Oxidized Bacterial Cellulose Membranes Immobilized with Papain for Dressing Applications: Physicochemical and In Vitro Biological Properties.

This research consolidates our group's advances in developing a therapeutic dressing with innovative enzymatic debridement, focusing on the physicochemical and in vitro biological properties of papain immobilized in wet oxidized bacterial cellulose (OxBC-Papain) dressing. OxBC membranes were produced with Komagataeibacter hansenii oxidized with NaIO4, and papain was immobilized on them. They were characterized in terms of enzyme stability (over 100 days), absorption capacity, water vapor transmission (WVT), hemocompatibility, cytotoxicity, and cell adhesion. The OxBC-Papain membrane showed 68.5% proteolytic activity after 100 days, demonstrating the benefit of using the OxBC wet membrane for papain stability. It had a WVT rate of 678 g/m2·24 h and cell viability of 99% and 86% for L929 and HaCat cells, respectively. The membranes exhibited non-hemolytic behavior and maintained 26% clotting capacity after 1 h. The wet OxBC-Papain membrane shows significant potential as a natural biomolecule-based therapeutic dressing for wound care, offering efficient debridement, moisture maintenance, exudate absorption, gas exchange, and hemostasis without cytotoxic effects or cell adhesion to the dressing. Further research, especially using in vivo models, is needed to assess its efficacy in inducing epithelialization. This study advances stomatherapy knowledge, providing a cost-effective solution for enzymatic debridement in healthcare.

Development of silanized bacterial cellulose aerogels for the incorporation of natural oils with healing properties: Copaiba (Copaifera officinalis), bourbon geranium (Pelargonium X ssp.) essential oils and buriti (Mauritia flexuosa) vegetable oil.

Bacterial cellulose (BC) represents a promising biomaterial, due to its unique and versatile properties. We report, herein, on purposely-designed structural modifications of BC that enhance its application as a wound dressing material. Chemical modification of the functional groups of BC was performed initially to introduce a hydrophobic/oleophilic character to its surface. Specifically, silanization was carried out in an aqueous medium using methyltrimethoxisilane (MTMS) as the silanizing agent, and aerogels were subsequently prepared by freeze-drying. The BC-MTMS aerogel obtained displayed a highly porous (99 %) and lightweight structure with an oil absorption capacity of up to 52 times its dry weight. The XRD pattern indicated that the characteristic crystallographic planes of the native BC were maintained after the silanization process. Thermal analysis showed that the thermal stability of the BC-MTMS aerogel increased, as compared to the pure BC aerogel (pBC). Moreover, the BC-MTMS aerogel was not cytotoxic to fibroblasts and keratinocytes. In the second step of the study, the incorporation of natural oils into the aerogel's matrix was found to endow antimicrobial and/or healing properties to BC-MTMS. Bourbon geranium (Pelargonium X ssp.) essential oil (GEO) was the only oil that exhibited antimicrobial activity against the tested microorganisms, whereas buriti (Mauritia flexuosa) vegetable oil (BVO) was non-cytotoxic to the cells. This study demonstrates that the characteristics of the BC structure can be modified, while preserving its intrinsic features, offering new possibilities for the development of BC-derived materials for specific applications in the biomedical field.

Bacteria-Derived Cellulose Membranes Modified with Graphene Oxide-Silver Nanoparticles for Accelerating Wound Healing.

This study reports on the modification of bacterial cellulose (BC) membranes produced by static fermentation of Komagataeibacter xylinus bacterial strains with graphene oxide-silver nanoparticles (GO-Ag) to yield skin wound dressings with improved antibacterial properties. The GO-Ag sheets were synthesized through chemical reduction with sodium citrate and were utilized to functionalize the BC membranes (BC/GO-Ag). The BC/GO-Ag composites were characterized to determine their surface charge, morphology, exudate absorption, antimicrobial activity, and cytotoxicity by using fibroblast cells. The antimicrobial activity of the wound dressings was assessed against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The results indicate that the BC/GO-Ag dressings can inhibit ∼70% of E. coli cells. Our findings also revealed that the porous BC/GO-Ag antimicrobial dressings can efficiently retain 94% of exudate absorption after exposure to simulated body fluid (SBF) for 24 h. These results suggest that the dressings could absorb excess exudate from the wound during clinical application, maintaining adequate moisture, and promoting the proliferation of epithelial cells. The BC/GO-Ag hybrid materials exhibited excellent mechanical flexibility and low cytotoxicity to fibroblast cells, making excellent wound dressings able to control bacterial infectious processes and promote the fast healing of dermal lesions.

Loading of bacterial cellulose dressing with frutalin, a lectin from Artocarpus incisa L.

Bacterial cellulose (BC), produced by bacterial fermentation, is a high-purity material. BC can be oxidized (BCOXI), providing aldehyde groups for covalent bonds with drugs. Frutalin (FTL) is a lectin capable of modulating cell proliferation and remodeling, which accelerates wound healing. This study aimed to develop an FTL-incorporated dressing based on BC, and to evaluate its physicochemical properties and biological activity in vitro. An experimental design was employed to maximize FTL loading yield onto the BC and BCOXI, where independent variables were FTL concentration, temperature and immobilization time. BCOXI-FTL 1 (44.96 % ± 1.34) had the highest incorporation yield (IY) at the experimental conditions: 6 h, 5 °C, 20 µg mL-1. The second highest yield was BCOXI-FTL 6 (23.28 % ± 1.43) using 24 h, 5 °C, 100 µg mL-1. Similarly, the same reaction parameters provided higher immobilization yields for native bacterial cellulose: BC-FTL 6 (16.91 % ± 1.05) and BC-FTL 1 (21.71 % ± 1.57). Purified FTL displayed no cytotoxicity to fibroblast cells (<50 µg mL-1 concentration) during 24 h. Furthermore, BCOXI-FTL and BC-FTL were non-cytotoxic during 24 h and stimulated fibroblast migration. BCOXI-FTL demonstrated neutrophil activation in vitro similar to FTL. These promising results indicate that the bacterial cellulose matrices containing FTL at low concentrations, could be used as an innovative biomaterial for developing wound dressings.

Emulgel based on fish skin collagen-microalgae-silver increased neovascularization and re-epithelialization of full thickness burn in rats.

Deep skin burn represents a global morbidity and mortality problem, and the limitation of topical treatment agents has motivated research to development new formulations capable of preventing infections and accelerating healing. The aim of this work was to develop and characterize an emulgel based on collagen (COL) and gelatin (GEL) extracted from fish skin associated with Chlorella vulgaris extract (CE) and silver nitrate (AgNO3). COL and GEL were characterized by physicochemical and thermal analyses; and CE by electrophoresis and its antioxidant capacity. Three emulgels formulations were developed: COL (0.5%) + GEL (2.5%) (E1), COL+GEL+CE (1%) (E2), and COL+GEL+CE + AgNO3 (0.1%) (E3). All formulations were characterized by physicochemical, rheology assays, and preclinical analyses: cytotoxicity (in vitro) and healing potential using a burn model in rats. COL and GEL showed typical physicochemical characteristics, and CE presented 1.3 mg/mL of proteins and antioxidant activity of 76%. Emulgels presented a coherent physicochemical profile and pseudoplastic behavior. Preclinical analysis showed concentration-dependent cytotoxicity against fibroblast and keratinocytes. In addition, all emulgels induced similar percentages of wound contraction and complete wound closure in 28 days. The histopathological analysis showed higher scores for polymorphonuclear cells to E1 and greater neovascularization and re-epithelialization to E3. Then, E3 formulation has potential to improve burn healing, although its use in a clinical setting requires further studies.

Extraction and characterization of hyaluronic acid from the eyeball of Nile Tilapia (Oreochromis niloticus).

Hyaluronic acid (HA) is a biopolymer of enormous value aggregation for in general industry. The vitreous humor of the eyeball from Nile tilapia contains appreciable amounts of hyaluronic acid. In this sense, the aim of this work was to extract and characterize hyaluronic acid from the eyeball of the Nile tilapia for biomedical applications, adding value to fish industry residues. The characterization by infra-red (FTIR), 13C nuclear magnetic resonance (NMR) and high performance liquid chromatography (HPLC) confirmed that hyaluronic acid was obtained. The gel permeation chromatography (GPC) showed that the obtained material presents a low molecular mass (37 KDa). Thermogravimetry (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD) analysis showed that the materials present a thermal stability superior to the commercial hyaluronic acid from Streptococcus equi, with a partially crystalline character. The cytotoxicity assay (MTT method) with fibroblast cells (L929) demonstrated that the extracted biopolymer besides not being cytotoxic, was able to stimulate cell proliferation. Therefore, the hyaluronic acid extracted from this source of residue constitutes a product with biotechnological potential, which has adequate quality for wide biomedical applications.

Virucidal PVP-Copper Salt Composites against Coronavirus Produced by Electrospinning and Electrospraying.

Electrospinning technology was used to produced polyvinylpyrrolidone (PVP)-copper salt composites with structural differences, and their virucidal activity against coronavirus was investigated. The solutions were prepared with 20, 13.3, 10, and 6.6% w/v PVP containing 3, 1.0, 0.6, and 0.2% w/v Cu (II), respectively. The rheological properties and electrical conductivity contributing to the formation of the morphologies of the composite materials were observed by scanning electron microscopy (SEM). SEM images revealed the formation of electrospun PVP-copper salt ultrafine composite fibers (0.80 ± 0.35 µm) and electrosprayed PVP-copper salt composite microparticles (1.50 ± 0.70 µm). Energy-dispersive X-ray spectroscopy (EDS) evidenced the incorporation of copper into the produced composite materials. IR spectra confirmed the chemical composition and showed an interaction of Cu (II) ions with oxygen in the PVP resonant ring. Virucidal composite fibers inactivated 99.999% of coronavirus within 5 min of contact time, with moderate cytotoxicity to L929 cells, whereas the virucidal composite microparticles presented with a virucidal efficiency of 99.999% within 1440 min of exposure, with low cytotoxicity to L929 cells (mouse fibroblast). This produced virucidal composite materials have the potential to be applied in respirators, personal protective equipment, self-cleaning surfaces, and to fabric coat personal protective equipment against SARS-CoV-2, viral outbreaks, or pandemics.

The effects of the molecular weight of chitosan on the tissue inflammatory response.

The molecular weight of chitosan (CS) may affect its physical properties and its ability to induce an appropriate host response. The biocompatibilities of CS membranes of low (LMWCS) and high (HMWCS) molecular weight were investigated by inserting these materials into the subcutaneous tissue of rats for 1-28 days and evaluating leukocyte infiltration, granulation tissue, fibrosis, arginase-1 immunostaining, as well as nuclear factor-κB (NF-κΒ) and fibroblast growth factor (FGF)-2 expressions. Both CS membranes induced a peak of leukocyte infiltration on the first day of insertion and stimulated granulation and fibrous tissue generation when compared to control. LMWCS induced more collagen deposition a week earlier, when compared to the control and HMWCS membrane. The membranes also increased arginase-1 immunostaining, a M2 macrophage marker. M2 macrophage is recognized as anti-inflammatory and pro-regenerative. NF-κB is an essential biomarker of the inflammatory process and induces the expression of several pro-inflammatory cytokines. The LMWCS membrane reduced inflammation, as indicated by a reduced nucleus/cytoplasm NF-κB ratio in surrounding tissue from days 7 to 14 when compared to control. On the first day, the expression of FGF-2, a biomarker of inflammatory resolution, was increased in the tissue of the LWMCS group, when compared with HMWCS, which was consistent with the type I collagen deposition. Thus, LWMCS was associated with a prior reduction of the inflammatory response and improved wound healing.

Anti-acetylcholinesterase and toxicity against Artemia salina of chitosan microparticles loaded with essential oils of Cymbopogon flexuosus, Pelargonium x ssp and Copaifera officinalis.

Essential oils (EOs) are bioactive compounds with therapeutic potential for use as alternatives or as support to conventional treatments. However, EOs present limitations, such as sensibility to environmental factors, which can be overcome through microencapsulation. The objective of this study was to produce, by spray drying, chitosan microparticles (CMs) loaded with EO of Lemongrass (Cymbopogon flexuosus), Geranium (Pelargonium x ssp) and Copaiba (Copaifera officinalis). Physicochemical and biological characterization of these microparticles showed that CMs presented spherical morphology, had an average size range of 2-3 µm with positive zeta potential (ZP) values, and enhanced thermal stability, compared to free EO. The encapsulation efficiency (EE) ranged from 4.8-58.6%, depending on the oil's properties. In vitro EO release from CMs was determined at different pHs, with 94% release observed in acid media. All microparticles were non-hemolytic at concentrations of up to 0.1 mg·mL-1. EOs and CMs presented acetylcholinesterase (AChE) inhibition activity (IC 50 ranged from 11.92 to 28.18 µg·mL-1). Geranium and Copaiba EOs presented higher toxicity against Artemia salina, and greater inhibition of acetylcholinesterase, indicating potential bioactivity for Alzheimer's disease (AD). Our findings demonstrate that CM systems may show promise for the controlled release of these EOs.

Papain immobilized on alginate membrane for wound dressing application.

Wound dressings based on natural polymers are of considerable interest in the pharmaceutical industry owing to their improved performance in the human body when compared to synthetic polymers. Alginate, a polysaccharide from brown algae, is commonly studied as a wound dressing owing to its biocompatibility and biodegradability. To improve its therapeutic features and thereby increase wound healing, papain (a proteolytic enzyme from Carica papaya latex) was proposed to be incorporated. Papain is capable of promoting the debridement of devitalized or necrotic tissues. The development of dressing based on alginate and papain aggregates the healing properties of both materials. In addition, the adsorption on a support can stabilize the enzyme structure and permits its release in a controlled manner. The optimal conditions for immobilization were evaluated (initial concentration, temperature, and pH), and the amount immobilized was measured by Bradford assay. The enzyme activity stability over 28 days was measured. The release profile was determined using Franz cell. In vitro cytotoxicity assays were performed using fibroblasts and keratinocytes. Optimal immobilization conditions were identified in a neutral medium at a papain concentration of 20 mg/mL and temperature of 25 °C. The enzyme remained active after immobilization (80 % of its initial activity), and the matrix protected the enzyme from deactivation (70 % reduction on the matrix compared to 94 % in a buffer solution). Franz cell displayed a release profile of 64.1 % of the enzyme after 24 h. The biological assays indicated a bioactive material with proteolytic properties.

Resorbable bacterial cellulose membranes with strontium release for guided bone regeneration.

Hybrid materials, based on bacterial cellulose (BC) and hydroxyapatite (HA), have been investigated for guided bone regeneration (GBR). However, for some GBR, degradability in the physiological environment is an essential requirement. The present study aimed to explore the use of oxidized bacterial cellulose (OxBC) membranes, associated with strontium apatite, for GBR applications. BC membranes were produced by fermentation and purified, before oxidizing and mineralizing by immersing in strontium chloride solution and sodium bibasic phosphate for 5 cycles. The hybrid materials (BC/HA/Sr, BC/SrAp, OxBC/HA/Sr and OxBC/SrAp) were characterized for biodegradability and bioactivity and for their physicochemical and morphological properties. In vitro cytotoxicity and hemolytic properties of the materials were also investigated. In vivo biocompatibility was analyzed by performing histopathological evaluation at 1, 3 and 9 weeks in mices. Results showed that the samples presented different strontium release profiles and that oxidation enhances degradation under physiological conditions. All the hybrid materials were bioactive. Cell viability assay indicated that the materials are non-cytotoxic and in vivo studies showed low inflammatory response and increased connective tissue repair, as well as degradation in most of the materials, especially the oxidized membranes. This study confirms the potential use of bacterial cellulose-derived hybrid membranes for GBR.

Papain immobilization on heterofunctional membrane bacterial cellulose as a potential strategy for the debridement of skin wounds.

We combined the chemical and physical methods of papain immobilization through the aldehyde groups available on oxidized bacterial cellulose (OxBC) to provide high proteolytic activity for future applications as bioactive dressing. Bacterial cellulose (BC) was obtained by the fermentation of Komagataeibacter hansenii in Hestrin-Schramm medium for 5 days, followed by purification and oxidation using NaIO4. Surface response methodology was used to optimize papain immobilization (2%, w/v) for 24 h. The independent variables: pH (3-7) and temperature (5 to 45 °C) were investigated. The mathematically validated optimal conditions of 45 °C and pH 7 had a statistical effect on the immobilization yield (IY) of papain in OxBC (52.9%). These ideal conditions were also used for papain immobilization in BC (unoxidized). The IY of 9.1% was lower than that of OxBC. OxBC-Papain and BC-Papain were investigated using thermal analysis, confocal microscopy, and diffusion testing. The OxBC support exhibited a more interactive chemical structure than the BC support, and was capable of immobilizing papain by covalent bonds (-C-NHR) and adsorption (ion exchange), with 93.3% recovered activity, 49.4% immobilization efficiency, and better thermal stability. Papain immobilized to OxBC by adsorption displayed 53% widespread papain activity. The results indicate the potential of prolonged bioactivity in debrided chronic wounds.

In vitro degradability and bioactivity of oxidized bacterial cellulose-hydroxyapatite composites.

Hydroxyapatite-associated bacterial cellulose (BC/HA) is a promising composite for biomedical applications. However, this hybrid composite has some limitations due to its low in vivo degradability. The objective of this work was to oxidize BC and BC/HA composites for different time periods to produce 2,3 dialdehyde cellulose (DAC). The BC and oxidized BC (OxBC) membranes were mineralized to obtain the hybrid materials (BC/HA and OxBC/HA) and their physico-chemical, degradability, and bioactivity properties were studied. The results showed that OxBC/HA was more bioactive and degradable than BC/HA, which isa function of the degree of BC oxidation. High glucose levels in the BC degradation were observed as a function of oxidation degree, and other products, such as butyric acid and acetic acid resulted from DAC degradation. Therefore, this chemical modification reaction favors BC degradation, making it a good biodegradable and bioactive material with a potential for bone regeneration applications.

Bacterial cellulose nanocrystals produced under different hydrolysis conditions: Properties and morphological features.

Bacterial cellulose (BC) is a polymer with interesting physical properties owing to the regular and uniform structure of its nanofibers, which are formed by amorphous (disordered) and crystalline (ordered) regions. Through hydrolysis with strong acids, it is possible to transform BC into a stable suspension of cellulose nanocrystals, adding new functionality to the material. The aim of this work was to evaluate the effects of inorganic acids on the production of BC nanocrystals (BCNCs). Acid hydrolysis was performed using different H2SO4 concentrations and reaction times, and combined hydrolysis with H2SO4 and HCl was also investigated. The obtained cellulose nanostructures were needle-like with lengths ranging between 622 and 1322nm, and diameters ranging between 33.7 and 44.3nm. The nanocrystals had a crystallinity index higher than native BC, and all BCNC suspensions exhibited zeta potential moduli greater than 30mV, indicating good colloidal stability. The mixture of acids resulted in improved thermal stability without decreased crystallinity.