis, rspctivly, ar shown as xampls in Fig. 5. Undr in vitro conditions in both buffrd nutrint mdia, i.. in nutral or slightly acidic ph at 37 C, within on wk, all OC/Ag(I) sampls undrgo gradual physical changs and los thir fibrous natur and no noticabl solubilization taks plac. Although chmical modification of ths polymrs can also significantly affct thir rat of dgradation, in this spcial cas th obsrvations ar similar to thos prviously rportd 26 for unmodifid OC, whos structur dos not brak down at 37 C vn aftr svral wks unlss th ph is highr than 7, or cllulas nzym is usd. Figur 5: SEM imags of (a) initial OC/Ag(I) and of th sampls tratd with (b) C. albicans in MEB (ph = 5.4) and (c) S. pidrmidis in BHI (ph = 7.0) at 37 C for 7 days CONCLUSION In vitro invstigations of a novl typ of antimicrobial matrial basd on 6- carboxycllulos containing silvr chlorid salt (OC/Ag(I)) undr th influnc of A. fumigatus, C. albicans, P. aruginosa and S. pidrmidis at 37 C in th dark, in buffrd nutrint mdia at nutral or slightly acidic ph for th priod of 7 days rvald that significant silvr rlas into th liquid nutrint mdia is not dpndnt on th prsnc of th invstigatd microorganism strain and did not significantly chang during th 300 obsrvation priod. Additionally, Ag rlas was not dpndnt on th ph of th cultur mdium, bing 5.4 and 7.0. Howvr, if th solution contains an xcss of chlorid, thr is a tndncy toward diffrnt complx formation btwn silvr chlorid and chlorids, which influncs th solubility of AgCl, rsulting in an incras in th concntration of Ag in th analyzd solutions. As follows from th FTIR and chromatographic analysis data, non of th invstigatd microorganisms promotd any significant brakdown of th OC structur undr th st

11 Oxidizd cllulos xprimntal conditions. Th only noticabl changs obsrvd in th matrial structur wr du to th cration of K and Na salts of OC from ions in th nutrint mdia, as confirmd by th valuation of th nutralization dgr by th titrimtric mthod and by qualitativ and quantitativ analyss of both th liquid and th rsulting solid sampls by ICP-TOF-MS and AAS, rspctivly. Th contnt of Na and K was strongly dpndnt on th composition of th nutrint mdia. Additionally, physical changs as a consqunc of th loss of th fibrous charactr wr noticd. Howvr, xposing th sampls to light conditions may rsult in changs of th colour of both OC/Ag(I) and liquid nutrint mdia du to th photorduction of silvr ions. For th intndd clinical us of OC/Ag(I), it must b takn into considration that th wound surfac contains blood, whil physical fluid contains chlorids and som nzyms. This may thn influnc both th microbiological ffctivnss of th silvr ions and th dgradation of OC. Ths invstigations nd to b continud in a furthr study. ACKNOWLEDGEMENTS: Financial support from th Univrsity of Pardubic (projct no. SGFChT06/2013), from th Ministry ofindustry and Trad of th Czch Rpublic (projct no. 2A- 1TP1/073) and from th Czch Scinc Foundation (projct no. 203/08/1536) is gratfully acknowldgd. REFERENCES 1 A. D. Russll and W. B. Hugo, Prog. Md. Chm., 31, 351 (1994). 2 A. B. G. Lansdown, Br. J. Nurs., 13, S6 (2004). 3 H. J. Klasn, Burns, 26, 117 (2000). 4 C. Graham, Br. J. Nurs., 14, S22 (2005). 5 M. Rai, A. Yadav, and A. Gad, Biotchnol. Adv., 27, 76 (2009). 6 N. Simontti, G. Simontti, F. Bougnol, and M. Scalzo, Appl. Environ. Microbiol., 58, 3834 (1992). 7 A. Mlaiy and W. J. Youngs, Exprt Opin. Thr. Pat., 15, 125 (2005). 8 M. Ip, S. L. Lui, V. K. M. Poon, I. Lung, and A. Burd, J. Md. Microbiol., 55, 59 (2006). 9 S. Thomas and P. McCubbin, J. Wound Car, 12, 101 (2003). 10 A. B. G. Lansdown, A. Williams, S. Chandlr, and S. Bnfild, J. Wound Car, 14, 155 (2005). 11 S. Tmplton, Prim. Intntion, 13, 170 (2005). 12 J. Vytrasova, A. Tylsova, I. Brozkova, L. Crvnka, M. Pjchalova t al., J. Ind. Microbiol. Biotchnol., 35,1247 (2008). 13 P. Dinn, Surg. Gyncol. Obstt., 142, 481 (1976). 14 M. Prnt, Ann. Chir., 37,700 (1983). 15 D. Spanglr, S. Rothnburgr, K. Nguyn, H. Jampani, S. Wiss t al., Surg. Infct. (Larchmt), 4, 255 (2003). 16 R. L. Stilwll, M. G. Marks, L. Safrstin, and D. M. Wisman, in "Handbook of Biodgradabl Polymrs", ditd by A. J. Domb, J. Kost, and D. M. Wisman, Hardwood Acadmic Publishrs, 1997, pp E. D. Stakhva-Kavrznva and A. S. Salova, Zh. Anal. Khim., 8, 365 (1953). 18 L. Husakova, I. Urbanova, L. Audrlicka-Vavrusova, J. Sramkova, T. Crnohorsky t al., Microchim. Acta, 173,173 (2011). 19 V. Kumar and T. Yang, Int. J. Pharm., 184, 219 (1999). 20 Y. Wu, J. H, W. Chng, H. Gu, Z. Guo t al., Carbohyd. Polym., 88, 1023 (2012). 21 M. El-Sakhawy and M. Milichovsky, Polym. Int., 49, 839 (2000). 22 R. Ramtt, J. Chm. Educ., 37,348 (1960). 23 J.-Y. Maillard and S. P. Dnyr, EWMA J., 6, 5 (2006). 24 S. D. Dimitrijvich, M. Tatarko, R. W. Gracy, C. B. Linsky, and C. Olsn, Carbohyd. Rs., 195, 247 (1990). 25 S. D. Dimitrijvich, M. Tatarko, R. W. Gracy, G. E. Wis, L. X. Oakford t al., Carbohyd. Rs., 198, 331 (1990). 26 M. Bajrová, K. Krjčová, M. Rabišková, J. Gajdziok, and R. Mastiková, Adv. Polym. Tchnol., 28, 199 (2009). 301