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Soft Materials

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Efficient alternative of antimicrobialnanocomposites based on cellulose acetate/Cu-NPs

Hussein Abou-Yousef, Essam Saber, Mohamed S. Abdel-Aziz & Samir Kamel

To cite this article: Hussein Abou-Yousef, Essam Saber, Mohamed S. Abdel-Aziz & Samir Kamel(2018): Efficient alternative of antimicrobial nanocomposites based on cellulose acetate/Cu-NPs,Soft Materials, DOI: 10.1080/1539445X.2018.1457540

To link to this article: https://doi.org/10.1080/1539445X.2018.1457540

Published online: 02 Apr 2018.

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Efficient alternative of antimicrobial nanocomposites based on celluloseacetate/Cu-NPsHussein Abou-Yousef a, Essam Saber a, Mohamed S. Abdel-Azizb, and Samir Kamel a

aCellulose & Paper Department, National Research Centre, Giza, Dokki, Egypt; bMicrobial Chemistry Department, National Research Centre,Cairo, Egypt

ABSTRACTThe current research presents an efficient, cheap, and safe antimicrobial material for widespread usebased on copper nanoparticles (Cu-NPs) loaded on cellulose acetate (CA) matrix. A reduction processof CuSO4·5H2O has been performed to prepare Cu-NPs. The nanosized copper particles includedoxidized Cu (15–20 nm). Two different loads of Cu-NPs were used in this study, 2% and 6%mol.%. Thepresence of Cu-NPs incorporated with CA films slightly affected the tensile index of the films, wherelow and high-loaded Cu-NPs enhanced the tensile index by small values ranged from 0.640 to 0.650and 0.667, respectively. A study on the antibacterial activity of these nanocomposites was carried outfor Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. It has been found that CAcontaining Cu-NPs (2%) exhibited the highest antimicrobial activity against all test microbes includingS. aeureus (21mm), P. aeruginosa (18mm), C. albicans (19mm), and Aspergillus niger (15mm). Resultsalso revealed that CA film with 6% exhibited lower activity than film with 2% Cu-NPs. The morpho-logical properties of CA/Cu-NPs films were characterized by scanning electron microscopy andtransmission electron microscope in addition to X-ray diffraction. Low-loaded Cu-NPs showed homo-genous distribution through CAmatrix while, the high-loaded Cu-NPswere agglomerated through CAmatrix. Thermal properties illustrated the enhancement of thermal stability of the film with increasingthe loaded Cu-NPs.

ARTICLE HISTORYReceived 15 December 2017Accepted 22 March 2018

KEYWORDSAntimicrobial; bio-basedfilm; cellulose acetate;copper nanoparticles;mechanical properties

Introduction

Polymers have been receiving a great attention as alter-natives for the conventional packaging materials due totheir mechanical properties, low density, and relativelylow cost. Most of polymers used in the packagingindustry are nonbiodegradable materials, which gener-ate environmental problems causing air and soil pollu-tion, underground water contamination, and alsoparticipate the global warming effect (1,2).

An ultimate goal in packaging industry is the searchfor alternative materials having no negative environ-mental effects. Biodegradable polymer is used as apromising resource to replace the synthetic polymers(nonbiodegradable) (3,4).

One of the most abundant natural polymers is cel-lulose acetate (CA) which derived from cellulose. CA isprepared by the reaction of cotton linter or cellulosepulp with acetic anhydride. Because of its relatively lowcost, a variety of applications have stimulated speciallyin the field of membrane technology, drug release,textile, and related materials (5). The limited using of

biopolymers has been attributed to their poor mechan-ical properties, their tendency to be degradable bydifferent mechanisms such as biologically, chemically,or photochemically. In order to improve these deficien-cies, incorporation of metallic nanoparticles is an alter-native to produced bio-based composite materials withimproved mechanical and antimicrobial proper-ties (6,7).

Synthesis of copper nanoparticles (Cu-NPs) facesfew problems due to their high sensitivity to air, result-ing in high rate of oxidation of Cu-NPs. This leads tothe oxide phase of Cu (CuO) is more thermodynami-cally stable than Cu metal (8). Cu-NPs have high ten-dency of oxidation that leads to restriction for using invarious applications. The stabilization of Cu-NPs isenhanced by using capping agent to minimize the oxi-dation, but may not prevent it completely (9,10).

Cu-NPs and nanosilver nanoparticles were consid-ered as common nanomaterials that could be incorpo-rated as nanometals in packaging films (11–13). Cu-NPs were considered as good substances as proficientcatalysts in the production of hydrogen (14). Cu-NPs

CONTACT Essam Saber essamksu@yahoo.com Cellulose & Paper Department, National Research Centre, 33 El Bohouth St., Dokki, Giza, P.O. 12622,Egypt.Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lsfm.

SOFT MATERIALShttps://doi.org/10.1080/1539445X.2018.1457540

© 2018 Taylor & Francis Group, LLC

and their oxides exhibited broad antimicrobial activityas reported in studies of growth inhibition of bacteriaand fungi as well as their biocide effect against algae.Nanocoating of Cu-NPs on cellulose has been fabri-cated via electrostatic assembly and this compositeexhibited antimicrobial activity (15,16).

A new approach of packaging systems is undergoingdevelopment depending on absorbing of moisture,liquids, and gaseous scavengers to increase shelf life ofproceeded food stuffs (17). Other systems are thosewhich release antimicrobial agents, which is inhibitingor retarding the microbial growth and food spoilage.Cu has been of particular interest because, unlike otherantimicrobial metals, it presents a broad spectrum ofaction against bacteria and molds due to its ability toaccept or donate electrons having high levels of cataly-tic oxidation and a high reduction potential (18–20).

Reduction of Cu in its salt is one of the fundamentalrouts for preparation of Cu-NPs depending on reduc-tion reaction in various solvents. In the current work,Cu-NPs have been prepared by reduction of Cu cationsin CuSO4·5H2O. The produced Cu-NPs have been sta-bilized by starch in aqueous medium without inert gasprotection (21,22).

A new material based on CA biopolymer with anti-microbial activity using Cu-NPs was developed in thisresearch. The aim of this study was to evaluate differentproperties of films prepared from the synthesized Cu-NPs blended with CA. Cu-NPs were characterized bytransmission electron microscope (TEM), and energydispersive X-ray spectroscopy (EDX). The studyfocused on the antimicrobial properties of the preparedfilms.

Experimental

Materials

CA with acetyl content of 40.3% was used in this study.Plasticizer used was polyethylene glycol 400 (PEG

3350), Sigma-Aldrich, India. Copper (II) sulfate penta-hydrate and sodium hydroxide NaOH were purchasedfrom Sigma-Aldrich, India. High purity acetone wasused as solvent.

Synthesis of Cu-NPs

The Cu-NPs were synthesized by chemical reductionprocess using copper (II) sulfate pentahydrate as pre-cursor salt and starch as capping agent. The prepara-tion method starts with addition of 0.1 M copper (II)sulfate pentahydrate solution into 120 mL of starch(1.2%) solution with vigorous stirring for 30 min. Inthe second step, 50 mL of 0.2 M hydroxylamine hydro-chloride solution was added to synthesis solution undercontinuous rapid stirring. Subsequently, 30 mL of 1 Msodium hydroxide solution was slowly added to theprepared solution with constant stirring and heatingat 80°C for 2 h. The color of the solution turned toyellow. After the completion of reaction, the solutionwas taken from the heat and allowed to settle overnight.The precipitates were separated from the solution bytwo successive centrifugation steps (1,000 rpm) fol-lowed by filtration and washed thrice with deionizedwater and ethanol to remove excess starch bound to thenanoparticles. The obtained yellow color precipitates(Fig. 1) are dried at room temperature. After drying,nanoparticles were stored in glass vial for furtheranalysis.

Preparation of CA blended by Cu-NP films

In a typical experiment, the CA-free film was preparedusing a solvent evaporation method. PEG 400 of 0.35 gwas dissolved in water (20 mL) for 1.5 h, then acetone(50 mL) was added to the PEG/water solution, and CA(0.8 g) was gradually added under stirring to the sol-vent system. To allow CA to dissolve completely, stir-ring continued for another 2 h. The CA solution wasdegassed for about 3 h, and the solution was ready to be

Cu-NPs after reduction of CuSO4.

H2O removal and precipitation of Cu-NPs by centrifuge.

Precipitated Cu-NPs after washing by CH3OH.

Figure 1. Preparation of Cu-NPs from copper (II) sulfate.

2 H. ABOU-YOUSEF ET AL.

used to cast films in Teflon Petri-dish. Cu-NPs (2 and6 mol.%) based on the amount of CA were used forblending with CA film. The addition of Cu-NPs wascarried out through the step of dissolving of PEG.

X-ray diffraction (XRD)

The XRD patterns of CA and CA/Cu-NPs films werecarried out on a Diano X-ray diffractometer usingCu (Kα1/Kα2) radiation source energized at 45 kVand a Philips X-ray diffractometer (PW 1930 gen-erator, PW 1820 goniometer). The XRD patternswere recorded in a diffraction angle range fromθ = 10° to 802°.

Scanning electron microscopy (SEM)

The surface morphology of CA and CA/Cu-NPs filmswere analyzed using SEM, (JSM 6360LV, JEOL/Noran).The microscope was attached to a dispersive energyspectrometer. The images were obtained using an accel-erating voltage of 10–15 kV.

Thermogravimetric analyzer (TGA)

The thermal stability of the CA/Cu-NPs films and CAsample (control) was analyzed by using a TGA(Shimadzu DTG-60, Japan) and differential thermalanalysis. The samples were heated at room temperatureto 800°C with a heating rate of 10°C/min under oxygenatmosphere.

Assay of antibacterial activity

The disk diffusion method was used to determine theantimicrobial activity of CA and CA/Cu-NPs films. Avolume of 0.1 mL of the tested microorganisms grownin Brian Heart Infusion Broth (at 42°C for 24 h, 108–109 cells/mL) was inoculated on Brian Heart Infusionmedia, and then spread on the entire surface of the dishusing a sterile spatula. Subsequently, sterile disks wereplaced onto agar at certain intervals by passing gently.After the plates were incubated at 42°C for 24 h, theinhibition zones around the disks, where no growthoccurred, were measured in millimeters. The experi-ments were repeated in duplicates for all of the teststrains.

Antimicrobial activity

Disk agar plate method was used to estimate the antimi-crobial activities CA and cellulose incorporated with silvernanoparticles (23). Four different test microbes –

Staphylococcus aureus, Escherichia coli, Candida albicans,and Aspergillus niger – were selected to evaluate the anti-microbial activities as representatives of gram-positivebacteria, gram-negative bacteria, yeast, and fungal groups.The bacterial and yeast test microbes were grown on anutrient agar medium (DSMZ1) of the following compo-nents (g/L): peptone (5.0), meat extract (3.0), agar (20.0),distilled water (1,000.0 mL), and the pH was adjusted to7.0. On the other hand, the fungal test microbe wascultivated on Czapek-Dox medium (DSMZ130) of thefollowing ingredients (g/L): sucrose (30.00), NaNO3

(3.0), MgSO47 H2O (0.50), KCl (0.50), FeSO4·7H2O(0.01), K2HPO4 (1.0), agar (18.0), distilled water(1,000.0 mL), and the pH was adjusted to 7.2. The cultureof each test microbe was diluted by distilled water (ster-ilized) to about 107–108 cells/mL, then 1 mL of each wasused to inoculate 1 L-Erlenmeyer flask containing 250 mLof solidified agar media. These media were transferred topreviously sterilized Petri dishes (10 cm diameter having25 mL of solidified media). The cellulose sheet disks wereplaced on the surface of agar plates seeded with testmicrobes and incubated for 24 h at the appropriate tem-perature of each test organism. Antimicrobial activitieswere recorded as the diameter of the clear zones (includ-ing the film itself) that appeared around the films (24,25).

Results and discussion

Preparation of Cu-NPs

When a salt of hydroxylamine is added to a solution ofcopper sulfate and sodium hydroxide, the reactioncould take place as follows:

(1) CuSO4 + NH2OH → CuSO4·NH2OH(2) CuSO4·NH2OH + NH2OH → CuSO4·2NH2OH(3) CuSO4·2NH2OH+3NH2OH→CuSO4·5NH2OH(4) 2CuSO4·5NH2OH + H2O → Cu2OSO4·2NH2OH

+ (NH2OH)H2SO4 + 6NH2OH(5) Cu2OSO4·2NH2OH + 2NaOH → Cu2O +

Na2SO4 + 4H2O + N2

Continuous addition of hydroxyl amine completes thereduction Cu2O into metallic copper as follows:

6. Cu2o + 2NH2OH → 2Cu + 3H2O + N2

So, rapid evolution of nitrogen and the production ofmetallic copper are observed. The size and shape of theprepared Cu were investigated by TEM. TEM imagesdemonstrated that the produced Cu metal particleswere in the nanorange and they are approximatelyspherical in shape and the average particle size was inthe range of 15–20 nm (Fig. 2).

SOFT MATERIALS 3

Surface morphology

The preparation of CA/Cu-NPs composites in the form offilms in the current article was investigated by SEM andEDX as shown in Figs. 3 and 4. The representative SEMimage of film surface that is obtained from CA (Fig. 3) wassmoother than the surfaces of the other films (Fig. 4). CAhas an acetyl content equal to 40.3% (low degree of sub-stitution [DS]), so high number of free hydroxyl groupsparticipate in hydrogen bonding, which may favor thehomogeneity of the films (26). CA films with Cu-NPsexhibit differentmorphologies and different chemical com-positions (Fig. 4). The severe aggregation of Cu-NPs isformed on their surfaces as well as inside of the films. TheEDX analysis recorded the presence of Cu with 0.37 and

1.09 at.%, which proves the incorporation of Cu-NPs intoCA films. According to SEM, it is clear that the amount ofloaded Cu-NPs could be divided into two portions. Thefirst one concentrates in the bulk of CA film; this agreeswith the photograph of CA-loaded Cu-NPs (low and highconcentration), where the color of CA film depends on theconcentration of Cu-NPs loaded.

In case of low concentration, the Cu located on thesurface of the film are available to be oxidized, then intoCu(CO3)2 by reaction with CO2 in atmosphere (Fig. 5b).

The high-loaded Cu-NPs film was appeared with darkyellow (Fig. 5b), where Cu-NPs are agglomerated in thefilmbulk. So, there is a difficulty for the diffusion ofCu-NPsfrom the bulk to the surface.

Figure 2. TEM micrographs of Cu-NPs.

Figure 3. SEM of CA film.

4 H. ABOU-YOUSEF ET AL.

Thermogravimetric analysis for CA and CA/Cu-NPsfilms

Fig. 6a shows analysis of CA reveals a minor weight loss at~100°C due to loss of adsorbed water. The sample lost14.77% of its original weight. The second event was occur-ring at a temperature range 300–400°C, which indicated thethermal decomposition of CA. TGA thermogram exhibitedsingle-step degradation pattern at 360°C, where the samplelost 77.58% of its original weight. Thermal decomposition

of CA consists of a series of degradation reactions such asdehydration at temperature around 100°C, deacetylation at320°C, and thermal pyrolysis of themain cellulosic chain at370°C.

Fig. 6b shows that the sample lost 13.35% of itsoriginal weight during the first stage where 69.56%was lost at 358.6°C. The sample gained some ther-mal stability due to the presence of Cu-NPs. CAwith high load of Cu-NPs shows remarkable thermal

Figure 5. Photograph of CA films loaded with (a) 2% and (b) 6% Cu-NPs.

100

(a) (b) (c)

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Figure 6. TGA of (a) CA, (b) CA/Cu-NPs (2%), and (c) CA/Cu-NPs (6%).

576

1.00K0.90K0.80K0.70K0.60K

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Lsec: 30.0 0 Cnts 0.000 keV Det: Apollo X-SDD Det ResoLsec: 30.0 0 Cnts 0.000 keV Det: Apollo X-SDD Det Reso3.00 4.00 5.00 6.00 7.00 8.00 9.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

448

Figure 4. SEM with different magnification and EDX of CA/Cu-NPs films.

SOFT MATERIALS 5

stability, where the sample lost only 4.79% of itsoriginal weight during the first stage. Also, thebulk loss of sample is 50.62% of its original weightat 362°C (Fig. 6c).

X-ray diffraction

The diffraction patterns of the nanosized copper par-ticles loaded on CA film are shown in Fig. 7. Thediffraction pattern of nanosized Cu particles isobserved, where the most intense of them are2θ = 26.73°, 42.29°, 43.9°, and 50.3°. Thus, the nano-sized copper particles include oxidized Cu (34.52 and36.63). The diffraction pattern of the unloaded CAcontains the amorphous region located at 2θ = 22°.Based on the diffractogram, both of CA and CA/Cu-NPs diffractions have been interfered in their locu-tions (27).

Mechanical studies

Fig. 8 shows the relation between the loaded Cu-NPs andmechanical properties ofCA films. Thepresence ofCu-NPsincorporated with CA films slightly affected the tensileindex of the films, where low- and high-loaded Cu-NPsenhanced the tensile index by lower values that ranged from0.640 to 0.650 and 0.667, respectively.

The results of mechanical properties illustrated thatthe strain percentage increased considerably for bothlow and high Cu-NPs loading, resulting in decreasingof the values of Young’s modulus of both types ofcopper loading. These results could be explainedaccording to the areas under curves of CA and Cu-NPs-loaded CA films in XRD as shown in Fig. 7. Thearea under the curve in loaded CA film illustratedincrease of the amorphous region resulting in increaseof the deformation, strain percentage, and loaded cop-per CA film. Consequently, as the strain percentage

300

150

100

50

200

100

10 20 30 40Position [º2 Theta] (Copper (Cu))

50 60 70

10 20 30 40

Position [º2 Theta] (Copper (Cu))

50 60 70

Figure 7. XRD of (a) CA and (b) CA/Cu-NPs films.

6 H. ABOU-YOUSEF ET AL.

increased, the Young’s modulus decreased for both typeof loaded copper films.

Release of copper ions

Fig. 9 illustrates the release of Cu-NPs incorporated inCA films for different time intervals ranged from 0.5to 24 h. The rate of Cu-NPs releasing was rapid atshort time interval (0.5 h), where the amount of Cu-NPs release was 65.23 and 54.15 mg/L for low- andhigh-loaded Cu-NPs, respectively. For moderate timeintervals up to 4 h, the release of Cu-NPs was slightlyincreased, while for long time intervals the release ofCu-NPs was constant for both types of loading. It was

observed that the rate of release of low-loaded Cu-NPsfilm was faster than that of high-loaded one. Thesecan be explained from the point of view of the exis-tence of Cu-NPs in high-loaded film in two levels:onto the surface and the bulk of the CA matrix asshown in Fig. 4. So, the Cu-NPs in the bulk, whichwere agglomerated, take more time to be released incase of high-loaded film. These features were notemerged in case of low-loaded Cu-NPs film becausemost of the particles were located onto the film sur-face and more homogeneous distribution leading torapid release of Cu-NPs in early time interval. At longtime interval, both low and high loaded Cu-NPs filmsreleased Cu-NPs closely together.

4.5Tensile Index Strain % Young’s modulus (MPa)

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Figure 8. The mechanical properties of CA and CA/Cu-NPs films.

Figure 9. Cu release from CA/Cu-NPs as a function of time.

SOFT MATERIALS 7

Antimicrobial studies

It has been affirmed from the previous studies that thebiocidal effects observed in Cu–polymer nanocompositesare based on three phenomena: (1) release of copper ions,(2) release of Cu-NPs from nanocomposites, and (3)biofilm inhibition (28). In order to evaluate the antibacterialactivity of the CA/Cu-NPs, selected samples have beentested toward bacteria strains of S. aureus, Pseudomonasaeruginosa, and C. albicans. Table 1 lists the clear zone ofcellulose and nanocomposites against strains. Also, Fig. 10shows the results obtained for the antibacterial activity of

the distinct nanocomposites and for blank CA used ascontrol. For both bacteria strains, the control samples didnot exhibit antibacterial activity. CA film without Cu-NPsshould not have any antimicrobial activities, and the clearzones appeared in case of S. aeureus, P. aeruginosa, and C.albicans might be due to the formation of acid by alkalihydrolysis of the nutrient agar medium (pH 7.5) and thishas been supported by the truth that no antimicrobialactivity was observed with the fungus A. niger which culti-vated on Potato Dextrose Agar (pH 5.5). It has been foundthat CA containing Cu-NPs (2%) exhibited the highestantimicrobial activity against all test microbes including S.

Table 1. The antimicrobial activity of CA and CA/Cu-NPs films against different groups of test microbes.

Specimen

Clear zone (θ mm)

Pseudomonas aeuginosa Staphylococcus aureus Candida albicans Aspergillus niger

CA 18 13 14 00CA/Cu-NPs (2%) 21 18 19 15CA/Cu-NPs (6%) 12 11 11 00Neomycin (100 µg/100 µl) 27 25 19 00Cycloheximide (100 µg/100 µl) 00 00 00 33

Figure 10. Antibacterial activities of CA and CA/Cu-NPs films against Staphylococcus aureus, Pseudomonas aeruginosa, and Candidaalbicans.

8 H. ABOU-YOUSEF ET AL.

aeureus (21 mm), P. aeruginosa (18 mm), C. albicans(19 mm), and A. niger (15 mm). Results also revealed thatCA film with 6% exhibited lower activity than film with 2%Cu-NPs and this is due to the heterogeneous distribution ofCu-NPs onto the surface of low-loaded film,which enhancethe rate of diffusion the Cu-NPs to surrounding media.These results were demonstrated by the morphologicalfeatures of SEM illustrated in Fig. 4, which stated that thepresence of two levels of incorporation of Cu-NPs in thebulk of CA films and onto the film surface. The Cu-NPsincorporated in polymeric matrices to generate antimicro-bial Cu–polymer nanocomposites exhibited excellentresults in suppressing the growth of a wide range ofmicroorganisms.

Regarding the results shown in Table 1, CA exhib-ited antimicrobial activity which may result from resi-dual acidity (up to 0.5%) during the production of CAand consequently, the growth of Staphylococcus,Pseudomonas, and Candida was inhibited.

Conclusion

The use of prepared CA loaded with Cu-NPs films asantimicrobial was investigated.

The obtained results showed that the presence of Cuwith 1.09 and 1.58 at.%, according to the EDX analysis,proves the incorporation of Cu-NPs into CA films.

According to SEM, the Cu-NPs incorporated in CAmatrix could be divided into two portions. The firstone is concentrating in the bulk of CA film; this agreeswith the photograph of CA-loaded Cu-NPs (low andhigh concentration), where the color of the preparednanocomposite film depends on the concentration ofthe loaded Cu-NPs.

High load Cu-NPs of CA films revealed remarkablethermal stability. XRD has been used for detection ofthe size of copper particles.

For the mechanical properties, it was found that thetensile index and strain percentage were increased consid-erably for both low andhighCu-NPs loading, but the valuesof Young’s modulus for both types of copper loading weredecreased.

The Cu-NPs in CA matrices exhibited excellentresults in suppressing the growth of a wide range ofmicroorganisms.

Funding

The authors acknowledge the National Research Center,Egypt for financial support of this research activity (grantnumber: P11090110).

ORCID

Hussein Abou-Yousef http://orcid.org/0000-0002-4533-8457Essam Saber http://orcid.org/0000-0002-8332-9201Samir Kamel http://orcid.org/0000-0002-7971-4318

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