Chitosan-bacterial cellulose patch of ciprofloxacin for wound dressing: Preparation and characterization studies

Journal Pre-proofs Chitosan-bacterial cellulose patch of ciprofloxacin for wound dressing: Preparation and characterization studies Maximiliano L. Cacicedo, Guilherme Pacheco, German A. Islan, Vera A. Alvarez, Hernane S. Barud, Guillermo R. Castro PII: DOI: Reference:

S0141-8130(19)32200-7 https://doi.org/10.1016/j.ijbiomac.2019.10.082 BIOMAC 13576

To appear in:

International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

25 March 2019 23 August 2019 8 October 2019

Please cite this article as: M.L. Cacicedo, G. Pacheco, G.A. Islan, V.A. Alvarez, H.S. Barud, G.R. Castro, Chitosan-bacterial cellulose patch of ciprofloxacin for wound dressing: Preparation and characterization studies, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.082

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© 2019 Published by Elsevier B.V.

Chitosan-bacterial cellulose patch of ciprofloxacin for wound dressing: Preparation and characterization studies

Maximiliano L. Cacicedoa, Guilherme Pachecob, German A. Islana, Vera A. Alvarezc, Hernane S. Barudb, and Guillermo R. Castroa.

a Laboratorio

de Nanobiomateriales, CINDEFI, Departamento de Química, Facultad de Ciencias

Exactas, Universidad Nacional de La Plata-CONICET (CCT La Plata), Calle 47 y 115, B1900AJL La Plata, Argentina.

b Universidade

de Araraquara (UNIARA) - Laboratório de Biopolímeros e Biomateriais (BioPolMat), Rua Carlos Gomes 1217, 14.801-320, Araraquara, SP, Brazil.

c CoMP

(Grupo de Materiales Compuestos), Instituto de investigación en Ciencia y Tecnología de

Materiales (INTEMA) (CONICET, UNMdP), Solís 7575, B7608FDQ Mar del Plata, Argentina.

Corresponding author: Prof. Guillermo R. Castro E-mail: [email protected] Phone: +54-221-4833794 ext. 132 (office)

1

Abstract Biopolymeric blends based on bacterial cellulose (BC) films modified with low molecular weight chitosan (Chi) were developed for controlled release of ciprofloxacin (Cip). Biophysical studies revealed a compatible and cooperative network between BC and Chi including deep structural changes in the BC matrix shown by spectroscopic and thermal analyses (SEM, roughness analysis, FTIR, XRD, TGA, mechanical properties and water vapor transmission rate). Incorporation of chitosan to BC matrix generated a thickening scaffold with high permeability to water vapor from 0.7 to 3.2 g mm/m2 h. Cip loaded onto the BCChi film showed a hyperbolic release profile with a 30% decrease in antibiotic release mediated by the presence of Chi. BC-Chi blend films containing Cip tested against Pseudomonas aeruginosa and Staphylococcus aureus showed a synergic effect of chitosan on Cip antimicrobial activity. Besides, in vitro studies revealed the lack of cytotoxicity of BCChi-Cip films in human fibroblasts.

Keywords: Bacterial Cellulose; Chitosan; Ciprofloxacin.

1. Introduction The number of patients with chronic wounds, burns, scalds, and ulcers is rising worldwide, and it is a special challenge to beat. Also, wounds could allow pathogens to spread infections systemically and consequently could cause septicemia. The main therapies to treat wounds involve the systemic and local administration of antibiotics. Nevertheless, the usually high antibiotic doses could cause undesirable side effects on many organs (Singh et al., 2014). Transdermal patches are useful options since they are easy to manipulate, do not require specific infrastructure or trained personnel, and can be produced at low costs. Several models for local antibiotic administration and wound healing therapies including films and hydrogels were developed (Boateng and Catanzano, 2015). However, the increase of microbial resistance to common antibiotics created a global antibiotic emergency declared by WHO. Chitosan (Chi) is a linear biopolymer mostly obtained from crustacean shells composed of Nacetyl-D-glucosamine and β(1→4)-D-glucosamine randomly distributed. Chi leads the list of the most used polymers for wound dressings (Sahariah and Másson, 2017). Some properties, such as biocompatibility, biodegradability and accelerated wound healing, make chitosan an 2

excellent biomaterial for biomedical applications. Chi is an aminated biopolymer that can be used to make three dimensional structures by ionotropic gelation in presence of multivalent anions such as triployphosphate (Martins et al., 2012). In addition, chitosan has antibacterial and antifungal activities that contribute to preventing local wound infections during the healing process (Lopez-Moya et al., 2015). However, Chi low mechanical resistance and low oxygen permeability are limiting its use in wound healing patches. An alternative to develop Chi films is the formation of blends with other polymers (Cai et al., 2011; Jia et al., 2017). Bacterial cellulose (BC) has been intensively studied for many applications in the biomedical field, including artificial blood vessels, tissue engineering, wound dressing, and drug delivery (Cacicedo et al., 2016; de Oliveira Barud et al., 2016). BC is an extracellular polysaccharide synthesized at the air/liquid interface media of bacterial cultures. BC is produced in nanofibrils composed of β(1→4) glucose units stabilized by inter- and intrachain hydrogen bonds. In static microbial cultures, the BC nanofibril chains make selfassembled films of extremely pure cellulose with a high water content (about 99%), high mechanical strength and well-defined biocompatibility (Abeer et al., 2014). However, native BC films do not show antimicrobial activity. Also, BC are not able to entrap, keep and control the release of small molecules like antibiotics (Cacicedo et al., 2016). In order to develop controlled release devices, BC must be modified by using polymers that can alter pore size and interchain strenght (Abeer et al., 2014). In this sense, BC films doped with Chi could enhance the ability to entrap and sustain the release of an antibiotic. BC-Chi patches can be advantageous for wound healing since they could produce local biological effects such as antimicrobial, antifungal ones, wound healing acceleration and will be able to entrap and release an antibiotic generating a shock-like effect against the potential pathogens in the wounds. Among antibiotics, ciprofloxacin (Cip) is the fifth largest generic antibiotic produced in the world. Cip belongs to the fluoroquinolone family, a wide class of antibiotics with broad antibacterial spectrum currently used in many infections. In addition, Cip has been reported as an excellent antibiotic for wound dressing (Li et al., 2017). Cip antimicrobial activity is relevant for Gram-negative microorganisms including the promiscuous P. aeruginosa. Besides, the antimicrobial activity of Cip against Gram-positive microorganisms such as S. aureus is sometimes reduced compared to other fluoroquinolones (Islan et al., 2015).

3

The aim of the present work is to design a strategy for ex situ modification of BC films containing Chi and Cip with high antimicrobial activity for wound dressing. Hybrid BC-Chi films were characterized using several biophysical methods (i.e. SEM, roughness analysis, TGA, FTIR, XRD, mechanical properties and water vapor transmission rate). Cip release profiles were established, and antibacterial activity was assayed against relevant dermal associated Gram-negative and Gram-positive pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. The antimicrobial activities of the hybrid BC-Chi scaffold were analyzed by international protocols and fluorescent probes for live and dead microbes. Finally, the cytotoxicity of the hybrid films was evaluated in human fibroblasts.

2. Materials & Methods 2.1. Chemicals and media Ciprofloxacin

(1-cyclopropyl-6-fluoro-1,4-dihydro-4-ox-o7-(1-piperazinyl)-3-quinoline

carboxylic acid) and low molecular weight chitosan (MWavg= 120 KDa) were purchased from Sigma-Aldrich (Buenos Aires, Argentina). Cell culture materials were purchased from Coriell Cell Repositories (Coriell Institute for Medical Research, Camden, NJ, USA), Dulbecco´s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin) and fetal bovine serum were purchased from Vitrocel (Brazil). All other reagents used were of analytical or microbiological grade purchased from Merck (Darmstadt, Germany) or from Sigma Chemical Co. (St. Louis, MO) except when otherwise indicated. Cip was quantified at 280 nm spectrophotometrically (Perkin Elmer LS 50B, Japan) using appropriate calibration curves.

2.2. Bacterial cellulose 2.2.1. Bacterial cultivation Komagataeibacter xylinus (ATCC 23760) was maintained in Hestrin and Schramm (HS) agar culture medium at 4–8°C. One single colony from the solid medium was used to inoculate into 50 ml of modified HS liquid medium, composed of 50.0 g/L of glucose, 4.0 g/L of yeast extract, 0.73 g/L of MgSO4.7H20, 2.0 g/L of KH2PO4, 20.0 g/L of ethanol, and distilled water (1 L). Microbial cultures were incubated in static conditions at 28°C for 24 h. BC films of 11 mm diameter were produced in a 48-well plate for 48 h culture. 4

2.2.2. Bacterial cellulose purification Cellulose films were collected and incubated for disinfection in 0.1% sodium hypochlorite for 5 min. Next, films were washed at least three times with distilled water for 30 min. Later, films were immersed in 0.1 N NaOH solution at 60°C for 24 h. After that, repetitive washes with distilled water were performed until neutral pH. Finally, films were autoclaved at 121°C for 20 min.

2.2.3. Ex situ modification of bacterial cellulose with chitosan A 2.0% (w/v) chitosan solution was prepared in 3.0% acetic acid and 10.0% (w/v) glycerol. Purified and sterile BC films were previously dried and then incubated by immersion in chitosan solution at 30°C with shaking (250 rpm) for 24 h. In the following step, films and 100 µl of the chitosan solution were incorporated and mixed in each well of a 48-well plate. Finally, a 5.0% (w/v) solution of tripolyphosphate (TPP) as cross-linking agent was added, and the plates were incubated at 4°C for 2 h. BC films were weighted before and after modification with chitosan. Weight differences allowed quantifying the amount of chitosan that has been incorporated into the network. Chitosan constitutes 36.8 ± 3.2 % of the total mass of the hybrid films. After the modification process, films were dried by the solvent-cast method. Plates were incubated at 30°C for 12 h until constant mass.

2.2.4. Cip incorporation and payload evaluation For the incorporation of the antibiotic to BC-Chi films, a 10 mg/ml solution of Cip was prepared in 3.0% acetic acid. Then, Cip stock solution was added to chitosan solution to reach a drug concentration of 0.5 mg/ml. Finally, films and 100 µl of the chitosan-Cip solution were incorporated and mixed in each well of a 48-well plate. The next steps were the same as mentioned in section 2.2.3. The total payload of each BC-Chi-Cip film was evaluated by immersing the membrane in 3.0% acetic acid. The mixture was sonicated using an ultrasonic processor (40% amplitude, 130 W, Cole-Parmer, USA) equipped with a 6 mm titanium tip for 30 min. Later, the mixture was centrifuged, and Cip was spectrophotometrically quantified in the supernatant. This method was performed for two independent batches and for five membranes of each one. The total payload was evaluated as follows: 5

Cip incorporation =

(Cip)supernatant WBC

where (Cip)supernatant is the mass (g) of Cip in the supernatant and WBC is the mass of BC film (grams). Values were expressed as Cip grams per 100 grams of film. Control assays of films without Cip and free Cip were performed. No absorbance interference, Cip degradation, or UV maximum shift or intensity decrease were observed.

2.3. BC-Chi film characterization 2.3.1. Scanning electron microscopy (SEM) Freeze-dried samples were sputtered on the surface with gold using a metalizer (Balzers SCD 030) obtaining a layer thickness between 15 and 20 nm. Film surfaces and morphologies were observed by SEM (Philips SEM 505 model, Rochester, NY, USA) and the images were processed by a digitizer program (Soft Imaging System ADDA II).

2.3.2. Roughness analysis SEM images were analyzed by ImageJ software (NIH, USA). Surface parameters such as roughness and polymer distribution were determined by the mean and standard variation of the gray values of all the pixels of the image respectively. Histograms of SEM images (2,500x) were performed three times in duplicate.

2.3.3. Thermogravimetric analysis (TGA) Dynamic thermogravimetric measurements of native and hybrid BC membranes were performed using a Shimadzu TGA-50 instrument. Tests were run in the range of 20°C to 800°C with 10°C/min heating rate under N2 atmosphere.

2.3.4. Vibrational spectroscopic analysis (FTIR) FTIR spectra of the lyophilized BC and BC-Chi samples were recorded in a spectrometer (Thermo Scientific Nicolet, model 6700, CT, USA) coupled to an ATR (attenuated total reflectance) accessory for all measurements. Each sample was scanned 32 times in the 600 to 4000 cm−1 range with a resolution of 4 cm−1. 6

2.3.5. X-ray diffraction (XRD) XRD patterns of cellulose film samples were collected in reflection mode on a glass substrate. The measurement was performed with an Analytical Expert instrument using CuKα radiation (λ= 1.54 Å) from 2θ= 10° to 40° in continuous mode with 0.07° step size. The results were analyzed using Origin software.

2.3.6. Water vapor transmission rate (WVTR) Firstly, composite hydrogel samples with circular shape (radius of 14 ± 1 mm) were put as a cap with an adhesive on the mouth of a bottle containing 20 ml of distilled water (based on ASTM E96. Standard Test Methods for Water Vapor Transmission of Materials). Then the system (bottle + hydrogel sample) was kept for 3 days in a constant temperature-humidity homemade chamber (37°C at 75% RH). The water vapor transmission rate (WVTR) of each nanocomposite sample was calculated by using the following equation (Gonzalez et al., 2016):

where A is the bottle mouth area (mm2), M0 and M1 are the mass of the system before and after placing it in the chamber, respectively. The WVTR was normalized to sample thickness (tk) to obtain the specific water vapor transmission rate:

R2 = WVTR x tk in

2.4. In vitro drug release BC membranes were placed in 1.5 ml acetate buffer (pH 5.5) in 2.0 ml plastic vials at 37°C. Samples of 500 µl were withdrawn and replaced with equal volumes of fresh buffer at defined intervals. Cip concentrations in the samples were spectrophotometrically determined as mentioned before. Kinetic release experiments were performed in quadruplicate.

2.5. Antimicrobial assays 7

Pseudomonas

aeruginosa

ATCC

15442

(non-fermenting

Gram-negative

bacteria)

and

Staphylococcus aureus ATCC6538 (Gram-positive bacteria) were cultured in nutrient broth medium incubated at 37°C and 100 rpm for 12 h. Mueller Hinton agar was inoculated with microbial samples at 0.5 McFarland scale and poured into Petri dishes. Later, disks with empty and Cip loaded BC-Chi films were placed on the surface of Petri dishes and incubated at 37°C for 24 h. The inhibition halos against P. aeruginosa and S. aureus were determined by using the modified disk diffusion method according to international clinical standards (CLSI/NCCLS).

2.6. Biofilm staining with Live / Dead kit The commercial Live/Dead® BacLightÔ kit is composed of two fluorescent dyes: green (SYTO9Ô) and red (propidium iodide) to stain live and dead cells respectively. For microbiological assays, P. aeruginosa and S. aureus growing at late exponential phase were inoculated into a soft nutrient agar, and a drop of 20 µl was placed on the surface of a glass slide under sterile atmosphere, followed by incubation for 24 h to allow biofilm formation. Subsequently, biofilms were completely covered with empty and Cip loaded BC-Chi films for 10, 30 and 60 min. After treatment, biofilms were carefully washed with deionized water. Controls with untreated bacteria (live) and HClO treated biofilm (dead) were performed. Biofilm staining was performed with both dyes mixed in equal proportions (0.75 µl of each one in 0.5 ml of sterile deionized water) and applied above the entire biofilm and held in darkness for 20 min. Then, biofilms were washed with deionized water and observed in a Leica DM 2500 epifluorescence microscope (Germany) equipped with UV filters (495–505 nm) at 400x to determine the viability of the bacteria. The filters used were U-MWG2 (excitation between 510 nm and 550 nm, emission at 590 nm), which show live bacteria in green, and U-MWB2 (excitation 460 nm and emission 490-520 nm) showing the damaged bacteria in red. Images were overlapped with the image program GIMP 2 (free software kindly provided by S. Kimball, P. Mattis and GIMP team).

2.7. Cytotoxicity studies The cytotoxicity measurements were performed with weighed, milled samples immersed in cell culture Dulbecco’s Modified Eagle's Medium supplemented with 10% fetal bovine serum and antibiotics (penicillin and streptomycin) for 24 h. Later, the samples were removed, and the resultant medium was placed in cell culture plate wells (96 wells) containing human fibroblast cells (GM07492) with cell density 1x104 cells/well. The plates were kept in a cell culture incubator 8

(Panasonic-CO2 incubator MOC-19 AIC-UV) at 37°C with a humidified atmosphere containing 5% CO2 and 95% air atmosphere for 24 h. After that period, cell viability was determined by MTT (tetrazolium 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) colorimetric method. Subsequently, the culture medium was removed from the wells, which were washed with phosphate buffered saline (PBS). Aliquots of 50 µl of MTT were added to each well, and the cells were kept in culture conditions for 4 h. After this period, 100 µl of isopropyl alcohol was added, and the well content was mechanically homogenized until complete formazan solubilization. The optical density (OD) at 570 nm was determined spectrophotometrically (Polaris-CELER). The measurement was performed in triplicate for each sample and converted into percentages of cell viability related to the control group and subjected to analysis of variance (one-way ANOVA) at 5% significance level. The control group consisted of cultured cells in standard medium not exposed to the films.

2.8. Statistical analysis All experiments were carried out three times with a minimum of three replicates each one. Analyses of data were performed by Student T-test or by analysis of variance (ANOVA, followed by Tukey’s HSD test) with a significance level of 0.05.

3. Results and discussion 3.1. BC-Chi film characterization The surface and cross-section of native and modified BC films were observed by SEM. Figures 1A to 1C show the open microfibril network inside unmodified BC. The addition of Chi to BC films produces strong changes in the polymeric network (Figures 1D to 1F). The surface of BC-Chi film was smoother compared to BC, but the addition of Chi causes deep changes inside the BC network. BC-Chi images showed the presence of a closed network where BC fibrils were not detected. The empty space found in between BC fibrils was filled by Chi molecules that work as structural interference (Figure 1C). The images confirmed that Chi is able to penetrate into the BC network and interact with BC microfibrils changing the physicochemical properties of the BC film as previously suggested (Lin et al., 2013). Surface analysis of BC and BC-Chi films was carried out by ImageJ processing software to determine semiquantitative differences. Changes in the mean values of BC-Chi films indicate strong modifications in the spatial structure alignment of the biopolymeric chains. Also, the significant decrease in the standard deviation of BC-Chi films can be correlated 9

with the presence of a smoother surface observed by SEM. These results were also evidenced by the modification of the surface profiles after Chi incorporation into the BC network (Figure 1S, Supplementary material). The interactions between BC and Chi are certainly due to the formation of an intertwined network between both biopolymers with similar structural and chemical characteristics (Cacicedo et al., 2016a). Analysis of film surfaces by ImageJ program showed significant differences between the BC and BC-Chi films (Figure 1S, Supplementary material). This results is in agreement with previous report in where changes in the physicochemical properties of the BC-Chi structures were correlated with the increase of Chi in the composite (Cai et al., 2008). Also, the biocide activity of hybrid films was demonstrated by the incorporation of different quantities Chi in PLA and starch films which showed antimicrobial activity when the polymer content is higher than 10 to 15% (Bonilla et al., 2013; Shen et al., 2010). Chi modification of BC films displayed smooth surface characteristics observed in the 2D and 3D graphs and quantified by the decrease of the mean value and standard deviation (Figure 1S, Supplementary material). The results are opposite to the BC-alginate or BC-pectin films, where the addition of alginate or pectin to BC films makes uneven surfaces (Cacicedo et al., 2016b and 2018). Thermal degradation studies were performed to get better understanding of the Chi modification nature and how it affected the physicochemical properties of the film. The thermal properties of native BC, Chi and BC–Chi films were studied by thermogravimetry. The tests were carried out in a nitrogen atmosphere to avoid thermo-oxidative processes. Two-step decomposition curves were observed for all samples (Figure 2a). The first step was attributed to water evaporation with a different type of interaction within the polymers in the temperature range between 30°C and 150°C. Since film samples were previously freeze-dried, only water strongly bound to the biopolymers was expected to be in the films. BC and Chi showed similar low water amount values of 6.6% and 9.1%, respectively (Table 1). In contrast, BC-Chi film exhibited a more than twice increase in water content, which was about 20.8%. The higher water content for the hybrid matrix could be attributed to the very closed BC-Chi network where water is trapped in between the polymer chains. It is generally accepted that water acts as plasticizer in polymeric matrices (Lourdin et al., 1997). The second step in the curves, which was attributed to thermal decomposition of the biomaterials and the weight loss, in the range 150-400°C, was almost equal for each sample (Figure 2a). BC and Chi TGA curves showed a similar behavior until 325.8°C, where both curves separate. In comparison, BC-Chi exhibited a higher degradation rate that was better observed in DTGA curves and with the maximum thermal degradation temperature (Tp) (Figure 2a, Table 1). Tp is considered 10

a structural parameter related to the molecular weight, crystallinity and orientation of the polymers (Ouajai and Shanks, 2005). Particularly, the Tp for the hybrid BC-Chi film shifted down from 299.7°C-330.7°C for Chi and BC respectively to 197.5°C, and a shoulder was located at 208.2°C after Chi modification, indicating a strong interaction between BC fibrils and Chi chains. Essentially, the thermogravimetric analysis of the BC-Chi film clearly showed strong intermolecular interactions between BC and Chi. The molecular interactions between BC and Chi were further confirmed by spectroscopic analyses using XRD and FTIR (Figure 2b). Since BC and Chi have similar structures, good compatibility and interaction at molecular level between both polymers were expected. Particularly, the FTIR spectrum of plain BC films revealed peaks centered at 3340 cm-1 and 2895 cm-1 assigned to O-H and aliphatic C-H stretching, respectively (Figure 2b). Another strong peak at 1650 cm-1 was assigned to the glucose carbonyl of cellulose (Barud et al., 2008). Also, a peak centered at 1060 cm-1 was assigned to C-O stretching in agreement with previous report (Cacicedo et al., 2018). Meanwhile, Chi exhibited absorption bands at 1655 cm-1 (amide I), 1593 cm–1 (-NH2 bending) and 1375 cm–1 (amide III). The absorption bands at 1153 cm–1 (antisymmetric stretching of the C-O-C bridge), 1086 cm-1 and 1035 cm–1 (skeletal vibrations involving the C-O stretching) are characteristic of chitosan saccharide structure (Povea et al., 2011). After Chi incorporation, hybrid BC-Chi films exhibited all peaks described for BC and additional peaks indicating the presence of chitosan in contact with BC network. Peaks at 1647 cm1,

1575 cm-1 and 1975 cm-1 were observed in the composite and attributed to chitosan amide I,

amide II and amide III, respectively (Cai et al., 2011; Lin et al., 2013). Also, the absorption bands of Chi at 1655 cm-1 and 1593 cm-1 were shifted to 1647 cm-1 and 1575 cm-1 in BC-Chi films suggesting an interaction between BC and Chi chains (Aranaz et al., 2016). Additionally, the peak assigned to O-H stretching became wider, suggesting that chitosan molecules interfered with the hydrogen bonds between cellulose chains (Jia et al., 2017). The characteristic bands of Chi were shifted in presence of BC suggesting an intermolecular bonding between the Chi amino groups and the hydroxyl residues of BC by hydrogen bonds. Similar observations were previously reported for Chicellulose ethers and Chi-BC interactions (Yin et al., 2006; Phisalaphong & Jatupaiboon, 2008). The analysis of Chi-Cip FTIR spectrum showed great similarity with the Chi spectra and no significant new peaks and/or peak-shifts compared to the Chi spectrum were observed. Also, The Chi-Cip spectrum showed a very different profile compared to the Cip spectrum (Figure 2S, Supplementary material). These results could be the interpreted based on the relative low antibiotic concentration compared to Chi in the formulation and low sensitivity of the FTIR-ATR 11

technique. In general, it is assumed that molecular concentrations lower than 1.0% cannot be detected by FTIR-ATR technique (Rogachev et al., 2013). Weaker hydrogen bonds could point out a crystallinity change of the network structure. In this sense, the structure of BC and BC-Chi films was studied by XRD to analyze the changes caused by chitosan interpenetration into BC network (Figure 2c). The XRD profiles of BC showed characteristic Bragg’s angles of 2θ= 14.61°, 16.91° and 22.81° indexed as the (1 1 0), (0 1 0) and (0 0 2) reflection planes respectively (Cacicedo et al., 2018). Meanwhile, the XRD profile of Chi displayed a maximum Bragg's angle values of 2θ at 20.37° indexed as (1 1 0) and slight shoulder at 10.65° indexed as (0 0 2) reflection planes respectively (Figure 3S, Supplementary material). Similar values of Chi reflection planes were reported previously (de Queiroz et al., 2017). However, BC-Chi exhibited a strong reduction in the (0 0 2) peak and the disappearance of the (1 1 0) and (0 1 0) peaks; these changes indicate a significant structural modification in the polymeric network. Although the crystallinity index was difficult to quantify, XRD spectra of BC-Chi film allowed to understand the strong decrease in the crystallinity of the polymeric network. A similar behavior of BC after in situ modifications with polymers was previously reported (Lin et al., 2013; Cacicedo et al., 2016). A decrease in the crystallinity index is generally associated with an increment in the amorphous phase of the matrix, related to an increase in water absorption capacity. These findings confirm the assumptions from the FTIR analysis where it was suggested that the presence of chitosan partially interferes with the self-assembly of BC fibers and therefore strongly reduces the cellulose crystalline structure. Young’s modulus (E), maximum tension (σmáx) and elongation at break (ε) of hybrid BC-Chi films were lower than those of plain BC films (Table 1S, Supplementary material). As was observed in the XRD analysis, in the BC network the amorphous phase increased significantly after chitosan incorporation. Therefore, the results suggest that Chi weakens the BC film mechanical properties. The interactions of the Chi aminated groups with hydroxyl residues of BC observed by FTIR can cause a weakness of the BC interchain hydrogen bonds with the consequent decrease of its mechanical properties. Nevertheless, the values for BC-Chi film are still in the range of those reported for skin and wound dressings (Wang et al., 2002).

3.2. Water vapor transmission rate evaluation The water vapor transmission rate is a key parameter for wound dressing because suitable WVTR values can prevent wound dehydration and enhance healing capacity (Wu et al., 2018). The WVTR 12

values of skin can vary from 11.6 g/m2h for normal skin to 214 g/m2h for first-degree burns (Lamke et al., 1977). On the other hand, BC has shown its potential as a wound healing biomaterial (Cacicedo et al., 2016; Ye et al., 2018). Normalized WVTR values (Ɍ) indicated that thickening of BC membranes produced by the presence of Chi increased the water vapor permeability by more than four times in comparison with the control group (Table 2). This result showed the enhanced property of the films to balance wound humidity for an optimum healing process. These results are in agreement with those reported for wound dressings (Gonzalez et al., 2016).

3.3. Drug loading and release studies Wound dressing is based on the concept of creating a perfect environment to allow epithelial cell growth during the development of the healing process. Optimum healing conditions include moisture around the wound, excellent oxygen transference and avoidance of bacterial proliferation. Particularly, the purpose of applying antibiotics to wounds is mainly to prevent and combat potential infections, especially in some complex cases such as diabetic patients, severe burns and accidental wounds (Falanga, 2005). Additionally, local antibiotic release could help overcome the inefficiency of systemic antibiotic administration in diabetic patients due to poor blood circulation at the extremities (Yazdanpanah et al., 2015). Local antibiotic administration has the advantage of decreasing the antibiotic concentration circulating in the body and increases local concentrations, which could enhance the treatment efficiency and reduce the risks of microbial drug resistance and avoid undesirable toxic events. During the development of a hybrid system based on BC, Chi was chosen to provide local antibacterial activity and Cip to improve antibacterial effects, especially during the first hours after dressing application. The amount of incorporated Cip was 53.37 ± 2.99 µg of drug, expressed as 0.1% (w/w). Since the device was designed for wound healing applications and to prevent infections, the Cip incorporated into the hybrid scaffold was at low concentration compared with other antimicrobial films used for the treatment of dermal infections (Roy et al., 2015; Wang et al., 2007). The Cip release data were plotted as percentage of drug released versus time in hours. Approximately, within the first hour after immersion in acetate buffer, 60% of the total payload was released (Figure 3). Plain bacterial cellulose films rapidly released almost the complete payload (90%) after the first hour. The presence of Chi in the BC scaffold reduces the pore diameter and the molecular diffusion of Cip through the films consequently the mean free path decreased as 13

previously reported (Islan et al., 2013). Thereafter, cumulative release from BC-Chi films stayed almost constant ranging between 67.0 ± 0.7% and 74.2 ± 3.5%. These results guarantee a quick drug release at the beginning of the envisioned treatment producing a strong antibacterial effect. Then, a sustained release profile of the antibiotic from the hybrid scaffold could allow the prophylaxis of the treated wound at least during the first 6 h after application. The Cip release kinetics was characterized by fitting the experimental data with the standard release mathematical models. According to the r2 values in Table 3, the best fit for Cip release from plain BC films was with the first-order model, which states that drug release depends on its concentration inside the polymeric matrix. First-order release kinetics is related to the release of water-soluble molecules from insoluble matrices. On the other hand, the best fit for Cip release from BC-Chi hybrid films was the Korsmeyer–Peppas model, used when more than one type of mechanism is involved. This system exhibited an n value smaller than 0.43, indicating that the release rate was also significantly dependent on the rate of Cip diffusion (Fickian diffusion) through the cross-linked polymeric network.

3.4. Antimicrobial activity The antibacterial activity of BC-Chi and BC-Chi-Cip films was tested against Pseudomonas aeruginosa and Staphylococcus aureus, which are potential microorganisms causing wound infections (Serra et al., 2015). A good inhibitory effect was observed in both films. The presence of chitosan in BC matrix caused a local antibacterial effect following the film circumference (Figure 4S, Supplementary material). The inhibition halo was bigger in the case of P. aeruginosa, from 1.9 mm to 1.2 mm, in comparison with the halo observed in S. aureus. The local antimicrobial effect can be explained because of chitosan cross-linking and its limited availability to leave the polymeric network. After Cip incorporation, the antibacterial effect of the BC-Chi film increased synergically. The diameter of inhibition halos was more than three times bigger for both microorganisms, ranging between 4.3 and 5.5 mm. Additionally, a strong inhibition effect against Ps. aeruginosa was also observed when Cip was present (Figure 4S, Supplementary material). The time dependence of microbial cell viability was established with the Live/Dead® BacLightÔ kit (Figure 4). Untreated bacteria were able to produce a biofilm after 24 h of incubation, and a high number of living bacteria appeared in green for both P. aeruginosa and S. aureus. By carefully covering the biofilm surface with BC-Chi-Cip film, red zones began to appear after a 10-min incubation period due to the presence of damaged cells. The total amount of bacteria composing 14

the biofilm was killed one hour later. Slight cell damage was noticed after incubation with BC-Chi for 1 h. The results agree with those of the inhibition assays, where the strongest antibacterial effect was observed after Cip incorporation to the films. Similar results were found with levofloxacin, another antibiotic belonging to the fluoroquinolone family (Islan et al., 2017).

3.5. Cytotoxicity studies The compounds that can be toxic to bacterial cells could also be toxic to the skin cells themselves and delay healing. In this sense, the in vitro biological properties of BC and BC-Chi-Cip films were assessed by determining their cytotoxicity. The results obtained from incubation in human fibroblast cells (GM07492) are shown in Figure 5S (Supplementary material). Cell viability was found to be 95.0% for the BC group and 84.2% for the BC-Chi-Cip group after 24-h incubation. In terms of cell viability, no significant differences were found between the negative control and BC group (p≥ 0.05). These results showed the absence of toxicity from the extract of the BC membranes analyzed. Cip loaded BC-Chi samples exhibited a significant but slight decrease in the metabolic activity of cells. This effect can be classified as moderate cytotoxicity with comparable values to commercial wound dressings (Kempf et al., 2011).

4. Conclusions BC films produced by K. xylinus after 48-h cultures, purified and later sterilized were modified ex situ with Chi. Chi incorporation thickened the BC matrices. SEM images revealed the presence of an interconnected network of BC and Chi biopolymers, forming homogeneous and stable gels. TGA and FTIR studies confirmed a collaborative interaction between Chi and BC, suggesting a possible mechanism of chitosan integration in the BC network. XRD analysis showed a profound structural change in the hybrid scaffold and a strong decrease in the crystalline structure of the BC network due to the presence of Chi. Also, hybrid BC-Chi films exhibited optimum water vapor permeability suggesting a correct balance for humidity control of the wound for optimum healing. The incorporation of ciprofloxacin to hybrid BC-Chi films enabled a sustained release of the antibiotic for more than 6 h. The presence of Chi in the film allowed a controlled release of the antibiotic at a slower rate than with plain BC. The antimicrobial activity of BC-Chi films with and without Cip was also assayed. BC-Chi exhibited local and peripheral inhibition halos. Meanwhile, the presence of the antibiotic effectively produced a stronger inhibition effect on both tested bacteria. Also, hybrid BCChi membranes were able to destroy biofilms of common pathogen microorganisms that may cause 15

wound infections, such as P. aeruginosa and S. aureus strains, in about 1 h. A stronger antimicrobial effect was also observed when Cip was incorporated to the BC-Chi film. Finally, the BC-Chi scaffold was incubated with fibroblasts and no relevant toxicity was observed. The integration between bacterial cellulose and chitosan proved to be successful, generating an excellent composite for wound dressing applications. The antimicrobial activity of chitosan to prevent wound infections was enhanced by the presence of the antibiotic ciprofloxacin. A sustained release of the drug during the initial period of the treatment may contribute to pathogen eradication. Depletion of film payload after this period contributes to the avoidance of undesirable side effects caused by excess drug exposure. Then, wound prophylaxis could be guaranteed by chitosan, and appropriate wound healing would take place. Finally, the BC-Chi system displayed dual antimicrobial capacity with excellent structural properties for wound healing applications.

Acknowledgements The present work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP 0498), Universidad Nacional de La Plata (Grants X701 and I159) and Agencia Nacional de Promoción Científica y Técnica (ANPCyT, PICT2016-4597) of Argentina.

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20

Transdermal delivery

BC-Chi patch Cip Cip Cip p

Cip p

Cip ip p

Cip Cip p Cip

Cip

Cip Ci ip

Highlights

·

Bacterial cellulose films dipped into chitosan solution displayed deep structural modifications revealed by spectroscopic and thermogravimetric analyses.

·

Incorporation of chitosan to BC matrix generated a thickening scaffold with high permeability to water vapor

·

Ciprofloxacin loaded onto the BC-Chi film showed enhanced antimicrobial activity.

·

Presence of Chi in BC films allowed to get a controlled release of Cip.

21

Figures

Figure 1. SEM images of BC (A) and BC-Chi (D) surfaces, and BC (B and C) and BC-Chi (E and F) cross sections at 1,000x and 2,500x magnifications, respectively.

A

B

22

C

D

E

23

F

24

Figure 2a Thermal degradation analysis for BC, Chitosan and BC-Chi. Main graph corresponding to DTGA curves and secondary graph to TGA curves.

1.5 0

––––––– BC-Chi ––––––– Chitosan ––––––– BC Weight Change (%)

-20

Deriv. Weight (%/°C)

1.0

-40

-60

-80

-100 0

200

400

Temperature (°C)

600

800 Universal V4.5A TA Instruments

0.5

0.0 0

200

400

Temperature (°C)

600

800 Universal V4.5A TA Instruments

25

Figure 2b. FTIR spectra corresponding to BC, Chi and BC-Chi films.

26

Figure 2c. XRD spectra for BC and BC-Chi films.

27

Figure 3. Ciprofloxacin release from BC (○) and BC-Chi (●) films in 190 mM acetate buffer (pH 5.5). Experimental data was fitted to First order model for BC films (blue line) and Korsmeyer–Peppas model for BC-Chi films (red line), according to the highest correlation coefficient (r2) values.

100

Cip released (%)

80

60

40

20

0 0

1

2

3

4

5

6

Time (hours)

28

Figure 4. Pseudomonas aeruginosa and Staphylococcus aureus biofilms dyed with the Live/Dead® BacLightÔ kit and observed with an epifluorescent microscope at 400x. Overlay images of untreated biofilm and biofilm after treatment with BC-Chi and BC-Chi-Cip.

Staphylococcus aureus

BC/Chi/Cip 60 min

BC/Chi 60 min

BC/Chi/Cip 10 min

Control Live

Pseudomonas aeruginosa

29

TABLES Table 1. Relevant values from thermal degradation analysis of BC, chitosan and BC-Chi films.

Films Parameter

BC

Chitosan

BC-Chi

Mass loss in 30-150°C range (%)

6.6

9.1

20.8

Mass loss in 150-400°C range (%)

66.5

49.3

64.7

Tp (°C)

330.7

299.6

197.5

Residue at 800°C

16.4

28.9

12.7

Table 2 Thickness, area and WVTR values of BC and BC-Chi films. Studies were performed with five samples for each group.

Thickness

Area*

(mm)

(mm2)

Sample

Ɍ WVTR (g/m2h)

(g mm/m2 h)

BC

0.050 ± 0.010

297.08 ± 15.27

14.38 ± 0.46

0.714 ± 0.175

BC-Chi

0.175 ± 0.037

530.93 ± 0.05

12.34 ± 0.10

3.287 ± 0.436

30

Table 3. Mathematical models and respective parameters (correlation coefficients and release constants) obtained from the fitting of the experimental data corresponding to a Cip release from BC and BCChi films. r2: correlation coefficient; k1: first-order release constant; kH: Higuchi constant; kKP: Korsmeyer–Peppas constant; n: release mechanism exponent.

Mathematical models Samples

First order

Higuchi

Korsmeyer - Peppas

r2

k1(h-1)

r2

kH(h-0.5)

r2

kKP(hn)

n

BC

0.98

1.305

0.78

51.745

0.87

65.255

0.313

BC-Chi

0.66

0.504

0.71

38.460

0.94

50.063

0.237

31