JDDST D 21 02132

Journal of Drug Delivery Science and Technology
Bacterial cellulose-based device for sustained release of the antibacterial drug

rifampicin for topical use in the treatment of skin wounds infection
--Manuscript Draft--
Manuscript Number: JDDST-D-21-02132
Article Type: Research Paper
Keywords: Bacterial cellulose; biofilm-producing, sustained drug release; rifampicin; wound

infection, molecular dynamic simulation
Corresponding Author: Wilton Rogério Lustri

Centro Universitário de Araraquara: Universidade de Araraquara
Araraquara, São Paulo BRAZIL
First Author: Wilton Rogério Lustri
Order of Authors: Wilton Rogério Lustri
Caroline Yamada
Silmara Cristina Lazarini Frajácomo
Nayara C. Amaral
Hernane S. Barud
Adriano Marques Gonçalves
Rafael Mariano-Neto
Guedmiller S. Oliveira
Abstract: Skin constitutes the principal physical barrier for defense of the body against the entry
of microorganisms. When a disruption occurs, due cutaneous trauma or surgery, the
result can be a skin and soft-tissue infections (SSTI) and skin wound infection (SWI)
disorder by several bacteria Gram-positive and Gram-negative biofilm-producing as
Staphylococcus aureus, that can progress to bacteremia, pneumonia, further causes
metastatic infections such as infective endocarditis, osteomyelitis, systemic infections,
through the blood migration, being an important cause of morbidity and mortality
among hospitalized patients and a major therapeutic challenge for clinicians. The
present work had the aims the production of bacterial cellulose membrane (BC), by K.
hansenii ATCC 23769 and the surface modifications by methanol (MetOH) and heat
treatment to produce BC (BC-MetOH) with different fiber densities, thickness and
entangling that allow the use as support for sustained release of the antibacterial drug
RIF by BC-MetOH-RIF aiming the future use as topical dressing for infected wound
with biofilm-producing bacteria, using S. aureus ATCC 25923 biofilm-producing as a
model. BC treated with dH2O (BC-dH2O) was used as control. The obtained results
demonstrated efficiency of sustained release of antibiotic RIF, by BC-MetOH-RIF, with
the great possibility of adding to the therapeutic arsenal for the treatment of bacterial
infections of skin and soft tissues, including the bacteria biofilm-producing, since RIF is
a broad-spectrum, capable of penetrates readily through the biofilm, and not
commercially available for topical use.
Suggested Reviewers: Agnieszka Tercjak

University of the Basque Country: Universidad del Pais Vasco
agnieszka.tercjaks@ehu.es
Juncal Gutierrez
Eskola Politeknikoa de Donostia-San Sebastián: Universidad del Pais Vasco Escuela
Universitaria Politecnica de San Sebastian
juncal.gutierrez@ehu.es
Guillermo Raúl Castro

National University of La Plata: Universidad Nacional de la Plata
grcastro@gmail.com
Suresh Narine
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Trent University
sureshnarine@trentu.ca
Opposed Reviewers:
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Cover Letter
UNIVERSITY OF ARARAQUARA – UNIARA

Laboratory of Medical Chemistry and Regenerative Medicine-QUIMMERA
14801-320 - Araraquara, São Paulo, Brazil
_______________________________________________________________________
November 10th, 2021.
To
Professor Florence Siepmann
Editor-in-Chief, Journal of Drug Delivery Science Technology
University of Lille Faculty of Pharmacy, Lille Cedex, France
We are sending you the manuscript entitled “Bacterial cellulose-based device for
sustained release of the antibacterial drug rifampicin for topical use in the treatment of skin
wounds infection” to be published as Original Research in Journal of Drug Delivery Science
Technology. This manuscript is original and unpublished, and it is not being considered for
publication elsewhere. The motivation and scientific interest of this work is centered obtains BC,
by the bacterial strain Komagataeibacter hansenii ATCC 23769, aimed surface modification by
methanol and heat treatment to obtention BC-MetOH-RIF with release capacity of rifampicin
(RIF), a semi-synthetic antibiotic with a broad spectrum of action with great possibility of
adding, in a short period of time, to the therapeutic arsenal for the treatment of bacterial
infections of skin and soft tissues, including the biofilm-producing, since RIF is not
commercially available for topical use. The BC-MetOH-RIF obtained were characterized,
by scanning electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric,
and by derivative thermogravimetry analyses. Were obtained BC-MetOH-RIF with high
sustained release capacity of RIF (696 hours) by disc diffusion methods, demonstrating the
potential of the BC-MetOH-RIF to designs of new drug release systems with biomedical
applications. Since the manuscript is in a multidisciplinary field and focused on the production of
BC-MetOH-RIF, we believe it fits the scope of this journal.
Hoping to hear from you soon and thanking you in advance,
Sincerely yours,
Wilton R. Lustri
Professor Wilton Rogério Lustri, Ph.D.
Department of Biological and Health Sciences
University of Araraquara 14801-320, Araraquara-SP, Brazil
E-mail: wrlustri@uniara.edu.br
Graphical Abstract
Yamada et al., 2021
Graphical Abstract
Manuscript File Click here to access/download;Manuscript File;Manuscript
Yamada et al. 2021 (2).docx
Bacterial cellulose-based device for sustained release of the antibacterial

drug rifampicin for topical use in the treatment of skin wounds infection
Caroline Yamada1a, Silmara C. Lazarini1a, Nayara C. do Amaral1, Hernane S. Barud1,

Adriano Marques Gonçalves1, Rafael Mariano-Neto2, Guedmiller Souza de Oliveira2,
Wilton R. Lustri1a.
1
Department of Biological and Health Sciences - University of Araraquara – UNIARA,
14801-320 Araraquara, SP, Brazil.
2
Instituto de Química, Universidade Federal de Uberlândia, Av. João Naves de Ávila,
2121 Uberlândia, MG, Brazil
*Corresponding author: Wilton R. Lustri
Address: Laboratory of Bacterial Cellulose and Medicinal Chemistry, University of

Araraquara - SP – Brazil
Telephone: +55(16) 99154-7476
E-mail address: wrlustri@uniara.edu.br
a
These authors contributed equally to the manuscript.
Abstract
Skin constitutes the principal physical barrier for defense of the body against the entry of
microorganisms. When a disruption occurs, due cutaneous trauma or surgery, the result
can be a skin and soft-tissue infections (SSTI) and skin wound infection (SWI) disorder
by several bacteria Gram-positive and Gram-negative biofilm-producing as
Staphylococcus aureus, that can progress to bacteremia, pneumonia, further causes
metastatic infections such as infective endocarditis, osteomyelitis, systemic infections,
through the blood migration, being an important cause of morbidity and mortality among
hospitalized patients and a major therapeutic challenge for clinicians. The present work
had the aims the production of bacterial cellulose membrane (BC), by K. hansenii ATCC
23769 and the surface modifications by methanol (MetOH) and heat treatment to produce
BC (BC-MetOH) with different fiber densities, thickness and entangling that allow the
use as support for sustained release of the antibacterial drug RIF by BC-MetOH-RIF
aiming the future use as topical dressing for infected wound with biofilm-producing
bacteria, using S. aureus ATCC 25923 biofilm-producing as a model. BC treated with
dH2O (BC-dH2O) was used as control. The obtained results demonstrated efficiency of
sustained release of antibiotic RIF, by BC-MetOH-RIF, with the great possibility of
adding to the therapeutic arsenal for the treatment of bacterial infections of skin and soft
tissues, including the bacteria biofilm-producing, since RIF is a broad-spectrum, capable
of penetrates readily through the biofilm, and not commercially available for topical use.
Keywords: Bacterial cellulose; biofilm-producing, sustained drug release; rifampicin;

wound infection, molecular dynamic simulation
1. Introduction
Skin constitute the principal physical barrier for defense of the body against the
entry of microorganisms [1] and when a disruption occurs, due cutaneous trauma or
surgery, the result can be initially a skin and soft-tissue infections (SSTI) or skin wound
infection (SWI) by microorganisms penetration, being mostly caused by Gram-positive
and Gram-negative microorganisms [2–5], which can progress to systemic infections [3],
increased healthcare costs, and a deterioration in the overall treatment results [6]. In
addition, according to data of the World Health Organization until 2050, bacterial
infections will be greater than cancers cases [7,8].
Due to the fact that the topical application of antimicrobials promotes higher
concentration at the site, less systemic adverse effects and less selection of resistant
bacteria, this route of administration has advantages in the control of wound infections
compared to systemic antibiotics [9]. Recent publications also cite several relevant results
from the topical use of antimicrobials [10–14].
Nevertheless, wounds infection, especially considered chronic. that do not heal

within 4 to 8 weeks, are difficult to treat [15], being highly susceptible (more than 80%)
to contamination by bacterial pathogens that produce biofilms [16–18] resistant to
antibiotics of different classes [19] being a of significant causes of difficulty in healing
and can progress to fatal systemic infections [9].
S. aureus, an important human Gram-positive pathogen [20] biofilm-producing

[2,3], is frequently associated to SSTI is related as of serious infections as bacteremia,
pneumonia, further causes metastatic infections such as infective endocarditis,
osteomyelitis, through the blood dissemination [20].
In addition to producing resistance by hindering the penetration of antibiotics, the

S. aureus biofilm-producing [21] (Fig. 1) promotes bacterial adhesion to wound surfaces
resulting in chronic infection [8,22], forming a matrix with antiphagocytic activities
promoting evasion of the immune response [23]. S. aureus is also capable of producing
several virulence factors, including secretion of toxins and enzymes, which lead to
additional tissue damage [24].
Fig. 1 - Schematic illustration of skin wound infection by biofilm-producing S.
aureus.
Wound dressings impregnated with antimicrobial agents have emerged as

options to reduce colonization and bacterial infections of wounds and to improve the
healing process, since dressings currently available are only applied to protect the wound
from contamination, but not for treatment of the wound infected wounds [10]. Thus, it is
evident the importance of producing advanced dressings that allow the incorporation of
different classes of antibiotics [11–14] being able to locally release high concentrations
of antibiotics [25], in addition to preventing or treating infections, promote local moisture
maintenance, allow oxygen exchange, adsorb wound exudate, accelerate healing, reduce
pain [26].
Several publications cite the relevant results of topical use of antimicrobials [10–
14] available in semi-solid forms. However, even with the availability of several semi-
solid pharmaceutical forms for topical treatment of skin SSTI, such as ointments, creams,
gels, that have sustained release characteristics, there is low patient compliance, due to
the need for repetitive applications, especially in the treatment of chronic infections [27].
Thus, the development of sustained-release antibiotics, such as RIF, a semisynthetic
antibiotic obtained from rifamycin [28], capable of penetrating and biofilm eradicator
[29] become necessary.
Besides the semi-solid pharmaceutical forms for topical treatment of skin SSTI
[27], various types of biomaterials are used in the production of dressings, as cross-linked
biopolymers are in use as base materials for hydrogel dressings like, carboxymethyl
cellulose, chitosan, collagen, hyaluronic acid, and alginate [30–32], as specially bacterial
cellulose (BC) [33–35]
BC, as it is a natural polymer, endowed with peculiar characteristics such as

similarity to natural fabrics [36], high mechanical resistance, hypoallergenicity,
biocompatibility and high degree of liquid absorption [37,38], are widely studied as
controlled drug delivery systems, by our research group and many international groups
[10,38–45].
RIF antibiotic used as a first line treatment for tuberculosis, as well as in the
treatment of other infectious diseases [28] constitute one of the most potent and broad-
spectrum antibiotics against others Gram-positive bacterial pathogens, being its
antibacterial activity of is due to specific inhibition of bacterial RNA polymerase [46,47]
Although pristine BC has peculiar characteristics such as highly porous structure,

the inability to resist the free movement of gases, solvents and other small molecules,
makes this pristine biopolymer inappropriate for sustained drug delivery systems [49,50].
The drug release efficiency by BC is influenced by factors such as porosity,

surface area, crystallinity, and dilation kinetics [51,52]. Thus, chemical and physical
modifications of the BC surface become inevitable for successful applications in
controlled drug release systems [49,50,53].
Different physical, chemical and mechanical modification techniques have been

reported using polymers, solvents and biosynthetic pathways to prepare advanced BC
based functional materials with superior properties [45,54]. The used processes most
frequently for chemical modification are related to creating free hydroxyl groups [39] and
surface acetylation which presented greater advantages with the ability to maintain a large
modified surface area with controlled hydrophobic properties and compatibility with
other materials[55].
Li et al. [56] reported that using compatible solvents with low surface tension, as
alcohols (ethanol, isopropanol and n-butanol), exhibited highly porous structures in foam
materials with controllable pore structure prepared from nanofibrillated cellulose.
Lazarini et al. [57] reported obtaining BC produced by K. hansenii variants obtained after
selective pressures (exposure to UV radiation, different temperatures and different pH).
The results obtained demonstrate that the membranes synthesized by the variants
presented differences in thickness, degree of interlacing of the fibers and in pore size.
In addition, BC has been investigated as a support for delivery of antibacterial
agents as silver nanoparticles [58], silver metal complex [59,60], anesthetics as lidocaine
[61], analgesics as ibuprofen and diclofenac [61,62], antibiotics as tetracycline,
amoxicillin and levofloxacin [63], and anti-cancer drugs doxorubicin and methotrexate
[43,64]. Furthermore, the RIF has flexible backbone with two phenolic and two aliphatic
OH groups in addition to nitrogen and oxygen donor atoms [47] which allows the
interaction, through hydrogen bonds with BC. However, the use of BC for sustained
release of RIF has in the literature are scarce [6,65] .
The present work had the aims of the production of BC, by K. hansenii ATCC
23769, and the BC surface modifications by methanol (MetOH) and heat treatment to
produce BC-MetOH, with different fiber densities, thickness and entangling that allow
the use of support for sustained release of the antibacterial drug RIF by BC-MetOH-RIF
aiming for the future use as topical dressing for infected wound with biofilm-producing
bacteria, using S. aureus ATCC 25923 biofilm-producing as a model. BC treated
distillated water (BC-dH2O) was impregnated with RIF (BC-dH2O-RIF) and used as
control. The obtained BC-MetOH, BC-dH2O, BC-MetOH-RIF, and BC-dH2O-RIF were
characterized by field mission gun-scanning electron microscopy, thermogravimetric
analysis, derived thermogravimetry and Fourier transform infrared spectroscopy.
Interaction analysis of the RIF and BC in aqueous and methanolic solution through
simulations of molecular dynamics were also performed.
2. Materials and Methods
2.1. Materials
Anhydrous glucose and ethanol were purchased from Synth®, yeast extract,
bacteriological agar and Müller-Hinton agar were purchased from Kasvi. The bacteria for
BC production K. hansenii ATCC 23769 and S. aureus ATCC 25923 biofilm productor
were purchased from André Tosello Foundation. RIF (C43H58N4O12) was purchased from
Sigma-Aldrich.
2.2. Methods
2.2.1. BC production and processing
K. hansenii ATCC 23769 was reactivated from stock in glycerol stored at -80ºC,
in a medium used for maintenance, being kept in a B.O.D. oven at 28ºC until the growth
of a BC film, being used as pre-inoculum. BC production was performed using 100 mL
of culture medium compost by anhydrous glucose 30gL, yeast extract 5.6 gL and ethanol
50 mLL (G30), and 50 mL of pre-inoculum the cultures were adjusted to McFarland
scale 1.0 using a spectrophotometer reading. The cultivation was incubated in a
Biological Oxygen Demand (B.O.D.) oven at 28ºC for 7 days. After the 7 days the
membranes were initially washed in distilled water until cleared and then treated with
0.5M NaOH for 30 minutes in a water bath at 80ºC, for removal of bacterial strains. After
treatment with NaOH 0.5M the membranes were kept in a water bath at 65ºC, with
periodic water changes until the pH was neutralized. After the treatment and processing
the BC were dried, being kept in a ventilated oven at 65°C, until completely drying.
Posteriorly the dried BC were used for characterization and all tests. Elemental analysis
of carbon and hydrogen was performed using a Perkin-Elmer analyzer, model 2400
CHNS / O. The analytical result determined that the BC membrane produced showed C
(%) 42.66 and H (%) 6.92, thus compared with the theoretical calculation analysis for BC
(C% = 44.4; H% = 6.72) confirms the pure composition of the BC membrane obtained.
2.2.2. Treatment of BC with Methanol (MetOH)
The BC membranes produced and processed as described on item 2.2.1. were cut
into discs as 10mm in diameter and separately placed in 24-well plates, where they were
kept submerged in 100 µL of MetOH for 24 hours and kept refrigerated (4ºC). After 24
hours, the membranes were subjected to thermal treatment at 65 ºC in ventilated oven.
After thermal treatment the residual methanol was completely removed. The obtained
BC-MetOH and BC-dH2O were used for RIF impregnation. The BC-dH2O was also
produced. BC-MetOH and BC-dH2O were characterized by TGA / DTG, FEG-SEM, and
FTIR.
2.3. Treatment of BC-MetOH and BC-dH2O with RIF
The BC discs obtained as described on item 2.2.2. were placed in 24-well plates
and kept submerged separately in 180 µL of a 20mgmL-1 methanolic RIF solution (BC-
RIF-MetOH) and 180 µL of a 20 mgmL-1 aqueous RIF solution (BC-RIF-dH2O). BC-
MetOH and BC-dH2O were used as control. The BC-RIF-MetOH and BC-RIF-dH2O
were incubated for 24 hours under refrigeration at 4ºC until complete swelling. After this
procedure, BC-RIF-MetOH and BC-RIF-dH2O were subjected to thermal treatment in a
ventilated oven at 35ºC until total removal of the solvents (H2O and MetOH).
2.4. Determination of volume/area ratio of swelling of BC-MetOH and BC-dH2O
The volume/area ratio of swelling was performed using methodology described

by Lazarini et al. (2016) [33]. To determine the swelling mass (Sm), membranes with an
area of 4 cm2 were dehydrated as described on item 2.2.1. and submerged separately in
distilled water and 50 µL methanol for 4h at 4ºC to reach swelling equilibrium. The water
and MetOH mass of the different BC-MetOH and BC-dH2O after swelling was measured
in a Shimadzu analytical, and calculated applying the Eq. 1.:
(1) 𝑆𝑚 = 𝑀𝑆 − 𝑀𝑑
where Sm corresponds to the swelling mass, MS to the BC-MetOH and BC-dH2O swollen
mass after 4 hours and Md to the dry mass of the BC membrane (g)
In the sequence, under the same conditions, 180 µL of RIF solution (20mgmL)
using the methanol and deionized water as solvents was weighted. After obtaining the
mass of 180 µL of a RIF solution, the corresponding mass of RIF adsorbed on each of the
BC-MetOH and BC-dH2O was calculated using Eq. 2.:
180µLx Sm
(2) 𝑆𝑣 = Mr
where Sv corresponds to the volume of RIF solution (20mgmL-1) adsorbed and Mr is the
mass of 180 µL of RIF solution.
Determination of the volume/area ratio of swelling was performed using Eq. 3.:
Sv
(3) 𝑉𝐴 = Abc
where VA corresponds to the volume/area ratio of swelling in the membrane after

swelling, and Abc is the area (0.7854 cm2) of BC membranes.
Determination of the mass RIF/disc (RIFM) was performed using Eq. 4:
𝑉𝐴 −3.6
(4) 𝑅𝐼𝐹𝑀 = 180
2.5. Analysis of the RIF sustained release capacity of BC-MetOH-RIF and BC-
dH2O-RIF
BC-RIF-MetOH and BC-RIF-dH2O were used for disc diffusion analysis using
an adapted methodology described by CLSI [55]. The discs were placed on Mueller
Hinton agar (MH), inoculated with S. aureus ATCC 25923. The plate was incubated in a
bacteriological oven at 37ºC for 24 hours. After this period, the inhibition zone was
measured, and the BC discs were transferred to a new MH agar plate with S. aureus
ATCC 25923. This process was repeated until there was no more formation of inhibition
zone. BC-MetOH and BC-dH2O were used as control. All the experiments were
performed in triplicate.
2.6. Characterization of BC-RIF-MetOH, BC-RIF-dH2O, BC-MetOH, BC-dH2O
The characterizations of BC-RIF-MetOH, BC-RIF-dH2O, BC-MetOH, BC-dH2O

were performed by FEG-SEM were in the JEOL JSM-6360 LV device with carbon
coating, vibrational absorption spectroscopy in the infrared region performed on the FTIR
spectrophotometer Cary 630 Agilent (ATR). The measurements were performed in ATR
(attenuated total reflectance) mode, from 4000 to 600 cm-1 scans, and thermogravimetric
and derived thermogravimetry (TGA/DTG) analysis using TA Instruments equipment
and SDT Q600 cell. The conditions employed were synthetic air atmosphere with
continuous flow of 100 mL / min and heating rate of 10° C per minute. The temperature
used in the experiment was between 30-600° C, using an alumina crucible as a reference.
2.7. Molecular dynamic simulation
All simulated system were molecular dynamic simulated within the NAMD 2.9
program [67] using CHARMM36 force field [68] parameters. Before obtaining the final
MD trajectory, systems were energy minimized in NVT ensemble using the following
parameters: time step of 2 fs; cutoff: 12 Å for describing short-range interactions as well
as particle-mesh Ewald (PME) formalism was applied. To reach 310 K of temperature,
the system was gradually warmed up and equilibrated during 1 ns. A 10 ns of MD
simulation was sufficient to obtain characterize the interference of the solvent in the
studied molecular structures. Hydrogen bonds (H bonds) were computed during the
trajectories using H bonds plugin implemented in Visual Molecular Dynamics (VMD)
program [69]. Another important analyzed parameter was the number of solvent
molecules between RIF and cellulose. In this case, an algorithm was created to be used
in VMD to account it using TCL language programming [70]. The tridimensional models
and interactions between molecules were analyzed with Discovery Studio, version
20.1.0.192955.
3. Results and discussion
3.1. FEG-SEM of dry BC treated with methanol (BC-MetOH) and deionized water
(BC-dH2O)
The FEG-SEM comparative analysis dry BC-MetOH and BC-dH2O discs

showed significative difference in relation to fiber interlacing, thickness, arrangement,
and pore size as can observed in the Fig. 2.
A B
Fig. 2. BC - FEG-SEM comparative analysis BC-MetOH (A) and BC-dH2O (B). The
Fig. show the difference in relation to fiber interlacing, thickness, arrangement, and pore
size. (10,000x).
Several authors described different techniques to produce physical, chemical, and

mechanical modifications, using polymers, solvents, and biosynthetic pathways. This, is
to prepare advanced BC-based functional materials with superior properties for drug
delivery application [42,54,71]. However, information regarding methanol usage to
produce physical modification in BC to application as drug release system is scarce.
3.3. FTIR analysis
The FTIR spectra of pristine BC are shown in Fig. 3, bands in the range 3350-
3500 cm-1 of pure cellulose are attributed to O–H stretching, while bands from 2800-2900
cm-1 are assigned to C–H stretching. The band at 1160 cm-1 is attributed to C–O–C
stretching [72,73].
The FTIR analysis shows a difference between pristine BC and the BC-MetOH-
RIF and BC-RIF-dH2O. However, FTIR spectra curves suggest that there was no change
in the chemical composition of RIF, both in BC-MetOH-RIF and BC-RIF-dH2O, because
the absorption peaks of pure RIF were maintained equally in BC-MetOH-RIF and BC-
RIF-dH2O. These results demonstrate the incorporation of RIF into the BC-MetOH and
BC-dH2O.
Fig. 3. FTIR spectra of commercial RIF (A), BC-MetOH-RIF (B), BC-dH2O-RIF (C) and
BC-dH2O (D). The spectra confirm the incorporations of RIF in the BC using both of
solvents.
The FTIR spectrum of the commercial sample of RIF (Fig.3, Panel A) is similar
to that published by Agrawal et al. [74], demonstrating the incorporations of RIF in the,
BC-MetOH, and BC-dH2O using both solvents.
3.4. TGA/DTG analysis
According to the TGA / DTG curves (Fig. 4), and with the data compiled in Table
1 presented below, the pristine BC curve (Fig. 4A) shows three mass loss events. The first
loss event, approximately 2% mass, occurs between 30 °C and 100 °C and is available
for loss of water molecules adsorbed on the material surface, subsequently, two
significant mass loss events are observed: the first is about 70% in the 160-360°C range;
the second is approximately 26% in the range of 360-500°C. These events are associated
with the thermal degradation of cellulose, which concerns the depolymerization
processes, the decomposition of the glycosidic units and the formation of gaseous
residues as already observed by De Salvi et al. [75].
The MetOH-treated sample (Fig. 4C) showed a profile similar to the characteristic
profile of BC, with three significant mass loss events and low residue at 600 °C (about
2%). The difference observed between the treated sample and the pure BC sample is that
the BC-MetOH sample showed an increase in mass loss in the first event (4%) compared
to the pure BC sample (2%). This event, as already presented, refers to the loss of water
adsorbed on the surface of the material and is related to the fact that the treatment makes
the BC more porous and its fibers less dense, as pointed out in the FEG-SEM analysis,
facilitating the evaporation of water molecules.
It is observed that the RIF sample (Fig. 4B) presents the characteristic profile of
the drug, with mass loss of about 2% up to 100 °C, another loss between 200-250 °C with
about 10%, a third loss in the range 250-450 °C having lost about 29% and finally a fourth
loss between 450-600°C losing about 33% of the mass, as already demonstrated by Alves
et al. [76]. In addition, the RIF sample has about 22% residue at 600°C, demonstrating
that its degradation is not complete at this temperature.
The samples containing RIF, on the other hand, have different characteristics. The
BC-dH2O-RIF (Fig. 4E) sample has a profile similar to pure BC, however, the last event
of thermal BC loss of mass at approximately 500°C, while the sample with RIF extends
to 600°C, thus demonstrating a possible increase in degradation temperature. While the
BC-MetOH-RIF (Fig. 4D) sample has an intermediate degradation profile, showing
characteristic peaks of BC and RIF. Furthermore, it is verified that RIF increased the
stability of BC-MetOH, passing the initial temperature of the first mass loss event from
30°C for BC-MetOH to 45°C for BC- MetOH-RIF, which may to suggest that the RIF
was in fact impregnated on the BC membrane treated with MetOH, thus corroborating
the other analyzes presented.
Table. 1. TGA results of the samples, showing mass loss events, their temperatures, and
the final residue at 600°C.
First mass Second mass Third mass Fourth mass Residue

loss event loss event loss event loss event at
Sample (%)/ (%)/ (%)/ (%)/ 600°C
temperature temperature temperature temperature
range (°C) range (°C) range (°C) range (°C)
Pure BC 2/30-100 70/160-360 26/360-500 -/- 2
RIF 3/50-160 13/190-250 25/260-430 32/440-600 22
BC-MetOH 4/30-100 73/230-350 21/360-505 -/- 2
BC-RIF-MetOH 4/45-100 5/160-220 39/230-350 50/355-600 1
BC-RIF-dH2O 4/30-100 65/150-345 28/350-590 -/- 2
From the data obtained by the DTG curves, it can be confirmed what was said
from the TGA curves: the treatment with MetOH increased the thermal stability of the
membrane at ~9°C, while the RIF decreased the stability of the membranes, both the
MetOH-treated and the untreated membrane. Despite the decrease in thermal stability,
there will be no loss in the use of this material in any case since it will be used at body
temperature (~37°C).
In addition, it is possible to verify in the BC-MetOH-RIF sample DTG curves
(Fig. 4D) the appearance of the RIF characteristic curves, confirming again that the RIF
was in fact incorporated in the treated sample, corroborating with the other analyses.
While this profile is not found in the untreated membrane (BC-dH2O-RIF), demonstrating
that there was not the expected interaction for this material. As observed in Fig 4C, the
MetOH was completely removed. The total remove of MetOH was confirmed using the
diffusion disc test with BC-MetOH and BC-dH2O after drying. The results presented in
Fig 13.
Fig. 4. TGA and DTG curves of the BC-dH2O (A), RIF (B), BC-MetOH (C), BC-MetOH-
RIF (D), and BC-RIF-dH2O (E).
3.5. Analysis FEG-SEM of BC-MetOH-RIF and BC-RIF-dH2O
The SEM images of BC-MetOH-RIF and BC-dH2O-RIF are showed in Fig. 5. As

can observed It is possible to see that this difference is related to the network interlacing,
and density of the fibers. Although the membranes were produced under the same
conditions, it is possible to report the structural difference with treatment of BC and the
solubilization of the RIF drug using MetOH, as can observed in the Fig. 2A, that showed
the membrane treated with MetOH.
A B
Fig. 5. SEM comparative analysis BC-MetOH-RIF (Panel A) and BC-dH2O-RIF (Panel

B). The Fig. show the difference in relation to fiber interlacing, thickness, arrangement,
and pore size and chemical and physical changes in the RIF drug. (10,000x).
The Fig. 5 demonstrates that, in addition, to the change in the physical
characteristics of the BC by with MetOH solvent and heat treatment. The solvent also
produced chemical and physical changes in the RIF drug, demonstrated by the RIF crystal
structure shown in Fig. 5B, characteristic of RIF after water dilution and drying [77].
As can be observed, the BC-dH2O-RIF showed RIF in crystal forms and

superficial distribution, while the RIF (amorphous structure) contained in the BC-
MetOH-RIF support is more integrated into the BC membrane, which probably promoted
greater capacity retention, providing a sustained and controlled release of the antibiotic,
when compared to the BC-dH2O-RIF support.
3.6. Determination of volume/area ratio of swelling and RIF/disc mass
The volume / area of swelling allows the approximate calculation of the amount
of drug that was adsorbed by the BC discs used in the release test. The results obtained
(Table 2) showed that there was a difference of approximately 1mg of incorporated RIF
mass between the disks with BC-MetOH-RIF (3.45 mg) and BC-dH2O-RIF (2.44 mg),
considering that, in both conditions, the stock concentration of methanolic and aqueous
RIF solutions was 20mgmL-1.
Table 2. Determination of volume/area ratio of swelling and RIF/disc mass
Ms Md Sm Sv VA 𝑅𝐹𝑀𝑀
Sample
(mg) (mg) (mg) (µL) (µLcm2) (mg)
86.93 3.39 83.54 95.78 121.95 2.44

BC-dH2O-RIF
(+5.3*) + + + + +
( 0.30*) ( 5.56*) ( 6.37*) ( 8.07*) ( 0.16*)
60.30 4.63 55.67 135.41 172.40 3.45

BC- MetOH-RIF + + + + + +
( 3.40*) ( 0.47*) ( 3.15*) ( 7.67*) ( 9.70*) ( 0.19*)
Ms = BC membrane mass swollen; MD = BC membrane dry mass; Sm = swelling mass;

Sv = volume of RIF solution (20mgmL-1) adsorbed; VA volume/area ratio of swelling.
* Standard deviation
3.7. Interaction analysis of the BC-MetOH-RIF and BC-dH2O-RIF by using
molecular dynamic simulations
During the equilibration step, water molecules are capable of separate BC from
RIF, while methanol ones not. As a result, the value of the interaction energy in methanol
is more favorable than water (Table 3).
Table 3. Average interaction energies and average number of hydrogen bonds formed
between RIF and BC and with the solvents.
Interaction Energy
(kcal/mol)/
BC-dH2O BC-MetOH H2O C2H6OH
Hydrogen bonds
formed
RIF -14.9909 (0) -17.486 (~0.5) -98.9638 (~1.7) -52.5644 (~2.5)
BC + 0.0000 (0) -2.818 (~1.0) -51.1307 (~1.1) -28.2307 (~2.0)
As can be seen in the above table, the interaction energy related to the BC-RIF
interaction is stronger in methanol solution than in water. This fact is attribute to the
average hydrogen bond formed during the system trajectory in methanol where at least
HB ~0.5 is formed and no HB in water was noticed. Additionally, the distance observed
in the last frame of the trajectory shows that cellulose is totally surrounded by water
molecules, this fact was not observed in MetOH.
Interactions analysis of BC-RIF in MetOH (Fig. 6) showed that in this solvent

hydrogen bonds between RIF and BC molecules are formed, while in water environment,
interactions between RIF and the solvent are more likely to occur (Fig. 7). Besides, the
alignment of RIF structures after MD simulation showed an overlay of 74%, with 1.8
RMSD (Fig. 8). In this sense, the difference observed in experimental RIF release, in both
environments, are probably due to solvent and RIF interactions, rather than major
structural changes in RIF.
Fig. 6. Predicted MD interactions between BC and RIF in methanol system. Conventional

hydrogen bonds are shown in green and carbon hydrogen bonds are shown in blue.
Fig. 7. Predicted MD interactions between BC and RIF in water system. Conventional
Fig. 8. Alignment of RIF molecules after MD simulation. Carbons of RIF in water system
are shown in green and carbons of RIF in MetOH system are shown in gray. Hydrogens
were omitted.
3.8. Sustained RIF delivery by disc diffusion methods
The results showed that the BC-MetOH-RIF showed 696 hours of uninterrupted
release, and the BC-dH2O-RIF were able to maintain the release for 120 hours. The Fig.
9 show the results of the release with BC- MetOH-RIF. The Fig. 10 shows the obtained
results using BC-dH2O-RIF as support of the release.
Although, as described in the results of determination of volume/area ratio of

swelling, where there was a mass of about 1mg of incorporated RIF in the BC-MetOH-
RIF in relation to BC-dH2O-RIF, this difference in mass does not justify the maintenance
of the RIF release by BC-MetOH-RIF for an additional 576 hours. Thus, it can be inferred
that the greater capacity for RIF release by BC-MetOH-RIF is due to changes in the
physical characteristics of BC, by MetOH and by the change in the physical
characteristics of RIF impregnated in the BC-MetOH-RIF membrane as can be observed
in the Fig. 2 and 5.
The results obtained in this work demonstrate that, even the BC-MetOH-RIF
containing 1 mg more RIF than BC-dH2O-RIF the release maintenance for 696 hours was
due to the treatment of BC membrane with MetOH and the use of methanolic solution of
RIF, also demonstrating that the treatment with MetOH provided an increase in the
surface area of the membrane, allowing an increase in the volume of swelling and greater
RIF interaction with the BC membrane, as can be seen in Fig. 9 and Fig. 10.
*Standard deviation
Fig. 9. Sustained/controlled release using BC-MetOH-RIF by disc diffusion assay. The

Fig. shows the averages of the measures of the zones of inhibition by time of diffusion.
Fig. shows the triplicates of the diffusion tests every 24 hours.
The Fig. 11 and 12 showed the Sustained / controlled release using BC-dH2O-RIF
by disc diffusion assay.
*Standard deviation
Fig. 11. Sustained/controlled release using BC-dH2O-RIF by disc diffusion assay. The
Fig. 12. Sustained/controlled release using BC-dH2O-RIF by disc diffusion assay – The
The Fig. 13 shows the results of diffusion disc test evaluating the toxicity of BC-
MetOH compared to BC-dH2O after heat treatment. The results demonstrate that BC-
MetOH did not show bacterial growth inhibitory activity, as well as the BC-dH2O control.
The absence of an inhibition zone around the BC-MetOH disc demonstrates that MetOH
was completely removed after heat treatment, does not present toxicity risks.
A B
Fig. 13. Diffusion disc test for evaluating the toral remove solvent of BC discs after heat
treatment. Panel A: BC-MetOH and Panel B: BC-dH2O.
4. Discussion
In the present work was produced surface modifications of BC by methanol and

heat treatment to produce membranes with different fiber densities, thickness and
entangling that allow the use of device for sustained release of the antibacterial drug RIF
in vitro, using the S. aureus ATCC 25923 biofilm-producing, for future use as topical
dressing in wound infected treatment.
RIF, a semisynthetic antibiotic, constitute one of the most potent and broad-
spectrum antibiotics against others Gram-positive bacterial pathogens, being its
antibacterial activity [46,47] capable of penetrates readily through the S. aureus biofilm-
producing without significant impact on cellular viability [21]. RIF release systems as
topic wound dressing would constitute an ideal procedure prevention, and treatment of
surgical site infections and wounds infected from biofilm-producing microorganisms
constitute a major healthcare problem that requires new and innovative treatment
methods.
According to Pötzinger et al. [78], the selection of the drug-loading method for a
certain application should be based on the physicochemical characteristics of the drug,
including molar mass, solubility, stability during each process step, therapeutic dose and
the characteristics of BC as pristine wet, semidried, freeze-dried and structural
modifications da BC membrane or the drug loading procedure. Despite substantial
structural variabilities of the loaded drugs regarding size, hydro-/lipophilicity and
stability, the published drug-loading strategies for BC are almost comparable and quite
limited up to now.
Results obtained by Demir et al. [79] was demonstrated that the topical of RIF was
an easy and effective method for reducing surgical site infections SSIs and meningitis/
VP shunt infections related to neural tube defects surgery.
Hartinger et al. [6] developed two types (homogeneous and sandwich structured)
carbodiimide-cross-linked fresh water fish collagen wound dressings for in vivo use as
RIF-release device and were tested by means of a series of incubations in phosphate-
buffered saline. The microbiological activity was tested against methicillin-resistant S.
aureus employing disc diffusion tests. The results demonstrated that RIF release devices
delayed the release of RIF in vitro, which translated into at least 22 hours of RIF release
in the rat model.
Aguilar-Colomer et al. [65] showed the impact of an initial burst effect followed
by a RIF sustained release above the minimum inhibitory concentration (MIC) up to 96
h using mesoporous silica nanoparticles (MSNs) on Gram-positive and Gram-negative
bacterial biofilms. Such doses reduced by 99.9% bacterial biofilm of S. aureus and
remained active up to 72 h with no emergence of bacterial resistance.
Results recently obtained by Ngwabebhoh et al. [80] reported the development of

self-crosslinked chitosan/dialdehyde xanthan gum blended hypromellose hydrogel for the
controlled delivery of RIF. The results of this study shown more than 50% cumulative
release within 24 h for all investigated antibiotic drugs.
As described by CLSI 2019 [66], the sensibility of S. aureus for RIF, by disc
diffusion, is  20mm, with the RIF concentration per disc being 5 µg. The obtained results
in the present work showed that the inhibition zone, in triplicate, for each 24 hours were
greater than 20 mm, suggesting that the concentration of RIF released, every 24 hours,
BC-MetOH-RIF devices were able to maintain the release of concentrations above that
considered to be the minimum for inhibiting the growth of S. aureus for 576 hours. These
results demonstrate the effectiveness of retention and sustained release of RIF by the BC-
MetOH-RIF device, which may increase the therapeutic arsenal in the treatment and
prevention of SSTIs and SWI.
In this work we demonstrated that MetOH and heat treatment promoted superficial
changes in BC, allowing to obtain BC-MetOH-RIF reproducibly that promoted the
sustained release of the antibiotic at concentrations higher than the MIC, according to
CLSI [66] for 576 hours (inhibition zone above 20mm), 504 hours longer than BC-dH2O-
RIF used as a control (inhibition zone above 20mm up to 72 hours).
These results demonstrated that the efficiency of sustained and controlled release
of antibiotic RIF is due to treatment with methanol and heat and impregnation of BC-
MetOH with the methanolic solution of RIF, when compared to the membrane without
methanol treatment (BC-dH2O) impregnated with solution aqueous containing the same
antibiotic. The absence of an inhibition zone around the BC-MetOH disk used as a control
demonstrates that methanol was completely removed after the heat treatment, presenting
no toxicity risks demonstrate the great possibility of adding, in a short period of time, to
the therapeutic arsenal for the treatment of bacterial infections of skin and soft tissues,
including the biofilm-producing, since RIF is not commercially available for topical use.
5. Conclusions
The BC with methanol and heat treatment presented physical changes in its
structure that allowed a greater volume of swelling, in addition to the use of methanolic
RIF solution providing a greater interaction of the antibiotic with the BC membrane (BC-
RIF-MetOH). These results showed that methanol treatment and the use of the RIF
methanolic solution produced change in the physical and chemical characteristics of RIF
better capacity to maintain a sustained / controlled release of antibiotic RIF by 576h
uninterruptedly, in concentrations above the MIC, demonstrated by the maintenance of
inhibition zone around of the BC-MetOH-RIF membranes.
Thus, the results obtained in this work demonstrate that a product was obtained with
a great possibility of adding, in a short period of time, to the therapeutic arsenal for the
treatment of bacterial infections of skin and soft tissues, including the biofilm-producing,
since there are no commercially available topical formulations, using the RIF, an
antibiotic of broad spectrum, approved by global health agencies for systemic use, with
known therapeutic and adverse effects, which would certainly be reduced with topical use
and, because BC It is a biomaterial known to be hypoallergenic, biocompatible,
nanostructured, with staggered and reproducible productivity, including the MetOH
treatment procedure, not requiring extensive studies of development phases applied to
new drugs release systems.
Credit authorship contribution statement
Caroline Yamada: Investigation, Writing, Validation.

Silmara C. Lazarini Frajácomo: Investigation, Writing, Validation.
Hernane S. Barud: Investigation, Resources, Funding Acquisition.
Nayara C. do Amaral: Investigation, Writing - Review & Editing.
Adriano Marques Gonçalves: Investigation of Interaction analysis using molecular
dynamic simulations
Guedmiller Souza de Oliveira: Investigation of Interaction analysis using molecular
dynamic simulations
Rafael Mariano-Neto: Investigation of Interaction analysis using molecular dynamic
simulations
Wilton R. Lustri: Conceptualization, Methodology, Investigation, Validation, Writing -
Original Draft, Visualization, Project administration, Supervision and Funding
Acquisition.
Declarations of Conflicts of Interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This study was supported by grants from the Brazilian Agencies FAPESP (São
Paulo Research Foundation, Grants# 2015/09833-0, 2018/12590-0, 2018/25512-8 and
no. 2013/07793-6 and CNPq (National Council of Scientific and Technological
Development, PQ Grant # 300968/2016-7, 407822/2018-6; INCT-INFO), and TA
Instruments Brazil. Also, this study was financed in part by the Coordination for the
Improvement of Higher Education Personnel - Brazil (CAPES) - Finance Code 001 and
FUNADESP (National Foundation for Development of Private Superior Education -
Grant # 2700375). We are grateful to the High-Resolution Scanning Microscope
Laboratory of the Chemistry Institute of the State University of Araraquara - IQ-UNESP-
Araraquara, São Paulo, Brazil and TA Instruments Brazil and Laboratório de Computação
Científica Aplicada e Tecnologia de Informação from the Federal University of
Uberlândia (UFU), Campus Pontal, financial supported by FINEP.i.
6. References
[1] L. Rahman, R.S. Lembang, S. Lallo, S.R. Handayani, Usmanengsi, A.D. Permana,
Bioadhesive dermal patch as promising approach for improved antibacterial
activity of bioactive compound of Zingiber cassumunar Roxb in ex vivo
Staphylococcus aureus skin infection model, J. Drug Deliv. Sci. Technol. 63
(2021) 102522. https://doi.org/10.1016/j.jddst.2021.102522.
[2] D.L. Stevens, A.L. Bisno, H.F. Chambers, E.P. Dellinger, E.J.C. Goldstein, S.L.
Gorbach, J. V Hirschmann, S.L. Kaplan, J.G. Montoya, J.C. Wade, Practice
Guidelines for the Diagnosis and Management of Skin and Soft Tissue Infections :
2014 Update by the Infectious Diseases Society of America, 59 (2014).
https://doi.org/10.1093/cid/ciu296.
[3] R.R. Watkins, M.Z. David, Approach to the Patient with a Skin and Soft Tissue
Infection, Infect. Dis. Clin. North Am. 35 (2021) 1–48.
https://doi.org/10.1016/j.idc.2020.10.011.
[4] Z. Khatoon, C.D. Mctiernan, E.J. Suuronen, T. Mah, Bacterial bio fi lm formation
on implantable devices and approaches to its treatment and prevention, Heliyon.
(2018) e01067. https://doi.org/10.1016/j.heliyon.2018.e01067.
[5] N. Goel, S. Warisul, S. Kumar, R. Sinha, S.K. Khare, Antimicrobial resistance in
bio fi lms : Exploring marine actinobacteria as a potential source of antibiotics and
bio fi lm inhibitors, Biotechnol. Reports. 30 (2021) e00613.
https://doi.org/10.1016/j.btre.2021.e00613.
[6] J.M. Hartinger, P. Lukáč, M. Mlček, M. Popková, T. Suchý, M. Šupová, H. Chlup,
L. Horný, J. Závora, V. Adámková, O. Slanař, P. Kozlík, K. Molnarova, E.
Honsová, L. Lambert, T. Grus, Rifampin-Releasing Triple-Layer Cross-Linked
Fresh Water Fish Collagen Sponges as Wound Dressings, Biomed Res. Int. 2020
(2020). https://doi.org/10.1155/2020/3841861.
[7] J. O’Neill, Antimicrobial Resistance : Tackling a crisis for the health and wealth
of nations, Rev. Antimicrob. Resist. (2014).
[8] Y. Liu, Y. Li, L. Shi, Controlled drug delivery systems in eradicating bacterial
biofilm-associated infections, J. Control. Release. 329 (2021) 1102–1116.
https://doi.org/10.1016/j.jconrel.2020.10.038.
[9] A. Punjataewakupt, S. Napavichayanun, P. Aramwit, The downside of
antimicrobial agents for wound healing, Eur. J. Clin. Microbiol. Infect. Dis. 38
(2019) 39–54. https://doi.org/10.1007/s10096-018-3393-5.
[10] I. Negut, V. Grumezescu, A.M. Grumezescu, Treatment Strategies for Infected
Wounds, (2018) 1–23. https://doi.org/10.3390/molecules23092392.
[11] S. Ye, L. Jiang, J. Wu, C. Su, C. Huang, X. Liu, W. Shao, Flexible Amoxicillin
Grafted Bacterial Cellulose Sponges for Wound Dressing : in Vitro and in Vivo
Evaluation Flexible Amoxicillin Grafted Bacterial Cellulose Sponges for Wound
Dressing : in Vitro and in Vivo Evaluation, (2018).
https://doi.org/10.1021/acsami.7b16680.
[12] S. Anjum, A. Arora, M.S. Alam, B. Gupta, Development of antimicrobial and scar
preventive chitosan hydrogel wound dressings, Int. J. Pharm. 508 (2016) 92–101.
https://doi.org/10.1016/j.ijpharm.2016.05.013.
[13] A. Anjum, C. Sim, S. Ng, Hydrogels Containing Antibiofilm and Antimicrobial
Agents Beneficial for Biofilm-Associated Wound Infection : Formulation
Characterizations and In vitro Study, (2017). https://doi.org/10.1208/s12249-017-
0937-4.
[14] R. Marius, A.M. Holban, G.D. Mogos, O. Fuf, D. Savu, R.C. Popescu, A.M.
Grumezescu, E. Bezirtzoglou, V. Lazar, Bionanocomposites Based on Natural
Polymers and Antibiotics for Wound Healing Applications, (2016).
https://doi.org/10.3390/molecules21060761.
[15] B. Buyana, B.A. Aderibigbe, D.T. Ndinteh, Y.T. Fonkui, P. Kumar, Alginate-
pluronic topical gels loaded with thymol, norfloxacin and ZnO nanoparticles as
potential wound dressings, J. Drug Deliv. Sci. Technol. 60 (2020) 101960.
https://doi.org/10.1016/j.jddst.2020.101960.
[16] A.D. Permana, M. Mir, E. Utomo, R.F. Donnelly, Bacterially sensitive
nanoparticle-based dissolving microneedles of doxycycline for enhanced treatment
of bacterial bio fi lm skin infection : A proof of concept study, Int. J. Pharm. X. 2
(2020) 100047. https://doi.org/10.1016/j.ijpx.2020.100047.
[17] C.E. Edmiston Jr., A.J. McBain, C. Roberts, D. Leaper, Clinical and
Microbiological Aspects of Biofilm-Associated Surgical Site Infections, Adv. Exp.
Med. Biol. I (2015) 69–84. https://doi.org/10.1007/978-3-319-11038-7.
[18] S.M. Shiels, D.J. Tennent, J.C. Wenke, Topical rifampin powder for orthopedic
trauma part I: Rifampin powder reduces recalcitrant infection in a delayed
treatment musculoskeletal trauma model, J. Orthop. Res. 36 (2018) 3136–3141.
https://doi.org/10.1002/jor.24055.
[19] D.Z. Zmejkoski, N.M. Zdravkovi, D.D. Trisic, M.D. Budimir, Z.M. Markovic,
N.O. Kozyrovska, B.M.T. Markovic, Chronic wound dressings – Pathogenic
bacteria anti-biofilm treatment with bacterial cellulose-chitosan polymer or
bacterial cellulose-chitosan dots composite hydrogels, 191 (2021) 315–323.
https://doi.org/10.1016/j.ijbiomac.2021.09.118.
[20] H. Ma, J. Cheng, L. Peng, Y. Gao, G. Zhang, Z. Luoi, Adjunctive rifampin for the
treatment of staphylococcus aureus bacteremia with deep infections: A meta-
analysis, PLoS One. 15 (2020). https://doi.org/10.1371/journal.pone.0230383.
[21] C.W. Hall, T.-F. Mah, Molecular mechanisms of biofilm-based antibiotic
resistance and tolerance in pathogenic bacteria, FEMS Microbiol. Rev. 41 (2017)
276–301. https://doi.org/10.1093/femsre/fux010.
[22] S. Roy, S. Santra, A. Das, S. Dixith, M. Sinha, S. Ghatak, N. Ghosh, S. Khanna,
S. Mathew-steiner, P. Das, B.N. Blackstone, H.M. Powell, K. Valerie, D.J.
Wozniak, C.K. Sen, Staphylococcus aureus Biofilm Infection Compromises
Wound Healing by Causing Deficiencies in Granulation Tissue Collagen, Ann
Surg. 271 (2021) 1174–1185. https://doi.org/10.1097/SLA.0000000000003053.
[23] H.T.T. Nguyen, T.H. Nguyen, M. Otto, The staphylococcal exopolysaccharide PIA
– Biosynthesis and role in biofilm formation , colonization , and infection, Comput.
Struct. Biotechnol. J. 18 (2020) 3324–3334.
https://doi.org/10.1016/j.csbj.2020.10.027.
[24] Z. Versey, W.S. da Cruz Nizer, E. Russell, S. Zigic, K.G. DeZeeuw, J.E. Marek,
J. Overhage, E. Cassol, Biofilm-Innate Immune Interface: Contribution to Chronic
Wound Formation, Front. Immunol. 12 (2021) 1–25.
https://doi.org/10.3389/fimmu.2021.648554.
[25] R. Da, V. Smith, F. Hayden, P. Cronin, Systemic antibiotics for treating malignant
wounds ( Review ), (2017).
https://doi.org/10.1002/14651858.CD011609.pub2.www.cochranelibrary.com.
[26] R. Portela, C.R. Leal, L. Pedro, Minireview Bacterial cellulose : a versatile
biopolymer for wound dressing applications, 2019 (2020).
https://doi.org/10.1111/1751-7915.13392.
[27] K. Frederiksen, R.H. Guy, K. Petersson, Formulation considerations in the design
of topical, polymeric film-forming systems for sustained drug delivery to the skin,
Eur. J. Pharm. Biopharm. 91 (2015) 9–15.
https://doi.org/10.1016/j.ejpb.2015.01.002.
[28] I. Sutradhar, M.H. Zaman, Evaluation of the effect of temperature on the stability
and antimicrobial activity of rifampicin quinone, J. Pharm. Biomed. Anal. 197
(2021). https://doi.org/10.1016/j.jpba.2021.113941.
[29] C.J.S. Jr, S.M. Shiels, D.J. Tennent, S.K.H. Bs, C.K. Murray, J.C. Wenke,
Rifamycin Derivatives Are Effective Against Staphylococcal Biofilms In Vitro
and Elutable From PMMA, Clin. Orthop. Relat. Res. (2015) 2874–2884.
https://doi.org/10.1007/s11999-015-4300-3.
[30] A. Gupta, D.J. Keddie, V. Kannappan, H. Gibson, I.R. Khalil, M. Kowalczuk, C.
Martin, X. Shuai, I. Radecka, Production and characterisation of bacterial cellulose
hydrogels loaded with curcumin encapsulated in cyclodextrins as wound dressings,
Eur. Polym. J. 118 (2019) 437–450.
https://doi.org/10.1016/j.eurpolymj.2019.06.018.
[31] H. Hamedi, S. Moradi, S.M. Hudson, A.E. Tonelli, Chitosan based hydrogels and
their applications for drug delivery in wound dressings : A review, Carbohydr.
Polym. 199 (2018) 445–460. https://doi.org/10.1016/j.carbpol.2018.06.114.
[32] S.U. Islam, M. Ul-Islam, H. Ahsan, M.B. Ahmed, A. Shehzad, A. Fatima, J.K.
Sonn, Y.S. Lee, Potential applications of bacterial cellulose and its composites for
cancer treatment, Int. J. Biol. Macromol. 168 (2021) 301–309.
[33] L. Zhang, Y. Yu, S. Zheng, L. Zhong, J. Xue, Preparation and properties of
conductive bacterial cellulose-based graphene oxide-silver nanoparticles
antibacterial dressing, Carbohydr. Polym. 257 (2021) 117671.
https://doi.org/10.1016/j.carbpol.2021.117671.
[34] O. Asanarong, V. Minh Quan, S. Boonrungsiman, P. Sukyai, Bioactive wound
dressing using bacterial cellulose loaded with papain composite: Morphology,
loading/release and antibacterial properties, Eur. Polym. J. 143 (2021) 110224.
https://doi.org/10.1016/j.eurpolymj.2020.110224.
[35] D. Ciecholewska-Juśko, A. Żywicka, A. Junka, R. Drozd, P. Sobolewski, P.
Migdał, U. Kowalska, M. Toporkiewicz, K. Fijałkowski, Superabsorbent
crosslinked bacterial cellulose biomaterials for chronic wound dressings,
Carbohydr. Polym. 253 (2021). https://doi.org/10.1016/j.carbpol.2020.117247.
[36] M. Pang, Y. Huang, F. Meng, Y. Zhuang, H. Liu, M. Du, Application of bacterial
cellulose in skin and bone tissue engineering, 122 (2020).
https://doi.org/10.1016/j.eurpolymj.2019.109365.
[37] S.C. Lazarini, C. Yamada, H.S. Barud, E. Trovatti, P.P. Corbi, W.R. Lustri,
Influence of chemical and physical conditions in selection of Gluconacetobacter
hansenii ATCC 23769 strains with high capacity to produce bacterial cellulose for
application as sustained antimicrobial drug-release supports, J. Appl. Microbiol.
125 (2018) 777–791. https://doi.org/10.1111/jam.13916.
[38] S.C. Lazarini, R. de Aquino, A.C. Amaral, F.C.A. Corbi, P.P. Corbi, H.S. Barud,
W.R. Lustri, Characterization of bilayer bacterial cellulose membranes with
different fiber densities: a promising system for controlled release of the antibiotic
ceftriaxone, Cellulose. 23 (2016) 737–748. https://doi.org/10.1007/s10570-015-
0843-4.
[39] H. Ullah, F. Wahid, H.A. Santos, T. Khan, Advances in biomedical and
pharmaceutical applications of functional bacterial cellulose-based
nanocomposites, Carbohydr. Polym. 150 (2016) 330–352.
https://doi.org/10.1016/j.carbpol.2016.05.029.
[40] M.C.I. Mohd Amin, N. Ahmad, M. Pandey, C. Jue Xin, Stimuli-responsive
bacterial cellulose-g-poly(acrylic acid-co-acrylamide) hydrogels for oral
controlled release drug delivery, Drug Dev. Ind. Pharm. 40 (2014) 1340–1349.
https://doi.org/10.3109/03639045.2013.819882.
[41] G. Juncu, A. Stoica-Guzun, M. Stroescu, G. Isopencu, S.I. Jinga, Drug release
kinetics from carboxymethylcellulose-bacterial cellulose composite films, Int. J.
Pharm. 510 (2016) 485–492. https://doi.org/10.1016/j.ijpharm.2015.11.053.
[42] M. Badshah, H. Ullah, A. Rahman, S. Khan, J. Kon, T. Khan, Surface modi fi
cation and evaluation of bacterial cellulose for drug delivery, Int. J. Biol.
Macromol. 113 (2018) 526–533. https://doi.org/10.1016/j.ijbiomac.2018.02.135.
[43] M.L. De Fontes, A.B. Meneguin, A. Tercjak, J. Gutierrez, B. Stringhetti, F. Cury,
A. Martins, S.J.L. Ribeiro, H.S. Barud, Effect of in situ modification of bacterial
cellulose with carboxymethylcellulose on its nano/microstructure and
methotrexate release properties, Carbohydr. Polym. (2017).
[44] W. Treesuppharat, P. Rojanapanthu, C. Siangsanoh, H. Manuspiya, S.
Ummartyotin, Synthesis and characterization of bacterial cellulose and gelatin-
based hydrogel composites for drug-delivery systems, Biotechnol. Reports.
(2017). https://doi.org/10.1016/j.btre.2017.07.002.
[45] H.G. de Oliveira Barud, R.R. da Silva, H. da Silva Barud, A. Tercjak, J. Gutierrez,
W.R. Lustri, O.B. de Oliveira, S.J.L. Ribeiro, A multipurpose natural and
renewable polymer in medical applications: Bacterial cellulose, Carbohydr.
Polym. 153 (2016). https://doi.org/10.1016/j.carbpol.2016.07.059.
[46] P. Alifano, C. Palumbo, D. Pasanisi, A. Talà, Rifampicin-resistance, rpoB
polymorphism and RNA polymerase genetic engineering, J. Biotechnol. (2014).
https://doi.org/10.1016/j.jbiotec.2014.11.024.
[47] A.D. Politano, K.T. Campbell, L.H. Rosenberger, R.G. Sawyer, Use of Silver in
the Prevention and Treatment of Infections : Silver Review, 14 (2013) 8–20.
https://doi.org/10.1089/sur.2011.097.
[48] E.A. Saad, H.A. Kiwan, M.M. Hassanien, H.E. Al-adl, Synthesis , characterization
, and antitumor activity of a new iron-rifampicin complex : A novel prospective
antitumor drug, J. Drug Deliv. Sci. Technol. 57 (2020) 101671.
https://doi.org/10.1016/j.jddst.2020.101671.
[49] U. Beekmann, L. Schmölz, S. Lorkowski, O. Werz, J. Thamm, D. Fischer, D.
Kralisch, Process control and scale-up of modified bacterial cellulose production
for tailor-made anti-inflammatory drug delivery systems, Carbohydr. Polym. 236
(2020) 116062. https://doi.org/10.1016/j.carbpol.2020.116062.
[50] J. Salonen, M. Shahbazi, H. Ullah, M. Badshah, T. Khan, Fabrication ,
characterization and evaluation of bacterial cellulose-based capsule shells for oral
drug delivery, (2017). https://doi.org/10.1007/s10570-017-1202-4.
[51] S. Adepu, M. Khandelwal, Ex-situ modification of bacterial cellulose for
immediate and sustained drug release with insights into release mechanism,
Carbohydr. Polym. 249 (2020) 116816.
https://doi.org/10.1016/j.carbpol.2020.116816.
[52] S. Adepu, M. Khandelwal, Materialia Bacterial cellulose with microencapsulated
antifungal essential oils : A novel double barrier release system, Materialia. 9
(2020) 100585. https://doi.org/10.1016/j.mtla.2020.100585.
[53] M. Ul-islam, S. Khan, M.W. Ullah, J.K. Park, Bacterial cellulose composites :
Synthetic strategies and multiple applications in bio-medical and electro-
conductive fields, (2015) 1847–1861. https://doi.org/10.1002/biot.201500106.
[54] G.F. Picheth, C.L. Pirich, M.R. Sierakowski, M.A. Woehl, C.N. Sakakibara, C.F.
de Souza, A.A. Martin, R. da Silva, R.A. de Freitas, Bacterial cellulose in
biomedical applications: A review, Int. J. Biol. Macromol. 104 (2017) 97–106.
[55] W. Hu, S. Chen, J. Yang, Z. Li, H. Wang, Functionalized bacterial cellulose
derivatives and nanocomposites, Carbohydr. Polym. 101 (2014) 1043–1060.
[56] J. Li, T. Song, H. Xiu, M. Zhang, R. Cheng, Q. Liu, X. Zhang, E. Kozliak, Y. Ji,
Foam materials with controllable pore structure prepared from nanofibrillated
cellulose with addition of alcohols, Ind. Crops Prod. 125 (2018) 314–322.
https://doi.org/10.1016/j.indcrop.2018.09.016.
[57] S.C. Lazarini, C. Yamada, H. da S. Barud, E. Trovatti, P.P. Corbi, W.R. Lustri,
Influence of chemical and physical conditions in selection of Gluconacetobacter
hansenii ATCC 23769 strains with high capacity to production bacterial cellulose
for application as sustained antimicrobial drug release supports, J. Appl. Microbiol.
33017300 (2018) 0–1. https://doi.org/10.1111/jam.13916.
[58] S. Adepu, Biomaterials Broad-spectrum antimicrobial activity of bacterial
cellulose silver nanocomposites with sustained release, J. Mater. Sci. 53 (2018)
1596–1609. https://doi.org/10.1007/s10853-017-1638-9.
[59] N.A.S. Aquaroni, D.H. Nakahata, S.C. Lazarini, F.A. Resende, A.L.P. Cândido,
H. da Silva Barud, A.M. Claro, J.E. de Carvalho, C.M. Ribeiro, F.R. Pavan, B.C.
Lustri, T.R.M. Ribeiro, C.G. Moreira, T.Z. Cândido, C.S.P. Lima, A.L.T.G. Ruiz,
P.P. Corbi, W.R. Lustri, Antibacterial activities and antiproliferative assays over a
tumor cells panel of a silver complex with 4-aminobenzoic acid: Studies in vitro
of sustained release using bacterial cellulose membranes as support, J. Inorg.
Biochem. 212 (2020) 111247. https://doi.org/10.1016/j.jinorgbio.2020.111247.
[60] W.R. Lustri, S.C. Lazarini, P.P. Lustri, Bruna Cardinali Corbi, M.A.C. Silva, F.A.
Resende Nogueira, R. Aquino, A.C. Amaral, O. Treu Filho, A.C. Massabni, H. da
Silva Barud, Spectroscopic characterization and biological studies in vitro of a new
silver complex with furosemide: Prospective of application as an antimicrobial
agent, J. Mol. Struct. 1134 (2017) 386–394.
https://doi.org/10.1016/j.molstruc.2016.12.056.
[61] E. Trovatti, C.S.R. Freire, P.C. Pinto, I.F. Almeida, P. Costa, A.J.D. Silvestre, C.P.
Neto, C. Rosado, Bacterial cellulose membranes applied in topical and transdermal
delivery of lidocaine hydrochloride and ibuprofen: In vitro diffusion studies, Int.
J. Pharm. 435 (2012) 83–87. https://doi.org/10.1016/j.ijpharm.2012.01.002.
[62] N.H.C.S. Silva, A.F. Rodrigues, I.F. Almeida, P.C. Costa, C. Rosado, C.P. Neto,
A.J.D. Silvestre, C.S.R. Freire, Bacterial cellulose membranes as transdermal
delivery systems for diclofenac: In vitro dissolution and permeation studies,
Carbohydr. Polym. 106 (2014) 264–269.
[63] R.D. Pavaloiu, A. Stoica, M. Stroescu, T. Dobre, Controlled release of amoxicillin
from bacterial cellulose membranes, Cent. Eur. J. Chem. 12 (2014) 962–967.
https://doi.org/10.2478/s11532-014-0541-3.
[64] M.L. Cacicedo, M.C. Castro, I. Servetas, L. Bosnea, K. Boura, P. Tsafrakidou, A.
Dima, A. Terpou, A. Koutinas, G.R. Castro, Bioresource Technology Progress in
bacterial cellulose matrices for biotechnological applications, Bioresour. Technol.
213 (2016) 172–180. https://doi.org/10.1016/j.biortech.2016.02.071.
[65] A. Aguilar-Colomer, M. Colilla, I. Izquierdo-Barba, C. Jiménez-Jiménez, I.
Mahillo, J. Esteban, M. Vallet-Regí, Impact of the antibiotic-cargo from MSNs on
gram-positive and gram-negative bacterial biofilms, Microporous Mesoporous
Mater. 311 (2021). https://doi.org/10.1016/j.micromeso.2020.110681.
[66] CLSI, Performance standards for antimicrobial susceptibility testing, Clin. Lab.
Stand. Inst. 29 (2019).
[67] L. V. Kalé, M. Bhandarkar, R. Brunner, N. Krawetz, J. Phillips, A. Shinozaki,
NAMD: A case study in multilingual parallel programming, J. Comput. Chem.
1366 (1997) 367–381. https://doi.org/10.1007/BFb0032705.
[68] B.R. Brooks, C.L. Brooks III, A.D.M. Jr, L. Nilsson, R.J. Petrella, B. Roux, Y.
Won, G. Archontis, C. Bartels, S. Boresch, A. Caflisch, L. Caves, Q. Cui, A.R.
Dinner, M. Feig, S. FischeR, J. Gao, M. Hodoscek, W. Im, K. Kuczera, T.
Lazaridis, J. Ma, V. Ovchinnikov, E. Paci, R.W. Pastor, C.B. Post, J.Z. Pu, M.
Schaefer, B. Tidor, R.M. Venable, H.L. Woodcock, X. Wu, W. Yang, D.M. York,
M. Karplus, CHARMM: The Biomolecular Simulation Program, J. Comput.
Chem. 32 (2012) 174–182. https://doi.org/10.1002/jcc.21287.
[69] W. Humphrey, A. Dalke, K. Schulten, VMD: Visual molecular dynamics, J. Mol.
Graph. 14 (1996) 33–38. https://www.tapbiosystems.com/tap/products/index.htm.
[70] C.J. Nelson, Tcl/Tk Programmer’s Reference, in: Agroecol. Bases Científicas Para
Una Agric. Sustentable, 1999: p. 538. http://www.leisa-
al.org/web/images/stories/revistapdf/vol22n2.pdf#page=30.
[71] S.A. de Oliveira, B.C. da Silva, I.C. Riegel-Vidotti, A. Urbano, P.C. de Sousa
Faria-Tischer, C.A. Tischer, Production and characterization of bacterial cellulose
membranes with hyaluronic acid from chicken comb, Int. J. Biol. Macromol. 97
(2017) 642–653. https://doi.org/10.1016/j.ijbiomac.2017.01.077.
[72] P. Kotcharat, P. Chuysinuan, T. Thanyacharoen, S. Techasakul, S. Ummartyotin,
Development of bacterial cellulose and polycaprolactone (PCL) based composite
for medical material, Sustain. Chem. Pharm. 20 (2021).
https://doi.org/10.1016/j.scp.2021.100404.
[73] S. Leal, C. Cristelo, S. Silvestre, E. Fortunato, A. Sousa, A. Alves, D.M. Correia,
S. Lanceros-Mendez, M. Gama, Hydrophobic modification of bacterial cellulose
using oxygen plasma treatment and chemical vapor deposition, Cellulose. 27
(2020) 10733–10746. https://doi.org/10.1007/s10570-020-03005-z.
[74] S. Agrawal, Y. Ashokraj, P. V. Bharatam, O. Pillai, R. Panchagnula, Solid-state
characterization of rifampicin samples and its biopharmaceutic relevance, Eur. J.
Pharm. Sci. 22 (2004) 127–144. https://doi.org/10.1016/j.ejps.2004.02.011.
[75] D.T.B. De Salvi, H.S. Barud, J.M.A. Caiut, Y. Messaddeq, S.J.L. Ribeiro, Self-
supported bacterial cellulose/boehmite organic-inorganic hybrid films, J. Sol-Gel
Sci. Technol. 63 (2012) 211–218. https://doi.org/10.1007/s10971-012-2678-x.
[76] R. Alves, T.V.D.S. Reis, L.C.C. Da Silva, S. Storpírtis, L.P. Mercuri, J.D.R. Matos,
Thermal behavior and decomposition kinetics of rifampicin polymorphs under
isothermal and non-isothermal conditions, Brazilian J. Pharm. Sci. 46 (2010) 343–
351. https://doi.org/10.1590/S1984-82502010000200022.
[77] J.N. Schianti, N.N.P. Cerize, A.M. de Oliveira, S. Derenzo, A.C. Seabra, M.R.
Góngora-Rubio, Rifampicin nanoprecipitation using flow focusing microfluidic
device, J. Nanomedicine Nanotechnol. 4 (2013). https://doi.org/10.4172/2157-
7439.1000172.
[78] Y. Pötzinger, D. Kralisch, D. Fischer, Bacterial nanocellulose: the future of
controlled drug delivery?, Ther. Deliv. 8 (2017) 753–761.
[79] N. Demir, E. Peker, İ. Gülşen, S. Kocaman, O. Tuncer, E. Kırımi, Powder Topical
Rifampin for Reducing Infections After Neural Tube Defect Surgery in Infants,
World Neurosurg. 95 (2016) 165–170.
https://doi.org/10.1016/j.wneu.2016.07.092.
[80] F.A. Ngwabebhoh, O. Zandraa, R. Patwa, N. Saha, Z. Capáková, P. Saha, Self-
crosslinked chitosan / dialdehyde xanthan gum blended hypromellose hydrogel for
the controlled delivery of ampicillin , minocycline and rifampicin, Int. J. Biol.
Macromol. 167 (2021) 1468–1478.
Figure Captions
Fig. 1. Schematic illustration of skin wound infection by biofilm-producing S. aureus.
Fig. 2. BC - FEG-SEM comparative analysis BC-MeTOH (A) and BC-dH2O (B). The
size. (10,000x).
Fig. 3. FTIR spectra of RIF, BC-RIF-METOH, BC-RIF-dH2O and pure BC. The spectra
confirm the incorporations of RIF in the BC using both of solvents.
Fig. 4. TGA and (d) DTG curves of the pristine BC (A), RIF (B), BC-MeTOH (C), BC-
MetOH-RIF (D), and BC-RIF-dH2O (E).
Fig. 5. SEM comparative analysis BC-MetOH-RIF (Panel A) and BC-RIF-dH2O (Panel


are shown in green and carbons of RIF in methanol system are shown in gray. Hydrogens
were omitted.

Fig. shows the averages of the measures of the zones of inhibition by time of diffusion
Fig. 11. Sustained/controlled release using BC-RIF-dH2O by disc diffusion assay – The
Fig. 12. Sustained/controlled release using BC-RIF-dH2O by disc diffusion assay – The
Fig. 13. Control of diffusion test after 24 hours. Panel A: BC-MetOH and Panel B: BC-
dH2O.
Credit authorship contribution statement
Caroline Yamada: Investigation, Writing, Validation.

Silmara C. Lazarini Frajácomo: Investigation, Writing, Validation.
Hernane S. Barud: Investigation, Resources, Funding Acquisition.
Nayara C. do Amaral: Investigation, Writing - Review & Editing.
Adriano Marques Gonçalves: Investigation of Interaction analysis using molecular
dynamic simulations
Guedmiller Souza de Oliveira: Investigation of Interaction analysis using molecular
dynamic simulations
Rafael Mariano-Neto: Investigation of Interaction analysis using molecular dynamic
simulations
Wilton R. Lustri: Conceptualization, Methodology, Investigation, Validation, Writing -
Original Draft, Visualization, Project administration, Supervision and Funding
Acquisition.
Figure 1 Click here to access/download;Figure;Fig 1. Yamada et al
2021.docx
Yamada et al. 2021
Fig. 1. Schematic illustration of skin wound infection by biofilm-producing S. aureus.

2021.docx
Yamada et al. 2021

Fig. 2. BC - FEG-SEM comparative analysis BC-MetOH (A) and BC-dH2O (B). The
size. (10,000x).
A B
2021.docx
Yamada et al. 2021

Fig. 3. FTIR spectra of RIF, BC- MetOH-RIF, BC-dH2O -RIF and pure BC. The spectra
confirm the incorporations of RIF in the BC membranes using both of solvents.
2021.docx
Yamada et al. 2021

Fig. 4. TGA and (d) DTG curves of the pristine BC (A), RIF (B), BC-MetOH (C), BC-
MetOH-RIF (D), and BC- dH2O-RIF (E).
2021.docx
Yamada et al. 2021

Fig. 5. SEM comparative analysis BC-MetOH-RIF (Panel A) and BC-dH2ORIF (Panel
A B
2021.docx
Yamada et al. 2021

2021.docx
Yamada et al. 2021

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Yamada et al. 2021
are shown in green and carbons of RIF in methanol system are shown in gray. Hydrogens
were omitted.
2021.docx
Yamada et al. 2021

Fig. 9. Sustained/controlled release using BC-MetOH -RIF by disc diffusion assay. The
Fig. shows the averages of the measures of the zones of inhibition by time of diffusion
*Standard deviation
2021.docx
Yamada et al. 2021
Fig. 10. Sustained/controlled release using BC-MetOH -RIF by disc diffusion assay. The
2021.docx
Yamada et al. 2021

*Standard deviation
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Yamada et al. 2021
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Yamada et al. 2021

Fig. 13. Diffusion disc test for evaluating the toral remove solvent of BC discs after heat
treatment. Panel A: BC-MetOH and Panel B: BC-dH2O.
A B
Table 1 Click here to access/download;Table;Table 1 Yamada et al
2021.docx
Yamada et al. 2021

Table. 1. TGA results of the samples, showing mass loss events, their temperatures and the final
residue at 600°C.
First mass Second Third mass Fourth Residu

loss event mass loss loss event mass loss e at
Sample (%)/ event (%)/ (%)/ event (%)/ 600°C
temperature temperature temperature temperature
range (°C) range (°C) range (°C) range (°C)
Pure BC 2/30-100 70/160-360 26/360-500 -/- 2
RIF 3/50-160 13/190-250 25/260-430 32/440-600 22
BC-MetOH 4/30-100 73/230-350 21/360-505 -/- 2
BC-RIF-MetOH 4/45-100 5/160-220 39/230-350 50/355-600 1
BC-RIF-dH2O 4/30-100 65/150-345 28/350-590 -/- 2

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Yamada et al. 2021
Table 2. Determination of volume/area ratio of swelling and RIF/disc mass

Ms Md Sm VA 𝑅𝐹𝑀𝑀
Sample Sv (µL)
(mg) (mg) (mg) (µLcm2) (mg)
86.93 3.39 83.54 95.78 121.95 2.44

BC-dH2O-RIF
( 5.3*) ( 0.30*) ( 5.56*) ( 6.37*) (+8.07*)
+ + + + +
( 0.16*)
60.30 4.63 55.67 135.41 172.40 3.45

BC- MetOH-RIF
( 3.40*) ( 0.47*) ( 3.15*) (+7.67*) (+9.70*)
+ + + +
( 0.19*)
Ms = BC membrane mass swollen; MD = BC membrane dry mass; Sm = swelling mass;

Sv- volume of RIF solution (20mgmL) adsorbed; VA volume/area ratio of swelling.
*Standard deviation
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Yamada et al. 2021
Table 3. Average interaction energies and average number of hydrogen bonds formed
between RIF and BC and with the solvents.
Interaction Energy
(kcal/mol)/
BC-dH2O BC-MetOH H2O C2H6OH
Hidrogen bonds
formed
RIF -14.9909 (0) -17.486 (~0.5) -98.9638 (~1.7) -52.5644 (~2.5)
BC + 0.0000 (0) -2.818 (~1.0) -51.1307 (~1.1) -28.2307 (~2.0)

Conflict of Interest
UNIVERSITY OF ARARAQUARA – UNIARA

Laboratory of Medical Chemistry and Regenerative Medicine-QUIMMERA
14801-320 - Araraquara, São Paulo, Brazil
_______________________________________________________________________
November 10th, 2021.
To
Professor Florence Siepmann
Editor-in-Chief, Journal of Drug Delivery Science Technology
University of Lille Faculty of Pharmacy, Lille Cedex, France
Declarations of Conflicts of Interest
The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper.
Wilton R. Lustri
Professor Wilton Rogério Lustri, Ph.D.
Department of Biological and Health Sciences
University of Araraquara 14801-320, Araraquara-SP, Brazil
E-mail: wrlustri@uniara.edu.br

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Journal of Drug Delivery Science and Technology

Bacterial cellulose-based device for sustained release of the antibacterial drug

Manuscript Number: JDDST-D-21-02132

Article Type: Research Paper

Keywords: Bacterial cellulose; biofilm-producing, sustained drug release; rifampicin; wound

Corresponding Author: Wilton Rogério Lustri

First Author: Wilton Rogério Lustri

Order of Authors: Wilton Rogério Lustri

Silmara Cristina Lazarini Frajácomo

Adriano Marques Gonçalves

Suggested Reviewers: Agnieszka Tercjak

Guillermo Raúl Castro

UNIVERSITY OF ARARAQUARA – UNIARA

November 10th, 2021.

Hoping to hear from you soon and thanking you in advance,

Yamada et al., 2021

Bacterial cellulose-based device for sustained release of the antibacterial

Caroline Yamada1a, Silmara C. Lazarini1a, Nayara C. do Amaral1, Hernane S. Barud1,

*Corresponding author: Wilton R. Lustri

Address: Laboratory of Bacterial Cellulose and Medicinal Chemistry, University of

Telephone: +55(16) 99154-7476

E-mail address: wrlustri@uniara.edu.br

Keywords: Bacterial cellulose; biofilm-producing, sustained drug release; rifampicin;

Nevertheless, wounds infection, especially considered chronic. that do not heal

S. aureus, an important human Gram-positive pathogen [20] biofilm-producing

In addition to producing resistance by hindering the penetration of antibiotics, the

Wound dressings impregnated with antimicrobial agents have emerged as

BC, as it is a natural polymer, endowed with peculiar characteristics such as

Although pristine BC has peculiar characteristics such as highly porous structure,

The drug release efficiency by BC is influenced by factors such as porosity,

Different physical, chemical and mechanical modification techniques have been

2. Materials and Methods

2.2.1. BC production and processing

2.2.2. Treatment of BC with Methanol (MetOH)

2.3. Treatment of BC-MetOH and BC-dH2O with RIF

2.4. Determination of volume/area ratio of swelling of BC-MetOH and BC-dH2O

The volume/area ratio of swelling was performed using methodology described

where VA corresponds to the volume/area ratio of swelling in the membrane after

Determination of the mass RIF/disc (RIFM) was performed using Eq. 4:

2.6. Characterization of BC-RIF-MetOH, BC-RIF-dH2O, BC-MetOH, BC-dH2O

The characterizations of BC-RIF-MetOH, BC-RIF-dH2O, BC-MetOH, BC-dH2O

2.7. Molecular dynamic simulation

3. Results and discussion

The FEG-SEM comparative analysis dry BC-MetOH and BC-dH2O discs

Several authors described different techniques to produce physical, chemical, and

3.3. FTIR analysis

3.4. TGA/DTG analysis

First mass Second mass Third mass Fourth mass Residue

Pure BC 2/30-100 70/160-360 26/360-500 -/- 2

RIF 3/50-160 13/190-250 25/260-430 32/440-600 22

BC-MetOH 4/30-100 73/230-350 21/360-505 -/- 2

BC-RIF-MetOH 4/45-100 5/160-220 39/230-350 50/355-600 1

BC-RIF-dH2O 4/30-100 65/150-345 28/350-590 -/- 2

3.5. Analysis FEG-SEM of BC-MetOH-RIF and BC-RIF-dH2O

The SEM images of BC-MetOH-RIF and BC-dH2O-RIF are showed in Fig. 5. As

Fig. 5. SEM comparative analysis BC-MetOH-RIF (Panel A) and BC-dH2O-RIF (Panel

As can be observed, the BC-dH2O-RIF showed RIF in crystal forms and

3.6. Determination of volume/area ratio of swelling and RIF/disc mass

86.93 3.39 83.54 95.78 121.95 2.44

60.30 4.63 55.67 135.41 172.40 3.45

Ms = BC membrane mass swollen; MD = BC membrane dry mass; Sm = swelling mass;

RIF -14.9909 (0) -17.486 (~0.5) -98.9638 (~1.7) -52.5644 (~2.5)

BC + 0.0000 (0) -2.818 (~1.0) -51.1307 (~1.1) -28.2307 (~2.0)