Applied Microbiology and Biotechnology

https://doi.org/10.1007/s00253-019-09699-x

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Novel polymeric dressing to the treatment of infected chronic wound

Bruna Cambraia Garms1 • Felipe Azevedo Borges1 • Natan Roberto de Barros1 • Mônica Yonashiro Marcelino2 •
Marcel Nani Leite3 • Marina Constante Del Arco4 • Sérgio Luiz de Souza Salvador4 • Giovana Sant’Ana Pegorin2 •
Kassandra Sussi Mustafé Oliveira5 • Marco Andrey Cipriani Frade3 • Rondinelli Donizetti Herculano2

Received: 10 November 2018 / Revised: 11 February 2019 / Accepted: 12 February 2019

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Natural rubber latex (NRL) is a natural polymer which has arisen large interest in the biomedical field, mostly, due to its
ability to facilitate angiogenesis and therefore, tissue repair. Moxifloxacin (MXF) is a broad-spectrum antibiotic orally
administrated. Considering the biological properties of the NRL and its ability to deliver a wide range of compounds, the
present study aimed to develop a novel device for infected chronic wound treatment. MXF-loaded NRL was obtained by a
casting method. The results demonstrated that the incorporation of MXF in NRL did not promote any molecular interac-
tion, preserving the integrity of the compounds. The mechanical properties of the biomaterial did not show any significant
change, indicating enough elasticity for dermal application. The microbiological assays confirmed the ability of the polymer
to deliver the drug without influencing its pharmacological properties. Moreover, it has expressed activity against major
bacterial strains presented in wound infections. Finally, the biomaterial shown biocompatibility from the in vitro study.
Thus, the present work has shown that MXF-loaded NRL membrane is a promising biomaterial to infected wound treatment.

Keywords Natural rubber latex · Moxifloxacin · Antibiotic · Skin infection · Wound healing

Introduction epidemiology forecast, based on data from seven countries,

it is expected to achieve the number of 1.5 million cases by
Infected chronic wounds represent a significant expense to 2025 (Gomes et al. 2017). Some of these unhealed lesions
the public health service. In the USA, an annual cost of 25 may persist for a long period of time which makes them
billion dollars for the treatment of this pathology is esti- more susceptible to entry of external particles and micro-
mated. In addition, considering only diabetic foot ulcers organism, causing infections. Furthermore, over 90% of
the chronic wounds present microorganism colonization
(Alvarado-Gomez et al. 2018). Most of the dermal infec-
 Bruna C. Garms
tions are caused by Staphylococcus aureus, Staphylococcus
b.garms@uq.edu.au epidermidis, Pseudomonas aeruginosa, and Escherichia
 Rondinelli D. Herculano
coli. Additionally, it is worth to mention that the growth of
rond@fcfar.unesp.br microorganisms has a negative effect in the tissue repair, and
it can delay the healing process (Lam et al. 2018).
1
Institute of Chemistry, São Paulo State University (UNESP), The standard of care for infected wound consists of surgi-
Araraquara, São Paulo, Brazil cal debridement followed by oral administration of broad-­
2
School of Pharmaceutical Sciences, São Paulo State University spectrum antibiotic, such as moxifloxacin (MXF) (Han and
(UNESP), Araraquara, São Paulo, Brazil Ceilley 2017; Marra et al. 2017). However, the prolonged
3
School of Medicine, São Paulo University (USP), Ribeirão Preto, use of MXF can lead to side effects and antibiotic resistance.
São Paulo, Brazil In order to overcome these issues, sustained delivery system
4
School of Pharmaceutical Science, São Paulo University (USP), (SDS) has been proposed as a strategy to promote a more
Ribeirão Preto, São Paulo, Brazil efficient therapy. The ideal delivery device should be able
5
Faculty of Agricultural Sciences, São Paulo State University to reach and maintain therapeutic concentrations of drug
(UNESP), Botucatu, São Paulo, Brazil

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 Appl Microbiol Biotechnol

in the wound site avoiding systemic side effects (Lulinski In a clinical trial using NRL for wound healing, reepi-
2017). Also, the SDS using polymers as scaffold/matrix has thelization, reduction of tissue necrosis areas, and increase
the ability to reduce antimicrobial and antibiolfilm activi- of vascular permeability even in diabetic patients were
ties (Otto et al. 2008; Jennings et al. 2016; Ambrogi et al. observed. Moreover, the study showed that the polymeric
2017). Scaffaro et al. (2018) showed that the gradual release dressing enables to maintain the moisture of the wound
of Carvacrol incorporated in membranes of poly (lactic and facilitates the debridement and angiogenesis process
acid) reduced the activity against Staphylococcus aureus (Cursi et al. 2004). In SDS application, the NRL has been
and Candida albicans within 144 h after treatment. Besides reported as an effective device to release several compounds,
that, the authors observed a decrease in the development including natural extract, nicotine proteins, molecules, cal-
of S. aureus and C. albicans biofilms in single and mixed cium phosphate, peptides, glycerol, and drugs (Barros et al.
culture (Scaffaro et al. 2018). 2016; Miranda et al. 2017; Carvalho et al. 2018; Marcelino
Several polymers have been used as scaffold with the et al. 2018; Barros et al. 2019; Zancanela et al. 2019).
purpose of controlling the bioavailability and drug release Furthermore, the release profile can be modified by chang-
(Park 2014; Miranda et al. 2017; Vilela et al. 2017). A highly ing the material porosity (Herculano et al. 2010).
interesting biopolymer that has attracted attention in the bio- Considering the biological properties of the NRL and its
medical field is natural rubber latex (NRL). This is extracted ability to deliver a wide range of compounds, the present
from Hevea brasiliensis rubber tree and consists of a poly- study aimed to develop a novel polymeric device to promote
disperse system formed by 40–45% of rubber compound, simultaneously the infection treatment and tissue repair.
50% of water and 4–5% of protein, lipids, and carbohydrates Hence, the present work explored the biomaterial efficacy
(Ferreira et al. 2009; Neves-Junior et al. 2006). in strains of Staphylococcus aureus, Staphylococcus epider-
Reports in the literature showed that 15 NRL proteins are midis, Pseudomonas aeruginosa, and Escherichia coli for
responsible for latex allergies. These are denominated by the infected chronic wound treatment. In this study, we devel-
World Health Organization and International Nomenclature oped a novel polymeric dressing for infected wounds.
Union of Imunological Societies (WHO/IUIS) Allergen
Nomenclature as Hev b 1–15, being the major allergens the
Hev b 1, 3, 5, and 6 (molecular weight between 14 and 24 kDa). Materials and methods
Additionally, in order to obtain latex serum, membranes-­based
latex, and manufactured latex products, the use of chemical sub- Membranes manufacture
stances, as preservatives, vulcanizing agents, and accelerators,
are necessary, which can be cytotoxic and allergenic, (Brehler Latex from Hevea brasiliensis, produced by the crossing
et al. 2002; Nettis et al. 2002; Reunala et al. 2004; Miranda et al. clones RRIM 600 e PB 235 (Lot: 01703/13), was purchased
2017; Vandenplas and Raulf 2017; Raulf et al. 2018). from BDF Latex Company (Guarantã, São Paulo, Brazil).
Herculano et al. (2011) developed an alternative man- The polymer content was about 60% of dry rubber, 4–5% of
ufacturing process of NRL membrane, which the high-­ weight of non-rubber components, such as protein, lipids,
molecular-­weight proteins were removed in the centrifu- and 35% of water (Marcelino et al. 2018). In order to avoid
gation process. In addition, enzyme treatment, leaching, coagulation and correct the pH of the mixture, ammonia
creaming, chlorination, miscellaneous, and fumed silica was added to the material until reaching a pH value of 10.2.
have been used (Suksaeree et al. 2014; Miranda et al. 2017), Then, mixture was centrifuged at 8000g for 2 h (Herculano
instead of the common chemical reagents use. et al. 2011). The centrifugation method used does not elimi-
In the last decade, the interest of the natural polymer in nate all the proteins present in NRL, only high-­molecular-­
the medical field has increased due mostly to its biological weight proteins responsible for allergic and cytotoxic
properties. Studies have shown the ability to promote neoan- reactions obtaining the Natural Rubber Latex Biomedical
giogenesis, cell adhesion, and extracellular matrix formation, (Barros et al. 2017).
facilitating the healing process (Kotake et al. 2018). All of MXF-loaded membranes were produced by a casting
these properties make the polymer a versatile material for a method, using 3 mL of latex and 3 mL of MXF solution
wide range of applications. Dias et al. (2019) used low-level (1.67 mg mL−1) in a circular plate of 60-mm diameter and
laser and F1 natural latex protein on crush-type injuries to the let dry at 37 °C for 24 h to guarantee full polymerization
sciatic nerve. In other studies, the NRL have been employed before use. These membranes were utilized to the physical-­
on bone regeneration (Issa et al. 2012; Moura et al. 2014; chemical characterization (mechanical, morphological, and
Borges et al. 2017; Kotake et al. 2018), treatment of wounds infrared).
(Mendonca et al. 2010) and ulcers (Frade et al. 2011), mold for To the antimicrobial activity, cell viability assay, and drug
neovaginoplasty (Carvalho et al. 2018), and reconstruction of release profile, the membranes were produced in the same
iatrogenics abdominal wall defects (Paulo et al. 2005). proportion, but in a smaller area (28.26 mm2). To create

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the biopolymers, 60 μL of latex solution and 5 or 20 μg of

MXF (labeled as NRL + MXF 5 μg and NRL + MXF 20
Sigma-­Aldrich®, St. Louis, MO, USA) supplemented with

μg) were mixed. USA), 100 IU mL−1 penicillin and 100 μg mL−1 streptomy-
10% fetal bovine serum (Sigma-Aldrich®, St. Louis, MO,

cin (Sigma-Aldrich, Saint Louis, MO, USA) and 3 g L−1

Membrane characterization of glucose and incubated at 37 °C in 5% CO2. Cells were
maintained in culture and dissociated with trypsin (Gibco,
Physical-chemical properties of membranes were analyzed New York, USA) when reached 60–80% of confluence to
by a Fourier transform infrared spectrometer (ATR FTIR the culture dish area.
spectrometer—Bruker Tensor 27 source: HeNe laser; detec-
tor: DLaTGS with a resolution of 4 cm−1 and 32 scans) Cell viability
between 400 and 4000 cm−1. This technique was used to

loaded NRL membranes (containing 5 μg, 10 μg, and 20

identify functional groups in membrane surface, aiming to Samples of pure NRL membrane, three samples of MXF-­

μg of MXF), and three concentrations of MXF (5, 10, and

verify possible interactions between NRL and MXF powder

20 μg mL−1) were tested. The eluate obtained from the

(Murbach et al. 2014).
Surface and bulk morphology of membrane were exam-
ined by high-resolution scanning electron microscopy (FEG-­ samples was extracted according to ISO 10993-5. Each
SEM) JEOL, model 7500F (2 kV), with gold as conductor extract was incubated in DMEM medium for 24 h at 37
material and a take-off angle of 35°. Three random areas °C in 5% CO2.
were selected to perform the analysis and the membranes The cell viability as the indicator of cytotoxicity was
were analyzed at ×300 and ×5000 magnification (Herculano determinate by MTT assay (reduction of (3-(4,5-­dimethylth
et al. 2009). iazol-­2-yl)-2,5-diphenyltetrazolium bromide) to a formazan
Mechanical strength testing was applied in membranes product), as described by Mosmann (1983) using the
(44.8 mm×26.19 mm×0.84 mm) by the mechanical testing Vybrant MTT Cell Proliferation assay kit V-13154 (Gibco,
machine (EMIC DL 2000) with 10 kgf load cell at 500 mm New York, USA) (Mosmann 1983). A cell suspension was

microplate containing 200 μL of medium and incubated for

min−1 (according to ASTM D412) at 28 °C (Murbach et al. seeded at a concentration of 1×105 cells mL−1 in a 96-well
2014). All experiments for membrane characterization were

exposed to the compounds for 24 h and then, 10 μL of MTT

performed in triplicate. 24 h at 37 °C in 5% CO2. After cell adhesion, cells were

Release assay (5 μg mL−1) was added to each well and incubated for 4 h.
Thereafter, the precipitate was discarded and washed with

formazan crystals were dissolved with 100 μL of dimethyl-

In order to set the analytical curve, MXF solution was phosphate-buffered saline solution (PBS). The produced
prepared with NaOH (0.1 M) and concentrations in the
range of 2.5–100 ng mL−1. The MXF concentrations were sulfoxide (DMSO), and its concentration was measured in a
assayed by the spectrofluorometric technique (λ excita- spectrophotometer at 570 nm.
tion=291 nm; λ emission=462 nm; 20 kW; PerkinElmer, All the experiments were performed in triplicate. The
model LS 45). The data were compiled using FL-Winlab results expressed the mean percentage of viable cells. The
software. mean value of the optical density of the control group (posi-
To the release assay, MXF-loaded NRL membranes tive control, cells + culture medium) was considered to be
were placed individually in 100 mL of phosphate-buffered 100% cell viability, and the results of the experimental
saline (PBS) solution with pH 7.4 and 5.6, to mimic the groups were compared to this value. As a negative con-
pH of the injured and healthy skin, respectively (Watters trol, cell + DMSO 50%, representing 0% cell viability, was
et al. 2015). The drug release was measured and moni- adopted.
tored by the spectrofluorometric technique. The data were
compiled using Origin Pro 8 SR 4 software, from Origin Bacterial cultures
Corporation.
The antibacterial activity of MXF against Escherichia coli
Cell cultivation (ATCC 8739), Pseudomonas aeruginosa (ATCC 2327),
Staphylococcus aureus (ATCC 6538), and Staphylococcus
Monolayers of keratinocyte cell line HaCat (Rio de Janeiro epidermidis (ATCC 14990) was tested by microdilu-
Cell Bank, Brazil) and the Swiss 3T3 albino mouse fibro- tion assay. The microorganisms were sub-cultured onto
blasts (ATCC CCL-92) were cultivated to a final con- Brain Heart Infusion broth (BHI, Sigma-Aldrich, USA) to
centration of 1×10 5 cell/cm 2 and maintained in high- ensure purity and viability and incubated at 37 °C for 24 h
glucose Dulbecco’s Modified Eagle’s Medium (DMEM, (Cockerill 2011).

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MIC measurements Fourier transform infrared spectrometer (FTIR)

The assay was performed according to recommendations of The major functional groups of NRL and MXF were iden-
the Clinical and Laboratory Standards Institute (CLSI 2009) tified by FTIR. The expected peaks of the polymer were as
with modifications. Colonies’ suspensions were grown in follows: from 2960 to 2852 cm−1, 1446 and 1375 cm−1, and
Mueller Hinton broth (35 °C±2 °C), until turbidity equiva- 835 cm−1. Moreover, MXF powder has absorption bands at
lent reaches 0.5 McFarland standard. Concentrations of MXF 1704 cm−1 and 1454 cm−1. Figure 3 shows the spectrum of
solutions were adjusted from 2.5 to 320 mg L−1. Microdilution NRL (I), MXF (II), and MXF-loaded NRL (III) using the
plates were incubated at 37 °C for 24 h. MIC was considered attenuated total reflectance (ATR) mode obtained in the region
as the lower concentration of the drug that prevented growth. of 400–4000 cm−1 (Borges et al. 2017; Floriano et al. 2017).
In the spectrum (I), it is possible to identify characteris-
tic absorption for poly(cis-1,4-isoprene): between 2960 and
MBC measurements
2852 cm−1 (=CH stretching), 1446 cm−1 and 1375 cm−1
(deformation of CH2 and CH3), 1662 cm−1 (elongation
Cell suspensions used to perform the microdilution sensi-
C=C). Finally, the most representative absorption for NRL,
tivity test were sub-cultured into 90×15-mm Petri dishes
835 cm−1 (CH out of plan) was also observed. This spectrum
containing Mueller Hinton broth (Sigma-Aldrich, USA).
also showed the vibrations of proteins and phospholipids that
Concentrations of MXF solutions were adjusted from 2.5 to
stabilize the rubber molecules at 3537 cm−1 (stretching of OH
320 mg L−1. After that, they were incubated for 24 h at 37
and NH), 1542 cm−1 (NH), and between 1130 and 1010 cm−1
°C to verify the MBC, determined by the lowest concentra-
indicate compounds with oxygen (C–O and –O–O–).
tion of the drug that prevented the growth of the subculture
It was noted in the MXF spectrum that this compound
(Clinical and Laboratory Standards Institute 2009).
exhibits C=O stretching represented by the absorption of
1704 cm−1, the presence of monofluorobenzene (1183 cm−1)
 ethod for antibacterial disk diffusion
M and deformation of C=C (1454 cm−1) (Mudgil and Pawar
susceptibility test 2013). Nevertheless, the MXF-loaded NRL spectrum
showed the absorption bands at 1704 cm−1 and 1454 cm−1,
The disk diffusion test was performed in sterile Mueller demonstrating the presence of the antibiotic. Furthermore,
Hilton agar (Sigma-Aldrich, USA). Two milliliters con- the absorption bands found at 835 cm−1 and 2852 cm−1
taining 1.5×108 cells mL−1 from the cultures of E. coli, P. represented the characteristic bands of NRL. Therefore, the

Subsequently, membranes of NRL, NRL + MXF (5 μg),

aeruginosa, S. aureus, and S. epidermidis was inoculated. results have shown no covalent interaction between NRL and

NRL + MXF (20 μg), and commercial disk of MXF (5 μg)

MXF since the characteristic bands of both compounds were
maintained after the drug incorporation.10
were dispensed in the center of the inoculated agar plates.
The diameter of the circular zones of inhibition was meas-
ured after 24 h of incubation (Cockerill 2011). Mechanical strength testing

In order to evaluate whether the incorporation of the drug

Results influenced on the elasticity of the biomaterial, membranes of
MXF-loaded NRL and of NRL + NaOH 0.1 M (1:1, used as
Scanning electron microscopy (SEM) was performed in order the control) were submitted to the mechanical strength test.
to analyze the morphology of the biomaterial. Figure 1a illus- The mean of the triplicate of samples was 27.470±0.10 mm
trates the micrograph of MXF powder, which is observed in in height, 27.180±0.13 mm in width, and 0.876±0.02 mm
different dimensions of drug crystal. The crystals, when incor- in thickness. The values obtained are described in Table 1.
porated into the latex, appear to be distributed on the surface The curve profile presented the three stages of elastomers;
of the NRL membrane (Fig. 1b). On the other hand, the NRL elastic, plastic, and rupture zone (Fukahori et al. 2017.) In the
membrane (Fig. 1c) presents a homogeneous surface. first region, the stress was directly proportional to the elonga-
Cross-section images of the samples have shown the pres- tion, and the material still had the capacity to return to the
ence of the drug in the bulk of the material. Figure 2a illustrates original shape since the applied tension has been withdrawn.
the NRL without the presence of MXF, where it is possible The subsequent region referred to the plastic behavior of the
to see only the flaw of the material suffered at the moment of material, where occurred tightening, corresponds to a decrease
the fracture. In Fig. 2b, the image of the material bulk showed in the diameter of the specimen. At this stage, the deformation of
MXF crystal into the pores of the membrane. The same crystal the material is permanent and only part of its size is able to return
was seen in Fig. 2c, with a magnification of ×5000. to the original form. Lastly, the curve named voltage-induced

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Fig. 1 Surface micrographs of MXF powder samples (a), NRL + MXF (b), and NRL (c) membranes. Note the presence of drug crystals on the
surface of the material. The images have a magnification of ×300 and 2 KV

crystallization leads to sample rupture. Additionally, the data cellular viability for NRL membranes tested in 3T3 cells,
showed that the incorporation of the drug in the NRL promoted indicating no toxic effect. Similar result was observed for
a decrease of 47.2% of rupture stress. Meanwhile, the material keratinocytes (Fig. 5a), with nearly 80±1.51% of cellular

NRL in the lowest concentration of the drug (5 μg) in HaCat,

deformation suffered a decrease of 8.63%. viability for NRL membrane. For samples of MXF-loaded

sent toxicity in 3T3. The viability of NRL + MXF 10 μg

Drug release profile it is shown over 83.60±11.39% of viability and did not pre-

The MXF release profile through the NRL is represented was around 66.3±2.00% in HaCat; however, higher viability

+ MXF 20 μg were 92.79±2.55% in 3T3 and higher than

in Fig. 4, which was seen two-step kinetics: (i) initial burst was found in 3T3, 83.97±7.4%. The percentages of NRL
release and (ii) stable profile. The MXF was mainly released
by diffusion; however, another mechanism of release was by 71.8±1.04% in HaCat.

concentration (5 μg mL−1) achieved nearly 80±3.67% of cell

bulk degradation. After 48 h, 20% of MXF was released in In the MXF dilution, we found that the drug at the lowest
pH 5.6, and 32.4% of the antibiotic was carried out the NRL
at the pH of a healed skin. viability in HaCat and for 3T3 lines, almost no cytotoxicity

for MXF 10 μg mL−1, fibroblasts 92.25±5.7% of viability

The release was sustained for 295 h when the poly- was presented, 95.77±4.0%. Samples of viability for 3T3,
meric membrane was able to release 65.44% of MXF at

tration of 20 μg mL−1, MXF showed nearly 70±5.14% of

pH 7.4, and at pH 5.6, the percentage was 53.13% (Fig. 4). while in HaCat, approximately 71.26±1.88%. In a concen-
The release kinetics obeys the bi-exponential function y
(t)=y0+A1e−t/t1+A2e−t/t2, where y (t) is the amount of MXF viability in HaCat, while in 3T3 it was 84.81±13.20%. The
present in NRL at a given period, t. viability decreased proportionally as the concentration of
MXF increased, although maintained a no toxic effect.
Cell viability
Antibacterial susceptibility test
The cytotoxic effects of the samples (MXF, NRL, and NRL
+ MXF) in fibroblasts and keratinocytes cell lines were Using susceptibility testing methodology as recommended
observed in Fig. 5. The results showed 90.30±12.95% of by CLSI, the results for the in vitro antibacterial activity of

Fig. 2 The cross-sectional micrographs (a and b) of the NRL and NRL + MXF respectively, in magnification of ×300. Panel (c), of ×5000 mag-
nification, seeking a better visualization of the drug present inside the material

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Fig. 3 FTIR spectrum of NRL (I), MXF powder (II), and NRL + MXF (III) samples

the MXF were evaluated. The antibiotic showed the strong- correlation of the zone of inhibition expressed by the sam-

epidermidis, with an MIC of 2.5 μg mL−1, 2.5 μg mL−1, 5

est activity against S. aureus, E. coli, P. aeruginosa, and S. ples on agar surface.

μg mL−1, and 10 μg mL−1, respectively. The MBC and MIC disk with 5 μg of MXF was 30.00±1.0 mm, 10.00±1.00
The zone of inhibition measured for the commercial

values were similar, except to P. aeruginosa (MBC=7.5 μg mm, 27.00±0.70 mm, and 35.00±0.50 mm to S. aureus,

was observed that the membranes containing 5 μg of MXF

mL−1), indicating that generally, the same concentrations E. coli, P. aeruginosa, and S. epidermidis, respectively. It
of antibiotic were able to both inhibit bacterial growth and
kill the microorganisms. The results of MIC and MBC cor- inhibited the growth of S. aureus, E. coli, P. aeruginosa,
roborate with the reference values described by the CLSI and S. epidermidis with inhibitions zones of approximately
(Cockerill 2011). 30.00±0.90 mm, 28.20±0.20 mm, 20.00±0.30 mm, and

20 μg of MXF had an inhibition halo higher than the NRL

30.10±0.20 mm, respectively. The membranes containing
Antibacterial disk diffusion susceptibility test
+ MXF 5-μg membranes because the zone of inhibition is
We evaluated the antibacterial activity of the biomaterial proportional to the quantify of drug into polymeric matrix
using the disk diffusion test. The method allows investi- (Table 2).
gating microorganism susceptibility to antibiotics by the The results have shown the activity of NRL + MXF
against S. aureus, P. aeruginosa, E. coli, and S. epidermidis.
Table 1 Values obtained by the mechanical strength test NRL alone did not show any antibiotic effect against the

20 μg of MXF, the membranes inhibited the growth ofmi-

tested strains. On the other hand, when loaded with 5 and
Samples Young Modulus Stress (MPa) Deformation (%)

The zone of inhibition of the commercial disk with 5 μg of

(MPa) croorganisms that are commonly found in infected wounds.
NRL + NaOH 0.80 ± 0.04 0.53 ± 0.03 985 ± 50
MXF showed that S. aureus, P. aeruginosa, and S. epider-
NRL + MXF 0.80 ± 0.05 0.28 ± 0.04 900 ± 103
midis were sensitive to the concentration of the drug. The

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Fig. 4 Kinetic release of MXF carried by NRL membrane for 295 h

performed test showed that latex has the ability to release indicates that there was no chemical interaction between
MXF and therefore inhibit the growth of all of the microor- NRL and the drug (Garms et al. 2017). In this study, it is
ganisms tested (Fig. 6). evident that the integrity of the MXF structure was pre-
served after the association of the compounds, in according
to results of Barros et al. (2015) and Zancanela et al. (2019).
Discussion Considering that the biomaterial has been developed for
dermal purpose, its mechanical properties should be suit-
The tests performed for the biomaterial characterization have able for this application. It has been previously reported
shown that the integrity of both compounds was maintained. that although the incorporation of compounds to the NRL
In FTIR analysis, we observed the presence of the transmit- membrane can influence the modulus of elasticity of the
tance peaks used to identify both MXF and NRL. The results material, it still presents sufficient mechanical resistance
corroborate with Garms et al. (2017) that, incorporating a for biomedical applications (Floriano et al. 2017, 2016).
different antibiotic in the NRL membranes, observed that Moreover, in this study, we did not observe any alteration in
both spectra (drug and polymer) were not modified, which the Young Modulus of the polymer after MXF incorporation,

Fig. 5 Cell viability of NRL, NRL + MXF, and MXF samples in: 3T3 cell line (a) and HaCat cell line (b)

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Table 2 Measure of halos formed in S. aureus, E. coli, P. aeruginosa, and S. epidermidis strain in the disk diffusion technique

Microorganisms NRL (mm) Commercial disk MXF 5 μg (mm) NRL + MXF 5 μg (mm) NRL + MXF 20 μg (mm) Reference values (mm)

S. aureus 0 30.00 ± 1.00 30.00 ± 0.90 32.00 ± 0.70 28–35

E. coli 0 10.00 ± 1.00 28.20 ± 0.20 29.00 ± 0.40 28–35

≥24
P. aeruginosa 0 27.00 ± 0.70 20.00 ± 0.30 29.00 ± 0.80 17–25
S. epidermidis 0 35.00 ± 0.50 30.10 ± 0.20 30.40 ± 0.30

maintaining 0.80 MPa for both samples. The results corrobo- moment, as observed in previous studies (Barros et al. 2015,
rate with a previous study of Marcelino et al. (2018), which 2016; Marcelino et al. 2018). This behavior refers to the
have shown similar elasticity behavior when incorporated stable profile effect since the release occurs by diffusion
an antifungal agent in NRL membranes. The values of the from the more concentrated to lower concentration medium
modulus of elasticity observed in this work (0.8 MPa) were (Barros et al. 2015, 2016).
in accordance with the value of Young’s modulus of native NRL is a versatile polymer that presents attractive charac-
human skin ranges from 0.01 to 50 MPa (Zhao et al. 2017). teristics for SDS application, e.g., its hydrophobicity. Being
The SEM technique allowed to visualize the drug distri- a hydrophobic device enables its use in combination with
bution on the material and its modifications. The presence of several drugs, since the dispersion control of the bioactive
the drug crystals on the surface of the membrane indicated occurs mainly through diffusion. It probably explains the
the release profile of the antibiotic through the polymeric initial burst release, where the diffusion of MXF occurred
matrix (Fig. 2). These crystals tend to rapidly solubilize dur- through the pores located in the NRL surface. Additionally,
ing the initial release, promoting the burst effect (Barros the membrane works as a reservoir where the higher the
et al. 2016). It is also possible to visualize in Fig. 3 the amount of MXF incorporated in the biomaterial, the greater
drug in the bulk of the membrane. In this case, NRL works the release of it (Ashok et al. 2017; Zhang et al. 2017).
as a reservoir. Thus, we can assume that the crystals pre- Besides the drug diffusion, the release is also attributed to
sent in the material bulk would only be released in a second the polymer erosion. During this process, as the material is

20 μg; 3 NRL+MXF 5 μg; 4 commercial disk MXF 5 μg

Fig. 6 Halo inhibition presented by strains of a E. coli, b P. aeruginosa, c S. aureus, d S. epidermidis, and the samples 1 NRL; 2 NRL + MXF

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degraded, the associated compounds are dispersed in the into the NRL. NRL without MXF did not demonstrate anti-
medium, resulting in stable release profile (Carvalho et al. microbial activity, corroborating with the results presented
2018; Liu et al. 2018; Zhang et al. 2017). in our previous work, where the polymer has shown no activ-
During the healing process, the skin pH changes from ity for the microorganism tested in a broth dilution sensitiv-
the physiological pH (around 7.4) of the wound to a more ity test (Garms et al. 2017). The microorganisms that were
acid pH of the healthy skin, which is around 5.6 (Watters sensitive to the MXF-loaded NRL membranes at the two
et al. 2015). Nevertheless, the pH changes can influence concentrations tested were S. epidermidis, E. coli, S. aureus,
the release rate of the drug. In a recent study evaluating and P. aeruginosa. The results corroborate with studies of
the release of an antitumor drug through polymeric matrix, the use of NRL as an antibiotic delivery vehicle (Marcelino

E. coli, the commercial disk with 5 μg of MXF presented a

Kostkova et al. (2017) observed that at pH 7.4, the release et al. 2018; Garms et al. 2017; Phaechamud et al. 2016). For
was considerably lower than at pH 5.0. This fact was associ-
ated to the degradability of the material under these condi- low inhibition (intermediate), with diameter <28 mm. The
tions. These results corroborate to a similar work using NRL inhibition zone depends not only on the intrinsic antimi-
as release matrix, which was observed a higher release rate crobial activity but also on the diffusion rate of the antimi-
at pH values 7.4 and 7.6 due to the breakage of cross-linked crobial agent (Li et al. 2016). Therefore, the samples used
NRL membranes destabilizing the membrane (Alberto et al. (polymeric membrane disks) could demonstrate different
2014). Additionally, Mahor et al. (2016) using a different diffusion profile than the commercial disk (cellulose).

the membranes containing 20 μg of MXF, it is also possible

matrix to release MXF was able to promote 60–81% of drug Considering that the E. coli have shown susceptibility to
delivered during 12 h, showing a high delivery rate of the
antibiotic and a similar release profile as obtained in our that it was related to antibiotic resistance of the strain in this
study (Mahor et al. 2016). concentration (Clinical and Laboratory Standards Institute
Cytotoxicity analysis showed low toxicity of MXF-loaded 2009). Considering these results, we can assume that the

would be the NRL + MXF 20 μg. In a previous publication

NRL membranes in fibroblasts and keratinocytes lines. Guo most suitable sample to be employed in infected wounds
et al. (2016) used the technique in a biocompatibility study
of thermosensitive polymers and evaluated the percentage from our research group, latex membrane loaded with flu-
of cell viability in different cell lines (Guo et al. 2017). The conazole was investigated for skin fungal infection therapy.

test) have shown that NRL loaded with 25 μg of flucona-

polymer showed low toxicity even at high concentrations. In Microbiological analyses (disk diffusion and microdilution
another study, cellulose membranes also presented high cell
viability indicating biocompatibility of the material (Souza zole was effective against Candida albicans (Marcelino et al.
et al. 2017). Barros et al. (2019) evaluated the cytotoxicity 2018).
of NRL-Glycerol patch in keratinocytes (HaCaT) and fibro- In a qualitative analysis, Garms et al. (2017) demon-
blasts (3T3) which are two cell types present in the skin. strated that the activity of the drug released by the mem-
They showed that glycerol did not present cytotoxicity in branes was maintained, inhibiting microbial growth. In a
the fibroblasts; however, it had a dose-dependent influence study of gentamicin-loaded NRL membranes, Phaechamud
in the keratinocyte. et al. (2016) have demonstrated its activity against S. aureus
Studies have reported high cellular viability of NRL and P. aeruginosa strains. They observed an increase in the
membranes under similar conditions to the present study inhibition zone of the membranes when compared to the
for osteoblast cell line (Borges et al. 2017; Miranda et al. commercial disks of gentamicin. Zancanela et al. (2019)
2017). Borges et al. (2017) showed NRL low toxicity in con- compared the potential antimicrobial of the three different
centrations lower than 80%. Additionally, they demonstrated types of propolis incorporated into NRL membranes against
that NRL could be employed as scaffold facilitating cellular Candida albicans. The authors observed that red propolis
growth. Thereby, considering the material biocompatibil- compounds released through membrane showed an inhibi-

olis extract (MIC of 78 μg mL−1). These results showed that

ity, we can restate the potential of the MXF-loaded NRL tory activity as strong as that observed for the pure red prop-
membranes for the treatment of infected wounds (Akturk
et al. 2016). NRL is able to potentialize the action of the incorporated
The in vitro method (disk diffusion) was used to dem- compound.
onstrate MXF-loaded NRL activity in four bacteria strains In summary, the present study has shown that the incor-
that are commonly related to the colonization of chronic poration of MXF in NRL did not promote any molecular
wounds (Biswas et al. 2018). The results of MIC and MBC interaction, preserving the integrity of the compounds.
corroborate with the reference values recommended by the Additionally, the MXF-loaded NRL membranes did not
CLSI (Cockerill 2011). present any significant change in the mechanical properties
The disk diffusion test was performed to demonstrate that of the material, showing enough elasticity for dermal appli-
the drug activity was maintained after being incorporated cation. Furthermore, the cytotoxicity assay demonstrated its

13
 Appl Microbiol Biotechnol

biocompatibility and the microbiological assays confirmed Barros NR, Miranda MCR, Borges FA, Gemeinder JLP, Mendonça RJ,
the ability of the polymer to deliver the drug without influ- Cilli EM, Herculano RD (2017) Natural rubber latex: develop-
ment and in vitro characterization of a future transdermal patch
ence its pharmacological properties. Moreover, it has dem- for enuresis treatment. Int J Polym Mater Polym 66(17):871–876.
onstrated activity against the major bacteria strain presented https://doi.org/10.1080/00914037.2017.1280795
in wound infections. Therefore, the present study has shown Barros NR, Santos RS, Miranda MCR, Bolognesi LFC, Borges FA,
that MXF-loaded NRL membrane is a promising biomaterial Schiavon JV, Marques RFC, Herculano RD, Norberto AMQ
(2019) Natural latex-glycerol dressing to reduce nipple pain and
to infected wound treatment. healing the skin in breastfeeding women. Skin Res Technol 0:1–8.
https://doi.org/10.1111/srt.12674
Acknowledgments We thank all partners and laboratory members for Biswas DP, O'Brien-Simpson NM, Reynolds EC, O'Connor AJ,
their kind help. Tran PA (2018) Comparative study of novel in situ decorated
porous chitosan-selenium scaffolds and porous chitosan-silver
Funding The authors gratefully acknowledge the support of CNPq, scaffolds towards antimicrobial wound dressing application.
National Council for Scientific and Technological Development (no. J Colloid Interface Sci 515:78–91. https://doi.org/10.1016/j.
470261/2012-9 and no. 134018/2016-8), and FAPESP, São Paulo jcis.2018.01.007
Research Foundation (no. 2014/17526-8 and no. 2011/17411-8). Borges FA, de Barros NR, Garms BC, Miranda MCR, Gemeinder JLP,
Ribeiro-Paes JT, Silva RF, de Toledo KA, Herculano RD (2017)
Application of natural rubber latex as scaffold for osteoblast to
Compliance with ethical standards guided bone regeneration. J Appl Polym Sci 134(39):10. https://
doi.org/10.1002/app.45321
Conflict of interest The authors declare that they have no conflict Brehler R, Rutter A, Kutting B (2002) Allergenicity of natural rub-
of interests. ber latex gloves. Contact Dermatitis 46(2):65–71. https://doi.
org/10.1034/j.1600-0536.2002.450201.x
Ethical statements This paper is our original work. It has not been Carvalho FA, Uchina HS, Borges FA, Oyafuso MH, Herculano RD,
submitted elsewhere, and it is not under consideration in any other Gremiao MPD, Santos AG (2018) Natural membranes of Hevea
Journal. This article does not contain any studies with human par- brasiliensis latex as delivery system for Casearia sylvestris leaf
ticipants or animals performed by any of the authors. All the authors components. Rev Bras Farmacogn-Braz J Pharmacogn 28(1):102–
have seen the manuscript and approved its submission to Applied 110. https://doi.org/10.1016/j.bjp.2017.10.007
Microbiology and Biotechnology. Clinical and Laboratory Standards Institute (2009) Clinical and
Laboratory Standards Institute CLSI; CLSI Publishes 2009
Antimicrobial susceptibility testing standards. Atlanta, p 12
Cockerill FR (2011) Performance standards for antimicrobial suscep-
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