Contents lists available at ScienceDirect [608920]

Licență

Contents lists available at ScienceDirect [608920]

Byadmin ianuarie 1, 2024

Contents lists available at ScienceDirect
Polymer Testing
journal homepage: www.elsevier.com/locate/polytest
Antibacterial properties of ﬁlms of cellulose composites with silver
nanoparticles and antibiotics
Tatiana G. Volovaa,b, Anna A. Shumilovaa,∗, Ivan P. Shidlovskiya, Elena D. Nikolaevab,
Alexey G. Sukovatiyb, Alexander D. Vasilieva,c, Ekaterina I. Shishatskayaa,b
aSiberian Federal University, 79 Svobodnyi Av., Krasnoyarsk 660041, Russia
bInstitute of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS ”, 50/50 Akademgorodok, Krasnoyarsk 660036, Russia
cKirensky Institute of Physics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS ”, 43/50 Akademgorodok, Krasnoyarsk 660036, Russia
ARTICLE INFO
Keywords:
Bacterial cellulose
CompositesSilver nanoparticlesAntibioticsPropertiesAntibacterial activityABSTRACT
The present study describes production of bacterial cellulose composites with silver nanoparticles and antibiotics
and compares their properties. Bacterial cellulose (BC) composites synthesized in the culture of the strain ofacetic acid bacterium Komagataeibacter xylinus VKPM B-12068 with silver nanoparticles, BC/AgNps, were pro-
duced hydrothermally, under di ﬀerent AgNO
3concentrations (0.0001, 0.001, and 0.01 M) in the reaction
medium. The presence of silver in the BC/AgNp composites was con ﬁrmed by elemental analysis conducted
using scanning electron microscopy with a system of X-ray spectral analysis. Analysis showed that the average
atomic number of silver particles in composite samples depended on the concentration of AgNO 3: as AgNO 3
concentration in the reaction solution was increased, silver content in the composites increased from 0.044 to
0.37 mg/cm2. BC composites with amikacin and ceftriaxone were prepared by immersing dry BC ﬁlms in so-
lutions containing di ﬀerent concentrations of the antibiotics. The surface structure and properties and physi-
cochemical and mechanical characteristics of composites were investigated using SEM, DSC, X-ray analysis, thesystem for measuring water contact angles, and electromechanical tensile testing machine. The disk-di ﬀusion
method and the shake- ﬂask culture method used in this study showed that all experimental composites had
pronounced antibacterial activity against E. coli, Ps. eruginosa, K. pneumoniae, and St. aureus, and the BC/anti-
biotic composites were more active than BC/AgNp ones; S. aureus was the most susceptible to the eﬀ ect of BC
composites. No potential cytotoxicity was detected in any of the BC/AgNp composites in the NIH 3T3 mouse
ﬁbroblast cell culture, in contrast to the BC/antibiotic composites. These results suggest that BC composites
constructed in the present study hold promise as dressings for managing wounds, including contaminated ones.
1. Introduction
Bacterial cellulose (BC) –a biopolymer synthesized by micro-
organisms –is a promising material for biomedical application. The
chemical structure of BC is similar to that of plant-derived cellulose, but
BC has unique physical, mechanical, and chemical properties. This
material shows high biocompatibility, without being cytotoxic or
causing any allergic reactions. Studies of BC suggest that this natural
polymer can be useful for cellular and tissue engineering as material for
constructing sca ﬀolds and for reconstructive surgery as material for
skin defect reconstruction and as a matrix for drug delivery [1,2] .
Physical and mechanical properties of BC can be enhanced by preparing
BC composites with various materials: chitosan [3], collagen [4], so-
dium alginate, gelatin, polyethylene glycol [5].
BC is not inherently antibacterial, but BC composites with chitosanand alginate inhibit growth of pathogenic microorganisms such as
E.coli, Candida albicans, and Staphylococcus aureus [3,6,7] . Therefore,
BC composite ﬁlms can be considered for treating contaminated
wounds. BS can be hybridized with metallic silver particles to produce
an antibacterial and wound-healing formulation. Metallic silver and
compounds thereof have a strong bactericidal e ﬀect, inhibiting devel-
opment of a wide range of pathogenic microorganisms. Silver ions reactwith cell membrane protein thiol groups, a ﬀecting bacterial respiration
and transport of substances through the cell membrane [8].
Production of BC composites with silver nanoparticles has been
extensively discussed in the literature. Various approaches have beenproposed of the in situ generation of Ag or Cu and other metals in
cellulose matrix and cotton fabrics, by using di ﬀerent reducing agents
and by hydrothermal methods, which are simpler to use and eco-friendly.
https://doi.org/10.1016/j.polymertesting.2017.10.023
Received 7 August 2017; Received in revised form 10 October 2017; Accepted 28 October 2017∗Corresponding author.
E-mail address: shumilova.ann@mail.ru (A.A. Shumilova).Polymer Testing 65 (2018) 54–68
0142-9418/ © 2017 Elsevier Ltd. All rights reserved.
MARK

Composites of silver nanoparticles and bacterial cellulose with high
antimicrobial activity were prepared using Tollens' reaction [9]with
silver reduced by polydopamine, which had been used as a medium for
immersion of homogenized cellulose [10]. Other authors [11] used a
more complex approach to producing silver nanoparticles: homo-genized cellulose was ﬁrst oxidized using 2,2,6,6-tetra-
methylpiperidine-1-oxyl (TEMPO), which created carboxyl groups onthe surface, and then Na
+-Ag+exchange reaction was performed; the
ions were reduced using NaBH 4or sodium citrate. The silver particles
produced by this method were of di ﬀerent sizes and e ﬀective against
Escherichia coli and Staphylococcus aureus .
Diﬀerent modi ﬁcations of hydrothermal methods for production of
nanosilver and other metals involve the use of bacterial cellulose andnatural plant-derived agents as reductants. G. Yang et al. [12] used a
hydrothermal method with cellulose employed as a reducing and sta-bilizing agent. By optimizing reaction parameters, the authors obtained
narrow distribution of Ag particles, thus achieving an e ﬀective and
durable antibacterial action. Composites of cellulose with silver sulfa-diazine dispersed to nanosize were used as antimicrobial wound dres-
sing [13], which facilitated the healing of model burns in rats. J. Wu
et al. [14] reported a study in which model burns were treated with
composites prepared by reducing Ag+ on the surface of cellulose ﬁbers.
The composites suppressed wound micro ﬂora and favored ﬁbroblast
attachment. Silver nanoparticles were produced in situ in cotton fabrics
by the hydrothermal method at 80 °C [15]. The composites showed
good antibacterial activity against Gram-negative and Gram-positivebacteria. A similar method was used to prepare a cotton composite with
copper nanoparticles [16]. Cotton nanocomposite fabrics exhibited
good antibacterial activity and were e ﬀective against both Gram-posi-
tive and Gram-negative bacteria. Hence, they can be used in medical
applications: as wound dressings, surgical aprons, materials for hospital
beds, etc. In another study [17], silver nanoparticles were produced in
situ in cellulose matrix using Ocimum leaf extract as a reducing agent.
Composite ﬁlms showed good antibacterial activity and, thus, can be
used for packaging and medical purposes. P. Sivaranjana et al. (2017)
[18] reported producing cellulose nanocomposite ﬁlms with in situ
generated silver nanoparticles using Cassia alata leaf extract as a re-
ducing agent. The nanocomposite cellulose/AgNP ﬁlms had good ten-
sile properties and showed antibacterial activity; therefore, they can beconsidered for medical applications as dressing materials.
A number of studies report successful uses of plant extracts as re-
ducing agents for silver ions. In a study by L. Muthulakshmi et al. [19],
copper nanoparticles (CuNPs) were generated in situ inside cellulosematrix using Terminalia catappa leaf extract as a reducing agent, and
their properties were investigated. The composite ﬁlms possessed suf-
ﬁcient tensile strength, and, thus, they can replace polymer packaging
materials like polyethylene. Further, the cellulose/CuNP compositeﬁlms exhibited good antibacterial activity against E.coli .
In another work [20], cellulose gel ﬁlms with nanosilver were
produced using a precooled mixture of 8 wt% lithium hydroxide and
15 wt% urea as a solvent and ethyl alcohol as a nonsolvent, with the
Terminus cattapa leaf
extract used as a reducing agent. Composite cel-
lulose/AgNP ﬁlms showed good antibacterial activity against E. coli and
Bacillus sp. Hydrothermal synthesis was used to produce nanoparticles
of other metals too. For instance, polyhedron-shaped hematite (a-
Fe2O3) nanoparticles were successfully synthesized via a facile hy-
drothermal method by mixing FeCl3 and NH 4OH at high temperature
[21].
Besides BC, recent publications demonstrated that cellulose nano-
crystals and hydrogels could also be used as reductant and support forthe synthesis of AgNPs. For example, Rui Xiong et al. [22] demon-
strated a facile and environmentally friendly approach to prepareFe
3O4/Ag/nano ﬁbrillated cellulose (NFC) nanocomposites, which en-
ables tunability from highly porous, ﬂexible aerogels to solid and stiﬀ
ﬁlms. NFC acts as a biocompatible support for the magnetic silver na-
noparticles and a reducing agent for the silver ions. The Fe 3O4/Ag/NFCnanocomposite aerogel exhibited excellent catalytic properties for thereduction of 4-nitrophenol, and showed high antibacterial activity
against the model microbe S. aureus .
Several studies showed advantages of using cellulose crystals as a
reductant for producing nanosilver. Rui Xiong et al. [23] described
production of well dispersed and stable silver nanostructures usingcellulose nanocrystals without employing other reductants or disper-
sing agents. Moreover, the authors showed that morphology of silver
nanostructures could be adjusted by changing AgNO
3concentration
and producing silver shaped as nanospheres or dendrites with di ﬀerent
antibacterial activity (more pronounced in dendritic silver) against
Escherichia coli and Staphylococcus aureus . Successful use of cellulose
crystals for producing hybrid silver/biodegradable polymer nano-composites as potential food packaging was reported by Yu et al. The
authors produced nanocomposites consisting of biodegradable nano-
hydrates poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) and
cellulose/silver nanocrystals (CNC-Ag) with improved thermal, me-chanical, and antibacterial properties [24]. The authors found that
homogeneously dispersed CNC-Ag could serve as a factor improving theproperties of the pristine polymer. In another study [25], the authors
described fabrication of multifunctional cellulose nanocrystals/poly(lactic acid) nanocomposites with silver nanoparticles by spraying
method. Deposited silver (Ag) nanoparticles and CNF e ﬀectively re-
inforced mechanical properties and antibacterial activity of polylactideas well as water vapor permeability of the composite ﬁlm, which was
important for the packaging material. In a study by W. Xu et al. [26],
cellulose nanocrystals (CNCs) produced by hydrolysis of sulfuric acid
were dispersed in polycarbonate (PC) in organic solution to prepare a
composite. Due to the good dispersion of nano ﬁllers in polymeric ma-
trix, strong hydrogen bonds were formed between carbonyl groups ofpolycarbonate and hydroxyl groups of cellulose, which caused im-
provement of thermal and mechanical properties of the composite
ﬁlms.
A novel and straightforward synthetic strategy was developed to
prepare silver nanoparticles-doped cellulose microgels (AgNPs/CMG)nanohybrids at room temperature [27]. The as-prepared AgNPs/CMG
nanohybrids exhibited excellent catalytic performance in reduction of4-nitrophenol and organic dyes. The simplicity, sustainability, and
straightforwardness of this approach to prepare a highly e ﬃcient cat-
alyst and functional membrane open up new possibilities for large-scaleproduction and application of bioresources/noble metal nanohybrids in
various ﬁelds.
Another approach to imparting antibacterial activity against pa-
thogenic micro ﬂora to BC is to prepare BC composites with antibiotics.
As a potential wound dressing for treating acute traumas, freeze-driedBCﬁlm was loaded with benzalkonium chloride –an antimicrobial
agent of cationic surfactant type. Antimicrobial activity of the compo-site was observed for 24 h against Staphylococcus aureus and Bacillus
subtilis , which were the major bacteria in the contaminated wound [28].
Composites of bacterial cellulose with tetracycline hydrochloride, BC-
TCH, were produced and characterized by other authors [29,30] . The
composites exhibited excellent antibacterial activity and good bio-compatibility and enabled controlled release of the antibiotic.
Analysis of the literature suggests that production of nanocompo-
sites based on bacterial cellulose is a promising and important subject
of research. However, most of the studies describe production of BC
composites with nanosilver and characterize them as dependent on the
technique of production and ratios of components in the composites.
Much less consideration has been given to the properties of BC com-
posites with antibiotics.
Therefore, the purpose of this study was to prepare bacterial cel-
lulose composites with silver nanoparticles and antibiotics and to
compare their properties and antibacterial activity.T.G. Volova et al. Polymer Testing 65 (2018) 54–68
55

2. Experimental
2.1. Materials
Bacterial cellulose ﬁlms were synthesized in the Komagataei bac-
terxylinus B-12068 culture. The strain was isolated from the fermented
tea (kombucha) Medusomyces gisevii J. Lindauon Hestrin-Schramm (HS)
medium [31]. The strain was identi ﬁed based on its morphological,
biochemical, genetic, and growth parameters. The strain Komagataei-
bacter xylinus was deposited in the Russian National Collection of In-
dustrial Microorganisms (VKPM) with registration number VKPM B-
12068 and patented [32] The collection culture of K. xylinus B-12068
was maintained on the Hestrin-Schramm (HS) agar medium. Thestandard HS medium contained (% w/v): glucose –2, peptone –0.5,
yeast extract –0.5, Na
2HPO 4–0.27, and citric acid –0.115. The pre-
culture was performed on the HS agar. Then, the colonies were trans-
ferred into the ﬂask containing liquid HS medium and cultivated for 7
days at a temperature of 30 °C under static conditions as describedelsewhere [33]. To remove remnants of bacterial cells and components
of culture medium, BC ﬁlms were treated with 1.0 M NaOH at 70 °C,
followed by washing in deionized water. The synthesized cellulose was
separated from the culture ﬂuid. Then, BC ﬁlms were placed in a 0.5%
solution of hydrochloric acid for 24 h for neutralization and, after-wards, rinsed in distilled water until pH 7. The BC ﬁlms were stored in
sterile solution or air dried until they reached a stable weight.
BC composites with silver nanoparticles and antibiotics were pre-
pared using AgNO
3(Uralkhiminvest, Russia) and ceftriaxone and ami-
kacin (Sintez, Russia).
2.2. Production of BC composites with silver nanoparticles, BC/AgNps
BC composites with silver nanoparticles, BC/AgNps, were produced
by a hydrothermal method without utilizing any catalysts, using the
disks of BC layer as a reducing and stabilizing agent [34]. Puriﬁed raw
BCﬁlms were cut into disks 1 cm in diameter, placed in ﬂasks with
0.0001, 0.001, and 0.01 M of AgNO 3, and heated for 60 min at a
temperature of 90 °C. Composite BC ﬁlms with silver nanoparticles
were lyophilized at a temperature of −40 °C and pressure of 0.12 mbar
for 24 h in a vacuum drying unit ALPHA 1-2/LD (Martin Christ GmbH,Germany) or kept at room temperature in a laminar ﬂow cabinet for
24 h. The parameters of the produced silver nanoparticles were in-vestigated with a Zetasizer Nano ZS particle analyzer (Malvern, U.K.),
employing dynamic light scattering, electrophoresis, and laser Doppler
anemometry.
2.3. Production of BC composites with antibiotics
In order to impart bactericidal properties to cellulose, we prepared
BC composites with antibacterial drugs. We used amikacin (Sintez,
Russia) and ceftriaxone (Sintez, Russia). The dried BC ﬁlms were im-
mersed in solutions of antibiotics of di ﬀerent concentrations. After 24 h
of immersion, the ﬁlms were removed from the antibiotic solutions,
washed in distilled water, and dried at room temperature. Then, theﬁlms were die-cut into disks 1 cm in diameter.
2.4. A study of structure and physical and mechanical properties of BC
composites
The thickness of BC ﬁlms was measured with a LEGIONER EDM-25-
0.001 electronic digital micrometer (Legioner, China); accuracy of
measurement was 1 μm. Surface properties of the BC ﬁlms and mem-
branes were examined using a DSA-25E drop shape analyzer (Krüss.Germany) and software DSA-4 for Windows. Drops of water and diio-
domethane, 1.5 μl each, were alternately placed on the sample surface
with microsyringes, and moments of interaction between each liquidand sample surface were video recorded. Contact angles of these liquidswere measured by processing the frame of a stabilized drop in asemiautomatic mode, by the “Circle ”method, which is embedded in the
software package. The results of measurements were used to calculatesurface free energy and its dispersive and polar components (mN/m) by
the Owens, Wendt, Rabel and Kaelble method [35,36] . A minimum of
six measurements were taken for each surface; means and standarddeviations were calculated.
The surface microstructure of the BC ﬁlms was analyzed using
scanning electron microscopy (S 5500, Hitachi, Japan). Prior to the
analysis, the ﬁlms were freeze-dried in an ALPHA 1-2/LD freeze dryer
(Martin Christ GmbH, Germany) for 24 h. Samples (5 × 5 mm) wereplaced onto the sample stage and sputter-coated with gold, using an
Emitech K575X sputter coater (10 mA, 2 × 40 s). Fiber diameters were
measured by analyzing SEM images with image analysis program Image
Processing and Data Analysis in Java (ImageJ). The diameters of 50
individual ultra ﬁneﬁbers were then measured in each SEM micro-
graph. Diameters were analyzed in 10 ﬁelds of SEM images in triplicate.
The elemental composition of the BC composites was examined
using scanning electron microscopy (a TM-3000 Hitachi microscopewith the QUANTAX 70 program).
Thermal analysis of samples was performed using a DSC-1 di ﬀer-
ential scanning calorimeter (METTLER TOLEDO, Switzerland).
Powdered samples (4.0 ± 0.2 mg each) were placed into the alu-
minum crucible and compressed prior to measurement. Every sample
was measured at least 3 times. Samples were preheated to 60 °C and
cooled to 25 °C. The samples were heated to temperatures from 25 °C to
300 °C, at 5 °C × min
−1(measurement precision 1.5 °C); melting point
(Tm) and thermal decomposition temperature (T d) were determined
from exothermal peaks in thermograms. The thermograms were ana-
lyzed using the STARe v11.0 software.
X-Ray structure analysis and determination of crystallinity of the BC
composites were performed employing a D8 ADVANCE X-Ray powder
diﬀractometer equipped with a VANTEC fast linear detector, using
CuKa radiation (Bruker, AXS, Germany). In order to determine the
crystallinity (C x) of BC, the spectra were collected from a V antec high-
speed detector, with exposure time of 3000. The detector was operatedat 40 kV and 40 mA.
Mechanical properties of the BC composites were investigated using
an electromechanical tensile testing machine Instron 5565 (U.K.).
Samples 75 mm long, 12 mm wide were prepared for studying physical
and mechanical properties of the ﬁlms.
At least ﬁve samples were tested for each type of ﬁlms.
Measurements were conducted at ambient temperature; the clamping
length of the samples was 50 mm. The speed of the crosshead was
3 mm/min at ambient temperature. Young's modulus (E, MPa), tensile
strength ( σ, MPa) and elongation at break ( ε, %) were automatically
calculated by the Instron software (Bluehill 2, Elancourt, France). Toobtain Young's modulus, the software calculated the slope of each
stress-strain curve in its elastic deformation region. Measurement error
did not exceed 10%.
BCﬁlms were sterilized with H
2O2plasma in a Sterrad NX medical
sterilizer (Johnson & Johnson, U.S.) and investigated in cell culture
assays.
2.5. Protein adsorption test
To estimate the antiadhesive e ﬀect of BC/AgNp and BC/antibiotic
ﬁlms towards protein, protein adsorption test was performed using
bovine serum albumin (BSA) ( “Amresco ”, U.S.). The BC composites
were incubated in 5 ml of the PBS solution with albumin concentration
of 1 mg/ml at 37 °C for 24 h. Then, the BC samples were removed, and
the remaining protein solution was analyzed. To determine protein
concentration, alkaline copper solution and Folin's reagent were added
to 1 ml of the protein solution. After 30 min exposure, which was ne-
cessary for color to develop, optical density was measured using a
spectrophotometer (Bio-Rad LABORATORIES Inc., U.S.) at aT.G. Volova et al. Polymer Testing 65 (2018) 54–68
56

wavelength of 570 nm, and residual protein content in the solution was
calculated from the standard curve.
2.6. In vitro release of nanosilver and antibiotics from BC composites
Sterile composite samples were exposed to phosphate-bu ﬀered
saline (PBS) to investigate release of silver and antibiotics. Under
aseptic conditions, the samples were placed into vials, each containing
50 ml saline at pH = 6.0 for silver and pH = 7 for antibiotics. The vials
were incubated in a thermostat for 72 h at a temperature of 37 °C. The
experiment was done with BCAgNP samples 1 cm in diameter prepared
at AgNO3 concentrations of 0.0001, 0.001, and 0.01 M and BC samples
with di ﬀerent percentages of antibiotics: amikacin (between 0.2 and
1%) and ceftriaxone (between 0.2 and 6%).
To estimate release kinetics, samples were periodically taken out of
the saline, and concentration of nanosilver was determined usingatomic absorption spectrophotometry (AAS), and concentrations of
antibiotics were measured using a Cary 60 UV-Vis spectrophotometer
(Agilent Technologies): amikacin at a wavelength of 210 nm and cef-triaxone at 304 nm.
2.7. In vitro antibacterial tests
The direct inhibitory e ﬀect of BC/AgNp and BC/antibiotic compo-
sites was tested on cultures of reference strains –Escherichia coli ATCC
25922, Pseudomonas eruginosa ATCC 27853, Klebsiella pneumoniae 204,
and Staphylococcus aureus ATCC 25923, using disk-di ﬀusion method in
agar (20 ml) on Petri dishes. The dishes were allowed to stay at room
temperature to solidify. The bacterial suspension was standard in-
oculum whose density corresponded to the 0.5 McFarland standard,
which contained approximately 1.5 × 10
8CFU/ml. The Petri dishes
were placed upside-down into an incubator and kept at a constanttemperature of 35 °C for 18– 24 h (depending on the microorganism
tested). The diameter of the growth retardation zones and the distancefrom the edge of the ﬁlm to the end of the absence zone were measured
by photographs of dishes, using the Image J. program. The results were
processed using the Microsoft Excel application package. The ar-
ithmetic mean and standard deviation were calculated.
Another test was conducted by cultivation of microorganisms using
the shake- ﬂask culture method, with BC/AgNp and BC/antibiotic ﬁlms
placed into the culture medium of Escherichia coli and Staphylococcus
aureus . BC composite disks 10 mm in diameter (n = 27) were placed in
test tubes, and 5 ml of microbial suspension of E. coli (0.6 × 10
8CFU/
ml) or St. aureus (0.6 × 108CFU/ml) prepared beforehand was added
to each test tube. These tubes were shaken at 250 rpm in incubators at aconstant temperature of 37 °C for 24 h. Then, the samples were read in
a KFK-2 photocolorimeter ( “ZOMZ ”, Russia) at the absorbance at
600 nm to measure the densities of the bacterial cultures. The cells werecounted using the standard curve constructed by the Breed method,relative to the initial values of the optical densities of the cultures.
2.8. Cytotoxicity assays
The ability of BC ﬁlms to facilitate cell attachment was studied using
NIH 3T3 mouse ﬁbroblast cells. The ﬁlms were placed into 24-well cell
culture plates (Greiner Bio-One, U.S.) and sterilized in a Sterrad NX
medical sterilizer (Johnson & Johnson, U.S.). Cells were seeded at
1×1 0
3cells/ml per well. Cells were cultured in DMEM medium
supplemented with 10% fetal bovine serum and a solution of antibiotics(streptomycin 100 μg/ml, penicillin 100 IU/ml) (Sigma) in a CO
2in-
cubator with CO 2level maintained at 5%, at a temperature of 37 °C.
The medium was replaced every three days. The number of cells at-tached to the ﬁlm surface was determined using DAPI.
Cell viability was evaluated using MTT assay at Day 7 after cell
seeding onto ﬁlms. Reagents were purchased from Sigma-Aldrich. A 5%
MTT solution (50 μl) and complete nutrient medium (950 μl) were
added to each well of the culture plate. After 3.5 h incubation, the
medium and MTT were replaced by DMSO to dissolve MTT-formazan
crystals. After 30 min, the supernatant was transferred to the 96-well
plate, and optical density of the samples was measured at wavelength
540 nm, using a Bio-Rad 680 microplate reader (Bio-Rad
LABORATORIES Inc., U.S.). Measurements were performed in tripli-
cate. The number of viable cells was determined from the standard
curve.
2.9. Statistics
Statistical analysis of the results was performed by conventional
methods, using the standard software package of Microsoft Excel.
Arithmetic means and standard deviations were found. The statistical
signiﬁcance of results was determined using Student's test (signi ﬁcance
level: P≤0.05). Statistical analysis of surface properties of the samples
was performed by using embedded methods of the DSA-4 software.
3. Results and discussion
3.1. Production and properties of BC
The dried BC ﬁlms synthesized in the Komagataeibacter xylinus B-
12068 culture had similar thickness (1.8 ± 0.2 mm) and density
(0.15 ± 0.01 cm
3) while their surface properties di ﬀered considerably,
as determined by water contact angle, which varied between 36° and57°. The ultrastructure and size of ﬁbrils in BC is a critical factor that
determines the unique properties of bacterial cellulose ﬁlms. SEM
images of the microstructure of BC ﬁlms show that BC ﬁlms were
layered nets of di ﬀerent densities composed of randomly oriented mi-
croﬁbrils ( Fig. 1). Whatever drying method was used, the average
diameter of BC ﬁlm micro ﬁbrils was 110 nm, the smallest and the
Fig. 1. SEM of pristine BCﬁlms and size distribution of
the diameters of the ﬁbers in the ﬁlms dried at room
temperature (A) and freeze-dried ones (B).
Bar = 10 μm.T.G. Volova et al. Polymer Testing 65 (2018) 54–68
57

largest diameters being 52 nm and 173 nm, respectively. However, in
freeze-dried ﬁlms,ﬁbrils were positioned more loosely, and the dis-
tance between them was 2.5 –3.0 times greater than in the ﬁlms dried at
room temperature, reaching about 1.6 μm.
3.2. Production and properties of BC composites with silver nanoparticles,BC/AgNps
During hydrothermal synthesis of silver nanoparticles, variations in
AgNO
3concentration in the reaction medium at 90 °C changed the
number of the nanoparticles synthesized ( Fig. 2A) without considerably
inﬂuencing their size ( Fig. 2B). For instance, the average sizes of Ag
nanoparticles at AgNO 3concentrations of 0.0001, 0.001, and 0.01 M
were 13, 23, and 12 nm, respectively.
The increase in the number of silver nanoparticles in BC ﬁlms is
illustrated by Fig. 2C. That was con ﬁrmed by quantitative elemental
analysis ( Fig. 3 andTable 1).
SEM images show that as AgNO 3concentration of the reaction
medium was increased, the number of silver nanoparticles adhering toBCﬁbrils and between them increased too ( Fig. 3). Ag nanoparticles
showed di ﬀerent aggregation behavior in freeze-dried BC ﬁlms com-
pared to the ﬁlms dried at room temperature. On the ﬁlms dried at
room temperature, the size of Ag particles was 25 –60 nm, the size of
their aggregates was 85 –350 nm, and the number of aggregates reached15 per 1 μm
2. On freeze-dried ﬁlms, particle aggregates were larger,
between 350 and 780 nm, and their number reached 19 per 1 μm2.
The presence of silver in the BC/AgNps was con ﬁrmed by elemental
analysis performed using scanning electron microscopy with a system
of X-ray spectral analysis ( Table 1 ). Analysis showed that the average
atomic number of silver particles in composite samples depended on theconcentration of AgNO
3; as AgNO 3concentration in the reaction solu-
tion was increased from 0.0001 to 0.01 M, silver content in the com-posites increased from 0.044 to 0.37 mg/cm
2.
Analysis of our results and the literature data shows that the main
factors determining the structure of BC/AgNp composites are the size ofnanoparticles, Ag content of the BC composites, method employed to
produce silver nanoparticles, and technique used to incorporate Ag
nanoparticles into BC ﬁlms. Silver concentration in BC ﬁlms, silver
nanoparticle size, and Ag particle aggregation between cellulose mi-croﬁbrils obtained in this study by using hydrothermal synthesis are
comparable with the data reported by G. Yang et al. [34] (the size of
silver particles of 14 –22 nm, silver content reaching 2.31% w/w) and
W. Shao et al. [29] (the size of particles of between 20 and 100 nm).
When silver nanoparticles were produced using reducing agents in the
reaction medium (sodium citrate, sodium borohydride, UV radiation,
triethanolamine, hydrazine, hydroxylamine, etc.), researchers
[10,37 –39]noted di ﬀerent sizes of Ag nanoparticles (8 –10 nm) and
their uniform distribution over the surface and between the ﬁbrils of
Fig. 2. The eﬀect of AgNO 3concentration on production of BC composites with silver nanoparticles, BC/AgNps: A –reaction medium; B –size distribution of Ag nanoparticles; C –a
photograph of composite ﬁlms.T.G. Volova et al. Polymer Testing 65 (2018) 54–68
58

BC.
Possible structural changes in the composites were determined by X-
ray diﬀraction analysis and di ﬀerential scanning calorimetry ( Fig. 4.)
The degree of crystallinity (C x) of BC varied considerably depending
on the carbon source used, the mode of cultivation of the strain, and the
structure and arrangement of the micro ﬁbrils. Samples of pristine BC
synthesized by the strain K. xylinus B-12068 di ﬀered substantially in
their degrees of crystallinity depending on the carbon source used. The
Cxof the samples synthesized on the HS medium with galactose was
45%, and the C xof the samples synthesized on the medium with glucose
and sucrose was 63– 68%; the samples synthesized in the medium with
the initial pH of 3.6 in the presence of citrate or acetate showed thehighest C
x(85–89%).
Fig. 4a shows results of X-Ray and di ﬀraction indices of BC com-
posites with nanosilver, indicating the main re ﬂexes corresponding to
crystalline cellulose and silver.In a study by French (2014) [40], three characteristic peaks were
identiﬁed in radiograms of freeze-dried BC ﬁlms –in the 14.60°, 16.82°,
and 22.78° ranges; the peaks corresponded to (110), (110), and (200)crystal planes of cellulose. This is in good agreement with the results of
X-ray di ﬀraction analysis obtained in this study and data in Fig. 5 a,
indicating the main re ﬂexes corresponding to cellulose crystal I αwith
the calculated parameters a= 6.72, b= 5.96, c= 10.40 Å;
α= 118.1°, β= 114.8°, γ= 80.4° and axis cparallel to the molecular
axis. Positions of these re ﬂexes di ﬀer between samples, e.g., (100)
varies between 14.24° and 14.52°. The presence of nanosilver in thecomposites is con ﬁrmed by the strong re ﬂexes with coordinates
2θ= 37.72° and 43.89° in radiogram 1 (a BC/AgNp sample produced at
the highest AgNO3 concentration in the reaction solution). Their posi-tions are consistent with re ﬂexes (111) and (002) of crystalline silver at
a cell parameter of 4.126 Å. Their large width must be due to the small
size of silver nanoparticles.
Analysis of radiograms did not show any signi ﬁcant changes in the
C
xvalue of BC composites with nanosilver. The degree of crystallinity of
the samples produced at the lowest AgNO 3concentration in the system
(0.0001 M), with the low content of silver in the composite (0.044 mg/
cm2), and with nanoparticles of an average size of 13 nm, was 61%.
With an increase in the content of silver nanoparticles in the compositefrom 0.16 to 0.37 mg/cm
2, the degree of crystallinity did not increase
signiﬁcantly; it was 73 and 78%, respectively, which was closer to the
Cxof pristine BC. Similar data were reported by other authors. The C xof
BCﬁlms varied considerably, between 46.7 and 91.62 [25,41 –43].[38]
reported the degree of crystallinity of BC/silver composites of
83.68 –86.21%, which was close to the C xof pristine BC. Comparable C x
Fig. 3. SEM images of the ﬁlms of BC composites with silver nanoparticles, BC/AgNps, and size distribution of silver nanoparticles in the ﬁlms produced under di ﬀerent AgNO 3
concentrations in the medium: ﬁlms dried at room temperature (A) and freeze-dried ﬁlms (B). Arrows denote aggregates of Ag nanoparticles. Bars = 200 and 300 μm.
Table 1
The in ﬂuence of conditions of the system on the elemental composition of BC/AgNps.
Samples Average atomic number (wt.%) Ag content (mg/cm2)
OC A g
BCﬁlm 59.8 40.1 –
Inﬂuence of AgNO 3concentration at 90 °C:
0.0001 M 53.03 45.9 1.08 0.044
0.001 M 54.2 41.7 4.10 0.16
0.01 M 51.2 40.1 9.1 0.37T.G. Volova et al. Polymer Testing 65 (2018) 54–68
59

values were obtained for the composites with nanosilver synthesized in
the presence of NaBH 4as a reductant [44,45] . Thus, the crystalline
structure and the degree of crystallinity of BC did not change con-siderably with silver nanoparticles synthesized in situ.
The maximal temperature of decomposition of a material is a cri-
terion of its thermal stability. Thermal decomposition of BC, as shown
in a number of studies, is determined by certain structural parameters
such as molecular weight, degree of crystallinity, and ﬁber alignment of
the BC [46,47] .D iﬀerences in thermal stability between pristine BC
samples are caused by di ﬀerent conditions of their synthesis. As the
region of degradation has no pronounced peaks, it seems reasonable tospeak of the decomposition onset temperature (T
dec. onset ). The BC
samples synthesized in the medium with galactose had the highestthermal stability, and T dec. onset was 284 °C. The BC samples synthesized
in the medium with sucrose had the lowest T dec. onset (220 °C). For all
samples, we observed two sections in the decomposition region. Theﬁrst section corresponded to a temperature range of between 220 °C
and 285 °C. In this section, the loss of BC mass was insigni ﬁcant. The
second section corresponded to a temperature range of between 360 °C
and 430 °C. The loss of BC mass in this section was more substantial, as
the rate of thermal decomposition processes increased. A study by F.
Mohammadkazemi et al. [41] also showed that the onset of thermal
decomposition of BC could occur in the range between 200 and 250 °C,but more noticeable decomposition, with the samples losing 70 –80% of
their weight, was observed at 360– 390 °C. This is consistent with the
data reported by other authors [48], showing the weight loss of the BC
Fig. 4. Physicochemical properties of composites: A –X-ray patterns of the BC/AgNp composite produced at di ﬀerent AgNO3 concentrations in the reaction solution: 1 –0.01 M;
4–0.001 M; 5 –0.0001 M, respectively, the degree of crystallinity (C x) 61, 83, and 86%; 2 and 3 –pristine cellulose (without silver), the degree of crystallinity 72 and 75%; B – DSC curves:
1–pristine cellulose, freeze-dehydrated; 2, 3 and 4 –the composite produced at 90 °C and di ﬀerent concentrations of AgNO3 (0.01 M, 0.001 M, and 0001 M, respectively); C –1, 2 –BC/
ceftriaxone 1% and 4%, 3 –BC/amikacin 1% the BC composite with, antibiotics, respectively.
Fig. 5. SEM images of BC/antibiotic composite ﬁlms: A
–BC/amikacin 1%; B –BC/ceftriaxone 1; C –BC/cef-
triaxone 6%; D –in vitro release of antibiotics to the
balanced phosphate-bu ﬀered saline: 1 –amikacin 1%; 2
–ceftriaxone 1%; 3 –ceftriaxone 6%.T.G. Volova et al. Polymer Testing 65 (2018) 54–68
60

sample during thermal decomposition at 300 °C and higher rates of this
process at 350– 370 °C.
Unlike BC, BC composites with silver nanoparticles produced under
diﬀerent conditions were not identical to each other and were char-
acterized by greater thermostability ( Fig. 4 B). Thus, composites pro-
duced at di ﬀerent temperatures but having similar contents of silver
had more pronounced peaks in the thermal decomposition region(415 –425 °C) than BC. The onset of the decomposition temperature for
the samples was in the region of 320– 325 °C ( Fig. 4 B), and that was
signiﬁcantly, almost 60– 80 °C, higher than in the BC samples produced
earlier. The composites produced by stabilizing the temperature butwith di ﬀerent concentrations of AgNO
3in the reaction medium also
showed higher thermal stability than the BC ( Fig. 4 B, curve 1). In all
composite samples, the regions of onset of thermal decomposition were
shifted to the right relative to the BC; at the same time, they were
characterized by the presence of two peaks with a gap between them of
between 40 and 120 °C ( Fig. 4 b, curves 2– 4). Analysis of BC composites
with antibiotics did not reveal any signi ﬁcant changes in temperature
parameters compared to pristine BC ( Fig. 4 C). Thus, incorporation of
silver nanoparticles into cellulose ﬁlms increased thermal stability of
BC. A similar enhancement of thermal stability of BC composites with
nanosilver was reported by other authors [24,25] .
An important property of a medical device is the ability to retain its
integrity throughout its lifetime. Mechanical properties of BC are lar-
gely determined by the BC producer used, conditions of synthesis, ﬁbril
thickness, and method of drying of the ﬁlms. The tensile strength of air-
dried BC ﬁlms varies between 129 and 198 MPa [49], while the tensile
strength of freeze-dried ones is an order of magnitude lower (8 –14 MPa)
[3,50] .
Results of measuring physical/mechanical properties of BC and BC
composites are listed in Table 2. The properties of the samples di ﬀer
substantially depending on the moisture content of the sample. Me-chanical parameters of wet pristine BC samples taken out of the fer-mentation medium and rinsed to remove the medium and bacterial
cells, with a moisture content of over 90%, were as follows: Young's
modulus 10.26 ± 0.35 MPa, tensile strength 0.75 ± 0.34 MPa, and
elongation at break 5.49 ± 1.21%. The dry ﬁlms, with a low moisture
content (5 –7%) only di ﬀered from the wet ones in their Young's mod-
ulus, which was 6 times higher. Composite ﬁlms with residual moisture
content of about 50% exhibited the highest mechanical strength para-meters, which were an order of magnitude higher than the corre-
sponding parameters of dry and wet BC samples: 47.60 ± 6.32 MPa,0.11 ± 0.13 MPa, and 4.35 ± 0.82%, respectively.
Loading of nanosilver into BC considerably changed mechanical
properties of BC, increasing the values of Young's modulus and elon-
gation at break by one order of magnitude and tensile strength by two
orders of magnitude compared to pristine BC ( Table 2). It is di ﬃcult to
compare results obtained in this study with the published data, as theliterature data vary considerably, which may be attributed to di ﬀerent
methods employed to produce BC composites and dissimilar propertiesof pristine BC. In a study by Y.Wan et al. [51], tensile strength of freeze-
dried BC samples with longitudinal ﬁbers was 1.2 MPa, and that was
somewhat higher than tensile strength of pristine BC (0.9 MPa) and BC
with transverse orientation of the ﬁbers (0.6 MPa). However, elonga-
tion at break of the BC with parallel ﬁbers was 22%, i.e. lower than the
elongation at break of pristine BC (30%) and BC with ﬁbers oriented
perpendicular to each other (39%). These data suggest that physical/mechanical parameters are determined by the orientation of ﬁbers in
BCﬁlms.
In a study by V. Sadanand et al. [17], parameters of me-
chanical strength of BC are considerably higher than the values re-
ported by Y.Wan et al. [51] but comparable to those obtained in the
present study: tensile strength of the composite BC/AgNp ﬁlms was
80 MPa, which was higher than that of pristine BC (50 MPa), while
elongation at break dropped from 11 to 5% as AgNO3 concentration
was increased. In another study V. Sadanand et al. [52], however,
strength parameters of the composite BC/CuNP ﬁlms were di ﬀerent:
their tensile strength was 100 MPa, which was lower than tensile
strength of pristine BC (125 MPa), and it decreased with an increase in
concentration of copper sulfate solution used to prepare CuNPs, but
elongation at break was higher in the composites (25%) than in BC
(20%). This could be explained by the properties of the ﬁller (CuNPs)
and a decrease in the size of composite crystals, but the authors did notreport moisture content of the samples. The authors of one more study
[19] investigated mechanical properties of BCAgNP composite pre-
pared by immersion of BC in 1– 5 mM AgNO
3solutions for 24 h, under
continuous stirring. The tensile strength of the composite was 108 MPa,and it was considerably higher than the tensile strength of pristine BC
(59 MPa). This parameter decreased with an increase in AgNP content
of the composite, to 70 MPa, which the authors attributed to agglom-
eration of AgNPs with an increase in AgNP content. These data are in
good agreement with the results obtained in the present study. Thus, in
most of the studies, loading of nanosilver into BC led to enhancement of
mechanical strength of the ﬁlms. Mechanical properties of BC compo-
sites with antibiotics were generally lower than those of BC/AgNpcomposites. Young's modulus of BC composites with amikacin tended to
decrease (from 110.19 to 63.24 MPa) with an increase in the content of
the antibiotic in the composite while tensile strength and elongation at
break ranged between 6.07 and 9.27 MPa and between 11.69 and
14.36%, respectively, and these values were comparable to the strength
parameters of BC composites with nanosilver. Tensile strength and
elongation at break of BC/ceftriaxone composites were similar to the
corresponding parameters of BC/amikacin. At the same time, Young's
modulus of BC/ceftriaxone was considerably lower than that of BC/
AgNps (8 –10 times lower) and BC/amikacin (1.5 –2.0 times lower).
Thus, mechanical properties of BC composites with antibiotics werecomparable with mechanical properties of pristine BC, and mechanical
properties of BC/AgNp composites were superior to them. That is, in-
corporation of nanosilver into BC reinforced mechanical strength of BC.
One of the factors determining biocompatibility of implants is
physicochemical reactivity of their surface. The main factors that in-
ﬂuence surface interaction with blood and tissue cells and components
of biological ﬂuids are surface topography, microstructure, and ad-
hesive properties. An indirect indicator of surface hydrophilicity is li-
quid contact angle. Properties of BC composites are listed in Table 3.B C
ﬁlms are inherently quite hydrophilic, with the water contact angle
below 50° (45.5 ± 17.6°). The surfaces of BC/AgNp composites tend to
become less hydrophilic (more hydrophobic) as their silver content
increases. The dispersive and polar components increase, too. SuchTable 2
Physical/mechanical properties of BC and BC/AgNp and BC/antibiotic composites.
Samples Young's modulus
(MPa)Tensile strength(MPa)Elongation atbreak (%)
Pristine BC (dry),
moisturecontent 5 –7%63.9 ± 8.52 0.52 ± 0.09 3.35 ± 0.61
Pristine BC (wet),
moisturecontent 95 –97%10.26 ± 0.35 0.75 ± 0.34 5.49 ± 1.21
BC (dry, moisture
content
50–55%)47.60 ± 6.32 0.11 ± 0.13 4.35 ± 0.82%
BC/AgNps (dry, moisture content 50 –55%)
BC/AgNps 0.0001 M 233 ± 5.21 14.96 ± 1.69 9.8 ± 2.57
BC/AgNps 0.001 M 218 ± 2.21 13.22 ± 2.05 7.66 ± 2.5BC/AgNps 0.01 M 99.3 ± 5.21 9.93 ± 1.27 11.73 ± 1.631BC/antibiotics (dry, moisture content 50 –55%)
BC+amikacin 0.2% 110.19 ± 13.5 7.18 ± 1.5 9.02 ± 2.1BC+amikacin 0.6% 90.16 ± 14.4 9.27 ± 1.4 14.36 ± 2.5
BC+amikacin 1% 63.24 ± 1.8 6.07 ± 1.8 11.69 ± 2.1
BC+ceftriaxone 1% 58.94 ± 6.9 5.3 ± 0.9 11.9 ± 2.7BC+ceftriaxone 4% 44.57 ± 4.2 6.44 ± 0.4 17.62 ± 1.1BC+ceftriaxone 6% 45.47 ± 8.1 6.03 ± 1.1 16.25 ± 2.5T.G. Volova et al.
Polymer Testing 65 (2018) 54–68
61

changes reduce fouling of the surfaces by proteins and cells, as de-
scribed in a study by Baoquan Jia et al. [53]. The authors of that study
observed an increase in water contact angle on BC/CuNp compositeﬁlms to 110 ± 0.4° –the value that was twice higher than that of the
water contact angle on the pristine BC (50.1 ± 1.8°).
3.3. Production and properties of BC composites with antibiotics
In order to impart bactericidal properties to cellulose, we prepared
BC composites with antibacterial drugs (amikacin and ceftriaxone).
Amikacin and ceftriaxone were chosen for the following reasons.
Ceftriaxone is a third-generation durable antibiotic, which is active in
vitro against most of the Gram-negative and Gram-positive microbes.
The antibacterial activity of cephalosporins and other β-lactam anti-
biotics is determined by inhibition of synthesis of peptidoglycane –the
structural basis of microbial wall. The mechanism of action of the
aminoglucoside antibiotic amikacin is based on its irreversible binding
to speci ﬁc receptors of bacterial ribosomes and inhibition of synthesis
of cytoplasmic membranes, which causes the death of bacterial cells.Amikacin is active against aerobic Gram-negative microorganisms,
especially Serratia spp., Enterococcus faecalis , and Staphylococcus aureus ,
but it is less e ﬀective against most of Gram-positive bacteria.
During preparation of the composites, concentrations of antibiotics
in the solutions varied between 0.1-0.2 and 1 –6%. SEM images show
that BC ﬁlms impregnated with antibiotics had a denser and more
uniform surface ( Fig. 5 ).
In contrast to silver nanoparticles, antibiotics impregnated into BC
did not signi ﬁcantly change the degree of crystallinity and temperature
properties of composite ﬁlms, but a ﬀected their surface properties
(Table 3). BC composites with antibiotics, like BC/AgNps, had more
hydrophobic surfaces than pristine BC ﬁlms; their dispersive compo-
nents and surface energies were higher too. SEM images showedchanges in the ﬁlm structure after addition of antibiotics: the distances
between BC ﬁbers decreased and they formed a uniform layer.
Release of antibacterial drugs from BC composites was investigated
in the phosphate-bu ﬀered saline (PBS) at 37 °C and pH = 7 for 72 h
(Fig. 5 D). No burst release of antibiotics from the BC matrix wasobserved, suggesting adhesion between BC and antibiotics. Antibioticswere being released from the composites during the whole 72 h ex-
periment, but release kinetics depended on the antibiotic type and the
level of loading of the BC matrix with it. The highest percentage of the
drugs were released over the ﬁrst ten hours. By the end of the 72 h
experiment, 49.5% of amikacin was released from the BC/amikacin 1%.Ceftriaxone was released at a higher rate, probably because of its
weaker adhesion to BC: its charge is −1, in contrast to the positively
charged amikacin. The 1%-ceftriaxone composite released 59.5% of theantibiotic and the 6%-ceftriaxone composite 64.5% over the 72 h ex-
periment. Thus, BC composites with antibiotics are rather stable in the
liquid medium, which may be important if they are used as wound
dressings for treating contaminated wounds and tissues. Our results are
consistent with the published data that showed the feasibility of loading
tetracycline into BC and slow release of the antibiotic from the dialysis
sac (80% for 3 h) compared to the release of the free drug. The authors
explained this result by the di ﬀerent charges of the drug and BC. BC is
charged negatively while the antibiotic has a positive charge, and theelectrostatic
interaction between BC and the drug slowed down tetra-
cycline release [29]. Slow release of tetracycline from nanocrystals was
described in another study [30]. The authors revealed the e ﬀect of pH
of the medium on this process: the highest release of tetracycline(82.21%) was observed at pH 7.2 and the lowest (25.1%) at pH 2.1. In
yet another study [28], 66% of benzalkonium chloride incorporated in
freeze-dried BC ﬁlms was released from the matrix over 24 h. The au-
thors suggested that the rate of drug release was determined by the
water content of the swollen hydrogel and the parameters of the net-
work such as the degree of crosslinking and the size of ﬁbers. In addi-
tion to that, drug release depended on the di ﬀusion coe ﬃcient of small
molecules or macromolecules through the gel network.
3.4. Protein adsorption on the surface of BC/AgNp and BC/antibiotic
composites
Nonspeci ﬁc protein adsorption on the surface is an important pro-
cess for implants, which come in contact with human body ﬂuids, as it
may cause thrombus formation, bacterial adhesion, and immune re-
sponses [54]. The generally accepted hypothesis of antiadhesive ac-
tivity of hydrated coatings is based on the ability of the hydrophiliccomponent to strongly bind water and, thus, create a hydrating layer,
which prevents adhesion of nonspeci ﬁc proteins and bacteria [55].A s
mentioned above, the magnitude of the water contact angle of BCcomposites with silver and antibiotics ranged between 50 and 70° de-
pending on the concentrations of the components. This is the inter-
mediate position between hydrophobic and hydrophilic surfaces.
Adhesive properties of the surface of BC composites were evaluated
in bovine serum albumin (BSA) protein assay. We measured the optical
density of protein solutions in which composite samples had been in-
cubated and ones used to incubate pristine BC ﬁlms. Initial optical
density of the protein solution was 0.3, which corresponded to proteinconcentration of 860 mg/ml ( Fig. 6).
Pristine cellulose, whose surface was more hydrophilic than the
surface of BC composites, had the most pronounced antiadhesiveproperties towards protein and did not adsorb it. Protein, taking intoTable 3
Surface properties of BC/AgNp and BC/antibiotic composites.
Samples Water contact
angle, degrees [°]Dispersive
component [mN/
m]Polar component
[mN/m]
Pristine BC 45.5 ± 17.6 28 ± 9.24 17.5 ± 8.37
BC/AgNpsBC/AgNps 0.0001 M 50.3 ± 4.61 42.6 ± 1.01 27.7 ± 3.6BC/AgNps 0.001 M 68.8 ± 1.76 46.2 ± 0.99 22.6 ± 0.77BC/AgNps0.01 M 69 ± 2.71 44.9 ± 1.85 25.1 ± 0.86BC/antibioticsBC+amikacin 0.2% 67.1 ± 2.12 42.2 ± 4.15 25.1 ± 2.04BC+amikacin 0.6% 66.5 ± 2.56 40.3 ± 12.69 25.8 ± 8.87
BC+amikacin 1% 70.4 ± 2.11 45.7 ± 1.11 24.7 ± 1
BC+ceftriaxone 1% 70.2 ± 2.40 44.3 ± 1.07 25.7 ± 1BC+ceftriaxone 4% 68.4 ± 1.10 42.5 ± 2.11 24.2 ± 2.21BC+ceftriaxone 6% 72.4 ± 1.26 47.3 ± 13.1 23.2 ± 1.13
Fig. 6. BSA adsorption A –relative value (optical den-
sity of protein solution),B –quantitative data (protein
concentration, mg/ml):1 –control (initial protein con-
tent); 2 –initial cellulose; 3 –BC/AgNps 0.01 M; 4 –BC/
AgNps 0.001 M; 5 –BC/AgNps 0.0001 M; 6 –BC/ami-
kacin 1%; 7 –BC/ceftriaxone 4%; 8 –BC/ceftriaxone
6% after 24 h.T.G. Volova et al. Polymer Testing 65 (2018) 54–68
62

account the isoelectric point 4.7, under physiological conditions, has
negative charge of the molecule; therefore, pristine cellulose, which
was also negatively charged, did not adsorb it. Adsorption of protein on
the pristine BC ﬁlms was low: protein concentration in the solution had
changed very little, measuring 800 mg/ml. This is consistent with thehypothesis suggesting that hydrophilic surfaces have the ability to
strongly bind water and, thus, create a hydration layer that prevents
adhesion of proteins and bacteria [56].
The amounts of protein adsorbed on composite samples were higher
and di ﬀered considerably depending on the type of the bactericidal
component (silver or antibiotics) and its concentration. The largestamounts of protein were adsorbed on the BC/AgNp composites:
250–320 mg/ml, with no substantial silver concentration dependence.
Hence, more than half of the protein from the solution was adsorbed onthe surface of BC/AgNp composites. This may be caused by protein-
nanoparticles hydrophobic interactions, which can be attributed to al-
bumin having eleven hydrophobic binding domains. These interactions
were previously described for gold nanoparticles [57].
Ad iﬀerent result was obtained for the BC/antibiotic composites.
Their adhesive properties towards BSA were considerably lower al-
though the ﬁlms of BC/AgNp and BC/antibiotic composites had similar
water contact angles ( Fig. 6). The composites produced in this study
had less hydrophilic (i.e. more hydrophobic) surface than BC and ad-
sorbed protein, but the degree of adsorption varied. Amikacin, being a
positively charged compound, adsorbed insigni ﬁcant amounts of pro-
tein. By contrast, ceftriaxone has a charge of −1 at neutral pH, and BC
is also negatively charged. Thus, the negatively charged bovine serum
albumin weakly adhered to the composite. As the concentration of the
negatively charged ceftriaxone in the composite was increased, the
antiadhesive properties of the composite decreased, which was con-
sistent with the decrease in protein sorption observed in our study. The
lowest protein adsorption was observed in the experiments with the BC
impregnated with 4 and 6% ceftriaxone, in which protein concentra-tions in the post-incubation solutions were 700 and 800 mg/ml, re-
spectively. These values were comparable to those obtained in the assay
of the pristine BC, which, together with rather low ceftriaxone retention
eﬃciency, suggested a modest e ﬀect of this antibiotic on the adhesive
properties of BC. The antiadhesive property of the surface is a favorablefactor, as implants with antiadhesive properties will be less prone to be
“coated ”with adsorbed proteins and other components of blood and
tissue ﬂuids, preventing microbial colonization and formation of bio-
ﬁlms on their surfaces. A search of the literature did not reveal any
studies on protein adsorption on ﬁlms of BC composites with silver and
antibiotics. X. Xu et al. [58] reported that as the surface hydrophilicity
of the mucin/poly(ethyleneimine) ﬁlms was increased, protein ad-
sorption decreased due to the ability of the hydrophilic component,
which strongly bound water and, thus, created a hydration layer, to
prevent adhesion of nonspeci ﬁc proteins and bacteria.
3.5. Antibacterial activity of BC/AgNp and BC/antibiotic composites
All samples of BC composites exhibited bactericidal activity against
test microbial cultures –the most common representatives of nosoco-
mial infection and pathogenic micro ﬂora of contaminated wounds.
However, the levels of antibacterial activity of the composites werediﬀerent ( Table 4 ,Fig. 7). BC/antibiotic composites had stronger in-
hibitory e ﬀect
on the growth of pathogenic microorganisms.
In experiments with BC/AgNps, the largest zone of inhibition
(15 ± 1.58 mm) was observed for St. aureus , at the highest con-
centration of silver nanoparticles (AgNps 0.01 M); the smallest zone ofinhibition (11 ± 0.20 mm) was created by BC/AgNps 0.0001 M for E.
coli. These di ﬀerences are caused by di ﬀerent susceptibility of bacteria
to BC composites with Ag particles, their cell structure and physiology.
Gram-positive bacteria are generally more susceptible to the bacter-
icidal e ﬀect of Ag nanoparticles [59]. Similar data were reported by W.
Shao et al. [29]: in their study, at a concentration of BC-Ag of 0.01 M,the diameters of the zones of inhibition of E.coli and St. aureus growth
were 11.7 ± 0.1 and 11.6 ± 0.1 mm, respectively J. Feng et al. [11],
reported a study of BC composites with silver reduced using NaBH4,which created zones of inhibition of diameter about 16.1 mm for E. coli
and 17.7 mm for S. aureus ; the BC composites with silver reduced with
sodium citrate created zones of inhibition with smaller diameters: 13.7
and 13.2 mm, respectively. The diameters of the zones of inhibition of
B.subtilis and E. coli by composites based on polydopamine magnetic
bacterial cellulose and Ag were 20 mm and 17 mm, respectively [10].
However, a study by S.H. Barud et al. [60] showed that composite
membranes of BC/Ag/TEA 1 mol/L
−1created inhibition zones for
P.aeruginosa ATCC-27853, E.coli ATCC 25922, and St.aureus ATCC
25923 reaching 20 mm. Di ﬀerences between results may be caused by
diﬀerent methods employed to produce composites, sizes of silver na-
noparticles, and their concentrations in BC ﬁlms. It has been assumed
that the lowest inhibitory concentration of Ag nanoparticles is about
0.05 –0.1 mg/ml [10]. In the present study, the strongest inhibition of
pathogenic micro ﬂora by BC/AgNp composites was observed under the
highest silver concentration in the reaction solution during composite
production, when silver content was 9.1 (or 0.37 mg/ml), i.e. these
results are consistent with the data reported by other authors. The zone
of inhibition of Klebsiella pneumoniae was 14 ± 0.36 mm at the highest
concentration of silver nanoparticles (AgNps 0.01 M).
Zones of inhibition by BC/antibiotic composites were generally
larger than those produced by BC/AgNp composites ( Table 4). It is well-
known that antibiotics penetrate through cell membrane and irrever-
sibly bind to speci ﬁc receptor proteins of bacterial cell, thus e ﬀectively
suppressing synthesis of bacterial membranes.
The strongest inhibition (reaching 30 –34 mm) by the BC composite
with amikacin (at concentrations of 0.6 and 1.0%) was observed in the
culture of P. aeruginosa ; somewhat smaller inhibition zones (20 –22 mm)
were formed in the two other cultures. Experiments with BC compositeswith ceftriaxone, especially those with low ceftriaxone concentrations,
did not suggest any deﬁ nite conclusions. The BC ﬁlms with low cef-
triaxone concentrations (0.6 and 1.0%) did not inhibit the growth of theGram-negative E. coli, Kl. pneumoniae and the Gram-positive S.aureus.
The reason why antibacterial activity of ceftriaxone was di ﬀerent
against the Gram-negative E.coli and the Gram-positive S.aureus may be
the di ﬀerence between the structures of the outer cell membranes of
these microorganisms. Cephalosporins penetrate through the outer cy-
toplasmic membrane of Gram-positive bacteria quite easily and sup-
press them. The membrane of Gram-negative bacteria has a more
complex structure, preventing cephalosporins from penetrating into the
periplasmic space of the cell. Cephalosporins penetrate the microbialTable 4
Inhibition of pathogenic bacteria on solid medium by BC/AgNp and BC/antibiotic com-posites.
Samples Diameter of inhibition zones (mm)
P. aeruginosa E.coli St.aureus K. pneumoniae
Pristine BC –– ––
BC/AgNps, M0.0001 M 12 ± 0.44 11 ± 0.20 15 ± 0.73 13 ± 0.44
0.001 M 13 ± 1.15 13 ± 0.50 14 ± 0.28 14 ± 0.64
0.01 M 14 ± 2.11 14 ± 0.61 15 ± 1.58 15 ± 0.36BC+amikacin,%0.2% 24 ± 0.34 15 ± 0.58 19 ± 0.6 23 ± 0.120.6% 30 ± 0.26 20 ± 0.9 22 ± 0.222 24 ± 0.321.0% 34 ± 0.49 21 ± 0.44 26 ± 0.8 25 ± 0.30BC+ceftriaxone, %0.2% 19 ± 0.64 –– –
0.6% 19 ± 0.44 – 16 ± 0.77 17 ± 0.50
1.0% 24 ± 0.33 – 18 ± 0.4 19 ± 0.36
4.0% 24 ± 0.31 25 ± 0.43 32 ± 0.32 23 ± 0.86.0% 33 ± 0.40 29 ± 0.35 34 ± 0.50 24 ± 0.34
“-”–no zone of inhibition.T.G. Volova et al.
Polymer Testing 65 (2018) 54–68
63

Fig. 7. Zones of inhibition of Klebsiella рneumoniae ,Pseudomonas aeruginosa ,Escherichia coli , and Staphylococcus aureus by the BC/AgNp 0.01 M and BC/amikacin 1% and BC/ceftriaxone
1% composites.T.G. Volova et al. Polymer Testing 65 (2018) 54–68
64

cell through the so-called porin channels. A possible reason why low
concentrations of ceftriaxone did not inhibit E. coli might be the di ﬃ-
culty of penetrating into the cell. At higher concentrations, this di ﬃ-
culty was alleviated, and the inhibitory e ﬀect became noticeable. At
ceftriaxone content of the composite increased to 4 and 6%, its e ﬀect
was similar to that of amikacin. Thus, the inhibitory e ﬀect of the
composites varied depending on the type of microorganisms and con-
centration of the inhibiting agent. B. Wei et al. [28] reported a similar
result. The authors used BC ﬁlms containing benzalkonium chloride at
diﬀerent concentrations (between 0.026 and 0.128% w/w). At the
highest concentration of the antibiotic, the zones of inhibition of E. coli,
St.aureus , and Bacilluss ubtilis were 15, 18.5, and 24.5 mm, respectively.
Antibacterial properties of all experimental BC composites were
conﬁrmed using the shake- ﬂask culture method. The inhibitory e ﬀects
of composites added to the culture medium were studied in two 24-hcultures ( E. coli andSt .aureus ). An indicator of antibacterial activity of
the composites was optical density of bacterial suspensions, whichcorresponded to cell concentration ( Fig. 8). BC/AgNp composites pro-
duced an inhibitory e ﬀect even at the lowest concentration of silver
nanoparticles in the composite (0.0001 M), and the strongest inhibition
was observed in St. aureus culture. At the highest silver concentration in
the composite, the optical density of either culture was no more than0.17 –0.20 (or 0.61 –1×1 0
3CFU/ml). That was 3.7 –4.0 times lower
than in the control and in the test with the pristine BC. Silver activity inliquid culture has been described in a number of papers. In a study by
G. Yang et al. [12], BC/AgNp composites prepared using the same
method as in this study –by immersion in St. aureus liquid culture for a
long time period (72 h) –caused optical density values close to zero
(0.08), while in the control (with pristine BC), optical density washigher –1.8. J. Wu et al. [14], after incubation of BC/AgNp 0.01 M
composites for 24 h, noted a 98.8 –100% decrease in E. coli ,S. aureus ,
and P. aeruginosa cells (8.2 × 10
2, 0, 1.1 × 103, respectively). The
authors of another study [43] proved that the increase in silver con-
centration (0.01– 0.03 M) in BC/AgNp composites produced using
NaBH4 caused inhibition of E. coli and S.aureus growth by a factor of
4–4.5 (2 × 106and 1 × 106) relative to their initial growth. An in-
crease in AgNO 3concentration to 0.04 –0.05 M completely suppressed
the growth of all cultures.Deﬁnite antibacterial activity of BC/antibiotic composites was ob-
served in liquid cultures ( Fig. 8), and it was also the highest in S.aureus
culture. The optical density was 4 times lower in both cultures with the
BC/amikacin 1% composite and with the composites containing higher
(4 and 5%) concentrations of ceftriaxone compared to the control. Asimilar e ﬀect was described by B. Wei et al. [28], in a study in which
optical density of St. aureus and B. subtilis cultures containing BC/
benzalkonium chloride composites decreased as concentration of the
inhibitory agent was increased, dropping to 0.077 and 0.003, respec-
tively, after 24 h. That was 17 times lower than the optical density of
the control medium (3.363 and 1.572). In a study by W. Shao et al.
[29],B Cﬁlms with tetracycline hydrochloride (0.05 –0.5 g/L) were
incubated at 37 °C in E. coli ,St. aureus ,B. subtilis , and C.albicans sus-
pensions for 6 min. Colony counts showed that BC/antibiotic ﬁlms in-
hibited growth of all microorganisms by 99 –100% ( E. coli by 99.98%, S.
aureus by 100%, B. subtilis by 100%, and C. albicans by 99.99%), in
contrast to pristine BC (2.6 × 10
8).
3.6. Cytotoxicity assays of BC/AgNp and BC/antibiotic composites inﬁbroblast cell culture
As the experimental BC composites were investigated as possible
candidates for wound dressings for treating skin defects and injuries,including those contaminated by pathogenic micro ﬂora, it was im-
portant to study the e ﬀect of silver and/or antibiotics impregnated into
BCﬁlms on dermal cells. BC composites were investigated for their
potential cytotoxicity in ﬁbroblast cell culture using DAPI stain –a
marker of nuclear DNA –and MTT assay, which determines the number
of viable cells.
The evaluation of the e ﬀect of BC composites on ﬁbroblasts using
DAPI staining was in good agreement with results of MTT assay ( Figs. 9
and 10). At Day 3, only one sample, which contained 0.2% amikacin,did not show any inhibitory e ﬀect. In all other tests, with higher ami-
kacin concentrations and all ceftriaxone concentrations, the number ofcells decreased. The most substantial decrease (by 35 –40% relative to
the control) was noted in the tests with composites containing cef-triaxone, irrespective of concentration.
A similar study in the ﬁbroblast cell culture was performed with BC/
Fig. 8. Optical densities of 24-h cultures of E.coli andSt.aureus exposed to e ﬀects of BC/AgNp (a, b) and BC/antibiotic (c, d) composites: 1 –control (suspension); 2 –pristine cellulose
(without silver); 3 –BC/amikacin 0.2%; 4 –BC/amikacin 0.6%; 5 –BC/amikacin 1%; 6 –BC/ceftriaxone 1%; 7 –BC/ceftriaxone 4%; 8 –BC/ceftriaxone 6%.T.G. Volova et al. Polymer Testing 65 (2018) 54–68
65

AgNp composites ( Fig. 9). In contrast to BC/antibiotic composites,
neither test (DAPI staining or MTT assay) revealed any signi ﬁcant in-
hibitory e ﬀect of silver nanoparticles on ﬁbroblast cell culture. While
inhibiting the growth of pathogenic micro ﬂora, the BC composites with
nanosilver produced a weak inhibitory e ﬀect on ﬁbroblasts. The slight
decrease in the number of viable ﬁbroblasts observed in MTT assay at
higher concentrations of nanosilver was not statistically signi ﬁcant. The
counts of viable ﬁbroblasts were similar on all BC/Ag ﬁlms:
6.95·103cells/cm2on the BC ﬁlm with the highest Ag concentration
(0.01 M), 7.30·103cells/cm2on the BC ﬁlm with an Ag concentration of
0.001 M, and 8.47·103cells/cm2on the BC ﬁlm with the lowest AgNO 3
concentration (0.0001 M). These values were comparable with the
control (8.85·103cells/cm2). Results obtained in the present study are
consistent with the published data suggesting that BC/AgNps produce astrong inhibitory e ﬀect on pathogenic and opportunistic pathogenic
microﬂora, without inhibiting the growth of epidermal cells [14,61] .4. Conclusion
New results were obtained on production and properties of bacterial
cellulose nanocomposites. Physical, mechanical, and biological prop-
erties of BC/AgNp and BC/antibiotic composites synthesized by the
hydrothermal method were investigated and compared. The ﬁlms of
bacterial cellulose composites with silver nanoparticles and antibioticsproduced in this study were comprehensively investigated, including
such parameters as their surface microstructure and properties, tem-
perature characteristics, degree of crystallinity, mechanical properties,
the ability of the surface to adsorb proteins, release of nanosilver and
antibiotics from composites, antibacterial activity against pathogenic
and opportunistic pathogenic micro ﬂora, and their e ﬀect on epidermal
cells. BC composite ﬁlms were found to have di ﬀerent structure, phy-
sicochemical properties and surface characteristics, and their behavior
towards pathogenic micro ﬂora and ﬁbroblast cell culture di ﬀered de-
pending on the type of antibacterial agent contained in them and itsconcentration in the composite. The disk-di ﬀusion method and the
shake- ﬂask culture method used in this study showed that all experi-
mental composites had pronounced antibacterial activity against E. coli,
Ps. eruginosa, K. pneumoniae, and St. aureus , and the BC/antibiotic
composites were more active than BC/AgNp ones; S. aureus was the
most susceptible to the e ﬀect of BC composites. No potential cytotoxi-
city was detected in any of the BC/AgNp composites in the NIH 3T3
mouse ﬁbroblast cell culture, in contrast to the BC/antibiotic compo-
sites. These results suggest that BC composites constructed in the pre-sent study hold promise as dressings for managing wounds, including
contaminated ones.
Acknowledgement
The reported study was funded by Russian Foundation for Basic
Research, Government of Krasnoyarsk Territory, Krasnoyarsk Region
Science and Technology Support Fund to the research project №16-43-
242024. The study was supported by the State budget allocated to thefundamental research at the Russian Academy of Sciences (project No
AAAA-A17-117013050028-8).
Fig. 9. The number of viable cells (MTT assay) NIH 3T3 mouse ﬁbroblast cells:on com-
posites at Day 3 of the culture: 1 –control; 2 –pristine cellulose (without silver); 3 –BC/
AgNps 0.0001 M; 4 –BC/AgNps 0.001 M; 5 –BC/AgNps 0.01 M; 6 –BC/amikacin 0.2%; 7
–BC/amikacin 0.6%; 8 –BC/amikacin 1%; 9 –BC/ceftriaxone 1%; 10 –BC/ceftriaxone
4%; 11 –BC/ceftriaxone 6%.
Fig. 10. The number of viable cells (MTT assay) of NIH 3T3 mouse on BC/antibiotic and BC/AgNps after 3 days: C –control; 1 –BC/amikacin 0.2%; 2 –BC/amikacin 0.6%; 3 –BC/
amikacin 1%; 4 –BC/ceftriaxone 1%; 5 –BC/ceftriaxone 4%; 6 –BC/ceftriaxone 6%; 7 –BC/AgNps 0.0001 M; 8 –BC/AgNps 0.001 M; 9 –BC/AgNps 0.01 M.T.G. Volova et al. Polymer Testing 65 (2018) 54–68
66

Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.polymertesting.2017.10.023 .
References
[1] X. Ma, R.M. Wang, F.M. Guan, T.F. Wang, Arti ﬁcial dura mater made from bacterial
cellulose and polyvinyl alcohol, CN Patent ZL200710015537 (2010).
[2] S. Saska, H.S. Barud, A.M.M. Gaspar, R. Marchetto, S.J.L. Ribeiro, Y. Messaddeq,
Bacterial cellulose-hydroxyapatite nanocomposites for bone regeneration, Int. J.
Biomater. 2011 (2011) 1 –8https://doi.org/10.1155/2011/175362 .
[3] W.C. Lin, C.C. Lien, H.J. Yeh, C.M. Yu, S.H. Hsu, Bacterial cellulose and bacterial
cellulose chitosan membranes for wound dressing applications, Carbohydr. Polym.94 (1) (2013) 603 –611https://doi.org/10.1016/j.carbpol.2013.01.076 .
[4] M. Culebras, C.J. Grande, F.G. Torres, O.P. Troncoso, C.M. Gomez, M.C. Bañó,
Optimization of cell growth on bacterial cellulose by adsorption of collagen and
poly-L-lysine, Int. J. Polym. Mater. Po. 64 (8) (2015) 411 –415https://doi.org/10.
1080/00914037.2014.958829 .
[5] N. Shah, M. Ul-Islam, W.A. Khattak, J.K. Park, Overview of bacterial cellulose
composites: a multipurpose advanced material, Carbohydr. Polym. 98 (2) (2013)
1585 –1598 https://doi.org/10.1016/j.carbpol.2013.08.018 .
[6] H. Kwak, J.E. Kim, J. Go, E.K. Koh, S.H. Song, H.J. Son, H.S. Kim, Y.H. Yun,
Y.J. Jung, D.Y. Hwang, Bacterial cellulose membrane produced by Acetobacter sp.
A10 for burn wound dressing applications, Carbohydr. Polym. 122 (2015) 387 –398
https://doi.org/10.1016/j.carbpol.2014.10.049 .
[7] W.S. Chang, H.H. Chen, Physical properties of bacterial cellulose composites for
wound dressings, Food Hydrocoll. 53 (2016) 75 –83https://doi.org/10.1016/j.
foodhyd.2014.12.009 .
[8] S.L. Percival, P.G. Bowler, D. Russell, Bacterial resistance to silver in wound care, J.
Hosp. Infect. 60 (1) (2005) 1 –7https://doi.org/10.1016/j.jhin.2004.11.014 .
[9] M.L. Dorbe, A. Stoica-Guzum, Antimicrobial Ag-Polyvinyl alcohol-bacterial cellu-
lose composite ﬁlms, J. Biobased Mater. Bioenergy 7 (2013) 157 –162https://doi.
org/10.1166/jbmb.2013.1272 .
[10] M. Sureshkumar, D.Y. Siswanto, C.K. Lee, Magnetic antimicrobial nanocomposite
based on bacterial cellulose and silver nanoparticles, J. Mater. Chem. 20 (2010)
6948 –6955 https://doi.org/10.1039/c0jm00565g .
[11] J. Feng, Q. Shi, W. Li, X. Shu, A. Chen, X. Xie, X. Huang, Antimicrobial activity of
silver nanoparticles in situ growth on TEMPO-mediated oxidized bacterial cellulose,
Cellulose 21 (2014) 4557 –4567 https://doi.org/10.1007/s10570-014-0449-2 .
[12] G. Yang, J. Xie, Y. Deng, Y. Bian, F. Hong, Hydrothermal synthesis of bacterial
cellulose/AgNPs composite: a «green» route for antibacterial application,Carbohydr. Polym. 87 (4) (2012) 2482 –2487 https://doi.org/10.1016/j.carbpol.
2011.11.017 .
[13] X. Wen, Y. Zheng, J. Wu, L. Yue, C. Wang, J. Luan, Z. Wu, K. Wang, In vitro and in
vivo investigation of bacterial cellulose dressing containing uniform silver sulfa-diazine nanoparticles for burn wound healing, Prog. Nat. Sci. Mater. Int. 25 (2015)197–203https://doi.org/10.1016/j.pnsc.2015.05.004 .
[14] J. Wu, Y. Zheng, W. Song, J. Luan, X. Wen, Z. Wu, X. Chen, Q. Wang, S. Guo, In situ
synthesis of silver-nanoparticles/bacterial cellulose composites for slow-released
antimicrobial wound dressing, Carbohydr. Polym. 102 (2014) 762 –771https://doi.
org/10.1016/j.carbpol.2013.10.093 .
[15] V. Sadanand, H. Tian, A. Varada Rajulu, B. Satyanarayana, Antibacterial cotton
fabric with in situ generated silver nanoparticles by one-step hydrothermal method,
Int. J. Polym. Anal. Charact. 22 (3) (2017) 275 –279https://doi.org/10.1080/
1023666X.2017.1287828 .
[16]
V. Sadanand, T.H. Feng, A. Varada Rajulu, B. Satyanarayana, Preparation and
properties of low-cost cotton nanocomposite fabrics with in situ-generated copper
nanoparticles by simple hydrothermal method, Int. J. Polym. Anal. Charact. (2017)1–8https://doi.org/10.1080/1023666X.2017.1344916 .
[17] V. Sadanand, N. Rajini, B. Satyanarayana, A. Varada Rajulu, Preparation and
properties of cellulose/silver nanoparticle composites with in situ-generated silver
nanoparticles using Ocimum sanctum leaf extract, Int. J. Polym. Anal. Charact. 21
(5) (2016) 408 –416https://doi.org/10.1080/1023666X.2016.1161100 .
[18] P. Sivaranjana, E.R. Nagarajan, N. Rajini, M. Jawaid, A. Varada Rajulu, Cellulose
nanocomposite ﬁlms with in situ generated silver nanoparticles using Cassia alata
leaf extract as a reducing agent, Int. J. Biol. Macromol. 99 (2017) 223 –232https://
doi.org/10.1016/j.ijbiomac.2017.02.070 .
[19] L. Muthulakshmi, N. Rajini, H. Nellaiah, T. Kathiresan, M. Jawaid, A.V. Rajulu,
Preparation and properties of cellulose nanocomposite ﬁlms with in situ generated
copper nanoparticles using Terminalia catappa leaf extract, Int. J. Biol. Macromol.95 (2017) 1064 –1071 https://doi.org/10.1016/j.ijbiomac.2016.09.114 0141 .
[20] L. Muthulakshmi, N. Rajini, H. Nellaiah, T. Kathiresan, M. Jawaid, A.V. Rajulu,
Experimental investigation of cellulose/silver nanocomposites using in situ gen-
eration method, J. Polym. Environ. 24 (2016), https://doi.org/10.1007/s10924-
016-0871-7 .
[21] M. Khalil, J. Yu, N. Liu, R.L. Lee, Hydrothermal synthesis, characterization, and
growth mechanism of hematite nanoparticles, J. Nanopart. Res. 16 (2014) 2362
https://doi.org/10.1007/s11051-014-2362-x .
[22] R. Xiong, C. Lu, Y. Wang, Z. Zhou, X. Zhang, Nanoﬁ brillated cellulose as the support
and reductant for the facile synthesis of Fe3O4/Ag nanocomposites with catalytic
and antibacterial activity, J. Mater. Chem. A 47 (2013) 14910 –14918 https://doi.
org/10.1039/c3ta13314a .
[23] R. Xiong, C. Lu, W. Zhang, Z. Zhou, X. Zhang, Facile synthesis of tunable silvernanostructures for antibacterial application using cellulose nanocrystals,Carbohydr. Polym. 95 (2013) 214 –219https://doi.org/10.1016/j.carbpol.2013.02.
077.
[24] H. Yu, B. Sun, D. Zhang, G. Chen, X. Yanga, J. Yao, Reinforcement of biodegradable
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with cellulose nanocrystal/silvernanohybrids as bifunctional nano ﬁllers, J. Mater. Chem. B 48 (2014) 8479 –8489
https://doi.org/10.1039/C4TB01372G .
[25] H.-Y. Yu, X.-Y. Yang, F.-F. Lu, G.-Y. Chen, J.-M. Yao, Fabrication of multifunctional
cellulose nanocrystals/poly(lactic acid)nanocomposites with silver nanoparticles by
spraying method, Carbohydr. Polym. 140 (2016) 209 –219https://doi.org/10.
1016/j.carbpol.2015.12.030 .
[26] W. Xu, Z. Qin, H. Yu, Y. Liu, N. Liu, Z. Zhou, L. Chen, Cellulose nanocrystals as
organic nano ﬁllers for transparent polycarbonate ﬁlms, J. Nanopart. Res. 15 (2013)
1562 –1570 https://doi.org/10.1007/s11051-013-1562-0 .
[27] Y. Han, X. Wu, X. Zhang, Z. Zhou, C. Lu, Reductant-free synthesis of silver nano-
particles-doped cellulose microgels for catalyzing and product separation, ACSSustain. Chem. Eng. 4 (12) (2016) 6322 –6331 https://doi.org/10.1021/
acssuschemeng.6b00889 .
[28] B. Wei, G. Yang, F. Hong, Preparation and evaluation of a kind of bacterial cellulose
dryﬁlms
with antibacterial properties, Carbohydr. Polym. 84 (1) (2011) 533 –538
https://doi.org/10.1016/j.carbpol.2010.12.017 .
[29] W. Shao, H. Liu, S. Wang, J. Wu, M. Huang, H. Min, X. Liu, Controlled release and
antibacterial activity of tetracyclinehydrochloride-loaded bacterial cellulose com-posite membranes, Carbohydr. Polym. 145 (2016) 114 –120https://doi.org/10.
1016/j.carbpol.2016.02.065 .
[30] C.J. Wijaya, S.N. Saputra, F.E. Soetaredjo, J.N. Putro, C.X. Lin, A. Kurniawan,
Y.H. Ju, S. Ismadji, Cellulose nanocrystals from passion fruit peels waste as anti-
biotic drug carrier, Carbohydr. Polym. 175 (2017) 370 –376https://doi.org/10.
1016/j.carbpol.2017.08.004 .
[31] S. Hestrin, M. Schramm, Synthesis of cellulose by Acetobacter xylinum. 2.
Preparation of freeze-dried cells capable of polymerizing glucose to cellulose,Biochem. J. 58 (2) (1954) 345 –352.
[32] S.V. Prudnikova, T.G. Volova, E.I. Shishatskaya, Shtamm bakterii Komagataeibacter
xylinus –produtsent bakterialnoi tsellulozy (A strain of bacterium Komagataeibacter
xylinus –a producer of bacterial cellulose). RF Patent for an invention No. 2568605.
Priority of 11 December 2014. Registered the RF State Register on 27 October 2015(in Russian).
[33] S.V. Prudnikova, I.P. Shidlovsky, The new strain of acetic acid bacteria
Komagataeibacter xylinus B-12068 –producer of bacterial cellulose for biomedical
applications, SibFU J. Biol. 10 (2) (2017) 246 –254https://doi.org/10.17516/1997-
1389-0017 .
[34] G. Yang, J. Xie, Y. Deng, Y. Bian, F. Hong, Hydrothermal synthesis of bacterial
cellulose/AgNPs composite: a «green» route for antibacterial application,Carbohydr. Polym. 87 (4) (2012) 2482 –2487 https://doi.org/10.1016/j.carbpol.
2011.11.017 .
[35] D.K. Owens, R.C. Wendt, Estimation of the surface free energy of polymers, J. Appl.
Polym. Sci. 13 (1969) 1741 –1747 https://doi.org/10.1002/app.1969.070130815 .
[36] D.H. Kaelble, Dispersion-polar surface tension properties of organic solids, J. Adhes.
2 (1970) 66 –81https://doi.org/10.1080/0021846708544582 .
[37] R. Jung, Y. Kim, H.S. Kim, H.J. Jin, Antimicrobial properties of hydrated cellulose
membranes with silver nanoparticles, J. Biomater. Sci. Polym. Ed. 20 (3) (2009)
311–324https://doi.org/10.1163/156856209X412182 .
[38] T. Maneerung, S. Tokura, R. Rujiravanit, Impregnation of silver nanoparticles into
bacterial cellulose for antimicrobial wound dressing, Carbohydr. Polym. 72 (1)(2008) 43 –51https://doi.org/10.1016/j.carbpol.2007.07.025 .
[39] H.G.O. Barud, R.R. Silva, H. Silva Barud, A. Tercjak, J. Gutierrez, W.R. Lustri,
O.B. Oliveira Junior, S.J.L. Ribeiro, A multipurpose natural and renewable polymer
in medical applications: bacterial cellulose, Carbohyd. Polym. 153 (2016) 406 –420
https://doi.org/10.1016/j.carbpol.2016.07.059 .
[40] A.D. French, Idealized powder di ﬀraction patterns for cellulose polymorphs,
Cellulose 21 (2) (2014) 885 –896https://doi.org/10.1007/s10570-013-0030-4 .
[41] F. Mohammadkazemi, M. Azin, A. Ashori, Production of bacterial cellulose using
diﬀerent
carbon sources and culture media, Carbohydr. Polym. 117 (2015)
518–523https://doi.org/10.1016/j.carbpol.2014.10.008 .
[42] C. Huang, H.J. Guo, L. Xiong, B. Wang, S.L. Shi, X.F. Chen, X.Q. Lin, C. Wang,
J. Luo, X.D. Chen, Using wastewater after lipid fermentation as substrate for bac-terial cellulose production by Gluconacetobacter xylinus, Carbohydr. Polym. 136
(2016) 198 –202https://doi.org/10.1016/j.carbpol.2015.09.043 .
[43] W. Shao, H. Liu, X. Liu, H. Sun, S. Wang, R. Zhang, pH-responsive release behavior
and anti-bacterial activity of bacterialcellulose-silver nanocomposites, Int. J. Biol.
Macromol. 76 (2015) 209 –217https://doi.org/10.1016/j.ijbiomac.2015.02.048 .
[44] Z. Yan, S. Chen, H. Wang, B. Wang, J. Jiang, Biosynthesis of bacterialcellulose/
multi-walled carbon nanotubes in agitated culture, Carbohydr. Polym. 74 (2008)659–665https://doi.org/10.3390/ma9030183 .
[45] Y. Zhang, H. Peng, W. Huang, Y. Zhou, D. Yan, Facile preparation and character-
ization of highly antimicrobial colloid Ag or Au nanoparticles, J. Colloid Interface
Sci. 325 (2008) 371 –376https://doi.org/10.1016/j.jcis.2008.05.063 .
[46] H.S. Barud, C.A. Ribeiro, M.S. Crespi, M.A.U. Martines, J. Dexpert-Ghys,
R.F.C. Marques, S.J.L. Ribeiro, Thermal characterization of bacterial cellulose –-
phosphate composite membranes, J. Therm. Anal. Calorim. 87 (3) (2007) 815 –818
https://doi.org/10.1007/s10973-006-8170-5 .
[47] A. Vazquez, M.L. Foresti, P. Cerrutti, M. Galvagno, Bacterial cellulose from simple
and low cost production media by Gluconacetobacter xylinus, J. Polym. Environ. 21(2) (2013) 545 –554https://doi.org/10.1007/s10924-012-0541-3 .
[48] B. Surma-Ś lusarska, S. Presler, D. Danielewicz, Characteristics of bacterial cellulose
obtained from Acetobacter xylinum culture for application in papermaking, FTEE 4T.G. Volova et al.
Polymer Testing 65 (2018) 54–68
67

(69) (2008) 108 –111https://doi.org/10.21833/ijaas.2017.03.004 .
[49] Y. Feng, X. Zhang, Y. Shen, K. Yoshino, W. Feng, A mechanically strong, ﬂexible and
conductive ﬁlm based on bacterial cellulose/graphene nanocomposite, Carbohydr.
Polym. 87 (1) (2012) 644 –649https://doi.org/10.1016/j.carbpol.2011.08.039 .
[50] A. Yoshino, M. Tabuchi, M. Uo, H. Tatsumi, K. Hideshima, S. Kondo, J. Sekine,
Applicability of bacterial cellulose as an alternative to paper points in endodontic
treatment, Acta Biomater. 9 (4) (2013) 6116 –6122 https://doi.org/10.1016/j.
actbio.2012.12.022 .
[51] Y. Wan, D. Hu, G. Xiong, D. Li, R. Guo, H. Luo, Directional ﬂuid induced self-
assembly of oriented bacterial cellulose nano ﬁbers for potential biomimetic tissue
engineering sca ﬀolds, Mater. Chem. Phys. 145 (2015) 7 –11https://doi.org/10.
1016/j.matchemphys.2014.10.037 .
[52] V. Sadanand, N. Rajini, A.V. Rajulu, B. Satyanarayana, Preparation of cellulose
composites with in situ generated copper nanoparticles using leaf extract and theirproperties, Carbohydr. Polym. 150 (2016) 32 –39https://doi.org/10.1016/j.
carbpol.2016.04.121 .
[53] B. Jia, Y. Mei, L. Cheng, J. Zhou, L. Zhang, Preparation of copper nanoparticles
coated cellulose ﬁlms with antibacterial properties through one-step reduction, ACS
Appl. Mater. Interfaces 4 (6) (2012) 2897 –2902 https://doi.org/10.1021/
am3007609 .
[54] L. Long, X. Yuan, Z. Li, K. Li, Z. Cui, X. Zhang, J. Sheng, Anti-fouling properties of
polylactic acid ﬁlm modi ﬁed by pegylated phosphorylcholine derivatives, Mater.
Chem. Phys. 143 (2014) 929 –938https://doi.org/10.1016/j.matchemphys.2013.
09.041 .
[55] B.L. Wang, Z. Ye, Y. Tang, H. Liu, Q. Lin, H. Chen, K. Nan, Loading of antibioticsinto polyelectrolyte multilayers after self-assembly and tunable release by catechol
reaction, J. Phys. Chem. C 120 (2016) 6145 –6155 https://doi.org/10.1021/acs.
jpcc.6b00957 .
[56] B.L. Wang, T.W. Jin, Y.M. Han, C.H. Shen, Q. Li, Q.K. Lin, H. Chen, Bio-inspired
terpolymers containing dopamine, cations and MPC: a versatile platform to con-struct a recycle antibacterial and antifouling surface, J. Mater. Chem. 3 (2015)
5501 –5510 https://doi.org/10.1039/C5TB00597C .
[57] S. Dominguez-Medina, J. Blankenburg, J. Olson, C.F. Landes, S. Link, Adsorption of
a protein monolayer via hydrophobic interactions prevents nanoparticle aggrega-tion under harsh environmental conditions, ACS Sustain Chem. Eng. 1 (7) (2013)
833–842https://doi.org/10.1021/sc400042h .
[58] Х. Xu, T. Jin, B. Zhang, H. Liu, Z. Ye, Q. Xu, H. Chen, B. Wang, In vitro and in vivo
evaluation of the antibacterial properties of a nisingrafted hydrated mucin multi-layerﬁlm, Polym. Test. 57 (2017) 270 –280https://doi.org/10.1016/j.
polymertesting.2016.12.006 .
[59]
J.P. Ruparelia, Strain speci ﬁcity in antimicrobial activity of silver and copper na-
noparticles, Acta Biomater. 4 (3) (2008) 707 –716https://doi.org/10.1016/j.actbio.
2007.11.006 .
[60] S.H. Barud, T. Regiani, R.F.C. Marques, W.R. Lustry, Y. Messaddeq, S.J.L. Ribeiro,
Antimicrobial bacterial cellulose-silver nanoparticles composite membranes, J.Nanomater. 2011 (2011) 1 –8https://doi.org/10.1155/2011/721631 .
[61] J. Zhang, P. Chang, C. Zhang, G. Xiong, H. Lio, Y. Zhu, K. Ren, F. Yao, Y. Wan,
Immobilization of lecithin on bacterial cellulose nano ﬁbers for improved biological
functions, React. Funct. Polym. 91 (2015) 100 –107https://doi.org/10.1016/j.
reactfunctpolym.2015.05.001 .T.G. Volova et al.
Polymer Testing 65 (2018) 54–68
68

Copyright Notice

© Licențiada.org respectă drepturile de proprietate intelectuală și așteaptă ca toți utilizatorii să facă același lucru. Dacă consideri că un conținut de pe site încalcă drepturile tale de autor, te rugăm să trimiți o notificare DMCA.

Acest articol: Contents lists available at ScienceDirect [608920] (ID: 608920)

Dacă considerați că acest conținut vă încalcă drepturile de autor, vă rugăm să depuneți o cerere pe pagina noastră Copyright Takedown.

Contents lists available at ScienceDirect [608920]

Copyright Notice

Rezidentiat 2004 251 PROFIL FARMACIE – INTREBARI – PARTEA a II a [613904]

Cap.1. Structura și construcția sistemelor de acționare electrică 1.1.Introducere Acționările electrice studiază conversia electromecanică a energiei… [617768]

Școala Națională de Sănătate Publică, Management și Prefecționare în Domeniul Sanitar București MANAGEMENTUL CALITĂȚII ÎN SPITALE ACREDITAREA… [605460]

Sistem wireless de monitorizare si control al proceselor din sere Lucrare de disertat ,ie Absolvent Lauren tiu Adrian Opincariu Conduc ator… [601810]

UNIVERSITY POLITEHNICA OF BUCHAREST FACULTY OF AUTOMATIC CONTROL AND COMPUTERS COMPUTER SCIENCE DEPARTMENT HBFXComputer Science – Logo Computer… [615852]

Arnau Folch1, Antonio Costa2 [631828]

Copyright Notice

Similar Posts