synthesis of silver nanoparticles using curcumin

Synthesis of Silver Nanoparticles Using Curcumin-CyclodextrinsLoaded into Bacterial Cellulose-Based Hydrogels for WoundDressing ApplicationsAbhishek Gupta Sophie M Briffa Sam Swingler Hazel Gibson Vinodh Kannappan Grazyna AdamusMarek Kowalczuk Claire Martin and Iza Radecka

Cite This Biomacromolecules 2020 21 1802minus1811 Read Online

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ABSTRACT Chronic wounds are often recalcitrant to treatment because of high microbial bioburden and the problem ofmicrobial resistance Silver is a broad-spectrum natural antimicrobial agent with wide applications extending to proprietary wounddressings Recently silver nanoparticles have attracted attention in wound management In the current study the green synthesis ofnanoparticles was accomplished using a natural reducing agent curcumin which is a natural polyphenolic compound that is well-known as a wound-healing agent The hydrophobicity of curcumin was overcome by its microencapsulation in cyclodextrins Thisstudy demonstrates the production characterization of silver nanoparticles using aqueous curcuminhydroxypropyl-β-cyclodextrincomplex and loading them into bacterial cellulose hydrogel with moist wound-healing properties These silver nanoparticle-loadedbacterial cellulose hydrogels were characterized for wound-management applications In addition to high cytocompatibility thesenovel dressings exhibited antimicrobial activity against three common wound-infecting pathogenic microbes Staphylococcus aureusPseudomonas aeruginosa and Candida auris

INTRODUCTION

Microbial colonization where it is not wanted can led tosevere infections which can result in disability disease andeven death Wound infections are one of the major factorsresulting in impaired healing1minus3 Wounds colonized byopportunistic microbes may fail to follow the natural reparativeand regenerative stages involved in the healing process4 Inaddition uncontrolled infections can prevent the restoration ofthe anatomical and physiological integrity resulting in chronicnonhealing wounds56 To prevent infections modern medi-cine is dependent on antimicrobial agents such as antibioticswhich can either destroy pathogens or inhibit their growthUnfortunately the misuse of antibiotics has resulted in theemergence of a number of multiresistant microbes Therefore

antibiotic therapy often proves to be ineffective in eradicating

infections in chronic nonhealing wounds3 Owing to the sitespecific delivery increased target site concentration reduced

adverse effects use of agents not suitable for oral and systemictherapy and low incidence of resistance78 antimicrobial-

loaded wound dressings have attracted wide interest as aconcurrent wound-management regime

Special Issue Anselme Payen Award Special Issue

Received December 13 2019Revised January 21 2020Published January 22 2020

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copy 2020 American Chemical Society1802

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This is an open access article published under a Creative Commons Attribution (CC-BY)License which permits unrestricted use distribution and reproduction in any mediumprovided the author and source are cited

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Hydrogels are one of the promising candidates as wounddressings because of their unprecedented properties includingtheir ability to maintain a moist microclimate at the wound sitewhich is proven to facilitate healing9minus11 A number of naturaland synthetic polymeric materials are in use to producehydrogel dressings1213 Bacterial cellulose (BC) is an exampleof a natural biosynthetic cellulose hydrogel which has beenwidely considered as a wound dressing because of its uniqueproperties14minus18 BC can be produced by several bacteria butGluconacetobacter xylinus (G xylinus) is considered as one ofthe best for BC production19 Our group has previouslyreported the findings on the physicochemical characterizationand suitability of BC hydrogels as wound dressings20 AlthoughBC does not have inherent antimicrobial activity however itscross-linked fiber network structure encouraged severalresearch attempts of loading antimicrobials anti-inflammatoryantioxidants and other healing agents for wound-dressingapplications172122 In continuation in the presented study aninvestigation has been made to produce biosynthetic hydrogelswith healing properties for chronic wound management Ournovel approach is consistent with the concept of the forensicengineering of advanced polymeric materials (FEAPM) whichcan help to understand the relationships between the structureof the biomaterial used its properties and behavior forpractical applications23

Currently the treatment of microbial infections is severelyaffected by the emergence of new multiresistant microbesincluding bacteria and fungi Gram-positive Staphylococcusaureus has a great ability to develop multiple drug resistancesSimilarly Gram-negative Pseudomonas aeruginosa a commonopportunistic pathogen shows resistance to most penicillinscephalosporins and carbapenems Pathogenic fungi such asCandida auris pose a significant health risk to patients sufferingfrom infected wounds and in particular to immunocompro-mised individuals which can result in candidiasiscandidemiaand invasive aspergillosis Recently a new multidrug-resistantstrain of C auris was discovered24 and has quickly become aprolific nosocomial pathogen in the UK2526 with diagnoseson all continents with 33 countries reporting incidents ofantifungal resistance C auris is resistant to most first- andsecond-generation antifungals with 90 of isolates beingresistant to fluconazole 40 resistant to amphotericin and 2to echinocandins such as caspofungin27minus29 The concernregarding these pathogens centers around the increasingresistance they are showing toward a very limited pool ofeffective antimicrobials30minus32 and hence the focus is ontargeted delivery by antimicrobial wound dressingsSilver is a well-known antimicrobial effective against fungi

yeast and bacteria including several antibiotic-resistantstrains33minus35 The emergence of nanotechnology enabling theproduction of silver nanoparticles has served a new therapeuticmodality Because of their characteristic broad-spectrumantimicrobial properties silver nanoparticles (AgNP) havereceived increased focus in biomedical applications includingfor wound management36minus38 Among the several differentapproaches to synthesize AgNP the use of natural substanceslike plant extracts has received wide research considerationbecause of the safe and eco-friendly procedure3739minus41

Curcumin (CUR) a natural polyphenol extracted fromturmeric is a well-known wound-healing agent with provenantimicrobial antioxidant and anti-inflammatory effects2242

In addition to its healing properties CUR has been used as areducing and capping agent to produce AgNP43 The main

challenge that limits the wider biomedical application of CURis its hydrophobic nature Several chemical mediators likedimethyl sulfoxide dichloromethane and sodium carbonateare employed in AgNP synthesis from CUR44minus48 Thesechemical mediators can have serious cytotoxic effects48

Selection of solvent medium reducing agent and nontoxicstabilizer are the main considerations in green nanoparticleproduction4950 The current study reports the production ofAgNP following a green chemistry approach using an aqueoussolution of curcuminhydroxypropyl-β-cyclodextrin(CURHPβCD) These AgNP produced using CURHPβCD(cAgNP) were characterized and loaded in the biosyntheticBC hydrogels to produce hydrogel dressings To the best ofour knowledge the protocol presented herein to producecAgNP is novel and this is the first time the production ofcAgNP-loaded BC has been reported This report presents theunique combination of silver nanoparticles with curcumin inthe biosynthetic BC to obtain the hydrogels for wound-management applications This study investigates the inherentantimicrobial antioxidant and cytocompatibility properties ofthese novel hydrogel dressings

MATERIALS AND METHODSMaterials Gluconoacetobacter xylinus (ATCC 23770) Pseudomo-

nas aeruginosa (NCIMB 8295) and Staphylococcus aureus (NCIMB6571) were obtained from the University of Wolverhampton culturecollection Candida auris (NCF 8971) was obtained from the PublicHealth England Culture Collection Porton Down UK A stockculture of P aeruginosa and S aureus were resuscitated at 37 degC ontryptone soy agar (TSA) (Sigma-Aldrich UK) G xylinus at 30 degCon mannitol agar and C auris at 37 degC on Sabouraud Dextrose Agar(SDA) HS media was prepared following the standard protocol51

Material for mannitol agar SDA and HS media were purchased fromLab M (UK) Tryptone soya broth (TSB) disodium phosphate andcitric acid were purchased from Sigma-Aldrich (UK) Overnightbroth cultures were prepared in appropriate medium using the stockplates

U251MG (U251) MSTO and Panc 1 cell lines were purchasedfrom ATCC (UK) Defibrinated horse whole blood was purchasedfrom TCS Biosciences Ltd

Silver nitrate was purchased from Fischer Scientific (UK)Hydroxypropyl-β-cyclodextrin (parenteral grade) was kindly providedby Roquette (France) Curcumin and dimethyl sulfoxide (DMSO)(spectrophotometric grade) were purchased from Alfa Aesar (UK)Ringer tablets were purchased from Lab M (UK) Thiazolyl BlueTetrazolium Bromide (MTT) sodium bicarbonate and 22-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (UK)Sodium chloride 09 (wv) (normal saline intravenous infusion)was purchased from Baxter UK Sodium hydroxide was purchasedfrom Acros Organics (UK) NaCl (58 gL) and glycine (76 gL)for preparing Sorensenrsquos glycine buffer were purchased from Sigma-Aldrich UK Trypsin was purchased from Lonza (Belgium)Dulbeccorsquos Modified Eaglersquos Medium (DMEM) fetal bovine serum(FBS) L-glutamine and antibiotic antimycotic were purchased fromGibco (UK)

Preparation of CURHPβCD Inclusion Complex Inclusioncomplex was synthesized by the solvent evaporation method followingthe protocol previously reported22 Briefly CUR solution (079 g in375 mL acetone) was added dropwise to the aqueous HPβCDsolution (30 g in 125 mL deionized water) under constant stirring atroom temperature for slow and complete evaporation of acetone Thesample was centrifuged and the aqueous supernatant containingCURHPβCD inclusion complex was frozen after filtration andlyophilized

Preparation and Characterization of cAgNP Preparation ofcAgNP Using CURHPβCD A novel green chemistry approach wasdeveloped for cAgNP synthesis by reducing silver nitrate (AgNO3)

Biomacromolecules pubsacsorgBiomac Article


1803

with an aqueous solution of CURHPβCD CURHPβCD (019 g)solution was prepared by dissolving in deionized water (18 mL)CURHPβCD aqueous solution was added dropwise with constantstirring to 1 mM AgNO3 aqueous solution (42 mL) under the boilingcondition in conical flasks The mixed solutions were boiled for 3 hfor reduction of Ag ions followed by cooling at room temperature for30 min The flask was covered with aluminum foil to maintain darkconditions throughout the reduction process to avoid any photo-chemical reactionsCharacterization of cAgNP Dynamic Light Scattering (DLS)

and Zeta Potential DLS measurements were performed on aMalvern Zetasizer (nano ZS) at the University of Birmingham Fiveconsecutive measurements were carried out at 25 degC with samplesequilibrated for 2 min before the measurements were started Theresults were averaged to calculate the mean size The same instrumentwas used to obtain the Zeta Potential values The same samples wereused for size measurements and equilibrated for 2 min beforemeasurements were started The results were obtained at 25 degC Fiveconsecutive measurements were taken and averaged to calculate thezeta potentialTransmission Electron Microscopy (TEM) The shape size and

distribution of cAgNP produced using CURHPβCD were inves-tigated by TEM Briefly a drop of aqueous colloidal cAgNP wascasted on a carbon-coated 300 mesh copper grid (agar scientific) leftfor 30 min rinsed off and allowed to dry at room temperature in acovered container TEM imaging was performed using JEOL 1400electron microscope operated at 80 keV Images were captured atdifferent range of magnifications A 100 cAgNP were randomlyselected and measured using ImageJ to obtain the size distributionPreparation of cAgNP-Loaded BC Hydrogels Production of

Bacterial Cellulose Hydrogels Bacterial cellulose (BC) hydrogelswere prepared by the protocol reported previously22 Briefly Gxylinus was selected for BC production and hydrogels werebiosynthesized in sterilized Hestrin and Schramm (HS) culturemedium under static condition BC pellicles were purified prior toexperimental useLoading cAgNP in BC Hydrogels BC pellicles afterpurification

were padded dry on sterilized filter paper and aseptically loaded withcAgNP by immersing in aqueous colloidal cAgNP overnight underagitated conditions at 4 degCCharacterization Studies Morphological Study by Scanning

Electron Microscopy (SEM) Morphology of BC (neat) has beenpreviously reported2252 BC before purification purified BC (neat)and cAgNP-loaded BC hydrogels were cut in to discs (8 mm) using abiopsy punch and lyophilized These discs were then coated withultrafine gold coating using SC500 fine coater (Emscope Kent UK)and placed on a carbon stub The morphology of the samples wasstudied using a Zeiss Evo 50 EP SEM (Carl Zeiss AG OberkochenGermany)Energy-Dispersive X-ray (EDX) Analysis Lyophilized cAgNP-

loaded BC hydrogel samples were analyzed by EDX (Zeiss Evo 50 EPSEM) coupled with X-MaxN 50 Silicon Drift Detector (OxfordInstruments) and the analysis was performed by Oxford InstrumentsINCA Energy Dispersive Spectroscopy Nanoanalysis software usingthe Point amp ID function to confirm the presence of silverMoisture Content (Mc) The wet mass (Ww) of BC (neat) and

cAgNP-loaded BC (test) hydrogels was recorded before lyophiliza-tion and the dry mass (Wd) was measured after 72 h of lyophilizationMc () was calculated using a formula

MW W

W

( )100c

w d

w=

minustimes

Cytocompatibility Study (In Vitro Cell Viability) Cytocompati-bility of BC (neat) has been previously tested and reported usingconditioned media20 and BC discs22 on different mammalian celllines To study the cytocompatibility of cAgNP-loaded BC an in vitrocell viability test was performed using Panc 1 (human pancreaticductal adenocarcinoma) U251 (human glioblastoma) and MSTO(human mesothelioma) following the protocol previously reported22

All cell lines were cultured in Dulbeccorsquos Modified Eaglersquos Medium(DMEM) supplemented with fetal bovine serum (FBS) antibioticantimycotic and L-glutamine at 37 degC in humidity incubator with 5CO2 BC pellicles after pad drying were either rehydrated with PBS(control) or cAgNP (test) overnight under agitated conditions (150rpm) at 4 degC These samples (control and test) were then asepticallycut in discs (8 mm) using a biopsy punch for experimental purposesand the temperature was adjusted to 37 degC before use

Briefly 25 000 cells were seeded per well (24-well plate) for 24 h at37 degC in 5 CO2 incubator The cells were exposed to either freecAgNP (equivalent amount) or cAgNP-loaded BC (8 mm discs) for24 h to investigate the effect on cell viability Cells without BC discsand PBS-loaded BC discs (8 mm) were used as controls respectivelyCell viability was evaluated following the standard MTT assaypreviously reported22 Data recorded was analyzed statistically by two-way analysis of variance (ANOVA) with a Tukeyrsquos multicomparisonstest using GraphPad Prism

Hemocompatibility The hemocompatibility of cAgNP-loaded BChydrogels was tested following the protocol previously reported22

Briefly defibrinated horse whole blood was washed twice usingcommercially available normal saline (pH 55) and blood cells wereresuspended in normal saline cAgNP-loaded BC discs (8 mm) werecut using a biopsy punch and incubated with 19 mL of horse bloodcells suspended in normal saline in test Eppendorf tubes The positive(+ve) control was blood cells suspended in distilled water andnegative (minusve) control was blood cells in normal saline All the testand control Eppendorf tubes were incubated at 4 degC for 2 h withperiodic inversion (every 15 min) Postincubation after the removalof BC discs from test Eppendorf tubes all the tubes were centrifugedat 3000 rpm for 10 min at 4 degC The supernatant was decanted andabsorbance was recorded (540 nm) to calculate percentage hemolysis( Hemolysis)

Hemolysis(Abs of sample) (Abs of ve control)

(Abs of ve control) (Abs of ve control)

100

=minus minus

+ minus minus

times

Antimicrobial Study (Disc Diffusion Assay) The antimicrobialactivity of cAgNP-loaded BC was investigated against P aeruginosa(Gramminusve) S aureus (Gram + ve) bacteria and C auris using thedisc diffusion assay PBS-loaded BC and HPβCD-loaded BC wereused as controls Discs (8 mm) of PBS-loaded BC HPβCD-loadedBC and cAgNP-loaded BC were placed aseptically on TSA platesspread with an overnight culture of P aeruginosa or S aureus andSDA plates were spread with an overnight culture of C auris Plateswere incubated at 37 degC for 24 h and zones of inhibition (ZOI) weremeasured Data is presented as mean plusmn standard deviation (SD) andanalyzed statistically by two-way ANOVA with a Tukeyrsquos multicomparisons test using GraphPad Prism

Antioxidant Study by DPPH Assay The antioxidant potential ofcAgNP produced using CURHPβCD was evaluated using 22-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay Theassay mixture with 1 mL of DPPH (80 μgmL) methanolic solutionand 1 mL of test colloidal cAgNP (including the blank) after mixingwere incubated for 30 min in the dark at room temperatureAbsorbance was recorded (517 nm) and the free radical scavengingpotential was calculated as percent antioxidant effect ( E) as

EAbs Abs

Abs100control sample

control=

minustimes

Transparency Test Monitoring the healing process without theneed of removing dressing could help minimize trauma to thegranulating tissue With the aim to use the hydrogel as a wound-dressing application the transparency level of cAgNP-loaded BChydrogels was assessed by simulation PBS-loaded BC and cAgNP-loaded-BC hydrogels were transferred on the laminated sheet of paperwith text typed in different colors The clarity of text underneath thehydrogel was examined to determine if the healing tissue at the woundsite could be observed through these hydrogels



1804

RESULTS AND DISCUSSION

Preparation of cAgNP and cAgNP-Loaded BC Hydro-gels Most of the AgNP synthesis methods involve the use oforganic solvents and toxic reducing agents with the potentialthreat to the environment and cytotoxic effect on mammaliancell lines The method reported herein involved bioreductionof AgNO3 with an aqueous solution of CURHPβCD resultingin nanoparticle formation The color changed from colorless topale yellow at the start of CURHPβCD addition whichintensified with increased dosage and subsequently changed toyellow-brown with time suggesting the production ofnanoparticles This could be due to the excitation of surfaceplasmon vibrations in AgNPs which is in accordance withliterature4053

G xylinus produced BC hydrogel pellicles which wereharvested and purified BC pellicles became clear afterpurification These results are in accordance with previousreports1522335254 When the purified padded dry BC wasrehydrated with the aqueous colloidal cAgNP the pelliclesreswelled and the color changed to yellow-brown ThesecAgNP-loaded BC hydrogels were stored at 4 degC throughoutthe experimental procedureCharacterization of cAgNP and cAgNP-Loaded BC

Hydrogels The formation of colloidal cAgNP produced wasconfirmed by testing its properties like size distribution andcharge After loading aqueous colloidal cAgNP in the paddeddry BC the physicochemical and in vitro hemocompatibilityand cytocompatibility hydrogels were investigatedParticle Size Distribution and Surface Charge by

TEM DLS and Zeta Potential The size and morphology ofcAgNP produced using CURHPβCD were studied from the

TEM images (Figure 1aminusc) TEM imaging showed that themorphology of the nanoparticles was mainly spherical in shapewith smooth edges The majority of bioreduced cAgNP were inthe diameter range of 20minus55 nm (80) and the average size of4271 plusmn 1797 nm (Figure 1aminusc) It emerged from the resultsthat nanoparticles were homogeneously surrounded by a thinlayer of capping material As the method employed in thecurrent work involved only AgNO3 and CURHPβCDwithout the use of any organic solvents and additional cappingagents this suggests that CURHPβCD was acting both as areducing and capping agent These results are in agreementwith literature reporting the use of curcumin as a reducing andcapping agent4043464853

DLS measurements identified nanoparticles with the hydro-dynamic diameter in the range of 18210 plusmn 883 nm (Figure1d) and the polydispersity index (PDI) value of 0196 plusmn 0009These results indicate both nucleation to form new nano-particles and aggregation could be happening consecutivelyThese findings are consistent with studies reported in theliterature5355 Low PDI indicates that the colloidal cAgNP wasnot very polydispersedThe zeta potential value is directly proportional to the

stability of nanoparticle dispersion55 Zeta potential studiesrevealed a negative charge on the synthesized nanoparticleswith the magnitude of minus201 plusmn 0702 mV These findings arein a close range to the previously reported values for AgNPbioreduced using curcumin43

Scanning Electron Microscopy (SEM) BC has a finefiber network structure with voids212252 SEM images revealedG xylinus trapped in the cellulose network before purification(Figure 2a) and that bacteria were successfully removed on

Figure 1 Characterization of cAgNP produced using CURHPβCD (ab) TEM photomicrographs (c) Size distribution as measured by TEManalysis and calculated with 100 nanoparticles (d) DLS data of cAgNP with size distribution



1805

purification (Figure 2b) The interspersed voids in the

interwoven cellulose ribbons of BC allow impregnation of

healing agents173356 SEM of lyophilized cAgNP-loaded BC

revealed that cAgNP penetrated through the voids during

rehydration of padded dry BC pellicles and got physically

trapped in the fiber network structure (Figure 2c) Also it

emerged that along with bioreduced cAgNP there was free

CURHPβCD trapped in the BC In addition to the reducing

Figure 2 SEM photomicrographs of (a) unwashed BC entangled G xylinus highlighted (b) BC after purification (cd) cAgNP loaded in BC fibernetwork

Figure 3 EDX spectrum of cAgNP-loaded BC



1806

and capping properties in cAgNP synthesis CURHPβCD hasbeen demonstrated to have wound-healing properties22 Theexcess of CUR inclusion complex in cAgNP-loaded BC maydeliver additional benefits contributing to wound healingMorphology and size of nanoparticles was also determined

in the SEM photomicrographs (Figure 2d) The averagediameter was in the range of 372minus651 nm and most of thenanoparticles appeared spherical These results are inaccordance with TEM results discussed in the section aboveEnergy-Dispersive X-ray (EDX) Analysis The elemental

analysis was carried out by EDX studies The results confirmedthat BC (neat) is composed mainly of carbon and oxygenwhich is in accordance with our previously reported data33 Inaddition to carbon and oxygen detection of silver in the EDXspectra of cAgNP-loaded BC (test) confirmed nanoparticleloading in the test samples (Figure 3)Moisture Content (Mc) BC hydrogels have been reported

to have a unique property of resistance to degradation despiteconsiderable moisture content which is several times its drymass14 This strength is contributed by its cross-linked fibernetwork structure In the current study the moisture contentof neat BC and cAgNP-loaded BC hydrogels was evaluatedThe results confirmed that neat BC pellicles imbibed 9968 plusmn009 (vw) (n = 3) water which is in accordance withpreviously reported data (gt995) for neat BC hydrogels2022

Moreover the study on cAgNP-loaded BC revealed that themoisture content in these hydrogels was 9886 plusmn 004 (n =3) These findings indicate that the difference in the mass ofneat BC and cAgNP-loaded BC is contributed by cAgNP and

CURHPβCD that gets physically trapped in the fiber networkof BC during the loading processThe high moisture content in BC can confer many benefits

such as increased malleability soft texture and creating a moistenvironment with increased dissolved oxygen to facilitateaerobic conditions at the wound-dressing interface In additionthese hydrogels may ease removal of the dressing reduce painsensation and therefore improve patient comfort Thesefeatures have been reported to facilitate the wound-healingprocess15215758 and therefore BC has attracted increasedinterest in the wound-care sector

Cytocompatibility (In Vitro Study) The cytocompatiblenature of BC is well documented in the literature20minus2259 It isone of the many intrinsic features that has resulted in BC-based proprietary products like Dermafill Biofill XCell andGengiflex5760minus62 In this study the cytocompatibility ofcAgNP-loaded BC hydrogels was evaluated using threedifferent mammalian cell lines The cytotoxicity as determinedby MTT assay revealed that cAgNP-loaded BC is cytocompat-ible as all the tested cell lines demonstrated good survival rate(Figure 4a)Furthermore we compared the cytocompatibility of free

cAgNP to cAgNP-loaded BC on U251 MSTO and Panc 1 celllines The results demonstrated that free cAgNP had acytotoxic effect on all the tested cell lines resulting in lowercell viability compared with cAgNP-loaded in BC (p lt 0001)(Figure 4ab) These results suggest that BC controls therelease of cAgNP thus minimizing the cytotoxic effect on themammalian cells These findings support the potential

Figure 4 Cytocompatibility test results (a) Bar graph showing the cell viability () after 24 h exposure to cAgNP-loaded BC and free cAgNP(equivalent amount) (n = 8) Representative photomicrographs of cells (10times magnification) after exposure to cAgNP-loaded BC and free cAgNPfor 24 h



1807

application of cAgNP-loaded BC for wound management ashydrogel dressingsHemocompatibility Hemocompatibility is an important

property for biomedical applications of a material BC has beenreported to be hemocompatible hence it has been used inproprietary wound dressings206364 In the current studycAgNPs were prepared in deionized water hence thehypothesis was drawn that cAgNP-loaded BC hydrogels mayhave hemolytic properties The test results revealed thatcAgNP-loaded BC hydrogels have a percentage hemolysis of685 plusmn 112 (n = 6) According to the ASTM F756 standardshemolytic indices this range is over the threshold value of 5hence cAgNP-loaded BC hydrogels would be classified as ahemolytic material65 The higher hemolysis () of the testedhydrogels could be attributed to the use of deionized waterinstead of the isotonic solution for the synthesis of cAgNPIn the case of a chronic wound there could be necrotic

tissue or slough at the wound bed and hence the hemolyticbehavior of these hydrogels may be minimal Further researchon the production of AgNP using CURHPβCD dissolved inisotonic solution may improve hemocompatibility of thesehydrogelsAntimicrobial Study Invasion of opportunistic microbes

could impair wound healing leading to chronic nonhealingwounds666 Silver nanoparticles have been intensively studiedas antimicrobial agents404953 Different mechanisms of actionof AgNPs have been proposed as their antibacterial andantifungal effect is not completely known67 In bacteria AgNPshave the ability to increase the permeability of the cellmembrane interfere with DNA replication denature bacterialproteins and release silver ions inside the bacterial cell4967

The antifungal action of silver nanoparticles are thought toincrease the reactive oxygen species within the fungal cell andincrease membrane permeability resulting in cell death68

In the current study PBS-loaded BC HPβCD-loaded BCand cAgNP-loaded BC hydrogels were tested against Paeruginosa S aureus and C auris using the disc diffusionassay at 24 h PBS-loaded BC and HPβCD-loaded BC did notexhibit antimicrobial activity however cAgNP-loaded BCdemonstrated significant antimicrobial activity (p lt 0001)against all three of the tested microbial strains (Figure 5)These results confirm the broad spectrum antimicrobial

activity of cAgNP-loaded in BC Moreover it is apparent thatcAgNP-loaded in BC has stronger antimicrobial activity(Figure 5) against P aeruginosa as compared with S aureus(p lt 0001) This could be explained by the difference in thecell structure of Gram-positive and Gram-negative bacteria

AgNPs have been reported to have the ability to separatecytoplasm from the bacterial cell wall (plasmolysis effect)leading to cell death in P aeruginosa In S aureus AgNPs actdifferently and cause the bacterial cell death by inhibiting thecell wall synthesis496769

Antioxidant Activity of cAgNP by DPPH AssayOxidative stress at the wound site may interfere with thehealing process70 hence a wound dressing with antioxidantproperties along with antimicrobial activity may prove to bebeneficial71 CUR has been reported to have healing propertiesincluding antimicrobial and antioxidant activities It has beenpreviously demonstrated that these properties were preservedin the inclusion complex of CUR in HPβCD22

In the current study cAgNP were successfully preparedusing CURHPβCD as reducing and stabilizing agent Thepercent antioxidant effect ( E) for aqueous cAgNP colloidalsuspension against DPPH was determined to be 7665 plusmn321 (n = 6) The test results confirmed that silver nitrate andHPβCD does not have antioxidant activity which is inaccordance with previously reported findings22 These resultsconfirmed that colloidal cAgNP aqueous medium producedusing CURHPβCD has antioxidant activity and when it isloaded in BC to produce hydrogels it could prove to bebeneficial in wound-healing process

Transparency Test Monitoring of wound-healing processis vital from a clinical perspective72minus74 Routinely this involvesthe removal of a dressing which can disturb the granulatingtissue and could cause trauma to the wound A dressing withthe feature enabling noninvasive monitoring could bebeneficial in wound healingThe transparent nature of BC has already been reported22

and in this study PBS-loaded BC hydrogels reconfirmed thesefindings (Figure 6a) In this study the transparency property of

cAgNP-loaded BC was evaluated by reading the text indifferent colors on white laminated paper sheet through thetest hydrogels The results (Figure 6b) demonstrate cAgNP-loaded BC hydrogels allow monitoring the wound without theneed for removal of the dressing This transparency testing is asimulation study and in order to confirm the results clinicalvalidation would be required and could be evaluated using invivo animal models

CONCLUSIONSThe development of advanced polymeric materials requiresprecise evaluation of structure properties and behavior toavoid potential failures of the commercial products manufac-tured from them The current study demonstrates the

Figure 5 Antimicrobial disc diffusion assay results for PBS-loadedBC HPβCD-loaded BC and cAgNP-loaded BC against P aeruginosaS aureus and C auris at 24 h (n = 10 error bars = SD)

Figure 6 Photomicrographs with the visual appearance of (a) BCloaded with PBS (b) cAgNP-loaded BC hydrogel pellicle



1808

production and both physicochemical and in vitro character-ization of cAgNP-loaded BC hydrogels It also increasesefficiency and minimizes the potential failure of preciselystructured biomaterials before during and after specificapplications The results confirmed that cAgNPs weresuccessfully synthesized following the green chemistryapproach using CURHPβCD and loaded in BC to producehydrogels with a potential wound-dressing application Thesehydrogels demonstrated broad-spectrum antimicrobial activityalong with antioxidant properties Moreover the hydrogelsshowed cytocompatibility with the tested cell lines The highmoisture content and the good level of transparency furtheradvocate their potential application in the management ofchronic wounds with high microbial bioburden

AUTHOR INFORMATIONCorresponding AuthorsAbhishek Gupta minus School of Pharmacy Faculty of Science andEngineering and Research Institute in Healthcare ScienceFaculty of Science and Engineering University ofWolverhampton WV1 1LY Wolverhampton UKorcidorg0000-0001-7901-1818 Email agupta

wlvacukMarek Kowalczuk minus Centre of Polymer and Carbon MaterialsPolish Academy of Sciences 41-819 Zabrze Polandorcidorg0000-0002-2877-7466

Email marekkowalczukcmpw-paneduplIza Radecka minus Wolverhampton School of Sciences Faculty ofScience and Engineering and Research Institute in HealthcareScience Faculty of Science and Engineering University ofWolverhampton WV1 1LY Wolverhampton UKorcidorg0000-0003-3257-8803 Email IRadecka

wlvacuk

AuthorsSophie M Briffa minus School of Geography Earth andEnvironmental Sciences University of Birmingham B15 2TTBirmingham UK

Sam Swingler minus Wolverhampton School of Sciences Faculty ofScience and Engineering University of Wolverhampton WV11LY Wolverhampton UK

Hazel Gibson minus Wolverhampton School of Sciences Faculty ofScience and Engineering and Research Institute in HealthcareScience Faculty of Science and Engineering University ofWolverhampton WV1 1LY Wolverhampton UK

Vinodh Kannappan minus Research Institute in Healthcare ScienceFaculty of Science and Engineering University ofWolverhampton WV1 1LY Wolverhampton UK

Grazyna Adamus minus Centre of Polymer and Carbon MaterialsPolish Academy of Sciences 41-819 Zabrze Poland

Claire Martin minus Department of Biological Sciences School ofSciences and the Environment University of Worcester WR13AS Worcester UK orcidorg0000-0002-5497-4594

Complete contact information is available athttpspubsacsorg101021acsbiomac9b01724

NotesThe authors declare no competing financial interest

ACKNOWLEDGMENTSThe authors would like to thank Professor Wang Weiguangand his team for their kind assistance Abhishek Gupta wouldlike to thanks the School of Pharmacy at the University of

Wolverhampton for the support towards his PhD researchThis paper is partly supported by European Unionrsquos Horizon2020 research and innovation programme under the MarieSkłodowska-Curie grant agreement No 872152 projectGREEN-MAP

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(17) Faisul Aris F A Mohd Fauzi F N A Tong W Y SyedAbdullah S S Interaction of silver sulfadiazine wıth bacterial cellulosevia ex-situ modification method as an alternative diabetic woundhealing Biocatal Agric Biotechnol 2019 21 101332(18) Khosravi K Koller M Akramzadeh N Mortazavian A MBacterial nanocellulose biosynthesis and medical application Biointer-face Res Appl Chem 2016 6 1511minus1516(19) Abeer M M Mohd Amin M C I Martin C A review ofbacterial cellulose-based drug delivery systems their biochemistrycurrent approaches and future prospects J Pharm Pharmacol 201466 1047minus1061(20) Gupta A Low W L Britland S T Radecka I Martin CPhysicochemical characterisation of biosynthetic bacterial cellulose asa potential wound dressing material Br J Pharmacol 2018 2 S37minusS38(21) Zmejkoski D Spasojevic D Orlovska I Kozyrovska NSokovic M Glamoclija J Dmitrovic S Matovic B Tasic NMaksimovic V Sosnin M Radotic K Bacterial cellulose-lignincomposite hydrogel as a promising agent in chronic wound healingInt J Biol Macromol 2018 118 494minus503(22) Gupta A Keddie D J Kannappan V Gibson H Khalil IR Kowalczuk M Martin C Shuai X Radecka I Production andcharacterisation of bacterial cellulose hydrogels loaded with curcuminencapsulated in cyclodextrins as wound dressings Eur Polym J 2019118 437minus450(23) Kowalczuk M M Forensic Engineering of AdvancedPolymeric Materials Mathews J Forensic Research 2017 1 No e001(24) Kean R Ramage G Combined Antifungal Resistance andBiofilm Tolerance the Global Threat of Candida auris mSphere 20194 No e00458-19(25) Ciric L Candida auris The new superbug on the block BBC[online] Available at httpswwwbbccouknewshealth-491708662019(26) Speare-Cole R Candida auris Eight Britons who died in UKhospitals were infected with Japanese super-fungus Evening Standard[online] Available at httpswwwstandardcouknewsukeight-britons-who-died-in-hospital-were-infected-with-japanese-superfungus-a4128901html 2019(27) Osei Sekyere J Candida auris A systematic review and meta-analysis of current updates on an emerging multidrug-resistantpathogen MicrobiologyOpen 2018 7 No e00578(28) Lockhart S Candida auris and multidrug resistance Definingthe new normal Fungal Genet Biol 2019 131 103243(29) Vallabhaneni S Jackson B Chiller T Candida auris AnEmerging Antimicrobial Resistance Threat Ann Intern Med 2019171 432minus433(30) Arikan-Akdagli S Ghannoum M Meis J AntifungalResistance Specific Focus on Multidrug Resistance in Candida aurisand Secondary Azole Resistance in Aspergillus fumigatus J Fungi2018 4 129minus142(31) Gohlar G Hughes S How to improve antifungal stewardshipPharmaceutical J 2019 303 7927(32) Srivastava V Singla R Dubey A Emerging Virulence DrugResistance and Future Anti-fungal Drugs for Candida PathogensCurr Top Med Chem 2018 18 759minus778(33) Gupta A Low W L Radecka I Britland S T Mohd AminM C I Martin C Characterisation and in vitro antimicrobial activityof biosynthetic silver-loaded bacterial cellulose hydrogels J Micro-encapsulation 2016 33 725minus734(34) Melaiye A Youngs W J Silver and its application as anantimicrobial agent Expert Opin Ther Pat 2005 15 125minus130(35) Brandt O Mildner M Egger A E Groessl M Rix UPosch M Keppler B K Strupp C Mueller B Stingl GNanoscalic silver possesses broad-spectrum antimicrobial activitiesand exhibits fewer toxicological side effects than silver sulfadiazineNanomedicine 2012 8 478minus488(36) Parveen A Kulkarni N Yalagatti M Abbaraju VDeshpande R In vivo efficacy of biocompatible silver nanoparticles

cream for empirical wound healing J Tissue Viability 2018 27 257minus261(37) Ravindran J Arumugasamy V Baskaran A Wound healingeffect of silver nanoparticles from Tridax procumbens leaf extracts onPangasius hypophthalmus Wound Medicine 2019 27 100170(38) Ahsan A Farooq M A Therapeutic potential of greensynthesized silver nanoparticles loaded PVA hydrogel patches forwound healing J Drug Delivery Sci Technol 2019 54 101308(39) Keshari A K Srivastava R Singh P Yadav V B Nath GAntioxidant and antibacterial activity of silver nanoparticlessynthesized by Cestrum nocturnum J Ayurveda and IntegrativeMedicine 2018 1minus8(40) Alsammarraie F K Wang W Zhou P Mustapha A LinM Green synthesis of silver nanoparticles using turmeric extracts andinvestigation of their antibacterial activities Colloids Surf B 2018171 398minus405(41) Hemmati S Rashtiani A Zangeneh A Zangeneh M MMohammadi P Veisi H Green synthesis and characterization ofsilver nanoparticles using Fritillaria flower extract and theirantibacterial activity against some human pathogens Polyhedron2019 158 8minus14(42) Akbik D Ghadiri M Chrzanowski W Rohanizadeh RCurcumin as a wound healing agent Life Sci 2014 116 1minus7(43) Song Z Wu Y Wang H Han H Synergistic antibacterialeffects of curcumin modified silver nanoparticles through ROS-mediated pathways Mater Sci Eng C 2019 99 255minus263(44) Pandit R Gaikwad S Agarkar G Gade A Rai MCurcumin nanoparticles physico-chemical fabrication and its in vitroefficacy against human pathogens 3 Biotech 2015 5 991minus997(45) Abdulwahab F Henari F Z Cassidy S Winser K Synthesisof Au Ag Curcumin AuAg and Au-Ag Nanoparticles and TheirNonlinear Refractive Index Properties J Nanomater 2016 20165356404(46) Yang X X Li C M Huang C Z Curcumin modified silvernanoparticles for highly efficient inhibition of respiratory syncytialvirus infection Nanoscale 2016 8 3040minus3048(47) Murugesan K Koroth J Srinivasan P P Singh AMukundan S Karki S S Choudhary B Gupta C M Effects ofgreen synthesised silver nanoparticles (ST06-AgNPs) using curcuminderivative (ST06) on human cervical cancer cells (HeLa) in vitro andEAC tumor bearing mice models Int J Nanomed 2019 14 5257minus5270(48) Khan M J Shameli K Sazili A Q Selamat J Kumari SRapid Green Synthesis and Characterization of Silver NanoparticlesArbitrated by Curcumin in an Alkaline Medium Molecules 2019 24719(49) Lyu Y Yu M Liu Q Zhang Q Liu Z Tian Y Li DChangdao M Synthesis of silver nanoparticles using oxidized amyloseand combination with curcumin for enhanced antibacterial activityCarbohydr Polym 2020 230 115573(50) Shameli K Ahmad M B Zamanian A Sangpour PShabanzadeh P Abdollahi Y Mohsen Z Green biosynthesis ofsilver nanoparticles using Curcuma longa tuber powder Int JNanomed 2012 7 5603minus5610(51) Hestrin S Schramm M Synthesis of cellulose by Acetobacterxylinum II Preparation of freeze-dried cells capable of polymerizingglucose to cellulose Biochem J 1954 58 345(52) Liu D Cao Y Qu R Gao G Chen S Zhang Y Wu MMa T Li G Production of bacterial cellulose hydrogels with tailoredcrystallinity from Enterobacter sp FY-07 by the controlled expressionof colanic acid synthetic genes Carbohydr Polym 2019 207 563minus570(53) Sathishkumar M Sneha K Yun Y Immobilization of silvernanoparticles synthesized using Curcuma longa tuber powder andextract on cotton cloth for bactericidal activity Bioresour Technol2010 101 7958minus7965(54) Jalili Tabaii M Emtiazi G Transparent nontoxic antibacterialwound dressing based on silver nano particlebacterial cellulose nano



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composite synthesized in the presence of tripolyphosphate J DrugDelivery Sci Technol 2018 44 244minus253(55) Srivatsan K V Duraipandy N Begum S Lakra RRamamurthy U Korrapati P S Kiran M S Effect of curcumincaged silver nanoparticle on collagen stabilization for biomedicalapplications Int J Biol Macromol 2015 75 306minus315(56) Wu J Zheng Y Song W Luan J Wen X Wu Z ChenX Wang Q Guo S In situ synthesis of silver-nanoparticlesbacterialcellulose composites for slow-released antimicrobial wound dressingCarbohydr Polym 2014 102 762minus771(57) Portela R Leal C R Almeida P L Sobral R G Bacterialcellulose a versatile biopolymer for wound dressing applicationsMicrob Biotechnol 2019 12 586minus610(58) Khalid A Ullah H Ul-Islam M Khan R Khan S AhmadF Khan T Wahid F Bacterial cellulose-TiO2 nanocompositespromote healing and tissue regeneration in burn mice model RSCAdv 2017 7 47662minus47668(59) Pertile R A N Moreira S Gil da Costa R M Correia AGuardao L Gartner F Vilanova M Gama M Bacterial CelluloseLong-Term Biocompatibility Studies J Biomater Sci Polym Ed2012 23 1339minus1354(60) Lee K Buldum G Mantalaris A Bismarck A More ThanMeets the Eye in Bacterial Cellulose Biosynthesis Bioprocessing andApplications in Advanced Fiber Composites Macromol Biosci 201414 10minus32(61) Lopes T D Riegel-Vidotti I C Grein A Tischer C AFaria-Tischer P C d S Faria-Tischer Paula Cristina de SousaBacterial cellulose and hyaluronic acid hybrid membranes Productionand characterization Int J Biol Macromol 2014 67 401minus408(62) Moniri M Boroumand Moghaddam A Azizi S AbdulRahim R Bin Ariff A Zuhainis Saad W Navaderi M MohamadR Production and Status of Bacterial Cellulose in BiomedicalEngineering Nanomaterials 2017 7 257(63) Andrade F K Silva J P Carvalho M Castanheira E M SSoares R Gama M Studies on the hemocompatibility of bacterialcellulose J Biomed Mater Res Part A 2011 98A 554minus566(64) Leitao A F Gupta S Silva J P Reviakine I Gama MHemocompatibility study of a bacterial cellulosepolyvinyl alcoholnanocomposite Colloids Surf B 2013 111 493minus502(65) Subcommittee F0416 on Biocompatibility Test MethodsStandard Practice for Assessment of Hemolytic Properties of MaterialsASTM F756-17 ASTM International West Conshohocken PA2017(66) Nagoba B Davane M Studies on wound healing potential oftopical herbal formulations- do we need to strengthen study protocolJ Ayurveda and Integrative Medicine 2019 10 316minus318(67) Guzman M Dille J Godet S Synthesis and antibacterialactivity of silver nanoparticles against gram-positive and gram-negativebacteria Nanomedicine 2012 8 37minus45(68) Khatoon U Rao G Mohan M Ramanaviciene ARamanavicius A Comparative study of antifungal activity of silverand gold nanoparticles synthesized by facile chemical approach JEnviron Chem Eng 2018 6 5837minus5844(69) Song H Y Ko K K Oh I H Lee B T Fabrication of silvernanoparticles and their antimicrobial mechanisms Eur Cells Mater2006 11 58minus59(70) Cano Sanchez M Lancel S Boulanger E Neviere RTargeting Oxidative Stress and Mitochondrial Dysfunction in theTreatment of Impaired Wound Healing A Systematic ReviewAntioxidants 2018 7 98(71) Ahmed O M Mohamed T Moustafa H Hamdy HAhmed R R Aboud E Quercetin and low level laser therapypromote wound healing process in diabetic rats via structuralreorganization and modulatory effects on inflammation and oxidativestress Biomed Pharmacother 2018 101 58minus73(72) Flanagan M Wound measurement can it help us to monitorprogression to healing J Wound Care 2003 12 189minus194

(73) Kenworthy P Phillips M Grisbrook T L Gibson WWood F M Edgar D W Monitoring wound healing in minorburnsA novel approach Burns 2018 44 70minus76(74) Malone M Schwarzer S Walsh A Xuan W Al GannassA Dickson H G Bowling F L Monitoring wound progression tohealing in diabetic foot ulcers using three-dimensional woundimaging J Diabetes Complications 2020 34 107471



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Hydrogels are one of the promising candidates as wounddressings because of their unprecedented properties includingtheir ability to maintain a moist microclimate at the wound sitewhich is proven to facilitate healing9minus11 A number of naturaland synthetic polymeric materials are in use to producehydrogel dressings1213 Bacterial cellulose (BC) is an exampleof a natural biosynthetic cellulose hydrogel which has beenwidely considered as a wound dressing because of its uniqueproperties14minus18 BC can be produced by several bacteria butGluconacetobacter xylinus (G xylinus) is considered as one ofthe best for BC production19 Our group has previouslyreported the findings on the physicochemical characterizationand suitability of BC hydrogels as wound dressings20 AlthoughBC does not have inherent antimicrobial activity however itscross-linked fiber network structure encouraged severalresearch attempts of loading antimicrobials anti-inflammatoryantioxidants and other healing agents for wound-dressingapplications172122 In continuation in the presented study aninvestigation has been made to produce biosynthetic hydrogelswith healing properties for chronic wound management Ournovel approach is consistent with the concept of the forensicengineering of advanced polymeric materials (FEAPM) whichcan help to understand the relationships between the structureof the biomaterial used its properties and behavior forpractical applications23

Currently the treatment of microbial infections is severelyaffected by the emergence of new multiresistant microbesincluding bacteria and fungi Gram-positive Staphylococcusaureus has a great ability to develop multiple drug resistancesSimilarly Gram-negative Pseudomonas aeruginosa a commonopportunistic pathogen shows resistance to most penicillinscephalosporins and carbapenems Pathogenic fungi such asCandida auris pose a significant health risk to patients sufferingfrom infected wounds and in particular to immunocompro-mised individuals which can result in candidiasiscandidemiaand invasive aspergillosis Recently a new multidrug-resistantstrain of C auris was discovered24 and has quickly become aprolific nosocomial pathogen in the UK2526 with diagnoseson all continents with 33 countries reporting incidents ofantifungal resistance C auris is resistant to most first- andsecond-generation antifungals with 90 of isolates beingresistant to fluconazole 40 resistant to amphotericin and 2to echinocandins such as caspofungin27minus29 The concernregarding these pathogens centers around the increasingresistance they are showing toward a very limited pool ofeffective antimicrobials30minus32 and hence the focus is ontargeted delivery by antimicrobial wound dressingsSilver is a well-known antimicrobial effective against fungi

yeast and bacteria including several antibiotic-resistantstrains33minus35 The emergence of nanotechnology enabling theproduction of silver nanoparticles has served a new therapeuticmodality Because of their characteristic broad-spectrumantimicrobial properties silver nanoparticles (AgNP) havereceived increased focus in biomedical applications includingfor wound management36minus38 Among the several differentapproaches to synthesize AgNP the use of natural substanceslike plant extracts has received wide research considerationbecause of the safe and eco-friendly procedure3739minus41

Curcumin (CUR) a natural polyphenol extracted fromturmeric is a well-known wound-healing agent with provenantimicrobial antioxidant and anti-inflammatory effects2242

In addition to its healing properties CUR has been used as areducing and capping agent to produce AgNP43 The main

challenge that limits the wider biomedical application of CURis its hydrophobic nature Several chemical mediators likedimethyl sulfoxide dichloromethane and sodium carbonateare employed in AgNP synthesis from CUR44minus48 Thesechemical mediators can have serious cytotoxic effects48

Selection of solvent medium reducing agent and nontoxicstabilizer are the main considerations in green nanoparticleproduction4950 The current study reports the production ofAgNP following a green chemistry approach using an aqueoussolution of curcuminhydroxypropyl-β-cyclodextrin(CURHPβCD) These AgNP produced using CURHPβCD(cAgNP) were characterized and loaded in the biosyntheticBC hydrogels to produce hydrogel dressings To the best ofour knowledge the protocol presented herein to producecAgNP is novel and this is the first time the production ofcAgNP-loaded BC has been reported This report presents theunique combination of silver nanoparticles with curcumin inthe biosynthetic BC to obtain the hydrogels for wound-management applications This study investigates the inherentantimicrobial antioxidant and cytocompatibility properties ofthese novel hydrogel dressings

MATERIALS AND METHODSMaterials Gluconoacetobacter xylinus (ATCC 23770) Pseudomo-

nas aeruginosa (NCIMB 8295) and Staphylococcus aureus (NCIMB6571) were obtained from the University of Wolverhampton culturecollection Candida auris (NCF 8971) was obtained from the PublicHealth England Culture Collection Porton Down UK A stockculture of P aeruginosa and S aureus were resuscitated at 37 degC ontryptone soy agar (TSA) (Sigma-Aldrich UK) G xylinus at 30 degCon mannitol agar and C auris at 37 degC on Sabouraud Dextrose Agar(SDA) HS media was prepared following the standard protocol51

Material for mannitol agar SDA and HS media were purchased fromLab M (UK) Tryptone soya broth (TSB) disodium phosphate andcitric acid were purchased from Sigma-Aldrich (UK) Overnightbroth cultures were prepared in appropriate medium using the stockplates

U251MG (U251) MSTO and Panc 1 cell lines were purchasedfrom ATCC (UK) Defibrinated horse whole blood was purchasedfrom TCS Biosciences Ltd

Silver nitrate was purchased from Fischer Scientific (UK)Hydroxypropyl-β-cyclodextrin (parenteral grade) was kindly providedby Roquette (France) Curcumin and dimethyl sulfoxide (DMSO)(spectrophotometric grade) were purchased from Alfa Aesar (UK)Ringer tablets were purchased from Lab M (UK) Thiazolyl BlueTetrazolium Bromide (MTT) sodium bicarbonate and 22-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (UK)Sodium chloride 09 (wv) (normal saline intravenous infusion)was purchased from Baxter UK Sodium hydroxide was purchasedfrom Acros Organics (UK) NaCl (58 gL) and glycine (76 gL)for preparing Sorensenrsquos glycine buffer were purchased from Sigma-Aldrich UK Trypsin was purchased from Lonza (Belgium)Dulbeccorsquos Modified Eaglersquos Medium (DMEM) fetal bovine serum(FBS) L-glutamine and antibiotic antimycotic were purchased fromGibco (UK)

Preparation of CURHPβCD Inclusion Complex Inclusioncomplex was synthesized by the solvent evaporation method followingthe protocol previously reported22 Briefly CUR solution (079 g in375 mL acetone) was added dropwise to the aqueous HPβCDsolution (30 g in 125 mL deionized water) under constant stirring atroom temperature for slow and complete evaporation of acetone Thesample was centrifuged and the aqueous supernatant containingCURHPβCD inclusion complex was frozen after filtration andlyophilized

Preparation and Characterization of cAgNP Preparation ofcAgNP Using CURHPβCD A novel green chemistry approach wasdeveloped for cAgNP synthesis by reducing silver nitrate (AgNO3)



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synthesis of silver nanoparticles using curcumin

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