Accepted Manuscript Title: Antimicrobial bacterial cellulose nanocomposites prepared by in situ polymerization of 2-aminoethyl methacrylate Author: Ana R.P. Figueiredo Andrea G.P.R. Figueiredo Nuno H.C.S. Silva Ana Barros-Timmons Adelaide Almeida Armando J.D. Silvestre Carmen S.R. Freire PII: DOI: Reference:

S0144-8617(15)00094-6 http://dx.doi.org/doi:10.1016/j.carbpol.2015.01.063 CARP 9651

To appear in: Received date: Revised date: Accepted date:

3-2-2014 11-9-2014 28-1-2015

Please cite this article as: Figueiredo, A. R. P., Figueiredo, A. G. P. R., Silva, N. H. C. S., Barros-Timmons, A., Almeida, A., Silvestre, A. J. D., and Freire, C. S. R.,Antimicrobial bacterial cellulose nanocomposites prepared by in situ polymerization of 2-aminoethyl methacrylate, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.01.063 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights (for review)

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Novel BC/PAEM nanocomposite membranes were prepared by in situ polymerization BC/PAEM nanocomposites showed improved thermal and mechanical properties BC/PAEM nanocomposites proved also to have antibacterial activity

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Antimicrobial bacterial cellulose nanocomposites prepared by in situ polymerization of

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2-aminoethyl methacrylate

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Ana R.P. Figueiredo,a Andrea G.P.R. Figueiredo,a Nuno H.C.S. Silva,a Ana Barros-Timmons,a Adelaide

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Almeida,b Armando J.D. Silvestrea* and Carmen S.R. Freirea*

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a

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Portugal

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b

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Portugal

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Department of Chemistry and CICECO, Campus de Santiago, University of Aveiro, 3810-193 Aveiro,

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Department of Biology and CESAM, Campus de Santiago, University of Aveiro, 3810-193 Aveiro,

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*Correspondence should be addressed to Carmen Freire e-mail: [email protected];

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Tel.: + 351-234-370-604; Fax: + 351-234-370-084.

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Received -- 2014; Revised

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2014

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2014; Accepted

Abstract

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Antimicrobial bacterial cellulose/poly(2-aminoethyl methacrylate) (BC/PAEM) nanocomposites

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were prepared by in situ radical polymerization of 2-aminoethyl methacrylate, using variable

18

amounts of N,N-methylenebis(acrylamide) (MBA) as cross-linker. The obtained nanocomposites were

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characterized in terms of their structure, morphology, thermal stability, mechanical properties and

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antibacterial activity. The ensuing composite membranes were significantly more transparent than

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those of pure BC and showed improved thermal and mechanical properties. The antibacterial activity

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of the obtained nanocomposites was assessed towards a recombinant bioluminescent Escherichia

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coli and only the non-crosslinked nanocomposite (BC/PAEM) proved to have antibacterial activity.

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Keywords: Bacterial cellulose; poly(2-aminoethyl methacrylate) (PAEM); in situ radical

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polymerization; nanocomposite membranes; antibacterial properties; bioluminescent Escherichia

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coli.

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1. Introduction

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Bacterial cellulose (BC), also named microbial cellulose, is an extracellular polysaccharide, with the

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same chemical structure as plant cellulose, produced by several bacteria namely those of the

32

Gluconacetobacter, Sarcina and Agrobacterium genera (Chawla, Bajaj, Survase, & Singhal, 2009;

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Klemm et al., 2011; Klemm, Heublein, Fink, & Bohn, 2005). The tridimensional nanofibrillar porous

34

structure of BC confers it unique properties such as high water holding ability, mechanical strength

35

and porosity (Klemm et al., 2011; Trovatti, Serafim, Freire, Silvestre, & Neto, 2011). These unique

36

properties, together with its natural biocompatibility and in situ moldability, encouraged the

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development of several advanced applications (Klemm et al., 2011). The main applications of pristine

38

BC are in the biomedical field (Fu, Zhang, & Yang, 2013), where it is employed as wound dressing

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material for the treatment of severe wounds (e.g. burns) (Czaja, Krystynowicz, Bielecki, & Brown,

40

2006), as temporary skin substitutes (Czaja et al., 2006), as potential artificial blood vessels for

41

microsurgery (Klemm, Schumann, Udhardt, & Marsch, 2001) and as potential topical drug delivery

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systems (Silva et al., 2013; Trovatti et al., 2012; Trovatti, Silva, et al., 2011).

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Besides the application of BC membranes in its native form, attempts have been made in order not

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only to take advantage of BC remarkable properties but also to produce nanocomposite materials

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with improved and novel functional properties.

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Pristine BC membranes lack intrinsic antimicrobial properties which would be very useful for instance

47

in the development of antimicrobial wound dressing materials in order to prevent wound infections.

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Examples of such materials include BC nanocomposites with antimicrobial properties achieved, for

49

instance, by the incorporation of silver nanoparticles (Barud et al., 2011; Pinto et al., 2009; Yang, Xie,

50

Deng, Bian, & Hong, 2012) or by adequate grafting of reactive groups onto BC nanofibers (Fernandes,

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Sadocco, Alonso-Varona, et al., 2013; Gao et al., 2013).

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Poly(2-aminoethyl methacrylate) (PAEM) is a synthetic biocompatible (Ji, Panus, Palumbo, Tang, &

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Wang, 2011) polymer that, as a result of its polycationic nature, shows antimicrobial properties

54

(Ionov, Synytska, Kaul, & Diez, 2009;), being more active against Gram-positive bacteria as compared

55

to Gram-negative ones (Ji, Panus, Palumbo, Tang, & Wang, 2011). In fact, polymers with pendant

56

ammonium groups are known to be effective against a broad spectrum of microorganisms. The

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mechanism by which PAEM and similar compounds kill bacteria has been associated with the

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interaction of the positively charged ammonium groups with the negatively charged bacterial cell

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membranes; that promotes the disruption of the cytoplasmatic membrane, the leakage of

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intracellular components and, ultimately, cell death (Geurts et al., 2001; Ionov et al., 2009; Li &

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Armes, 2009).

62

Following on what has been described above, as well as our interest on the preparation of bacterial

63

cellulose based nanocomposite materials by in situ polymerization (inside the BC network) of

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selected monomers (Fernandes, Sadocco, Aonso-Varona, et al., 2013; Figueiredo et al., 2013;

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Lacerda, Barros-Timmons, Freire, Silvestre, & Neto, 2013), the present work aims at imparting

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antimicrobial properties to the BC membranes, through the in situ radical polymerization of 2-

67

aminoethyl

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methylenebis(acrylamide) (MBA) as cross-linker. The obtained materials were characterized in terms

69

of their structure, morphology, thermal stability, mechanical properties and antibacterial activity.

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2. Material and Methods

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2.1. Chemicals and Materials

hydrochloride

(AEM)

inside

the

BC

network

using

N,N-

an

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2-Aminoethyl methacrylate in its hydrochloride form (AEM, 90%, stabilized), N,Nmethylenebis(acrylamide)

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dihydrochloride (ABMPA, 97%) were purchased from Sigma-Aldrich and used as received. All other

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solvents and reagents were of analytical grade and were also used as received.

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Bacterial cellulose (tridimensional network of nano and microfibrils with 10–200 nm width and 90%

77

water) in the form of wet membranes was produced in our laboratory using the bacteria

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Gluconacetobacter sacchari and conventional culture medium conditions (Trovatti, Serafim, et al.,

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2011).

stabilized)

and

2,2-azobis(2-methylpropionamidine)

te

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99%,

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(MBA,

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2.2. BC/PAEM nanocomposites preparation

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The present study describes the preparation of BC/PAEM nanocomposites with a BC:AEM ratio of 1:6,

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using variable amounts of cross-linker (0, 5, 10 and 20% w/w).

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Wet BC membranes (~100 mg dry weight, 4
4 cm) were weighted and 60% of its water content was

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removed by hand-pressing between two acrylic plates at room temperature. Drained BC membranes

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were placed in Erlenmeyers stopped with rubber septa and purged with N2. At the same time,

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aqueous solutions (5 ml) containing 700 mg of monomer (AEM) and the initiator ABMPA (1%

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winitiator/wmonomer) were prepared and also purged with nitrogen (in an ice bath) for 30 minutes. When

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the cross-linker was used, 5, 10 or 20% (wcross-linker/wmonomer) was added to the solution. Thereafter,

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the monomers aqueous solutions were added to the Erlenmeyers containing the BC membranes,

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with the aid of a syringe, and they were left to stand for 1 h at room temperature (25 ºC) until the 3 Page 4 of 27

complete absorption of the solution by the BC membrane. The reaction mixtures were then heated at

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70 ºC for 6 h. After that period, the septum was removed and the composite membranes were

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washed with 100 ml of water during 1 h. This procedure was repeated eight times. The washed

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membranes were placed over Petri dishes and dried at 40 °C overnight, and then kept in a desiccator

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until further use. All materials were prepared in triplicate. Table 1 summarizes the identification and

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some characteristics of the nanocomposite membranes prepared in this study.

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Samples of the PAEM and PAEM cross-linked with 20% MBA were prepared under the same conditions, using 500 mg of AEM, in the absence of BC, for comparison purposes.

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Nanocomposites

BC

Dry

membrane

BC

size (cm)

(mg)

AEM

MBA

(mg)

(%)

BC/PAEM

0

BC/PAEM/MBA (1:6:0.05)

BC/PAEM/MBA (1:6:0.20)

103 104 105 106 107 108 109 110

a

material (mg)

PAEMa,c

BCa,c

%

%

61.9±5.0

38.1±5.0

5

660

77.1±0.6

22.9±0.6

10

890

84.4±0.8

15.6±0.8

20

950

85.4±0.5b

14.6±0.5

700

PAEM and BC percent composition were estimated based on the mass difference between the

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Table 1- Identification of the nanocomposite membranes and component contents estimation

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nanocomposites and pristine BC membranes. b

The polymer content in the case of cross-linked nanocomposites corresponds to the PAEM/MBA

content in the material. c

Average of 3 replicates.

2.3. Nanocomposite membranes characterization All ensuing membranes were characterized in terms of structure (FTIR and

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C NMR),

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morphology (SEM), crystallinity (XRD), thermal stability and degradation profile (TGA), mechanical

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properties (tensile test), swelling behaviour and antibacterial properties.

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FTIR spectra were acquired using a Perkin Elmer FTIR System Spectrum BX spectrophotometer

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equipped with a single horizontal Golden Gate ATR cell. Thirty-two scans were acquired in the

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4000−500 cm−1 range with a resolution of 4 cm−1.

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CPMAS 13C NMR spectra were recorded on a Brüker Avance III 400 spectrometer operating at a B0

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field of 9.4 T using 9 kHz MAS with proton 90° pulse of 3 microseconds and time between scans of 3

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seconds.

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chemical shifts were referenced with respect to glycine (C=O resonance at δ 176.03 ppm).

C CPMAS NMR spectra were acquired using a contact time of 2000 microseconds.

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SEM micrographs of the nanocomposite membrane surfaces and cross-section were obtained on a

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HR-FESEM SU-70 Hitachi equipment operating at 1.5 kV and in the field emission mode. Samples

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were placed on a steel plate and coated with carbon before analysis.

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The X-ray diffraction (XRD) measurements were carried out with a Phillips X’pert MPD diffractometer using Cu Kα radiation.

TGA assays were performed using a Shimadzu TGA 50 analyser equipped with a platinum cell.

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Samples were heated at a constant rate of 10 °C/min, from room temperature to 800 °C, under a

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nitrogen flow of 20 mL/min. The thermal decomposition temperature was taken as the onset of

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significant (~0.5%) mass loss, after the initial moisture loss.

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Tensile tests were performed on an Instron 5564 tensile testing machine at a cross-head speed of 10

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mm/min using a 1 kN static load cell. The tensile test specimens were rectangular strips (30 mm×10

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mm) dried at 40 ºC and equilibrated in a 50% humidity atmosphere prior to testing. All

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measurements were performed for at least five replicates of each sample and the average value was

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recorded.

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The swelling ratio (SR) of the nanocomposite membranes was measured using the weighing

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method (Figueiredo et al., 2013; Khalil, Bhat, & Yusra, 2012). Triplicate 1 x 1 cm2 specimens of each

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material were immersed in distilled water at room temperature to study their swelling. The weight

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increase was periodically assessed for 48 hours. Samples were taken out of the water, their wet

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surfaces immediately wiped dry in filter paper, weighed and then re-immersed. The SR was

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calculated using the equation below:

 %  140 141 142 143

 
100% 

where Wd is the initial weigh of dry membrane and Ws is the weight of the membrane swollen in water. In order to verify if PAEM release from the membranes during this experiment, at the end the membranes were dried and weighed. 5 Page 6 of 27

144 2.4. Assessment of BC nanocomposites antimicrobial properties

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2.4.1. Bacterial strain and growth conditions

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The antimicrobial activity of the BC/PAEM and all BC/PAEM/MBA nanocomposites (and of the

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corresponding polymers PAEM and PAEM/20%MBA) was tested against the recombinant

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bioluminescent strain of Escherichia coli (Alves et al., 2008). Stock cultures were stored at -80 ºC in

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10% glycerol.

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Before each assay, an aliquot of E. coli was aseptically plated on tryptic soy agar (TSA, Merck)

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supplemented with 100 mg mL-1 of ampicilin (Amp) and 25 mg mL-1 chloramphenicol (Cm) and grown

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for one day at 25 °C. Subsequently, one colony was aseptically inoculated on tryptic soy broth (TSB,

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Merck) with both antibiotics and grown at 25 °C under stirring (120 rpm). Then, an aliquot of this

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culture was subcultured in 30 mL of TSB with Amp and Cm and grown overnight, under the same

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growth conditions, to reach an optical density (OD600) at 600 nm of 1.6 ± 0.1 corresponding to ∼ 108

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colony forming units (CFU) mL−1.

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To assess the antimicrobial activity of all nanocomposites ~50 mg of each material was placed in

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contact with 5 mL of a liquid bacterial suspension, prepared by tenfold diluting the overnight grown

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bacterial culture in TSB. Control bacterial cellulose and cross-linker samples were also run in each

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antibacterial test. All samples were incubated at 25 ºC. At time 0 and after 1, 2, 4, 6, 9, 12, 24, 36 and

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48 hours of incubation an aliquot (500 µl) of each sample and control was collected and the

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bioluminescence was measured in the luminometer (TD-20/20 Luminometer, Turner Designs, Inc.,

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USA). Three independent experiments were carried out and for each one two replicates were read on

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the luminometer.

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2.4.2. Bioluminescence versus colony forming units

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To evaluate the correlation between the CFU and the bioluminescence signal (measured in relative

169

light units, RLU) of E. coli an overnight culture of bioluminescent E. coli was serially diluted (10-1 - 10-7)

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in fresh phosphate buffered saline (PBS) 1x (8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4 and 0.24 g/L

171

KH2PO4; pH 7.4). Non-diluted and diluted aliquots were pour plated on TSA medium (1 mL) and,

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simultaneously, were read on the luminometer (50 μL) to determine the bioluminescence signal.

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Three independent experiments were carried out and for each one two replicates were plated and

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read on the luminometer.

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3. Results and Discussion

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BC/PAEM nanocomposites without and with 5, 10 and 20% (w/w) cross-linker (MBA) were

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prepared by in situ radical polymerization inside the drained BC membranes, previously soaked with

179

an AEM or AEM/MBA solution. The purified, wet BC/PAEM and BC/PAEM/MBA nanocomposite membranes showed a yellowish

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colour, in comparison with the milky-white BC membrane, as a result of the incorporation of PAEM

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inside the BC membranes. In addition, the BC/PAEM material was malleable, like BC, while the cross-

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linked nanocomposites were progressively tough with increasing cross-linker contents (Figure 1).

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Indeed, the BC/PAEM/MBA (1:6:0.20) was very difficult to remove from the Erlenmeyer where the

185

polymerization reaction took place. After air-drying, all nanocomposite membranes were visually

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very homogeneous, with a yellowish colour and considerably translucent whilst the pristine BC

187

membrane is opaque.

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The use of MBA as cross-linker allowed a higher retention of PAEM inside the BC membrane, as

189

observed by the stronger yellowish colour of the BC/PAEM/MBA membranes in comparison with

190

BC/PAEM counterparts (Figure 1). This was also confirmed by the gravimetric measurements (Table

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1) and is ascribed to the cross-linking reaction which hinders PAEM removal from the BC network

192

during the washing steps.

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Figure 1 - Visual aspect of the wet BC, BC/PAEM and all BC/PAEM/MBA nanocomposite membranes. 3.1. Structural Characterization

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The success of the polymerization of AEM into the BC network, either in the presence or absence of

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MBA, was assessed by FTIR-ATR and

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MBA, BC/PAEM and all BC/PAEM/MBA are shown in Figure 2.

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The FTIR spectrum of pure BC is characterized by a broad band at 3500-3000 cm-1, attributed to O–H

202

stretching vibrations; a band at 2892 cm-1 associated with C–H stretching vibration of CH2 groups, and

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a sharp and steep band at around 1100 cm-1 due to the presence of C–O-C stretching vibration of the

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ether linkage of cellulose (Amin, Ahmad, Halib, & Ahmad, 2012; Figueiredo et al., 2013; Goh et al.,

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2012).

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The main bands observed for PAEM could be assigned to the amine N-H stretching (3380 cm-1), C-H

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stretching from CH2 and CH3 groups (3300-2500 cm-1), carbonyl ester group stretching (1717 cm-1), N-

208

H bending (1602 cm-1), C-H asymmetrical bending from CH3 and CH2 groups (1457 cm-1), C-H

209

symmetrical bending from CH3 groups (1380 cm-1), CH2 twisting and wagging (1267 cm-1), C-O-C

210

stretching overlapped with C-N stretching (1134 cm-1), CH3 rocking (970 cm-1) and characteristic CH2

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rocking of the methacrylic polymers (746 cm-1). The FTIR spectrum of PAEM/20%MBA showed a

212

similar profile to that of PAEM, with the new vibrations resulting from MBA (specifically the amide

213

C=O stretching around 1630 cm-1) overlapped with those already present in the PAEM spectrum and

214

generating bands with increased relative area.

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In general, BC/PAEM and all BC/PAEM/MBA nanocomposite membranes showed FTIR spectra that

216

correspond to the sum of the spectra of its components (BC and PAEM or PAEM/20%MBA). The

217

success of the PAEM polymerization inside the BC network was mainly confirmed by the appearance

218

of an intense band at around 1717 cm-1, associated to the C=O stretching vibrations of PAEM, and by

219

the increase of the relative intensity of the bands on the region around 3500-2500 cm-1, as a result of

220

the increased number of aliphatic C-H vibrations resultant from the methacrylic polymeric matrix

221

and, in the particular case of BC/PAEM/MBA (1:6:0.05), (1:6:0.1) and (1:6:0.2) materials, due to the

222

cross-linker incorporation. This was further confirmed by the appearance of the methacrylic polymers

223

characteristic bands around 1457 and 746 cm-1, attributed to the CH2 bending and CH2 rocking

224

vibrations, as mentioned above.

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Moreover, the BC/PAEM/MBA nanocomposites specta (particularly those with higher MBA content)

226

showed a profile that is very similar to that of PAEM/20%MBA, as a result of the high polymer

227

content in this membrane.

C NMR analysis. The FTIR spectra of BC, PAEM, PAEM/20%

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Figure 2 – ATR-FTIR spectra of BC, PAEM, PAEM/20%MBA, BC/PAEM and all BC/PAEM/MBA nanocomposites.

9 Page 10 of 27

13

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Figure 3 displays the solid state

C NMR spectra of BC, PAEM and BC/PAEM and BC/PAEM/MBA

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(1:6:0.20) nanocomposites. The solid state NMR spectrum of BC was characterized by 13C resonances

234

at δ 65.4 (C-6), 71.9-74.7 (C-2,3,5), 89.0 (C-4) and 105.2 ppm (C-1); which is in agreement with

235

chemical shifts described in literature (Fink, Purz, Bohn, & Kunze, 1997; Lacerda et al., 2013;

236

Watanabe, Tabuchi, Morinaga, & Yoshinaga, 1998). PAEM, on the other hand, showed a NMR spectrum characterized by 13C resonances at δ 19.1 (α-

238

CH3), 40.0 (quaternary carbon), 45.2 (CH2 main chain), 54.4 (C-N) 62.5 (-O-CH2) and 178.4 ppm (C=O).

239

PAEM/20%MBA shows a

240

higher content of PAEM in the cross-linked material.

241

The 13C NMR spectra of BC/PAEM and BC/PAEM/MBA (1:6:0.20) nanocomposites are also a result of

242

the sum of the carbon resonances of BC and of the acrylic polymers. In fact, all nanocomposites have

243

a very similar profile. The major difference is the higher intensity of the resonances associated with

244

the synthetic polymer for the BC/PAEM/MBA nanocomposite in comparison with the cellulose C-1

245

resonance (which does not overlap with any PAEM or MBA resonance). This results from the higher

246

polymer content in this material due to the use of the cross-linking agent. These results are in perfect

247

agreement with the FTIR analysis discussed above.

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C NMR spectrum (not shown) very similar to that of PAEM, due to the

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248 10 Page 11 of 27

249 Figure 3 - CP-MAS 13C NMR spectra of BC, PAEM, BC/PAEM and BC/PAEM/MBA (1:6:0.20).

252

Furthermore, the absence of FTIR vibrations and carbon resonances characteristic of the monomer

253

(AEM), particularly those associated with double bonds, demonstrates the complete polymerization

254

of the monomer and/or the effective removal of unreacted monomer or by-products during the

255

washing procedure.

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256 3.2. Morphology

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Figure 4 shows a selection of SEM micrographs of BC, BC/PAEM and BC/PAEM/MBA nanocomposites

259

with 5 and 20% of MBA. The micrographs obtained for pure BC membranes revealed its well-known

260

ultrafine network structure, composed of a random assembly of nanofibers, as well as its lamellar

261

structure (cross-section images) (Hofinger, Bertholdt, & Weuster-Botz, 2011; Klemm et al., 2011; Wei,

262

Yang, & Hong, 2011)

263

BC and the nanocomposite membranes showed significant differences in terms of thickness;

264

therefore different magnifications were required to obtain images of all cross-sections. From these,

265

as expected, it is possible to conclude that increasing PAEM (or PAEM/20%MBA) content yields

266

thicker nanocomposite membranes.

267

From all SEM images acquired it was possible to further confirm the effectiveness of the

268

polymerization reaction, with the polymer formation not only on the surface but also on the interior

269

of the BC membranes. The cross-section micrographs of the nanocomposite membranes displayed

270

the typical lamellar morphology of BC completely impregnated with PAEM, particularly visible for

271

nanocomposites with higher contents of cross-linker. In fact, the BC/PAEM/MBA dry membranes are

272

much thicker than that prepared without cross-linker (BC/PAEM). Moreover, in the case of the

273

BC/PAEM nanocomposites the surface image showed the inexistence of a homogenous layer of

274

polymer, which in turn is clearly visible in the case of the BC/PAEM/MBA (1:6:0.05) and (1:6:0.20),

275

suggesting some surface lixiviation of the non-cross-linked polymer during the washing step. Indeed,

276

this difference may be the cause of the synthetic polymer mass difference between the two

277

nanocomposite materials (Table 1).

278

Finally, the conclusions drawn from the SEM micrographs are in agreement with the results

279

previously obtained by FTIR and

280

final material, yields spectra that are similar to those of the synthetic polymer.

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C NMR, according to which the increase in PAEM content in the

281 11 Page 12 of 27

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Figure 4 - SEM micrographs of the (A) surface, (B, C) cross-section of BC, BC/PAEM, BC/PAEM/MBA (1:6:0.05) and BC/PAEM/MBA (1:6:0.2).

286

3.3. X-Ray diffraction characterization

287

X-ray diffraction analyses have been performed on neat BC membranes, PAEM matrices (without and

288

with 20% w/w cross-linker) and BC/PAEM and all BC/PAEM/MBA nanocomposite membranes, in

289

order to assess the effect of the methacrylic polymers incorporation on the crystallinity of the

290

resulting materials.

291

As well known, BC exhibits a diffractogram typical of Cellulose I (native cellulose), with the main

292

peaks at 2θ 14.3, 16.8, 20.3, 22.6 and 34.0º (Ford, Mendon, Thames, & Rawlins, 2010; Klemm et al.,

293

2005; Lacerda et al., 2013), while PAEM and PAEM/20%MBA matrices display diffraction profiles

294

typical of amorphous polymers. The X-ray diffraction profiles of the nanocomposite membranes

295

showed merely the typical diffraction peaks of BC but with decreased intensity, being this more

296

evident for the peak at 2θ 14.3º. As expected, the introduction of the amorphous polymer into the

297

crystalline cellulose resulted in nanocomposite materials with decreased crystallinity. This is more

298

evident in the case of the BC/PAEM/MBA nanocomposites as a result of the higher synthetic polymer

299

content in this material.

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300 301

3.4. Swelling Behaviour

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Swelling studies were performed on BC membrane, BC/PAEM and all BC/PAEM/MBA

303

nanocomposites in order to evaluate their re-hydration ability (reverse swelling after drying) after 48

304

h of immersion in water (Figure 5). The studied samples showed quite distinct swelling behaviors;

305

whilst BC reaches a maximum swelling of 100% after 24h, all nanocomposites show much higher

306

swelling values, particularly BC/PAEM with a swelling ratio of 6200%. Furthermore, as expected,

307

increasing cross-linker content caused a decreasing on the swelling ability, specifically 870, 210 and

308

130 % for BC/PAEM/MBA (1:6:0.05), BC/PAEM/MBA (1:6:0.1) and BC/PAEM/MBA (1:6:0.2),

309

respectively. Nevertheless, in all cases a plateau was reached after 24 hours. .

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(b)

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(c)

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Figure 5 - (a) Graphical representation of the swelling ratio of BC/PAEM and all BC/PAEM/MBA nanocomposites and BC membrane as a function of time (0-48 h). (b) Expansion of the BC and of all BC/PAEM/MBA swelling ratio graphs. (c) Photographs of swollen BC, BC/PAEM and BC/PAEM/MBA (1:6:0.2) membranes.

322

The swelling behaviour of the BC/PAEM nanocomposites is attributed to the high hydrophilic

323

character of BC, and particularly of PAEM. The presence of PAEM inside the BC network prevents the

324

collapse of the BC structure during drying and the existence of a high number of hydrophilic

325

ammonium groups in its structure favours the water uptake. In the case of the BC/PAEM/MBA

326

nanocomposites, a progressively rigid and condensed polymeric network is formed as the amount of

327

cross-linker increases which restricts the inter-chain movement and possibly limits the swelling

328

capacity of the material (He et al., 2012; Levchik, Si, Levchik, Camino, & Wilkie, 1999; Marek, Conn, &

329

Peppas, 2010).

330

The high water swelling, mainly of BC/PAEM, is particularly important in biomedical applications of

331

this type of materials, namely as wound dressings, since they provide a wet environment that favours

332

tissue healing as well as the wound exudates absorption.

333

In addition, it was observed that some PAEM polymer released from the nanocomposites during

334

swelling, with BC/PAEM (without cross-linker) showing the highest polymer loss (around 54%).

335

Increasing the cross-linker content decreased considerably the polymer loss, precisely 20, 11 and 10

336

for BC/PAEM/MBA (1:6:0.05), BC/PAEM/MBA (1:6:0.1) and BC/PAEM/MBA (1:6:0.2), respectively.

337

This will also play an important role on the antimicrobial activity of the nanocomposites as will be

338

described below.

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3.5. Thermal Properties

14 Page 15 of 27

The TGA of BC/PAEM and all BC/PAEM/MBA nanocomposites was used to investigate their thermal

342

stability and degradation profiles. Reference BC membranes and PAEM and PAEM/20%MBA

343

polymeric matrices were also analysed for comparison purposes (Table 2, Figure 6).

344

Pristine BC membrane displayed a typical single mass-loss step degradation profile (Tomé et al.,

345

2010), initiating its thermal decomposition at 260 ºC and reaching a maximum decomposition rate at

346

350 ºC with 94% mass loss. The initial mass loss at around 100 ºC is associated with the volatilization

347

of residual water.

348

The TGA tracings of PAEM and PAEM/20%MBA reveal a three-step mass loss, occurring at 279, 429,

349

587 and 307, 431 and 585 ºC, respectively, in agreement with the thermal behavior described for

350

similar poly(methacrylates) containing amine side groups (Abdellaoui-Arous & Djadoun, 2011;

351

Cervantes-Uc, Cauich-Rodríguez, Herrera-Kao, Vázquez-Torres, & Marcos-Fernández, 2008)

352

Both PAEM and PAEM/20%MBA are less thermally stable than BC (Figure 6), and PAEM/20%MBA is

353

slightly more stable than PAEM, since its initial degradation temperature occurs at a temperature

354

~80ºC higher than that of PAEM, as expected, for cross-linked polymers (Uhl et al., 2001).

355

As regards the BC/PAEM and all BC/PAEM/MBA nanocomposites, all show a multi-step degradation

356

profile, very similar to that of the corresponding pristine synthetic polymer. The initial degradation

357

temperature of BC/PAEM increased by 67 ºC relative to its correspondent synthetic polymer, possibly

358

as a result of the establishment of interactions between the components (BC and methacrylic

359

polymer). However, the same trend was not observed between the BC/PAEM/MBA nanocomposites

360

and native PAEM/20%MBA. This may be due to the degradation of loose segments of PAEM present

361

on the external surface of the nanocomposite materials. Nevertheless, the rise of cross-linker content

362

employed resulted in the slight increase of the initial degradation temperature. In addition, BC/PAEM

363

and all BC/PAEM/MBA nanocomposite membranes are thermally stable up to around 120 ºC, the

364

temperature involved in typical sterilization procedures required for biomedical applications.

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15 Page 16 of 27

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(b)

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Figure 6 - TGA thermograms of (a) BC, PAEM and BC/PAEM and (b) BC, PAEM/20%MBA and all

371

BC/PAEM/MBA nanocomposites.

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16 Page 17 of 27

373 Table 2 - Thermal properties of pristine BC membranes, PAEM, PAEM/20%MBA and all BC/PAEM/MBA nanocomposites

Material

a

Tdmax1b

Tdmax2b

Tdmax3b

Tdmax4b

(ºC)

(ºC)

(ºC)

(ºC) -

Tdi (ºC) 260

353

-

-

PAEM

128

279

429

587

BC/PAEM

171

198

262

411

PAEM/20%MBA

210

307

431

585

-

182

222

277

419

-

188

241

286

410

-

182

240

391

410,418

BC/PAEM/MBA

an

(1:6:0.1) BC/PAEM/MBA

377

a

285

-

M

(1:6:0.2)

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BC/PAEM/MBA (1.6:0.05)

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BC

cr

374 375 376

Initial degradation temperature b Maximum degradation temperatures

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3.6. Mechanical Analysis

380

Tensile tests were performed, at room temperature, for BC and all nanocomposites. Figure 7 shows

381

the mechanical properties, including Young’s modulus, tensile strength and elongation at break,

382

determined from the typical stress-strain curves of the materials studied. The tensile tests of the

383

pristine polymers, PAEM and PAEM/20%MBA, were not performed due to their brittleness and

384

incapability to form membranes, respectively.

385

Pure BC membrane presents a tensile strength of 298 MPa and a Young’s modulus of 5.0 GPa. The

386

values of tensile strength and Young’s modulus observed for BC/PAEM and all BC/PAEM/MBA

387

nanocomposites are lower than those observed for pure BC as a result of the introduction of the

388

amorphous polymers into the crystalline BC membranes. However, these values are certainly higher

389

than those of the PAEM and PAEM/20%MBA matrices, whose mechanical properties could not be

390

assessed. Additionally, the BC/PAEM/MBA nanocomposites might be expected to have higher

391

Young’s modulus and tensile strength than those of BC/PAEM, due to the increasingly cross-linked

392

structure. This was observed for the BC/PAEM/MBA (1:6:0.05) but the same behavior was not

393

observed for the BC/PAEM/MBA (1:6:0.1) and (1:6:0.2) materials, which, considering its lower BC

394

content (Table 1), suggests that the mechanical performance of these materials is essentially

395

governed by their BC content. Concerning the elongation at break, the values obtained for both

396

BC/PAEM and all BC/PAEM/MBA nanocomposites were lower than those of pure BC. However,

397

BC/PAEM/MBA has higher elongation than BC/PAEM and the remaining cross-linked nanocomposites

398

which is in tune with its lower young modulus.

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(a)

400 401 402

(b)

18 Page 19 of 27

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403 (c)

405 406 407 408

Figure 7 - Young’s modulus (a) tensile strength (b) and elongation at break (c), of pristine bacterial cellulose (BC) and the BC-based nanocomposites: BC/PAEM and BC/PAEM/MBA (with 5, 10 and 20% of MBA).

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404

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3.7. Antibacterial properties

412

The presence of ammonium groups in the chemical structure of PAEM as well as the corresponding

413

nanocomposite materials turns them into potential biocidal agents. This hypothesis was evaluated by

414

placing BC, PAEM, PAEM/20%MBA and BC/PAEM and all BC/PAEM/MBA nanocomposites in contact

415

with a bacterial suspension of the bioluminescent Escherichia coli in a tryptic soy broth (TSB).

416

Prior to materials testing the correlation between the bioluminescent signal (RLU) and the viable

417

counts (CFU) of overnight cultures of bioluminescent E. coli was evaluated. A linear relationship

418

between the two variables was observed, revealing that 107 CFU mL-1 corresponds, approximately, to

419

104 RLU mL-1 (Figure 8 a).

420 421

(a) 19 Page 20 of 27

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424 425 426 427 428 429 430 431 432 433

Figure 8 – (a) Relationship between the bioluminescence signal and viable counts of overnight cultures of recombinant bioluminescent E. coli serially diluted in PBS. Viable counts are expressed in CFU mL-1 and bioluminescence in relative light units (RLU). Each value represents mean ± standard deviation of three independent experiments. (b) Bioluminescent signal of E. coli suspensions in TSB after 0, 1, 2, 4, 6, 9, 12, 24, 36 and 48 h of contact with BC, PAEM, PAEM/20%MBA, BC/PAEM, or BC/PAEM/MBA (1:6:0.05), (1:6:0.1) or (1:6:0.2). A control sample is also shown, for comparison, consisting of a tenfold diluted E. coli suspension in TSB. Each value represents mean ± standard deviation of three independent experiments.

434

The results of the antibacterial activity tests of BC, PAEM, PAEM/20%MBA and the corresponding

435

nanocomposites are shown in Figure 8 b. As expected, bacterial cellulose (BC) has no effect on the

436

bacterial viability, with the bioluminescence values being similar to those of the control sample (Pinto

437

et al., 2009). Similar results were also obtained for the cross-liker samples (results not shown).

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20 Page 21 of 27

The antibacterial activity of the tested materials is expected to be a result of the interaction between

439

the ammonium groups of the polymer with the bacteria. In the case of PAEM this appears to happen

440

as it causes total bacteria death in the first contact hours. And its incorporation inside the BC

441

network, creating the BC/PAEM nanocomposite, does not affect its ability to kill bacteria. In fact, this

442

activity is extended for more 18 hours. The high swelling ability of this material contributes to the

443

diffusion of the bacterial suspension into the nanocomposite material and together with the release

444

of PAEM into the bacterial suspension, promote the contact between the pendant ammonium

445

groups and bacteria and finally causing their death.

446

In the case of PAEM/20%MBA and the corresponding BC/PAEM/MBA nanocomposites no significant

447

activity is detected after 48 hours of contact with bacteria. Such reduced antibacterial activity seems

448

to be a result of the cross-linked structure which hinders its swelling ability and considerably reduces

449

the polymer dissolution and bacteria diffusion, and consequently the contact of the bulk of the

450

ammonium groups with bacteria.

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451 4. Conclusions

453

The present work describes the successful preparation of bacterial cellulose/poly(2-aminoethyl

454

methacrylate) (BC/PAEM) nanocomposite materials, with and without cross-linker, via in situ

455

polymerization.

456

The polymer incorporation into the BC membranes lead to the total filling of BC porous structure and

457

improved its mechanical properties as a result of the BC reinforcing ability. Furthermore, the

458

crystallinity of the ensuing materials is decreased, in comparison with that of pure BC, due to the high

459

amorphous polymer content in the material.

460

In addition, all BC/PAEM and BC/PAEM/MBA materials show increased swelling ability in comparison

461

with BC, being this more evident for BC/PAEM. This is due to the hydrophilic character of the polymer

462

and to its ability to prevent the collapse of the BC structure. The BC/PAEM is also the nanocomposite

463

showing antibacterial activity towards the bioluminescent E. coli, in contrast with its cross-linked

464

counterparts with no detectable antibacterial activity.

465

Following what was described before; imparting antimicrobial activity to BC membranes was

466

successfully achieved. Amongst the materials tested, the BC/PAEM nanocomposite proved to have

467

the best properties for potential application as wound dressing. Due to the fact that its characteristics

468

simultaneously allow a moist environment that favors wound healing whilst inhibiting the

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21 Page 22 of 27

469

development of bacterial infections. Its high swelling ability might also help the absorption of wound

470

exudates and thus further favoring wound healing.

471 472 Acknowledgements

474

The authors acknowledge Fundação para a Ciência e a Tecnologia (FCT) for CICECO (FCOMP-01-0124-

475

FEDER-037271; Refª. FCT PEst-C/CTM/LA0011/2013) NHCS Silva (SFRH/BD/85690/2012) funding.

476

C.S.R. Freire also acknowledges FCT/MCTES for a research contract under the Program "Investigador

477

FCT 2012".

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478 References

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Figure(s) Captions

Figure Captions

Figure 1 - Visual aspect of the wet BC, BC/PAEM and all BC/PAEM/MBA nanocomposite membranes.

Figure 3 - CP-MAS (1:6:0.20).

ip t

Figure 2 – ATR-FTIR spectra of BC, PAEM, PAEM/20%MBA, BC/PAEM and all BC/PAEM/MBA nanocomposites. 13

C NMR spectra of BC, PAEM, BC/PAEM and BC/PAEM/MBA

us

cr

Figure 4 - SEM micrographs of the (A) surface, (B, C) cross-section of BC, BC/PAEM, BC/PAEM/MBA (1:6:0.05) and BC/PAEM/MBA (1:6:0.2).

an

Figure 5 - ((a) Graphical representation of the swelling ratio of BC/PAEM and all BC/PAEM/MBA nanocomposites and BC membrane as a function of time (0-48 h). (b) Expansion of the BC and of all BC/PAEM/MBA swelling ratio graphs. (c) Photographs of swollen BC, BC/PAEM and BC/PAEM/MBA (1:6:0.2) membranes. Figure 6 - TGA thermograms of (a) BC, PAEM and BC/PAEM and (b) BC, PAEM/20%MBA

M

and all BC/PAEM/MBA nanocomposites.

Figure 7 - Young’s modulus (a) tensile strength (b) and elongation at break (c), of pristine

ed

bacterial cellulose (BC) and the BC-based nanocomposites: BC/PAEM and BC/PAEM/MBA (with 5, 10 and 20% of MBA).

Figure 8 – a) Relationship between the bioluminescence signal and viable counts of

Ac

ce pt

overnight cultures of recombinant bioluminescent E. coli serially diluted in PBS. Viable counts -1 are expressed in CFU mL and bioluminescence in relative light units (RLU). Each value represents mean ± standard deviation of three independent experiments. (b) Bioluminescent signal of E. coli suspensions in TSB after 0, 1, 2, 4, 6, 9, 12, 24, 36 and 48 h of contact with BC, PAEM, PAEM/20%MBA, BC/PAEM, or BC/PAEM/MBA (1:6:0.05), (1:6:0.1) or (1:6:0.2). A control sample is also shown, for comparison, consisting of a tenfold diluted E. coli suspension in TSB. Each value represents mean ± standard deviation of three independent experiments.

Page 27 of 27