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Antibacterial properties of a bacterial cellulose CQD-TiO2 nanocomposite

Samira Malmir, Atiyeh Karbalaei, Mehrab Pourmadadi, Javad

Hamedi, Fatemeh Yazdian, Mona Navaee

PII: S0144-8617(20)30009-6
DOI: https://doi.org/10.1016/j.carbpol.2020.115835
Reference: CARP 115835

To appear in: Carbohydrate Polymers

Received Date: 6 July 2019

Revised Date: 4 January 2020
Accepted Date: 6 January 2020

Please cite this article as: Malmir S, Karbalaei A, Pourmadadi M, Hamedi J, Yazdian F,
Navaee M, Antibacterial properties of a bacterial cellulose CQD-TiO2 nanocomposite,
Carbohydrate Polymers (2020), doi: https://doi.org/10.1016/j.carbpol.2020.115835

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 Antibacterial properties of a bacterial cellulose CQD-TiO2 nanocomposite

Samira Malmir1, Atiyeh Karbalaei2, Mehrab Pourmadadi3, Javad Hamedi4, Fatemeh Yazdian5*, Mona Navaee6
1
Department of Biotechnology, Faculty of Advanced Science and Technology, Tehran Medical Sciences,
Islamic Azad University, Tehran, Iran. Samira.malmir93@gmail.com
2
Department of Biotechnology, Faculty of Advanced Science and Technology, Tehran Medical Sciences,
Islamic Azad University, Tehran, Iran. At.karbalaee@gmail.com
3
Protein research center, Shahid Beheshti University, GC,Tehran, Iran. mehrabpourmadadi@gmail.com
4
Department of Microbial Biotechnology, School of Biology and Centre of Excellence in Phylogeny of Living
Organisms, College of Science, University of Tehran, Tehran, Iran;

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Microbial Technology and Products (MTP) Research Center, University of Tehran, Tehran,
Iran. jhamedi@gmail.com

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5
Department of Life Science Engineering, Faculty of New Science and Technologies, University of Tehran,
Tehran, Iran

6
Pharmaceutical Sciences Research Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran University
of Medical Science, Tehran, Iran -p
Corresponding authors: yazdian@ut.ac.ir. Tel/Fax: +982186093259
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Highlights
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 Cellulose produced by Gluconoacetobacter xylinum does not have antibacterial

properties.
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 CQD-TiO2 nanoparticles have antibacterial properties against Staphylococcus aureus

bacteria.
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 BC/CQD-TiO2 nanocomposite can be used as an antibacterial wound healing bandage.


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Scratch test indicated that the BC/CQD-TiO2 was effective in wound healing

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Abstract
Antibacterial dressing can prevent the occurrence of many infections of wounds. Bacterial
cellulose (BC) has the ability to carry and transfer the medicine to achieve a wound healing
bandage. In this study, Carbon Quantum Dots-Titanium dioxide (CQD-TiO2) nanoparticles
(NP) were added to BC as antibacterial agents. FTIR Spectroscopy illuminated that NPs were
well-bonded to BC. Interestingly, MIC test proved that BC/CQD-TiO2 nanostructure (NS) has
anti-bacterial properties against Staphylococcus aureus. The findings indicated that, CQD-
TiO2 NPs have stronger antibacterial properties with better tensile strength compared to CQD
NPs, in a concentration-dependent manner. Toxicity of CQD-TiO2 NPs on human L929

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fibroblast cells was also evaluated. Most importantly, the results of the scratch test indicated
that the NS was effective in wound healing in L929 cells. The approach in this study may

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provide an alternative to make an antibacterial wound dressing to achieve an effective drug-
based bandage.
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Key words: Bacterial cellulose; Cell Toxicity; CQD-TiO2 nanoparticles.

1. Introduction
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Wound dressing is used to expedite healing and prevent consequent infections
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(Mohammadnejad et al., 2018). Conventional dressings often have weaknesses that increase
the time of wound healing, cause painful swaddling of the dressing and many other problems
for the patient (Poormohammadi Mojaveri et al., 2011b). The discovery of bacterial cellulose
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(BC) has been an effective step in the production of wound dressing because of its interesting
mechanical characteristics in both dry and wet conditions, small size, porosity, water
absorption, ductility, biodegradability and high biocompatibility (Jia et al., 2009). additionally,
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BC as a hydrophilic biodegradable polymer has features in common with extracellular matrix

components making it effective in cell proliferation and tissue engineering scaffolds (Altun et
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al., 2019, Noorani et al., 2018, Khoshraftar et al., 2018). More importantly, high porosity of
the cellulosic membrane develops the ability to pass antibiotics or other drugs to the ulcer and
make an effective physical barrier against any foreign infection concurrently (Cai & Kim, 2010,
Rahmani et al., 2018). As a result, BC can serve as a suitable substrate for drug delivery.
Notably, BC does not have antibacterial activity by itself; and this has made the infection with
microorganisms a concern for the use of BC (Yang et al., 2012). Many studies have made use
of nanoparticles as antibacterial materials to solve this problem (Wen et al., 2015, Tashan et

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al., 2019). Studies show that nanoparticles appear to be a good drug against bacterial infections.
Because nanoparticles directly target the bacterial cell wall, unlike antibiotics that need to
penetrate the cell. Therefore, attention has been drawn to nanoparticles as substances that may
produce less drug resistance than antibiotics (Wang et al., 2017). Bacterial cellulose is made
by acetobacter as a biofilm. In this genus, the species of xylinum produce the highest amount
of cellulose (Hoenich, 2006). The most important feature of cellulose produced by acetobacter
xylinum is that, it adheres well to the intended surface and does not allow water to penetrate,
but gases and water vapor are easily transferred (Wen et al., 2015). This important feature has
made cellulose produced by acetobacter xylinum useful in medical applications and it is likely
to be used in artificial skin fabrication (Keshk, 2014). Some nanoparticles have antibacterial

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properties; therefore, are advantageous in making antibacterial BC composites
(Mohammadnejad et al., 2018, Mohammadrezaei et al., 2018, Mofradnia et al., 2018,

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Maghsoudi et al., 2017). Recent studies indicated the effectiveness of BC-based antibacterial
dressings with impregnated TiO2 NPs versus some strains of Gram-negative and Gram-positive
bacteria (Khan et al., 2015). TiO2 is one of the most widely used NPs in biological and medical
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applications due to its diverse properties including: biocompatibility, strong oxidation and
antibacterial properties having been on the spotlight in recent years. Some properties of TiO2
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are activated by irradiation. Because of the large band gap of TiO2 (Eg = 3.20 eV), it is often
excited under harmful UV light and can only use 5% of incoming solar energy to become
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activated (Gutiérrez et al., 2013). So, in order for TiO2 to take the full advantage of sun light,
its absorption must be shifted to the visible region of electromagnetic spectrum. Additionally,
high rate of recombination of photo generated electron-hole pair and low rate of electron
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transfer to oxygen result in low quantum efficiency. Therefore, researches have suggested that
doping TiO2 with other NPs makes improvement in the response of TiO2 to visible light and
enhances its separation of electron hole pairs (Harikishore et al., 2014).Today, with the aim of
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increasing the efficiency of TiO2 it is recommended to combine it with different metal elements
(Kitano et al., 2007, Macwan et al., 2011, Rupa et al., 2007, Shahmoradi et al., 2018).
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Fluorescent carbon nanoparticles or carbon quantum dots are new classes of carbon based
nanomaterials, catching lots of attention (Lim et al., 2015, Behboudi et al., 2019, Pourmadadi
et al., 2019). Carbon quantum dots (CQDs), in addition to inexpensiveness and availability,
have different functional groups on their surface such as: epoxy, amino, ether, hydroxyl,
carbonyl and carboxylic acid groups which increase the CQDs' reactivity and hydrophilicity
(Singh et al., 2018). It is worth noting that, CQDs chemical stability is much higher than other
quantum dots, and their superior biologic properties like: hydrophilicity, low toxicity and high
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aqueous stability (Singh et al., 2018) make them the best option to combine with TiO2 in
BC/CQD-TiO2NS. Interestingly, CQDs are very small in size (smaller than 10 nm), making
them essential tools for biomedical research, traceable targeted delivery and several therapy
applications (Singh et al., 2018). CQDs have shown great potential for having a wide range of
applications including drug delivery, photo catalysis, photodynamic therapy, electro catalysis,
bio sensing and chemical sensing (Lim et al., 2015). CQDs are also very appealing in Nano-
medicine because they could function as Nano-carriers for tracking and delivering of drugs or
genes (Kumar et al., 2013). In addition, they do not exhibit any clear sign of toxicity in animal
samples and could be used in invivo studies (Tao et al., 2012). Interestingly, researchers have
proven the antibacterial properties of CQDs and it has been proved in a study that Ag-CQDs

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NPs have noticeable antibacterial activities for Gram-positive S. aureus and Gram-negative E. coli
(Roy et al., 2015). According to the mentioned research, CQDs seem to be the best option for

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pairing with TiO2 because of their diverse functional groups, unique properties and small size.
In fact, the small size and functionality of the CQDs enable CQD-TiO2 to fit well into the BC
cavities, thereby providing a suitable base for CQD-TiO2 activity in BC as a wound healing
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material. TiO2's increased anti-cancer properties by CQDs doping, have been previously
proven (Li et al., 2010, Kang et al., 2009, Kang, Tsang, Zhang, et al., 2007, Kang, Tsang,
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Wong, et al., 2007). Furthermore, as mentioned above, their antibacterial properties have been
proven on their own, but the antibacterial properties of carbon quantum dots-Titanium dioxide
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(CQD-TiO2) NPs have not been investigated significantly yet. The novelty of this research is
the synthesis of CQD-TiO2 NPs impregnated with BC as wound healing materials. The small
size of the CQD-TiO2 NPs and various CQD functional groups make these NPs well fit and
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bond into the BC pores. As a result, the substrate is provided for the strong antibacterial
function of TiO2 and CQD.
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2. Materials and Methods

2.1. Preparation of microorganisms
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Gluconacetobacter xylinus (strain PTCC 1734), Staphylococcus aureus and Escherichia coli
were purchased from Iranian Scientific and Industrial Research Organization. All materials
were of analytical grade and used without further purification.

2.2. Production of bacterial cellulose

The Hestrin and Schramm (HS) medium was used for culture of growing G. xylinus containing
4 g/L glucose, 1 g/L peptone, 1 g/L yeast extract, 0/54 g/L Na2HPO4, 0/23 g/L Citric acid

4
monohydrate. HS medium was adjusted to pH 6.5 and autoclaved at 120℃ for 15 min. After
reaching the ambient temperature, 3 ml of a culture medium containing G. xylinus was added
to a 50 ml fresh culture medium and heated at 30 °C. After 3 days, 10 ml of cold sterilized
glycerol was added and homogenized. After homogenization of the culture medium, the
microbial suspension was divided into 1.5 mL micro-tubes. Micro-tubes containing microbial
suspension were first stored at 0 °C and after a few hours at -70 °C. To prepare the inoculum,
a 1.5 mL micro-tube from the microbial bank was added to 100 ml of the HS culture medium.
Cell suspension was then heated at 30 °C for 7 days to produce bacterial cellulose. After
incubation, bacterial cellulose produced on the surface of culture medium was harvested. The
BC was washed with plenty of distilled water and purified by boiling them in 1.0% NaOH for

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2 h, then washed in distilled water (Santos et al., 2015). Finally, BC dewatered by the hot
pressing device and dried at 40℃ for 72 h. In this research, the synthesized cellulose has been

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thoroughly crumbled to perform all cellulose tests (identification and molecular studies) except
for the determination of its mechanical strength.

2.3. Synthesis of nanoparticles

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We synthesized the CQD-TiO2 NPs based on hydrothermal method in the University of Tehran.
To prepare CQD-TiO2 NPs, 1 g of ascorbic acid was mixed with 20 ml of distilled water to let
it completely dissolve. In a separate container, we added 5 g of chitosan to 10 ml of acetic acid
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2% (poor acid dissolves chitosan) and put it on the styrene to thoroughly dissolve the chitosan
and become a clear gel of chitosan. Then, 20 ml of ascorbic acid solution was added to a 10 ml
clear chitosan gel and placed on the styrene to get fully mixed in order to obtain a homogeneous
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solution. The solution was then added to a volume of 0.8 g of TiO2 to be completely dissolved
and suitable for the proper doping of NaOH as an alkaline medium stabilizer. The required
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NaOH content was calculated by the following equation:

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TiO2 + 2 NaOH ----> Na2TiO3 + H2O

X gr NaOH = 1 gr TiO2 ×(1 mol 〖TiO2〗)/(79.866 gr 〖TiO2〗)×(2 mol NaOH )/(1mol 〖
TiO2〗 )× (40 gr NaOH )/(1 mol NaOH)=1 gr NaOH

1g of NaOH was added to the above solution and dissolved completely on the ester at room
temperature. The final solution was poured into a hydrothermal bomb and placed in an oven at

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140 °C for 24 hours to form CQD-TiO2. The final concentration of CQD-TiO2 was 76.66 mg /
ml.

2.4. Impregnation of CQD-TiO2 NPs into Bacterial Cellulose

To impregnate CQD-TiO2 NPs into BC, the CQD-TiO2 NPs solution was sprayed on wet BC
at room temperature. Then by using the heat press machine, the composite was totally
dewatered and dried for 3 days at 40°C.

2.5. Tensile strength and elongation tests

According to the iASTM standard method, the mechanical properties were appraised by
conducting elongation-at-break and tensile strength, by using a Texture Analyzer (Xu et al.,

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2005). In these tests, preconditioned BC/CQD-TiO2 composite cut into 1.0 cm (W) × 5.0 cm
(L) strips and mounted between the grips of the machine with initial grip gap of 50 mm. Sample

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broke at a cross-head speed of 2.0 mm/min; next uniaxially pulled. The results of elongation
and tensile experiments were exhibited by MPa and percentage (%), respectively. Young’s
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module of BC/TiO2-CQDs composite with different weight percentage of TiO2/CQD
nanohybrids are shown in Fig. 7c. Obviously, the addition of nanohybrid increased the Young's
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Modulus.
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2.6. Characterization
The characterization of the generated NPs and composites were defined by Dynamic Light
Scattering (DLS) technique and Scanning Electron Microscope (SEM). Other characteristics
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were distinguished by Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Diffraction
(XRD) (Golzar et al., 2018).
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2.7. Evaluation of antibacterial activity of nanoparticles

2.7.1. The MIC test of CQD-TiO2 NP was performed in three ways: In a solid plate containing
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bacterial culture, in a liquid test in 96 plates and in a disk test.

After checking the purity and carrying out biochemical tests, 24-hour culture was performed.
Then, from both S. aureus and E. coli bacteria with concentrations equal to 0.5 McFarland were
injected into the serum of sterilized physiology. While prepared, it was cultured into the Netrin
agar medium, the wells were then drilled in the medium. Concentrations of 1, 1/2, 1/4, 1/8
µg/ml of diluted CQD-TiO2 NPs were prepared and the amount of 100 µg/ml of them added to
each well. The plates were heated to 37 ° C for 24 hours. The doxycycline antibiotic disk was

6
used as a control to check the inhibition zone caused by the CQD-TiO2 NP. The diameter of
the no-growth halo around the well was investigated by ruler.
2.7.2. MIC liquid test was performed in a 96-well plate and in 3 independent experiments. 10
µg/ml of S. aureus bacteria and 90 µg/ml of Muller Hinton broth were added to each well, and
concentrations of 1, 1/2, 1/4, 1/8 µg/ml of diluted CQD-TiO2 NP were added to corresponding
wells. The plate was incubated at 37 ° C for 24 hours. Then its absorption by spectrophotometer
was investigated at 630 nm.
2.7.3. The lowest concentration of CQD-TiO2 NP having antimicrobial activities in the MIC
liquid method, was diluted. A sterile paper disk impregnated with 100 µg/mlCQD-TiO2 NP
and heated to 45 ° C for 1 hour. S. aureus and E. coli were uniformly cultivated in Netrin agar

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medium. The disk was then immersed in CQD-TiO2 NP at a given distance from the antibiotic
disc on the surface of the plate containing bacteria. The plates were heated to 37 ° C for 24

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hours. The antibiotic ceftriaxone for E. coli and ceftriaxone and amikacin for S. aureus were
used as control to examine the inhibition zone caused by the NP. Finally, by measuring the
diameter of the inhibition zone around the discs, the results were analyzed according to the
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standard Clinical and Laboratory Standards Institute (CLSI) table.
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2.8. Cell toxicity of nanoparticles

MTT test was performed to measure the toxicity of CQD-Tio2 in concentrations of 0 to 1/16
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µg/ml of L929 human cells. Fibroblasts were cultured in 96 well-plates at a concentration of 10

^ 6 µg/ml cells for 24hr. Then the culture medium in each well was replaced with 100μl of FBS
and DMEM. 20μl of nanoparticle solutions with specific concentrations were added to each
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well and incubated for 24 hours. Then, culture medium was replaced with 100μl fresh medium
containing 20μl of MTT (55 mg/ml in PBS) and further incubated for 4 hours. Finally, MTT
containing medium was replaced with 150μl DMSO and then shaken for 15 minutes. The
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absorption was then read at 500nm using a microplate reader. All of the tests were repeated
three times.
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2.9. In vitro wound healing assay

The effect of BC/CQD-TiO2 on cell migration was evaluated by scratch wound assay. This test
was similar to that of Liang et al., with minor modifications. Firstly, human L929 fibroblast
cells were seeded in 24 wells plates and were allowed to proliferate and adhere to the bottom
of the plate overnight. Then, cells were starved in 0.1% FBS medium for 2h and vertical
wounds were created at the cell surface by a sterile 10 μl pipette tip. BC/CQD-TiO2 was added

7
to cells and incubated at 37 °C for 48h. Each well contained 100 μM solution. Cells incubated
with the medium were used as negative controls. Nikon Eclipse Te-2000-U microscope (4X
magnification) was used to image the wounded cells, at the start of the incubations and again
after 48h. The surface of the scratch area (A) was measured using Image J software. Results
were expressed as percentages of wound area for each well using the formula: 100 x (At48 /
At0). Statistical analysis was performed with Sigma Plot software using a Student’s t-test.

3. Result

3.1. Conditions for the growth of Gluconoacetobacter xylinus

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By cultivating G. Xylinus bacteria for 200 hours and examining optical absorption at 600 nm
per 24 hours, the bacterial growth curve was plotted. According to the results, the culture time

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for G. Xylinus was about 168 hours (7 days). About 3 days after inoculation, the bacteria were
in their latent phase, and after 94 hours, the logarithmic phase passes through. According to the
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growth curve, the stagnation phase of the bacteria was short, and after passing less than 24
hours from the end of the logarithmic phase, the bacteria enter the death phase. The cause of
bacterial entry to the death phase may be the maximum cellulose growth on the culture medium
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on day seven.
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3.2. Characterization

3.2.1. Morphology of BC
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The results gained from the SEM analysis of BC produced by G. Xylinus are shown in Fig.2.
BC fibers are porous with interconnected pores and are well-organized in 3-D network
structures. A high rate of porosity was observed in a coherent texture of BC. Similar to this
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study, SEM analysis of BC sample obtained in Wen et al. showed fibrous network with highly
porous structure (Wen et al., 2015).
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3.2.2. Particle size of CQD-TiO2

Evaluation of the results of DLS analysis for CQD-TiO2 NPs in Fig.3, confirms the presence
of NPs with the size of 22.23 nanometers (with intensity of 39.81%).Nanoparticles of this
diameter are very suitable for combination with BC and easily fit into BC cavities (Wen et al.,
2015).

3.2.3. FTIR of CQD-TiO2 NPs

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Fig. 4 shows the FTIR of the CQD-TiO2 NPs. The absorption band is in the C=O region of
1600-1770 nm. The vibrations of the CH2 bond are also seen in the cm-1 region of 1350-1460.
These two couriers are related to CQDs NPs, which do not appear in CQD-TiO2 NPs. In CQD-
TiO2 NPs, all peaks of TiO2 and the Ti-O-Ti absorption band are seen in the range 800-400
nm. The absorption band in the region of 1054 is due to the tensile vibration of the O-O bond.
Absorption bonds in 1631 cm-1 and 3430 cm-1 are resulted from the formation of H-O-H bonds
and vibration of O-H water (Fig. 4).

3.2.4. Crystal structure of CQD-TiO2 NPs

The CQD-TiO2 NPs XRD pattern in Fig. 5, determined reflections at 2θ of 19/25°, 37/77°, and

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96/47°, which are the indicator reflections of this combination. On the other hand, the
reflections at 2θ of 73/19°, 27/52°, 29/60°, belongs to CQDs. FTIR from CQD-TiO2 NPs was

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performed to determine functional groups (Fig. 5). Peak results are roughly similar to the
results of Tian and colleagues' research, which represents peaks in 1050 cm-1 (C-O), 1218 cm-
1 (CO-C) and 1725 cm-1 (C=O) (Tian et al., 2014).

3.2.5. FTIR of the BC/CQD-TiO2

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Fig. 6 shows the FTIR of the BC/CQD-TiO2 produced composite. To prepare the sample, the
NPs were firstly sprayed onto the BC layer, and after drying, the BC was completely crushed
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and powdered; then FTIR was performed. The strong peak in the region of 13800-2995 cm-1 is
related to the tensile vibration of the O-H hydrogen bonding. The bending vibrations of the
HCH and OCH bonds are shown in 11378-1371 cm-1 and tensile vibrations of COC, while
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CCO and CCH bonds are found in 1897 cm-1.The bending vibrations of the C-OH bond are
also seen in 1675-673 cm-1.The peak occurred in 11300-1000 cm-1 is related to the tensile
vibration of C-OH in 1060 cm-1 and the flexural vibration of C-O-C in 1163 cm-1.The two
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indicator peak indices in 11642-1630 cm-1 correspond to link vibration of O = C, which is

caused by vibration of the Ti-O-C bond. The tensile vibration of C-H is also seen in 12910 cm-
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(Fig. 6).

3.3. Tensile strength and elongation trials

Mechanical strength and maximum BC/CQD-TiO2 tensile elongation values are shown in Fig.
7. The tensile elongation values of the BC (as control) and amplified BC with Sprayed CQD-
TiO2NPs are shown in Fig. 7A and Fig 7B respectively. Significantly, the addition of CQD-
TiO2 amplified the final tensile strength and enhanced the Young's Modulus (Fig. 7c), which

9
is similar to the results of Tian and colleagues' experiments (Tian et al., 2014). Increased tensile
strength of BC is due to the coherence between NPs and BC. Interestingly, these results are
consistent with the XRD and FTIR patterns.

3.4. Antibacterial Assay

3.4.1. Antibacterial properties of CQDs NPs
Antibacterial properties of CQDs NPs were examined by MIC test against two bacteria E. coli
and S. aureus as controls (Fig. 8). The results determined that CQDs prevented the growth of
both E. coli (Fig. 8A) and S. aureus (Fig. 8B) bacteria.

3.4.2. Antibacterial properties of CQD-TiO2 NPs

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Antibacterial properties of synthesized CQD-TiO2 NPs were evaluated against bacteria E. coli
and S. aureus by MIC test as shown in (Fig. 8).

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3.4.2.1. The results showed that CQD-TiO2 NPs had no effect on E. coli bacteria (Fig. 8D),
while the inhibition zone was observed around S. aureus bacteria. A 13 mm inhibition zone
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was seen around the disk of antibiotic doxycycline. While, inhibition zones around the disk of
the NPs in 1 µg/ml and ½ µg/ml were 15 mm and 13 mm respectively (Fig. 8C). As a result,
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the minimum inhibitory concentration of CQD-TiO2 for S. aureus bacteria is equal to ½ NP
concentrations. The experiment was carried out in three replicates and we observed that the
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NPs have no effect on the E. coli bacteria. It is noteworthy that the inhibition zone of S. aureus
bacteria around the CQD-TiO2 disk were more than the inhibition zone around the CQD disc.
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3.4.2.2. To ensure the results, liquid MIC test was performed for both bacteria. Concentrations
of 1, 1/2, 1/4, 1/8 µg/ml of diluted CQD-TiO2 NP were added to corresponding wells in 96-
well plate. The results showed that the lowest concentration of NPs with antibacterial activity
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was at ½ µg/ml.After 24 hours of incubation, the absorbance of each well was read by the
spectrophotometer so that the MacFarland 0.5 opacity for S. aureus was equal to 0.1 nm and
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optical absorption was recorded at 630 nm. According to the results of MIC, CQD-TiO2 NPs
did not have antibacterial effects on E. coli.

3.4.2.3. For further examination of antibacterial properties, comparing the inhibition zones
around Ceftriaxone, amikacin antibiotics and paper disk impregnated with CQD-TiO2 NPs
were used (Fig. 8E, F). The paper disk impregnated with the CQD-TiO2 NPs has no effect on
the growth of E. coli bacteria, like the MIC test (Fig. 8E). While paper disk impregnated with

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the CQD-TiO2 NPs caused a significant inhibition zone around the S. aureus bacteria. A 13
mm inhibition zone around the paper disk, 24 mm around the Ceftriaxone antibiotic disc and a
15 mm around amikacin antibiotic disc were observed (Fig. 8F).
The results exhibited that the antibacterial activity against E. coli was very lower than when it
was against S. aureus. This concept is because of the discrepancy in the structure of the cell
walls between gram-negative gram-positive bacteria (Wu et al., 2014).

3.5. Cytotoxicity of nanoparticle

The MTT test was performed to measure the toxicity of CQD-TiO2. The highest viability (over

98%) was at 1/8 concentration, which is shown in Fig.9 .The main factor for a material to have

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biocompatibility properties is such that, as a scaffold material it allows cell attachment,

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proliferation, differentiation ;and it is also non-toxic (Kiss & Brebbia, 2013). As shown in

Fig.9, CQD-TiO2 NPs did not exert destructive and toxic effects on L929 cells compared to
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control cells after 24h. Any material designed can be a candidate for wound dressing if it has

cell viability of above 85%, meaning that it is a non-toxic material and can be used in wound
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dressing (Altun et al., 2018).
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3.6. Cellular migration of human L929 fibroblast cells

The impact of CQD-TiO2 NPs and BC/CQD-TiO2 on L929 cell migration were investigated
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by vitro scratch assay in figure 10. When wounded monolayers of L929 cells were treated with

BC/CQD-TiO2, the cells like control group, easily closed the gap over 48h.
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4. Discussion
Generally speaking, conventional dressings have weaknesses including wound drying and
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sticking, leading them to become allergenic to some people. This increases the time of wound
healing resulting into painful dressing and many other problems for the patient (Härle, 1989,
Maghsoudi et al., 2017). For these reasons, researchers have been trying to produce dressings
with distinctive features compared to traditional ones for many years. Their research achieved
great results with the discovery of bacterial cellulose having many features of an ideal dressing
(Czaja et al., 2006, Khezri et al., 2018). In this research and many other studies, BC has been
used as a wound coating, due to high water holding capacity and proper exchange of water

11
vapor with the outside environment. The presence of pores in nanometer range allows entry
into molecules, high flexibility and compatibility with the immune system (Czaja et al., 2006,
Wang & Yi, 2008). It should be noted that the use of BC as a substance not being harmful to
the human body, has been documented in several studies, for example, Azeredo et al. used BC
for food packaging (Azeredo et al., 2019).The BC produced by G.Xylinus bacteria has porosity
and regular 3D network structure, which is suitable for adhesion, proliferation and cellular
activity (Standal et al., 1994). Wen et al, have shown such a structure for BC (Wen et al., 2015).
Therefore, BC has been used as a conduit for transporting and transferring drugs. It is
noteworthy to mention that in recent decades, different microbial and non-microbial polymers
have been used to transport and transfer drugs (Gombotz & Wee, 1998, Malekimusavi et al.,

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2019). Pour mohammadi et al. stated that BC, along with all unique properties that it has as a
dressing, can absorb antibiotic tetracycline hydrochloride and release it slowly

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(Poormohammadi Mojaveri et al., 2011a). Among the antibacterial materials, nanoparticles
play an important role (Hashemi et al., 2017, Tashan et al., 2019). TiO2 is a very popular NP
that its photocatalytic and antibacterial properties have been proven (Karbalaei et al., 2016),
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for instance, Amer et al, have reported that titanium-coated wound dressing can be very
effective in wound healing (Amer et al., 2018). In addition, researches have shown that the
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combination of TiO2 with other NPs increases its power and efficiency. It has been reported
that Ag-TiO2 has a stronger anti-bacterial effect against S. aureus than pure TiO2, and the main
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applications of Ag-TiO2 are in antibacterial agents, coatings and gas sensors production (Hsieh
et al., 2012, Ashkarran, 2011). Among NPs with antibacterial properties, CQDs have attracted
much attention due to numerous medical applications. CQDs pharmaceutical applications are
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in their unique physical and chemical properties, biocompatibility, small size and stability
(Zheng et al., 2015). Singh Saud et al. reported that the CQDs-TiO2 nanofibers exhibited
photocatalytic and antibacterial properties under visible light irradiation (against E. coli) more
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than TiO2 NPs alone (Saud et al., 2015).While in this study, antibacterial property of CQDs-
TiO2 NPs was also proved in non-irradiation of light against S. aureus bacteria. Some scholars
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found that the antibacterial mechanism of the CQDs-TiO2 NPs was such that the electron pair
cavity reacted with O2 or OH- to produce reactive oxygen species (ROS) and ROS could react
with DNA, cell membrane and cellular proteins that ultimately led to bacterial death (Ristic et
al., 2014, Han et al., 2015).
In general, it can be stated that, CQDs-TiO2 NP as an antibacterial agent, along with unique BC
specific properties, can be considered as a clear future for production of low-cost, bio-based
products with diverse applications (Czaja et al., 2007).
12
5. Conclusion
In order to achieve an active injectable coating and accelerate the wound healing, BC/CQD-
TiO2 composite was developed. SEM, FTIR and XRD from CQD-TiO2 NPs indicate the proper
connection of CQD to TiO2. CQD-TiO2 NPs have a variety of functional groups that can easily
react with BC and spread on it, which is proved by FTIR. The presence of CQD-TiO2 NPs
enhanced the mechanical strength and antibacterial activity of BC. According to the results, it
can be claimed that CQD-TiO2 NPs have anti-bacterial properties and their inoculation on
bacterial cellulose surface can be suitable for further studies in the field of producing an
appropriate coating for wound healing.

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Fig.1. Separation and purification steps of cellulose. A: wet cellulose and B: dried cellulose.
BC plate was dried in an oven at 40 ° C for 72 hours.

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Fig.2.SEM image of bacterial cellulose. Cellulose layer produced by G. Xylinus bacteria with
a regular tissue with large cavities.

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Fig.3. DLS diagram of TiO2-CQD NPs. The TiO2-CQD NPs can be seen at 22.23 nanometers
in size (with intensity of 39.81%). -p
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Fig.4. FTIR spectrum of TiO2-CQD NPs.

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Fig.5. X-ray diffraction pattern of CQD-TiO2 NPs.

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Fig.6. FTIR spectrum of the BC/CQD-TiO2 composite.

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Fig.7. Tensile elongation diagram of pure BC (A). Tensile elongation diagram of BC/CQD-
TiO2 nanocomposite (B). Young’s modulus (C).

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Fig.8. Plate containing E. coli bacteria (A). Distilled water was used as control and the
inhibition zone around E. coli bacteria treated with CQD NPs is significant (A). Plate
containing S. aureus bacteria (B). Distilled water was used as control and the inhibition zone
around S. aureus bacteria treated with CQD NPs is significant (B). Plate containing S. aureus
bacteria and doxycycline antibiotic (C). The diameter of the inhibition zone of the bacteria at
½ µg/ml concentration of CQD-TiO2 NPs is equal to the diameter of the inhibition zone around
the antibiotic disk. At concentrations of less than ½ µg/ml, no inhibition zone is observed and
there is no antibacterial activity (C). Plate containing E. coli bacteria and doxycycline antibiotic

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disc (D). There is no inhibition zone around the bacteria treated with CQD-TiO2 NPs at
different concentrations, while inhibition zone around the antibiotic disk are well seen (D).
Plate containing E. coli, antibiotic ceftriaxone disk and CQD-TiO2 NPs impregnated paper disk
(E). There is no inhibition zone around the CQD-TiO2 NPs disc (E), while there is an inhibition
zone around the antibiotic disc. Plate containing S. aureus bacteria, Ceftriaxone antibiotic disc,
amikacin antibiotic disc and CQD-TiO2 NPs impregnated paper disk (F). Inhibition zone
around the paper disk and the antibiotic disc was seen, but the diameter of the inhibition zone
of the bacteria around the CQD-TiO2 disk is less than the diameter of the inhibition zone around
the disk of antibiotics (F).

120

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Viability (% of control)

100
*
80 **
**

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***
60

40

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Control 15.332 7.666 3.833 mixture mixture mixture
(15.332) (7.666) (3.833)
Concentration (µg/mL)
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Fig.9. The viability of L929 human cells, 24 hours after treatment with CQD-TiO2 NPs. L929
human cells were treated with CQD-TiO2 NPs at concentrations of 0 to 1/16 μg/ml. Data are
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expressed in three independent experiments in duplicate. All tested samples were relative to
the control set at 100%. (*P<0.001,** P value <0.05 and ***P<0.0001).
Mixture: cellulose and composite
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 (A)

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(B) -p
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**
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Wound area (% of Time)

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80
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0
Control 15.332 7.666 3.833 mixture mixture mixture
(15.332) (7.666) (3.833)
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Concentration (µg/mL)
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Fig.10. Determination of the effect of the BC/CQD-TiO2 NS on L929 cell migration by in vitro
scratch assay. The cell monolayers were scraped and treated with different concentrations of
CQD-TiO2 NPs with and without BC. Cells were then photographed under an inverted
microscope at 0 h and 24 h after scraping (4X magnification). The dotted lines define the areas
lacking cells. (B) Quantification of cell migration according to percentage of wound area. Data
are expressed in five randomly selected fields of the denuded areas of triplicate experiments.
Difference between control and other groups is significant at p < 0.01(**), p < 0.001(***).
Mixture: cellulose and composite

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Standard Test Method for Tensile Properties

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