Modification of Bacterial Cellulose with Quaternary Ammonium

Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES

Article

Modification of bacterial cellulose with quaternary ammonium compounds based on fatty acids and amino acids and the effect on antimicrobial activity Anna Zywicka, Karol Fija#kowski, Adam F. Junka, Jakub Grzesiak, and Miroslawa El Fray Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00183 • Publication Date (Web): 26 Mar 2018 Downloaded from http://pubs.acs.org on March 27, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Modification of bacterial cellulose with quaternary ammonium compounds based on fatty acids and amino acids and the effect on antimicrobial activity

Anna Żywicka†, Karol Fijałkowski*,†, Adam F. Junka§,ȥ, Jakub Grzesiak£, Miroslawa El Fray*,¶

†

West Pomeranian University of Technology, Szczecin, Faculty of Biotechnology and Animal

Husbandry, Department of Immunology, Microbiology and Physiological Chemistry, Piastów 45, Szczecin, Poland §

Wrocław Medical University, Department of Pharmaceutical Microbiology and Parasitology,

Borowska 211A, Wrocław, Poland ȥ

Wrocław Research Centre EIT+, Laboratory of Microbiology, Stablowicka 147, Wrocław,

Poland £

Wrocław Research Centre EIT+, Laboratory of Electron Microscopy BIO, Stablowicka 147,

Wrocław, Poland ¶

West Pomeranian University of Technology, Szczecin, Faculty of Chemical Technology and

Engineering, Division of Functional Materials and Biomaterials, Al. Piastów 45, Szczecin, Poland

1 ACS Paragon Plus Environment

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: In the present work, bacterial cellulose (BC) membranes have been modified with bioactive compounds based on long chain dimer of C18 linoleic acid, referred to as the dilinoleic acid (DLA) and tyrosine (Tyr), a natural amino acid capable of forming noncovalent cation-π interactions with positively charged ethylene diamine (EDA). This new compound, [EDA][DLA-Tyr] has been synthesized by simple coupling reaction and its chemical structure was characterized by 1H NMR and FT-IR spectroscopy. The antimicrobial activity of a new compound against S. aureus and S. epidermidis, two cocci associated with skin and wound infections was assessed. The [EDA][DLA-Tyr] impregnated BC exhibited strong and long-term antimicrobial activity against both staphylococcal species. The results showed a 57 - 66% and 56 - 60% reduction in S. aureus and S. epidermidis viability, respectively, depending on [EDA][DLA-Tyr] concentration used. Importantly, [EDA][DLATyr] molecules were released gradually from the BC pellicle while a reference antibiotic, erythromycine (ER) did not show any antibacterial activity against S. aureus and S. epidermidis after 48 h of soaking in deionized water. Thus, a combination of [EDA][DLATyr] and BC could be a promising new class of wound dressing displaying both biocompatibility and antimicrobial activity.

KEYWORDS: bacterial cellulose; Komagataeibacter xylinus; modification; antibacterial activity; dimer fatty acid


Page 2 of 33

Page 3 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

INTRODUCTION The cellulose produced by certain species of bacteria (bacterial cellulose, BC) is considered an ideal wound dressing fulfilling the requirements of modern wound dressing materials, used for chronic traumas and extensive burns,1 presented in the so-called T.I.M.E. approach developed by the European Wound Management Association (EWMA). The acronym stands for Tissue (T), Infection (I), Moisture (M) and Epithelialization (E). Unlike past recommendations which specifically focused on infection prophylaxis, the present guidelines are addressing the maintaining of an appropriate wound moisture to enhance epithelialization and tissue growth and to promote wound healing.2 BC displays a set of features making it an ideal material for the management and care of chronic wounds regardless of their etiology. Bacterial cellulose demonstrates high tensile strength, flexibility and water holding capacity, a pronounced permeability to gases and liquids, and a great compatibility with living tissues.3 Taking into account the long-term use of dressings, it is important that BC is nontoxic and does not cause allergic reactions that might occur when dressings of xenogeneic origin are applied on a human body.4 The unique nano-morphology of BC results in a large surface area that can absorb a high amount of water and water-based solution, such as wound exudate. The high degree of hydration of BC accelerates wound healing by up to 40%.1 Moreover, BC dressings may be pre-formed to decrease the pain associated with healing; for example, they can be saturated with a cold, sterile water bringing relief to burn victims.5 Furthermore, BC dressings provide gas exchange with the environment and prevent secondary infection. Additionally, BC dressings are comfortably changed, because they do not adhere to



the skin3 and their fibrils do not remain in the wound as it happens in the case of filamentous dressings made of cotton.6 Despite many advantages, BC itself does not have antibacterial properties which is crucial to prevent wound infection during wound healing (“I” component of the T.I.M.E. approach). In order to equip a BC dressing with antimicrobial or healing properties, the polymer should be impregnated with drugs and/or agents accelerating angiogenesis.7,8 It was confirmed that BC can be effectively impregnated with antibacterial substances; several approaches using nanosilver,7 benzalkonium chloride,8 silver sulfadiazine,9 have already been described. BC's nano-porous structure allows the potential transfer of antibiotics or other medicinal products into the wound, while at the same time it serves as an efficient physical barrier against any external infection.3 Local application of antibiotics in wound treatment is presently prohibited due to the possible emergence of antimicrobial drug resistance. The use of antiseptics, due to their unspecific mechanism of action, does not lead to resistance acquisition. However, reports on isolation of chlorhexidine-resistant strains become more and more frequent.10,11 Quaternary ammonium compounds (QAC) are promising low-molecular weight biocides known of their excellent cell membrane penetration properties, low toxicity, good environmental stability, lack of skin irritation, and extended residence time and biological activity.12 QACs commonly possess both a positive charge and a hydrophobic segment. The antibacterial activity of QACs is strongly dependent on the overall molecular structure and chain length of the alkyl chain. An increase in the amphiphilic chain of the QAC’s alkyl structure increases the antibacterial activity of the compound against both Gram-negative and Gram-positive bacteria.13 Also, long-chain unsaturated fatty acids, such as oleic acid, linoleic acid, linolenic acid14 and their derivatives15 are gaining increased interest as promising antimicrobial agents


Page 4 of 33

Page 5 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

of natural origin. These natural substances can be used as potential therapeutics and antibacterial agents thanks to their potency and broad spectrum of activity.16 Unsaturated fatty acids exhibit a broad spectrum of antimicrobial activity against enveloped viruses, fungi and various bacteria including Neisseria gonorrhoeae,17 Helicobacter pylori,14 group B streptococci (GBS), group A streptococci (GAS) and also against methicillin-resistant Staphylococcus aureus.18 In the present work, the combination of a long-chain unsaturated fatty acid, dimer of C18 linoleic acid, referred to as dilinoleic acid (DLA) and tyrosine (Tyr), a natural amino acid capable of forming noncovalent cation-π interactions19 with positively charged ethylene diamine (EDA), was synthesized and used as an antimicrobial agent for the impregnation of bacterial cellulose membrane. The aim of the current study was to evaluate the antibacterial activity of bacterial cellulose impregnated with [EDA][DLA-Tyr] compound against S. aureus and S. epidermidis, the two cocci associated with skin and wound infections.

MATERIALS AND METHODS Preparation of quaternary ammonium compound of [EDA][DLA-Tyr]. The quaternary ammonium compound was synthesized by heating dilinoleic acid (DLA) (Pripol 1009, molecular weight ~570 g·mol−1, Croda, The Netherlands) and ethylenediamine (EDA) (Sigma Aldrich, Poznan) at 55 - 60°C in 1:1 mol ratio. The resulting waxy material was dissolved in distilled water and buffered to pH-12 with 0.1N KOH. Then, 1 eq of tyrosine (Sigma Aldrich, Poznan) was added and mixed for 30 min. The resulting product was washed with diethyl ether twice and lyophilized. The reaction yield was 65%. The compound structure was characterized with infrared spectroscopy (Thermo Nicolet NEXUS spectrometer, scanning between 500 and 4000 cm−1, resolution 4 cm−1, 32 scans) and 1H NMR (the spectra were obtained using the Bruker DPX 400 Hz, with CDCl3 as solvent; all



shifts were determined with reference to tetramethylsilane, TMS).

Preparation of BC membranes. For BC production, the reference strain of Komagataeibacter xylinus (Deutsche Sammlung von Mikroorganismen und Zellkulturen DSM 46602) was used. Initially, K. xylinus was cultivated in stationary conditions for 7 days at 28°C, using a Herstin-Schramm (H-S) medium.20 The 7 day cultures were shaken to remove the bacteria from the cellulose and used to inoculate the H-S medium. Stationary culture of K. xylinus was established in a 24-well culture plate (Becton Dickinson and Company, USA) at 28°C in order to obtain regular-shape BC membranes of the same diameter. The BC pellicles were harvested from the medium after 7 days of cultivation, purified by means of alkalyic lisis, ie. application of 0.1 M NaOH at 80°C for 90 min, washed with double-distilled water until the pH stabilizes, dried at 60°C overnight and sterilized at 121°C, 100 Pa for 15 min. The averaged diameter of BC pellicles used for impregnation was 1.5 cm. The average weight of a BC pellicle was approximately 0.90 g and 0.008 g for wet and dry BC, respectively.

Scanning Electron Microscopy analysis. The microstructure of the BC membrane was observed using Scanning Electron Microscope (SEM). Initially, the NaOH purified BC was fixed in glutaraldehyde (POCH, Gliwice) for 7 days and dried in critical point dryer, Leica. Subsequently, the cellulose was subjected to the sputtering with Au/Pd(60:40) using EM ACE600, Leicasputter. The sputtered samples were examined using scanning electron microscope (Auriga 60, Zeiss, Oberkochen, Germany). The cellulose’s microfibril diameters were analyzed using software integrated with Auriga 60 Scanning Electron Microscope, while pore size was analyzed using ImageJ software (NIH).


Page 6 of 33

Page 7 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Porosity, pore size and surface area analysis. The N2, adsorption/desorption measurements were used to investigate the porosity of BC. The N2 adsorption/desorption isotherms at 77 K was measured using a Micromeritics ASAP 2010 M instrument (Micromeritics, U.S.A), and the specific surface area was calculated by the BET (BrunauerEmmett-Teller) method. The pore volume and pore diameter were calculated by the BJH (Barrett-Joyner-Halenda) method.

Determination of water related properties of BC. The water retention value (WRV), was determined based on the weight loss of the BC samples during incubation at 37°C at different time intervals until a constant weight of the dry sample was achieved. The weights were taken in triplicate. The WRV values21 were calculated from Equation (1): %WRV =

( )

∗ 100%

(1)

where, Wdwet is the weight of the BC during drying and Wdry is the weight of the dry sample.

To examine the swelling properties, the dried BC samples were immersed in distilled water and weighed every 1 min until a constant weight of the wet sample was achieved. The weights were taken in triplicate. The results are expressed as a swelling ratio (SR),22 calculated from Equation (2): %SR =

( )

∗ 100%

(2)

where, Wwet is the weight of the BC during swelling and Wdry is the weight of the dry sample.

Mechanical strength. The tensile strength test was conducted using Instron - 3366 Dual Column machine (Instron, Massachusetts, USA). The BC samples were cut to make 30 × 10 mm (length × width) bars and the thickness was measured prior to loading the samples



for testing. The tests were carried out at room temperature, at a speed of 1 mm/min. All measurements were performed in six repeats.

The total crystallinity index. Fourier transform infrared (ATR-FTIR) spectra of BC were obtained using ALPHA FT-IR Spectrometer (Burker, Germany) equipped with a DTGS detector and the platinum-ATR-sampling module with the robust diamond crystal and variable angle incidence of the beam. For each sample 32 scans of the resolution of 2 cm−1 were recorded within the spectral range of 4000 - 400 cm−1. Omnic Software was applied to collect and process the spectra. The total crystallinity index was computed with use of the absorbance value ratio for peaks 1430/900 I.C. I and 1370/2900 I.C. II.

Impregnation of BC membrane. In the first step, the antimicrobial activity and minimum inhibitory concentration (MIC) of [EDA][DLA-Tyr] and ER which constituted a positive control was determined by broth microdilution test. [EDA][DLA-Tyr] and ER dilutions in Mueller–Hinton broth (Oxoid) in the range from 10 - 50 mg/L and 0.1 - 2 mg/mL respectively were prepared and dispensed in the volume of 0.1 mL into each of the 96 wells of a microtitre plate. The standardized inoculum was added to give a final concentration of 5×105 CFU/mL. After incubation at 37°C for 18 h in ambient air, the MIC was recorded as the lowest concentration of antimicrobial inhibiting growth as recorded by OD readings at 600 nm. In the second step, each BC pellicles were immersed in 5 mL of [EDA][DLA-Tyr] or ER solution at several concentrations selected based on the results from the analyses of antimicrobial activity, for 24 h at room temperature.8 Next, the BC pellicles were removed from the [EDA][DLA-Tyr] or ER, immersed in distilled water (for 10 s) and wiped with filter


Page 8 of 33

Page 9 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

paper to remove non-absorbed [EDA][DLA-Tyr] or ER. As a negative control, pure cellulose without immobilized [EDA][DLA-Tyr] or ER was used.

Microorganisms. The antimicrobial activity of impregnated BC was tested against S. aureus (American Type Culture Collection - ATCC 6538) and S. epidermidis (ATCC 35984). Prior to the experiment, the bacteria were plated onto the BHIA (brain heart infusion agar, Oxoid, UK) and cultivated overnight at 37°C. After the incubation, one colony forming unit (CFU) of each microorganism was transferred to 10 mL of BHI and incubated overnight in the same culture conditions while shaking.

Agar diffusion disc method. The BC pellicles impregnated with [EDA][DLA-Tyr] or ER were placed onto the surface of the Mueller-Hinton (M-H) agar medium (BioMaxima, Poland) seeded with the suspension of S. aureus and S. epidermidis at a density of 1.5 x 108 CFU/mL. Then, the cultures were carried out at 37°C for 24 h. The average diameters of the inhibition zone (in mm) were calculated for each tested sample. The tests were performed in triplicate.

MTT reduction cell viability assay. The microorganism suspensions were diluted to obtain a concentration of 5×105 CFU/mL. Next, 250 µL of the microorganism suspension was transferred to 25 mL BHI broth medium in 50 mL plastic tubes (Polypropylene Conical Centrifuge Tube, Becton Dickinson and Company, USA) containing one pellicle of impregnated BC. The cultures were incubated at 37°C under agitation (120 rpm). After 24 h, 100 µL of the bacterial suspension was transferred to a 96-well plate (Becton Dickinson and Co., USA) and simultaneously 10 µL of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (3mg/mL in PBS, Sigma-Aldrich, Poznan) was added into each well.



Page 10 of 33

The plate was incubated at 37°C for 30 min. In the next step, 100 µL of isopropanol was added to each well, and the plates were vigorously shaken for 15 min. The amount of MTT formazan formed during the incubation was measured at a wavelength of 540 nm and reference wavelength of 630 nm using microplate reader (Infinite 200 PRO NanoQuant, Tecan, Switzerland). The experiment was conducted in technical triplicates and repeated three times (three biological replicates). The results are shown as % of living cells in the presence of BC impregnated with [EDA][DLA-Tyr] and ER, compared to pure cellulose (negative control) calculated by the formula: % viable bacteria =

( !"# $" ) ( %# ")

∗ 100%

(3)

where, IBC – is the BC impregnated with [EDA][DLA-Tyr] or ER; NC – is pure cellulose (negative control); B is the blank (BHI medium).

Release rate analysis. Pellicles of impregnated BC were incubated at 37°C for 24 h and 48 h in sealed beakers containing 10 mL of deionized water. After incubation, each BC pellicle was transfer to 50 mL plastic tubes containing 25 mL of BHI broth medium inoculated with 1% of cell suspension prepared as indicated in the “Microbial viability assay” section. Cell viability was determined after 24 h of incubation at 37°C using the MTT assay. The experiment was conducted in technical triplicates and repeated three times (three biological replicates).

Statistical analysis. The statistical significance of the differences between living cells in the presence of [EDA][DLA-Tyr]- and ER-impregnated BC were determined by a one-way analysis of variance (ANOVA) and followed by Tukey-Kramer multiple comparison test for post-hoc comparisons. The experiment was conducted in technical triplicates and repeated four times. The differences were considered statistically significant when the P-value was less 10 ACS Paragon Plus Environment

Page 11 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

than 0.05. The statistical analyses were conducted using Statistica®10 PL (StatSoft, Inc. 2016, Poland).

RESULTS AND DISCUSSION Compounds. The chemical structure of the new compounds was verified with 1H NMR and IR spectroscopy. As can be seen from 1H NMR spectrum (Figure 1), the following shifts were identified: 9.28 ppm (s, Ar-OH), 6.96 - 6.60 ppm (m, Ar-H), 4.69 ppm (m, D2O), 3.43 ppm (dd, –CH– from Tyr), 3.12 - 2.90 ppm (s, –CH2–NH3+ from EDA), 2.75 ppm (ddd, –CH2– from Tyr), 2.03 ppm (–CH2– group in α position to carbonyl bond), 1.41 ppm (–CH2– group in β position to carbonyl bond), 1.17 ppm (strong, characteristic signal from long chain of linoleic acid), 0.76 ppm (terminal groups in DLA).



Figure 1. 1H NMR spectrum of [EDA][DLA-Tyr] and signal assignments.

Functional groups of the new compound were also verified with IR spectroscopy (Figure 2). The analysis of DLA-EDA spectrogram shows an intense band from stretching vibrations of methylene groups (2920 - 2850 cm-1) and an absence of stretching vibrations from C=O group in DLA, and intense band from the overlap of bending vibrations of ammonium ion NH3+ and asymmetric stretching vibrations from carboxylic ion COO- (1640 1525 cm-1). Asymmetric bending vibrations of the methyl group were also found at 1455 cm1

. After modification of ammonium salt with Tyr, a new ionic compound was created as

confirmed by 3201 cm-1 band characteristics for stretching vibrations of aromatic C-H bonds and aromatic C-C bonds at 1410 cm-1. An intense, narrow band ascribed to bending vibrations from C-O carboxylic group and bending vibrations from -NH2 group was found at 1561 cm-1. The band at 1243 cm-1 was ascribed to the stretching vibrations of C-O carboxylic group.


Page 12 of 33

Page 13 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 2. The FT IR spectra of initial product (DLA-EDA) and final product [EDA][DLATyr].

Microstructure of BC. In terms of structure, BC composes of nanofibrils and these nanofibrils subsequently crystallize into microfibrils forming a three-dimensional network structure that resembles a sponge.23 Such a BC matrix displays hierarchical structure of pores since it consists of macropores (>100 µm), micropores (

Modification of Bacterial Cellulose with Quaternary Ammonium

Recommend Documents