BR102019027533A2 - antimicrobial bacterial cellulose dressings containing silver nanoparticles for use in the healing of chronic wounds and burns, obtaining process and obtained products - Google Patents

Abstract

The present invention refers to dressings consisting of bacterial cellulose (CB) membranes containing silver nanoparticles (NPAg), to be used in the treatment of chronic wounds, obtained from the immersion of porous CB membranes in successive dilutions of NPAg, with concentration between 62.5 and 500 ppm. The proposed dressings present a homogeneous distribution of nano-sized silver particles on the surface of the CB membrane nanofibers; they are viable and nontoxic at the cellular level and promote inhibition of bacterial growth (against E. coli and S. aureus). Therefore, they solve the problems present in the technical field, with regard to the rapid and large release of NPAg and/or silver compounds, which can cause toxicity, and to heterogeneous impregnation; thus being efficient for the treatment of chronic wounds and burns.

Description

ANTIMICROBIAL BACTERIAL CELLULOSE DRESSINGS CONTAINING SILVER NANOPARTICLES FOR APPLICATION IN THE HEALING OF CHRONIC WOUNDS AND BURNS, OBTAINING PROCESS AND OBTAINED PRODUCTS BRIEF DESCRIPTION

[001] This application for a patent for the invention of unpublished "ANTIMICROBIAL CELLULOSE DRESSINGS CONTAINING SILVER NANOPARTICLES FOR APPLICATION IN THE HEALING OF CHRONIC WOUNDS AND BURNS, PROCESS OF PROCUREMENT AND OBTAINED PRODUCTS" which refers to the production of bacterial cellulose membranes impregnated with silver nanoparticles, obtained from the immersion of CB membranes in successive dilutions of the NPAg suspension. The proposed dressings were able to overcome the problems present in the technical field, such as the rapid and large release of NPAg and/or compounds of silver, which can cause toxicity; heterogeneous impregnation; in addition to being able to promote increased antimicrobial activity against gram-positive and gram-negative bacteria and, finally, increase the effectiveness of these biodressings in preventing infections in chronic wounds and burns.

APPLICATION FIELD

[002] The present invention belongs to the section of human needs, to the field of health; more specifically, preparations for medical purposes characterized by special physical forms; and the chemistry section, the field of macromolecular organic compounds, their preparation or chemical processing and compositions based on them; more specifically, to cellulose, for describing the obtainment of bacterial cellulose membranes impregnated with silver nanoparticles and being used as dressings in the healing of chronic wounds and burns.

CONVINENCE

[003] Bacterial cellulose (CB) consists of a natural biopolymer, synthesized by bacteria (such as Acetobacter xylinum) composed largely of water (98-99%) presenting unique properties such as high liquid adsorption, high chemical purity, biocompatibility , biodegradability, high mechanical strength, and can be sterilized without any change in its structure and properties (J. Wu, Y. Zheng, W. Song, J. Luan, X. Wen, Z. Wu, X. Chen, Q Wang and S. Guo, 2014: Carbohydrate Polymers; T. Maneerung, S. Tokura and R. Rujiravanit, 2008: Carbohydrate Polymers).

[004] Due to all its advantages, CB has been used in several applications as a raw material for the production of paper, textiles, coatings, cosmetics, OLEDs, among others (HS Barud, T. Regiani, RFC Marques, WR Lustri , Y. Messaddeq and SJL Ribeiro, 2011: Journal of Nanomaterials; W. Hu, S. Chen, J. Yang, Z. Li and H. Wang, 2014: Carbohydrate Polymers; ML Foresti, A. Vázquez and B. Boury, 2017: Carbohydrate Polymers).

[005] Recently, the use of BC in biomedical applications has grown exponentially, being used in tissue engineering, construction of artificial blood vessels and mainly in the development of dressings for wounds and burns (ML Foresti, A. Vázquez and B. Boury, 2017: Carbohydrate Polymers; HS Barud, C. Barrios, T. Regiani, RFC Marques, M. Verelst, J. Deexpert-Ghys, Y. Messaddeq and SJL Ribeiro, 2008, Materials Science and Engineering C).

[006] Among all these applications, the use of BC in wounds and burns provides a moist environment to the affected region, favoring healing and reducing pain in injured patients. However, CB membranes lack antimicrobial activity, which is one of the critical roles of the skin barrier in effective wound healing (J. Wu, Y. Zheng, W. Song, J. Luan, X. Wen, Z. Wu , X. Chen, Q. Wang and S. Guo, 2014: Carbohydrate Polymers; T. Maneerung, S. Tokura and R. Rujiravanit, 2008: Carbohydrate Polymers).

[007] To overcome this disadvantage, silver compounds (silver nitrate (AgNO3) and silver sulfodiazine) have been synthesized and incorporated into CB membranes, adding antimicrobial activity to these biomaterials (H. J. Klasen, 2000: Burns). However, different methods of synthesis (in situ) and incorporation of these silver (Ag) compounds lead to the obtainment of biocomposites with heterogeneous distribution in addition to rapid release of Ag+ ions, which can generate ineffectiveness for prolonged periods in addition to certain tissue toxicity alive (HJ Klasen, 2000: Burns).

[008] Particularly, the use of silver nanoparticles (NPAg) has been intensively studied due to its great antimicrobial activity for bacteria, fungi, viruses, among others (J. Wu, Y. Zheng, W. Song, J. Luan, X Wen, Z. Wu, X. Chen, Q. Wang and S. Guo, 2014: Carbohydrate Polymers;HS Barud, C. Barrios, T. Regiani, RFC Marques, M. Verelst, J. Deexpert-Ghys, Y. Messaddeq and SJL Ribeiro, 2008, Materials Science and Engineering C; Santa Maria LC, ALC Santos, PC Oliveira, HS Barud, Y. Messaddeq and SJL Ribeiro, 2009, Materials Letters) The antimicrobial properties of NPAg depend on their bioavailability (in regarding the wounds) of the large surface area they present (due to their nanometric size) and, consequently, of their non-aggregation (J. Wu, Y. Zheng, W. Song, J. Luan, X. Wen, Z. Wu, X Chen, Q. Wang and S. Guo, 2014: Carbohydrate Polymers; T. Maneerung, S. Tokura and R. Rujiravanit, 2008: Carbohydrate Polymers).

[009] In this sense, the present invention managed to combine and preserve the properties of CB with NPAg Nanox® through the incorporation of NPAg in dry porous CB membranes as a method for obtaining new biocuratives (CB@NPAg). The methodology managed to overcome problems present in the technical field, such as the rapid and large release of NPAg and/or silver compounds, which can cause toxicity; heterogeneous impregnation; in addition to promoting improved antimicrobial activity against gram-positive and gram-negative bacteria and, finally, increasing the effectiveness of these biodressings in preventing infections in wounds and burns.

BACKGROUND OF THE INVENTION

[010] In the current state of the art, there are some prior arts describing biocellulose-based materials used in wound treatment, the presence of silver nanoparticles in wound care products is also described. However, no precedents were found that described the obtainment of dressings based on biocellulose and silver nanoparticles obtained through a process that could overcome problems present in the technical field, such as the rapid and large release of NPAg and/or silver compounds , which can cause toxicity; heterogeneous impregnation; in addition to promoting improved antimicrobial activity against gram-positive and gram-negative bacteria and, finally, increasing the effectiveness of these biodressings in preventing infections in wounds and burns.

[011] Previously WO2013176633A1, entitled "Wound dressing comprising biocellulose and silver nanoparticles", describes the obtainment of wound dressings that comprise carboxymethylcellulose, silver nanoparticles with a concentration of about 1000g/100cm2 or less, about 1000g/cm3, where silver nanoparticles have a plasmonic resonance band between 600 and 800 nm (here you need to specify what the maximum of the band is, better than the range that is there, that's what's most important) . The dressing is obtained from the application of a suspension of silver nanoparticles to biocellulose, the suspension also has stabilizers or thickeners.

[012] Priority BRPI1005066, entitled "Process for obtaining antimicrobial material based on bacterial cellulose and other types of amines and antimicrobial material", refers to a process for obtaining antimicrobial material based on bacterial cellulose and silver nanoparticles using three types of amines as silver reducing agents, useful for the treatment of chronic wounds and burns. The obtainment of antimicrobial material based on bacterial cellulose begins with the culture of Acetobacter xylinum in trays, for a period of 12 to 120 h at a temperature of about 28 °C, in Schramm-Hestrín medium, being characterized by, after a period of period between 12 and 120 h, allow removal of bacterial cellulose from the medium; this cellulose being treated with strong bases for the complete removal of acetobacteria embedded in the cellulose chains; then, it is added to a solution of silver nitrate, in a concentration between 10 mM to 1M, for 10 min; then, a solution of amines, composed of ethanolamine, diethanolamine or triethanolamine (N-(CH2-CH2OH)3), in a concentration between 10mM and 1M, is added to the membrane, being left to rest for 12 h; after this period, the material was washed in deionized water and dried at a temperature between 50 and 80 °C for 24 h.

[013] Priority BR102012026425A2, entitled "Biocellulose-based composition and bioactive agents for the treatment of wounds", refers to the production process of a curative composition, based on bioactive compounds and bacterial cellulose, in membrane or powder (in nanometer to millimeter particle size). The bioactives used are turmeric extract (Curcuma longa), which may contain (curcumin, demethoxycurcumin, bis-demethoxycurcumin and/or cyclocurcumin); Aloe Vera; reduced silver; ascorbic acid and/or calcium ascorbate, calcium (Ca2+, which may come from any salt with low toxicity) and sodium alginate (which may be of low, medium or high viscosity).

[014] In the article "Biosynthesis and characterization of bacterial nanocellulose for tissue engineering" Fischer et al. (2018) report on bacterial nanocellulose (BNC) dressings demonstrating their great application potential due to their properties such as high purity content, high power of water absorption and excellent biological adaptability, and describe that the incorporation of metals in the nanocellulose membrane is very promising, silver nanoparticles (NPAg) have been the object of several studies due to their antibacterial properties; cerium nitrate, Ce(NO3 )3, in turn, increases the effectiveness of the treatment by presenting immunomodulatory properties. Thus, in the work carried out, bacterial cellulose membranes functionalized with cerium nitrate and silver nanoparticles were synthesized and characterized for application in the treatment of human skin injured by burns .

[015] Previously CN101509025A, entitled "Method of preparing bacteria cellulose composite material", describes the obtainment of a composite membrane consisting of bacterial cellulose, sodium alginate, collagen, soluble chitosan and gelatin or hyaluronic acid.

[016] Previous PI0601330-9, entitled "Topical composition of biocellulose in the form of gel, spray-aerosol, cream and/or aqueous suspension for the treatment of epithelial lesions", presents a topical composition of bacterial cellulose as a new pharmaceutical formulation in form of gel, spray-aerosol, cream and/or aqueous suspension for the treatment of epithelial lesions, in addition to the delivery of other pharmacological agents.

[017] Previous PI0000465-0, entitled "Process for obtaining porous and/or fenestrated cellulosic membranes for the treatment of wounds, and cellulosic membranes thus obtained", describes the transformation of zooglea formed by cellulose microfibrils produced by bacteria, in porous and/or fenestrated membranes with ideal characteristics for curative and preventive treatments for wounds, especially those with rapid evolution and progression; those with difficult tissue regeneration, because they exude too much; and in injury prevention.

[018] The scientific article entitled "Present status and applications of bacterial cellulose-based materials for skin tissue repair", by Fu et al. (2012), provides a literature review on the use of bacterial cellulose to repair damaged skin tissue.

[019] Previous BR102013010960-6, entitled "Bioactive membranes composed of cellulose nanofibers/hydrocolloids", presents the formation of films based on plant and bacterial cellulose impregnated with hydrocolloids of natural and synthetic origin, which may or may not contain collagen or appropriate drugs.

[020] Previously BR102014016341-7, entitled "Biocellulose nanocomposites and their use", a nanocomposite of biocellulose and diethyldithiocarbamate is described, that is, a polymeric matrix of bacterial cellulose coated with diethyldithiocarbamate derivatives. The described biocomposite has the following characteristics: good flexibility, easy application to skin lesions, leishmanicidal capacity, absence of toxicity and sustained release of the active compound, which reduces the need for dressing change.

[021] Despite the large number of references in the current state of the art describing the association of biocellulose and silver nanoparticles, it is noted that none of the above describes a process of obtaining that enables the resolution of problems present in the technical field, such as such as: the rapid and large release of NPAg and/or silver compounds, which can cause toxicity; heterogeneous impregnation; in addition to promoting increased antimicrobial activity against gram-positive and gram-negative bacteria and, finally, greater effectiveness of these biodressings in preventing infections in wounds and burns.

PURPOSE OF THE INVENTION

[022] The present invention aims to provide bacterial cellulose membranes containing silver nanoparticles to be used in the treatment of chronic wounds.

THE INVENTION

[023] The present invention relates to bacterial cellulose (CB) membranes containing silver nanoparticles (NPAg) to be used in the treatment of chronic wounds, obtained from the immersion of porous CB membranes in successive dilutions of NPAg, in this way , suspensions of 500, 250, 125 and 62.5 ppm of NPAg are added to the membranes.

[024] The results obtained from physicochemical analyzes of NPAg and dressings confirmed the homogeneous distribution of nanometer-sized silver particles on the surface of the CB membrane nanofibers. Biological tests were carried out and, based on the evaluation of cell viability, it was possible to determine the minimum amount of NPAg that would be non-toxic at the cellular level and it was determined that all dressings were viable at the cellular level. The antimicrobial activity tests for the dressings were performed via the contact method and all samples showed inhibition of bacterial growth (against E. coli and S. aureus). In addition, death assays show that the inhibition of the microbial environment of E. coli and S. aureus occurs for a period of up to 28 and 48 h, respectively, suggesting that these dressings can be used in these time intervals, that is, the switch could be suggested after reaching the times determined via death trials.

ADVANTAGES OF THE INVENTION

• ✔ Disponibilizar membranas de celulose bacteriana (CB) contendo nanopartículas de prata (NPAg) a serem empregados no tratamento de feridas crônicas obtidas por um processo com simples etapas de execução;
• ✔ Disponibilizar membranas de celulose bacteriana (CB) contendo nanopartículas de prata (NPAg) a serem empregados no tratamento de feridas crônicas com distribuição homogênea das partículas de prata em tamanho nanométrico na superfície das nanofibras das membranas;
• ✔ Disponibilizar membranas de celulose bacteriana (CB) contendo nanopartículas de prata (NPAg) a serem empregados no tratamento de feridas crônicas viáveis e atóxicas a nível celular;
• ✔ Disponibilizar membranas de celulose bacteriana (CB) contendo nanopartículas de prata (NPAg) a serem empregados no tratamento de feridas crônicas que promovem inibição do crescimento bacteriano, contra E. coli e S. aureus, por um período de até 28 e 48 h, respectivamente, e que, portanto, demanda sua troca apenas após esses períodos.

[025] The present invention has the following main advantages:

• ✔ Provide bacterial cellulose (CB) membranes containing silver nanoparticles (NPAg) to be used in the treatment of chronic wounds obtained by a process with simple execution steps;
• ✔ Provide bacterial cellulose (CB) membranes containing silver nanoparticles (NPAg) to be used in the treatment of chronic wounds with homogeneous distribution of nanometric-sized silver particles on the surface of the membrane nanofibers;
• ✔ Provide bacterial cellulose (CB) membranes containing silver nanoparticles (NPAg) to be used in the treatment of viable and non-toxic chronic wounds at the cellular level;
• ✔ Provide bacterial cellulose (CB) membranes containing silver nanoparticles (NPAg) to be used in the treatment of chronic wounds that promote bacterial growth inhibition, against E. coli and S. aureus, for a period of up to 28 and 48 h, respectively, and that, therefore, demands its exchange only after these periods.

DESCRIPTION OF FIGURES

• ✔ FIG 1: Espectro de absorção na região do ultravioleta-visível das NPAg em suspensão aquosa;
• ✔ FIG 2: Microscopia eletrônica de varredura das NPAg obtidas com distintos aumentos (5000, 10000, 25000 e 50000x);
• ✔ FIG 3: Ensaio de viabilidade celular em diferentes concentrações das NPAg. Controle positivo (CTRL+: DMEM + SBF + 30 % DE DMSO) e controle negativo (CTRL-: DMEM + SBF + Antibióticos + Antifúngico);
• ✔ FIG 4: Determinação da concentração inibitória mínima (CIM) das NPAg nas bactérias Staphylococcus aureus (gram-positiva) e Escherichia coli (gram-negativa). Controle positivo: ausência de NPAg.
• ✔ FIG 5: Curativos porosos contendo diferentes concentrações de NPAg. CB@NPAg1: 50 %, CB@NPAg2: 25 %, CB@NPAg3: 12,5 % e CB@NPAg4: 6,25 % de suspensão de NPAg concentrada (1000 ppm) adicionados na preparação;
• ✔ FIG 6: Microscopia eletrônica de varredura (MEV) da amostra CB@NPAg1;
• ✔ FIG 7: Microscopia eletrônica de varredura (MEV) da amostra CB@NPAg2;
• ✔ FIG 8: Microscopia eletrônica de varredura (MEV) da amostra CB@NPAg3;
• ✔ FIG 9: Microscopia eletrônica de varredura (MEV) da amostra CB@NPAg4;
• ✔ FIG 10: Curvas de ATG/DTA da amostra CB@NPAg1;
• ✔ FIG 11: Curva de calibração da prata para quantificação do sobrenadante das amostras CB@NPAgs;
• ✔ FIG 12: Ensaio de viabilidade celular das amostras CB@NPAgs e duas comerciais: Aquacel Ag e Mepilex Ag. Controle positivo (C+: DMEM + SBF + 30% DE DMSO) e controle negativo (C-: DMEM + SBF + Antibióticos + Antifúngico);
• ✔ FIG 13: Testes de atividade antimicrobiana (método de contato) realizados em Escherichia coli (108 CFU/mL) durante 24 h de incubação à 37 °C das amostras CB@NPAgs e controle (Nexfill@ puro poroso);
• ✔ FIG 14: Testes de atividade antimicrobiana (método de contato) realizados em Staphylococcus aureus (108 CFU/mL) durante 24 h de incubação à 37 °C das amostras CB@NPAgs e controle (Nexfill@ puro poroso);
• ✔ FIG 15: Ensaio de curva de morte dos curativos CB@NPAgs em E. coli (108 CFU/mL) durante 48 h de incubação à 37 °C;
• ✔ FIG 16: Ensaio de curva de morte dos curativos CB@NPAgs em S. aureus (108 CFU/mL) durante 48 h de incubação à 37 °C;
• ✔ FIG 17: Ensaio de curva de morte do branco (CB Nexfill pura) e da amostra CB@NPAg embebida com prata e amostra comercial Mepilex Ag em E. coli (102 CFU/mL) durante 48 h de incubação à 37 °C;
• ✔ FIG 18: Ensaio de curva de morte do branco (CB Nexfill pura) e dos curativos CB@NPAgs em E. coli (102 CFU/mL) durante 48 h de incubação à 37 °C;
• ✔ FIG 19: Ensaio de curva de morte do branco (CB Nexfill pura) e dos curativos CB@NPAgs em P. aeruginosa (102 CFU/mL) durante 48 h de incubação à 37 °C.

[026] The invention will be described in a preferred embodiment, so, for better understanding, references will be made to the figures:

• ✔ FIG 1: Ultraviolet-visible absorption spectrum of NPAg in aqueous suspension;
• ✔ FIG 2: Scanning electron microscopy of NPAg obtained with different magnifications (5000, 10000, 25000 and 50000x);
• ✔ FIG 3: Cell viability assay at different concentrations of NPAg. Positive control (CTRL+: DMEM + FBS + 30% DMSO) and negative control (CTRL-: DMEM + FBS + Antibiotics + Antifungal);
• ✔ FIG 4: Determination of the minimal inhibitory concentration (MIC) of NPAg in Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria. Positive control: absence of NPAg.
• ✔ FIG 5: Porous dressings containing different concentrations of NPAg. CB@NPAg1: 50%, CB@NPAg2: 25%, CB@NPAg3: 12.5% and CB@NPAg4: 6.25% suspension of concentrated NPAg (1000 ppm) added in the preparation;
• ✔ FIG 6: Scanning electron microscopy (SEM) of the CB@NPAg1 sample;
• ✔ FIG 7: Scanning electron microscopy (SEM) of the CB@NPAg2 sample;
• ✔ FIG 8: Scanning electron microscopy (SEM) of the CB@NPAg3 sample;
• ✔ FIG 9: Scanning electron microscopy (SEM) of the CB@NPAg4 sample;
• ✔ FIG 10: ATG/DTA curves of sample CB@NPAg1;
• ✔ FIG 11: Silver calibration curve for supernatant quantification of CB@NPAgs samples;
• ✔ FIG 12: Cell viability assay of CB@NPAgs and two commercial samples: Aquacel Ag and Mepilex Ag. Positive control (C+: DMEM + FBS + 30% DMSO) and negative control (C-: DMEM + FBS + Antibiotics + Antifungal);
• ✔ FIG 13: Antimicrobial activity tests (contact method) performed in Escherichia coli (108 CFU/mL) during 24 h of incubation at 37 °C of CB@NPAgs and control samples (pure porous Nexfill@);
• ✔ FIG 14: Antimicrobial activity tests (contact method) performed on Staphylococcus aureus (108 CFU/mL) during 24 h of incubation at 37 °C of CB@NPAgs and control samples (pure porous Nexfill@);
• ✔ FIG 15: Death curve assay of CB@NPAgs dressings in E. coli (108 CFU/ml) during 48 h incubation at 37°C;
• ✔ FIG 16: Death curve assay of CB@NPAgs dressings in S. aureus (108 CFU/ml) during 48 h incubation at 37°C;
• ✔ FIG 17: Death curve assay of blank (CB Nexfill pure) and CB@NPAg sample soaked with silver and commercial sample Mepilex Ag in E. coli (102 CFU/ml) for 48 h incubation at 37 °C;
• ✔ FIG 18: Death curve assay of blank (CB Nexfill pure) and CB@NPAgs dressings in E. coli (102 CFU/ml) during 48 h incubation at 37°C;
• ✔ FIG 19: Death curve assay of blank (CB Nexfill pure) and CB@NPAgs dressings in P. aeruginosa (102 CFU/ml) during 48 h incubation at 37°C.

DETAILED DESCRIPTION OF THE INVENTION

• ✔ CB@NPAg1: membranas de celulose bacteriana impregnadas com suspensão de nanopartículas de prata em concentração de 500 ppm;
• ✔ CB@NPAg2: membranas de celulose bacteriana impregnadas com suspensão de nanopartículas de prata em concentração de 250 ppm;
• ✔ CB@NPAg3: membranas de celulose bacteriana impregnadas com suspensão de nanopartículas de prata em concentração de 125 ppm;
• ✔ CB@NPAg4: membranas de celulose bacteriana impregnadas com suspensão de nanopartículas de prata em concentração de 62,5 ppm.

[027] The preparation of antimicrobial bacterial cellulose dressings containing silver nanoparticles, called CB@NPAg, consists of immersion of porous CB membranes, synthesized by Acetobacter xylinum and commercially purchased from Nexfill@, in successive dilutions of the aqueous suspension of nanoparticles of silver (NPAg) with a concentration of 1000 ppm, also commercially purchased from the company Nanox®. Therefore, aqueous suspensions of NPAg are prepared, with concentrations ranging between 500 and 62.5 ppm using distilled water to reach the desired concentrations. The CB membranes were added to the 250 ml Erlenmeyer bottom and then the different concentrations of prepared NPAg were added. Erlenmeyer flasks were closed and covered with aluminum to avoid contact with light. After that, the Erlenmeyer flasks were added to a mechanical stirring table (orbital agitation, 240 rpm) for a period of 24 h at 25 °C (room temperature). After this period, four versions of the antimicrobial dressings were obtained:

• ✔ CB@NPAg1: bacterial cellulose membranes impregnated with a suspension of silver nanoparticles at a concentration of 500 ppm;
• ✔ CB@NPAg2: bacterial cellulose membranes impregnated with a suspension of silver nanoparticles at a concentration of 250 ppm;
• ✔ CB@NPAg3: bacterial cellulose membranes impregnated with a suspension of silver nanoparticles at a concentration of 125 ppm;
• ✔ CB@NPAg4: bacterial cellulose membranes impregnated with a suspension of silver nanoparticles at a concentration of 62.5 ppm.

[028] The results obtained in the physical-chemical analyzes and in the biological tests carried out with the described dressings are presented below, which demonstrate that the invention solves in an innovative way problems present in the current state of the art. Tests were also carried out, and are presented, with the nanoparticles used, prior to obtaining the dressings.

[029] Physicochemical analysis of Nanox® silver nanoparticles (NPAg).

[030] Nanox® silver nanoparticles (NPAg) were acquired for the development of CB membranes with antimicrobial activity. In this sense, the nanoparticles were previously analyzed by absorption spectroscopy in the ultraviolet-visible region (UV-Vis) and scanning electron microscopy (SEM) to confirm the size and shape of these particles. In figure 1, the UV-Vis spectrum of NPAg has a broad band of approximately 350 to 700 nm, with a maximum at 437 nm, a spectral profile characteristic of a heterogeneous distribution of particle diameter or even a distribution of aggregated particles, however, of medium nanometric size.

[031] Figure 2 shows the SEM images of the particles at different magnifications (5000, 10000, 25000 and 50000x). These micrographs corroborate the UV-Vis data obtained, showing particles of nanometric size, however, with distinct shapes and partially aggregated into blocks of particles. NPAg with diameters less than 100 nm are seen more clearly on micrographs at 50000x magnification.

[032] Cell viability of NPAg.

[033] The cytotoxicity of NPAg was evaluated in human fibroblast cells (MCR-5 lineage cells) at 37 °C for 24 h. Figure 3 shows the viability data for NPAg at different concentrations in order to determine the maximum amount of NPAg that does not cause toxicity to healthy cells. Given the assumption, starting from the concentrated aqueous suspension of NPAg, which has a concentration of 1000 ppm (mg/L), serial dilutions were carried out and, according to the graph in Figure 3, concentrations less than or equal to 4 ppm of NPAg were considered non-toxic at the cellular level, that is, they have a viability greater than 70% for the cell used.

[034] Determination of the minimal inhibitory concentration (MIC) of NPAg.

[035] After determining the cytotoxicity of NPAg, the Minimum Inhibitory Concentration (MIC) test of NPAg necessary to optimize the preparation of CB@NPAg dressings was performed. According to Figure 4, MIC assays were performed for S. aureus ATCC 25923 (gram-positive) and E. coli ATCC 25922 (gram-negative) bacteria. The test was based on the introduction of the indicator dye resazurin (blue) into the bacterial medium, which, being metabolized by bacteria, is reduced to resorufin, a compound formed with a red color, which confirms that NPAg did not cause the bacteria to die. Thus, the MIC values determined for the bacteria S. aureus and E. coli were 125 and 62.5 μg/mL (ppm), respectively. Therefore, the MIC value to inhibit the growth of S. aureus bacteria was twice that obtained for E. coli. This result is attributed to the cell wall of S. aureus (gram-positive) being thinner and composed of layers of peptidoglycans and glycopolymers, which favors a strong interaction of NPAg with the available functional groups, while E. coli bacteria (gram- negative) presents a thick outer layer of lipopolysaccharides, lipoproteins and phospholipids, before reaching the peptidoglycan layer, thus being a barrier to the access of NPAg and other antibiotic compounds (LR Osório, AB Meneguin, HB da Silva, HM Barreto, JA Osajima , EC da Silva Filho, 2018: Journal of Thermal Analysis and Calorimetry).

[036] Preparation of porous Nexfill@ dressings containing NPAg (CB@NPAgs).

[037] From the results of physicochemical characterization, cell viability and the MIC necessary to inhibit the growth of the two bacteria (gram-negative and gram-positive), the dressings were prepared via immersion of the porous Nexfill@ dressing in diluted concentrations of NPAgs to obtain samples called CB@NPAgs. Figure 5 shows the four prepared dressings: CB@NPAg1, CB@NPAg2, CB@NPAg3 and CB@NPAg4, with additions corresponding to the 2, 4, 8 and 16-fold dilutions of the initial concentration of NPAg obtained from the company Nanox® ( 1000 ppm).

[038] According to the images presented in Figure 5, it can be concluded that, qualitatively, as the concentrations of NPAg added for dressing preparation decreased, the yellowish color of CB Nexfill™ membranes also decreased, suggesting lower concentrations of NPAg present in these dressings. To confirm what was observed, the dressings were characterized via physicochemical analyses.

[039] Physicochemical analysis of CB@NPAgs dressings.

[040] The physical-chemical properties of the dressings were evaluated using techniques such as Scanning Electron Microscopy with Field Emission (SEM-FEG), thermogravimetric analysis (ATG) and indirect quantification of the silver present in CB@NPAgs performed via spectrometry of atomic absorption with continuous source and high resolution (HR-CS FAAS). The samples were analyzed without modification in their final structure, in the form of films, making the results even more representative of the final material.

[041] The prepared CB@NPAg dressings were initially characterized using scanning electron microscopy (SEM) in order to observe whether there was a change in the morphology of the CB dressings and also how the NPAg are arranged, whether they are aggregated or dispersed on the surface of the band Aid. Figures 6, 7, 8 and 9 show the SEMs of samples CB@NPAg1, CB@NPAg2, CB@NPAg3 and CB@NPAg4, respectively, at different magnifications (10000, 25000 and 100,000 times). From the micrographs obtained, it was possible to verify that the three-dimensional network of bacterial cellulose nanofibers was intact after the impregnation method using NPAg. Furthermore, it was possible to distinguish and observe a homogeneous distribution of NPAg along the CB nanofibers, for all samples. It is important to emphasize that the NPAg aggregation observed in the sample prepared for analysis via SEM can be attributed to the preparation mode, where the aqueous suspension was dried quickly on the sample holder, which may favor their aggregation. Furthermore, in general, no aggregation of NPAg was observed in any of the dressings, indicative of the interaction of NPAg with active sites on bacterial cellulose nanofibers. These active sites present in bacterial cellulose nanofibers can act as nanoreactors, promoting electrostatic interactions with NPAg and preventing their aggregation (IMS Araújo, RR Silva, G. Pacheco, WR Lustri, A. Tercjak, J. Gutierrez, JRS Júnior, JRS Júnior , FHC Azevedo, GS Figuêredo, ML Vega, et al., 2017: Cabohydrate Polymers; HS Barud, A. Tercjak, J. Gutierrez, WR Viali, ES Nunes, SJL Ribeiro, et al., 2015: Journal of Applied Physics) . Another point to be highlighted for all dressings is that, as the concentration of NPAg added to the Nexfill™ porous dressings decreased, less NPAg was observed on the nanofiber surfaces.

[042] In order to determine the concentration of NPAg present in Nexfill@ porous dressings, thermogravimetric analysis was performed on the sample that possibly has the highest concentration of NPAg among the four samples, the CB@NPAg1, according to figure 10. Two losses of characteristic masses of CB membranes are observed. First, a tiny loss of mass up to 100 °C, attributed to dehydration (water loss) of the CB, even though it is a previously dried membrane (HS Barud, C. Barrios, T. Regiani, RFC Marques, M. Verelst, J Dexpert-Ghys, Y. Messaddeq and SJL Ribeiro, 2008: Materials Science and Engineering C).

[043] The second and main mass loss corresponds to dehydroxylation, degradation and decomposition of CB from 320 to 550 °C, an exothermic event (event that releases heat) with a maximum at 421 °C (HS Barud, C. Barrios, T Regiani, RFC Marques, M. Verelst, J. Deexpert-Ghys, Y. Messaddeq and SJL Ribeiro, 2008: Materials Science and Engineering C). It is important to note that the residual found in the value of 1.26% enters into the equipment error, which is around 1%. Therefore, from the thermal analysis it was not possible to detect silver in the Nexfill@ dressing prepared with the highest concentration of NPAg.

Tabela 1. Linha de prata utilizada, dados da média de absorbância e concentração obtidos para o sobrenadante da amostra CB@NPAg via HR-CS FAAS.[044] As ATG did not show sufficient sensitivity to detect silver in the dressings, atomic absorption spectrometry was used for indirect quantification of silver, performed in the remaining supernatant of the dressing preparation. A silver standard calibration curve was generated (figure 11) for quantification of CB@NPAgs sample supernatants according to table 1, which displays the mean absorbance and concentration data obtained using the Ag328nm line in the HR-CS FAAS:

Table 1. Silver line used, mean absorbance and concentration data obtained for the CB@NPAg sample supernatant via HR-CS FAAS.

Tabela 2. Concentrações de NPAg determinadas em todas as amostras CB@NPAgs.[045] Considering that the concentration of NPAg added in the preparation of the samples were 500, 250, 125 and 62.5 ppm for the samples CB@NPAg1, CB@NPAg2, CB@NPAg3 and CB@NPAg4, respectively, and that the results concentration found for supernatants via HR-CS FAAS were 156.70; 91.74; 24.49 and 14.94 ppm, respectively, can indirectly determine the concentrations of NPAg present in the samples, as shown in table 2:

Table 2. NPAg concentrations determined in all CB@NPAgs samples.

[046] Biological tests.

[047] Minimum Inhibitory Concentration (MIC) of NPAg.

[048] To perform the MIC determination, following the CLSI protocol (2016) 9, Gram-positive (S. aureus ATCC 25923) and Gram-negative (E. coli ATCC 25922) bacterial strains were inoculated in tubes containing 10 mL of BHI (Brain Heart Infusion) and incubated for 18 h at 35-37 °C. Sufficient inoculum of each bacterial suspension was added to new tubes of sterile BHI medium to reach 1.0 turbidity on the McFarland nephelometric scale (~3.0x108 CFU/mL). A volume of 50 μL of the samples suspended in 10% DMSO aqueous solution (10 mg/mL stock) were added to the second well (B) of the 96-well microplate added with 50 μL of sterile BHI medium. Then, in the remaining 6 wells (C-H) serial dilutions were carried out in an aqueous solution of 10% DMSO. Then, 50 μL of the microorganism suspensions, on the 1.0 McFarland scale, were added to each sequential dilution, reaching a turbidity of 0.5 McFarland (~1.5x108 CFU/mL), in a final volume of 200 μL/well at concentrations ranging from 250 to 0.122 ppm. The first well of the microplate was used as growth control, 50 μL of sterile BHI medium, 50μL of 10% DMSO aqueous solution and 100 μL of bacterial suspension, at 1.0 McFarland scale. The microplates were incubated for 18 h at 35-37 °C in a humid chamber under agitation at 100 rpm. After the incubation period, 15 μL of 0.02% resazurin in sterile aqueous solution were added to each plate hole. After 3 h of reincubation, reading was performed. Tests were performed in triplicate.

[049] Cell Viability.

[050] A suspension of MCR-5 and GM07492 lineage cells (human fibroblast cells) were used to evaluate the NPAg and CB@NPAgs, respectively, adjusted to 1x104 cells/well in 100 μL of DMEM culture medium (Dulbecco's Modified Eagle's Medium) supplemented with FBS (Fetal Bovine Serum), antibiotics and antifungal. These cells were seeded in 96-well cell culture plates and incubated at 37°C, under 5% CO2 atmosphere (overnight) for cell adhesion. After this period, the culture medium was replaced by 100 μL of extract from CB@NPAgs samples and pure porous CB, and subsequently, the plates were incubated at 37 °C, under 5% CO2 atmosphere for 24 h. After the experimental period, the wells were washed once with PBS (phosphate buffer) and 50 µL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was added. The culture plates were again incubated in an incubator at 37 °C for 4 h.

• ✔ Controle negativo: DMEM + SBF + antibióticos + antifúngico;
• ✔ Controle positivo: DMEM + SBF + 30 % de DMSO (dimetilsulfóxido).

[051] After incubation, 100 μL of isopropanol was added to the wells and gently homogenized for the solubilization of formazan crystals. Optical density (OD) values obtained at a wavelength (λ) of 570 nm in a spectrophotometer were converted into percentages of cell viability relative to the negative control sample:

• ✔ Negative control: DMEM + FBS + antibiotics + antifungal;
• ✔ Positive control: DMEM + SBF + 30% DMSO (dimethylsulfoxide).

[052] Cell viability of CB@NPAgs dressings.

[053] The cytotoxicity of CB@NPAgs dressings and pure porous Nexfill@ dressing was evaluated according to the method described above (in materials and methods), in human fibroblast cells (lineage GM07492 cells) at 37 °C for 24 H.

[054] Figure 12 presents the cell viability data for the samples mentioned above and also two commercial samples Aquacel Ag and Mepilex Ag, for comparison purposes. All prepared dressings were viable at the cellular level, with samples CB@NPAg1 and CB@NPAg2 being at the limit of cell viability, close to 70%. The samples CB@NPAg3 and CB@NPAg4 showed viability values greater than 80%, which implies that these dressings are not toxic to the healthy cells used. The two commercial samples (Aquacel Ag and Mepilex Ag) showed toxicity at the cellular level, with viability values below 30%, very close to the positive control, which corresponds to high toxicity at the cellular level. However, the four CB@NPAgs dressings prepared are suitable for evaluation of antimicrobial action and subsequent in vivo assays.

[055] Antimicrobial Activity.

[056] Trials evaluating the antimicrobial activity of CB@NPAgs dressings.

[057] From previous antimicrobial assays, the CB@NPAgs samples did not show silver release, that is, they did not show the formation of a diffusion halo using the Agar diffusion method, both for E. coli and S. aureus . In this sense, antimicrobial tests using the contact method were carried out, according to figures 13 and 14.

[058] Contact method.

[059] To perform the MIC determination, following the CLSI protocol (Clinical and Laboratory Standards Institute (CLSI), 2016: Performance Standards for Antimicrobial Susceptibility Testing), Gram-positive bacterial strains (S. aureus ATCC 25923) and Gram- negative (E. coli ATCC 25922) were inoculated in tubes containing 10 mL of BHI (Brain Heart Infusion) and incubated for 18 h at 37 °C. Sufficient inoculum of each bacterial suspension was added to new tubes of sterile BHI medium until it reached 0.5 turbidity on the McFarland nephelometric scale (~1.5.0x108 CFU/mL). In Petri dishes, Muller-Hinton agar (MH) was added and, after drying the agar surface, the dressings in a rectangular shape measuring 10x5 mm were placed on the surface of the dish containing the microorganisms. Bacterial samples were dropped in triplicate on the surface of the dressings for contact evaluation. The plates were incubated for 24 h at 37 °C in a bacteriological incubator.

[060] The antibacterial activity of the samples was analyzed by the presence (growth) or absence of microorganisms under the dressings, qualitatively. Antimicrobial assays using E. coli bacteria were performed for CB@NPAgs and pure porous Nexfill@ samples (standard for comparison called control). Figure 13 shows the tests performed according to the contact method, in E. coli (108CFU/mL) during 24 h incubation at 37 °C. From the tests carried out, it was possible to observe that for all CB@NPAgs dressings no microbial growth was observed, suggesting that in contact with the dressing there was inhibition of the growth of E. coli. This can be proven by the clear growth of E. coli in the control sample.

[061] Figure 14 shows the images of antimicrobial assays of the samples via the contact method in S. aureus (108 CFU/mL) incubated for 24 h at 37 °C.

[062] Similar to the antimicrobial assays in E. coli, all dressings tested inhibited the growth of S. aureus while the control showed high microbial growth. Therefore, the samples CB@NPAg1, CB@NPAg2, CB@NPAg3 and CB@NPAg4 were effective in inhibiting the growth of both E. coli (gram-negative) and S. aureus (gram-positive) bacteria.

[063] Death assays (E. coli and S. aureus).

[064] Reactivation of strains, inoculum preparation and death test.

[065] The strains were inoculated with the aid of a sterile loop in a test tube with a lid containing 5 mL of BHI broth (Brain heart infusion). The tube was incubated at 37 °C for 24 h and after this period turbidity in the culture medium was observed, indicating microbial viability. After the reactivation of the strains, sowing will be done using the depletion technique, to obtain isolated colonies, on BHI agar plates. Plates will be incubated at 37 °C for 24 h. After incubation, 3 to 5 colonies were selected and transferred to a tube with 2 mL of BHI broth, incubated at 37 °C for 24 h. The bacterial inoculum was prepared in BHI broth, in order to produce a slight turbidity, with optical density visually equivalent to the 0.5 tube on the McFarland scale (1x107 CFU/mL). This suspension was diluted to 1x105 CFU/mL. The CB@NPAgs dressings were placed in Falcon tubes, containing 20 mL of BHI broth and an initial suspension containing 5x105 CFU/mL, and then incubated at 37 °C. 100 μL aliquots were removed every 4h and serially diluted (up to 10-6, in Eppendorf tubes containing 900 μL of saline) and seeded in BHI agar and subsequently incubated at 37 °C for 24 h.

[066] In order to quantify the antimicrobial activity of the prepared dressings, death assays were performed for E. coli and S. aureus bacteria. The log reduction of the microbial medium was evaluated every 4 h, up to a period of 48 h. Figure 15 shows the log of E. coli reduction (in CFU/ml) as a function of time for all dressings. The highest values of log reduction were detected up to the 28 h period, suggesting a possible period of use of the dressings with efficacy in inhibiting the microbial growth of E. coli. Therefore, the log reduction found was close to 1 for E. coli (gram-negative bacteria).

[067] Figure 16 shows the log curves of dressing reduction for S. aureus as a function of time. From this result, it was possible to detect that the action of the dressings against the S. aureus bacteria was prolonged, showing inhibition of microbial growth for up to 48 h. These results are in agreement with the MIC data obtained for NPAg, which show microbial inhibition for S. aureus at concentrations lower than necessary to inhibit the growth of E. coli. The log reduction obtained for S.aureus was close to 0.9.

[068] Finally, the results obtained via death tests confirm that the action of the dressings is via contact, with a small amount of NPAg released, in addition to suggesting that, from the reduction logs obtained, it is possible to estimate the time in which dressings are effective in inhibiting microbial growth. These results of log reduction close to 1 are in agreement with some commercial dressings presented in the literature, mainly in the 24-hour period (JJ Castellano, SM Shafii, F. Ko, G. Donate, TE Wright, RJ Mannari, WG Payne, DJ Smith, MC Robson, 2007: International Wound Journal).

[069] After the results obtained, in order to evaluate a CFU of 102 (similar to that found in the skin) and to compare with other types of samples (for example commercial samples), death curve tests were performed (in E. coli) for the CB@NPAg sample starting from the wet membrane, soaking with NPAg, for the commercial sample Mepilex Ag (figure 17) and also for the samples CB@NPAg1, CB@NPAg2, CB@NPAg3 and CB@NPAg4 (figure 18).

[070] From the curves obtained as a function of time (48 h, for E. coli) it was possible to observe a log reduction of approximately 10 both for the CB@NPAg soaked sample and for the Mepilex Ag sample. 28 h, a greater decrease in the log reduction was observed for soaked CB@NPAg than for Mepilex Ag, indicating that the Mepilex Ag sample can be used for a longer time. The two analyzed samples showed a profile of high Ag release, being considered samples with high antimicrobial activity as a function of time. However, previously presented cytotoxicity results show that Mepilex Ag is extremely toxic to healthy cells.

[071] Figure 18 shows the curves obtained as a function of time (48 h, for E. coli in 102 CFU/mL). The results obtained were similar to those obtained previously (for 108 CFU/mL), showing a log reduction of 1.3. It is important to highlight that, for the samples CB@NPAg1 and CB@NPAg2 (samples with the highest concentration of NPAg) a log reduction above 1 was observed in the 32 h incubation period.

[072] As previously shown, this log reduction value close to 1 corroborates that the analyzed dressings can inhibit microbial growth. Triplicate analyzes are being performed to increase the significance of the results obtained from the curves shown in Figure 18.

[073] Figure 19 shows the log reduction curves as a function of time (48 h) for CB@NPAgs dressings in Pseudomonas aeruginosa medium. The log reduction obtained for the samples was close to 1.2. It is important to note that, in the first 4 h, there was no microbial growth in any of the samples. The samples CB@NPAg1 and CB@NPAgs2 showed higher log reduction values after 4 h, extending up to 48 h of evaluation. This can be explained by the dose-response phenomenon, where dressings containing the highest concentrations of NPAg offer a greater reduction in the growth of the microorganism in question (AJ Kora, J. Arunachalam., 2009: Assessment of antibacterial activity of silver nanoparticles on Pseudomonas aeruginosa and its mechanism of action).

[074] Physical-chemical analyzes of NPAg and dressings via vibrational absorption spectroscopy techniques in the ultraviolet-visible region, scanning electron microscopy, thermogravimetric analysis and high-resolution atomic absorption spectrometry were performed thus confirming the homogeneous distribution of the particles of silver in gauge size on the surface of the nanofibers of CB Nexfill™ membranes. Using high-resolution atomic absorption spectrometry, it was possible to indirectly quantify the concentration of NPAg present in the dressings. The concentrations of NPAgs determined were 343.30; 158.26; 100.51 and 47.56 ppm for CB@NPAg1, CB@NPAg2, CB@NPAg3 and CB@NPAg4, respectively. Biological assays were performed and, based on the assessment of cell viability, it was possible to determine the minimum amount of NPAg that would be non-toxic at the cellular level. Subsequently, the minimum inhibitory concentration (MIC) of NPAg was determined for E. coli and S. aureus bacteria, the NPAg concentration for the MIC being lower for S. aureus (62.5 ppm) than for E. coli (125 ppm). Cell viability for dressings was also evaluated and it was possible to determine that all dressings were viable at the cellular level, with viability values above 70%. Antimicrobial activity tests for the dressings were performed via the contact method and all samples showed inhibition of bacterial growth (against E. coli and S. aureus) without showing a diffusion halo, suggesting that NPAg are not released from the BC nanofibers. Death assays were also performed for E. coli and S. aureus and confirmed log reduction values close to 1 and 0.9, respectively. These results confirm the low release of NPAg to the microbial medium, confirming that the dressings act in the inhibition of the microbial medium via contact. The antimicrobial activity obtained only by contact, is an important observation that can minimize the possibilities of releasing NPAg present in dressings, which would minimize the penetration of NPAg in treated living organisms. Furthermore, the death assays show that the inhibition of the microbial environment of E. coli and S. aureus occurs for a period of up to 28 and 48 h, respectively, suggesting that these dressings can be used in these time intervals, that is, the switch could be suggested after reaching the times determined via death trials. For comparison purposes, death curves were obtained for the commercial sample (Mepilex Ag) and for the CB@NPAg membrane embedded in E. coli. Despite the great antimicrobial action shown by the samples (log reduction 10), the commercial sample has high Ag release and, consequently, high toxicity at the cellular level, which should make its use in wounds and burns unfeasible. These results demonstrate that the proposed dressings solve in an innovative way the problems present in the technical field, being, therefore, deserving of the patent of invention privilege.

Claims (8)

ANTIMICROBIAL DRESSINGS characterized by being composed of porous bacterial cellulose membranes, synthesized by Acetobacter xylinum, impregnated with a suspension of silver nanoparticles, with a concentration between 62.5 and 500 ppm. ANTIMICROBIAL DRESSINGS, according to claim 1, characterized in that the CB@NPAg1 version has bacterial cellulose membranes impregnated with a suspension of silver nanoparticles at a concentration of 500 ppm. ANTIMICROBIAL DRESSINGS, according to claim 1, characterized in that the CB@NPAg2 version has bacterial cellulose membranes impregnated with a suspension of silver nanoparticles at a concentration of 250 ppm. ANTIMICROBIAL DRESSINGS, according to claim 1, characterized in that the CB@NPAg3 version has bacterial cellulose membranes impregnated with a suspension of silver nanoparticles at a concentration of 125 ppm. ANTIMICROBIAL DRESSINGS, according to claim 1, characterized in that the CB@NPAg4 version has bacterial cellulose membranes impregnated with a suspension of silver nanoparticles at a concentration of 62.5 ppm. PROCESS FOR OBTAINING ANTIMICROBIAL DRESSINGS characterized by starting with the preparation of aqueous suspensions of NPAg, with concentrations ranging between 500 and 62.5 ppm using distilled water to reach the desired concentrations; the CB membranes were added to the 250 ml Erlenmeyer bottom and subsequently the different concentrations of prepared NPAg were added; Erlenmeyer flasks were closed and covered with aluminum to avoid contact with light; after that, the Erlenmeyer flasks were added to a mechanical stirring table (orbital agitation, 240 rpm) for a period of 24 h at 25 °C (room temperature); after this period, the four versions of the antimicrobial dressings were obtained. APPLICATION OF ANTIMICROBIAL DRESSINGS, described according to claim 1, characterized by being for the healing of chronic wounds. APPLICATION OF ANTIMICROBIAL DRESSINGS, described according to claim 1, characterized in that it is for the treatment of burns.

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