Exploring Microemulsion Systems for the Incorporation of Glucocorticoids into Bacterial Cellulose: A Novel Approach for Anti-Inflammatory Wound Dressings.

The effective pharmacological treatment of inflamed wounds such as pyoderma gangraenosum remains challenging, as the systemic application of suitable drugs such as glucocorticoids is compromised by severe side effects and the inherent difficulties of wounds as drug targets. Furthermore, conventional semi-solid formulations are not suitable for direct application to open wounds. Thus, the treatment of inflamed wounds could considerably benefit from the development of active wound dressings for the topical administration of anti-inflammatory drugs. Although bacterial cellulose appears to be an ideal candidate for this purpose due to its known suitability for advanced wound care and as a drug delivery system, the incorporation of poorly water-soluble compounds into the hydrophilic material still poses a problem. The use of microemulsions could solve that open issue. The present study therefore explores their use as a novel approach to incorporate poorly water-soluble glucocorticoids into bacterial cellulose. Five microemulsion formulations were loaded with hydrocortisone or dexamethasone and characterized in detail, demonstrating their regular microstructure, biocompatibility and shelf-life stability. Bacterial cellulose was successfully loaded with the formulations as confirmed by transmission electron microscopy and surprisingly showed homogenous incorporation, even of w/o type microemulsions. High and controllable drug permeation through Strat-M® membranes was observed, and the anti-inflammatory activity for permeated glucocorticoids was confirmed in vitro. This study presents a novel approach for the development of anti-inflammatory wound dressings using bacterial cellulose in combination with microemulsions.

In situ Formation of Polymer Microparticles in Bacterial Nanocellulose Using Alternative and Sustainable Solvents to Incorporate Lipophilic Drugs.

Bacterial nanocellulose has been widely investigated in drug delivery, but the incorporation of lipophilic drugs and controlling release kinetics still remain a challenge. The inclusion of polymer particles to encapsulate drugs could address both problems but is reported sparely. In the present study, a formulation approach based on in situ precipitation of poly(lactic-co-glycolic acid) within bacterial nanocellulose was developed using and comparing the conventional solvent N-methyl-2-pyrrolidone and the alternative solvents poly(ethylene glycol), CyreneTM and ethyl lactate. Using the best-performing solvents N-methyl-2-pyrrolidone and ethyl lactate, their fast diffusion during phase inversion led to the formation of homogenously distributed polymer microparticles with average diameters between 2.0 and 6.6 µm within the cellulose matrix. Despite polymer inclusion, the water absorption value of the material still remained at ~50% of the original value and the material was able to release 32 g/100 cm2 of the bound water. Mechanical characteristics were not impaired compared to the native material. The process was suitable for encapsulating the highly lipophilic drugs cannabidiol and 3-O-acetyl-11-keto-ß-boswellic acid and enabled their sustained release with zero order kinetics over up to 10 days. Conclusively, controlled drug release for highly lipophilic compounds within bacterial nanocellulose could be achieved using sustainable solvents for preparation.

Bacterial nanocellulose patches as a carrier for hydrating formulations to improve the topical treatment of nail diseases.

Bacterial nanocellulose has been widely investigated for wound healing applications, mainly due to its moisturizing capabilities and biocompatibility. Even though the topical therapy of nail diseases could benefit from these properties, this application has not yet been investigated. Therefore, actively hydrating nail patches based on bacterial nanocellulose were developed to improve the delivery of ciclopirox olamine and Boswellia serrata extract through the nail plate. The nanocellulose matrix was used to enable the application of hydration enhancing solutions based on glycerol and urea as a mechanically stable patch. While the favorable mechanical characteristics of the material remained unchanged, an increase of the incorporated glycerol concentration enhanced the transparency and wetting capacity of the patches. A biphasic drug release from the patches could be observed for drug and extract with a faster release for the hydrophilic ciclopirox olamine. High glycerol concentrations correlated with increased cumulative release and permeation through keratin films for drug and extract, demonstrating the hydration driven permeation enhancement. Patches containing ciclopirox olamine showed strong antimycotic effects against relevant pathogens for onychomycosis. The present finding proposed the combination of bacterial nanocellulose with glycerol, urea and different drug as a promising platform for the local treatment of nail diseases.

Bacterial Cellulose as Drug Delivery System for Optimizing Release of Immune Checkpoint Blocking Antibodies.

Immune checkpoint blocking therapy is a promising cancer treatment modality, though it has limitations such as systemic toxicity, which can often be traced to uncontrolled antibody spread. Controlling antibody release with delivery systems is, therefore, an attractive approach to reduce systemic antibody spread and potentially mitigate the side effects of checkpoint immunotherapy. Here, bacterial cellulose (BC) was produced and investigated as a delivery system for optimizing checkpoint-blocking antibody delivery. BC was produced in 24-well plates, and afterward, the edges were removed to obtain square-shaped BC samples with a surface of ~49 mm2. This customization was necessary to allow smooth in vivo implantation. Scanning electron microscopy revealed the dense cellulose network within BC. Human IgG antibody was included as the model antibody for loading and release studies. IgG antibody solution was injected into the center of BC samples. In vitro, all IgG was released within 24 to 48 h. Cell culture experiments demonstrated that BC neither exerted cytotoxic effects nor induced dendritic cell activation. Antibody binding assays demonstrated that BC does not hamper antibody function. Finally, antibody-loaded BC was implanted in mice, and serum measurements revealed that BC significantly reduced IgG and anti-CTLA-4 spread in mice. BC implantation did not induce side effects in mice. Altogether, BC is a promising and safe delivery system for optimizing the delivery and release of checkpoint-blocking antibodies.

Bacterial Cellulose-Adaptation of a Nature-Identical Material to the Needs of Advanced Chronic Wound Care.

Modern wound treatment calls for hydroactive dressings. Among the variety of materials that have entered the field of wound care in recent years, the carbohydrate polymer bacterial cellulose (BC) represents one of the most promising candidates as the biomaterial features a high moisture-loading and donation capacity, mechanical stability, moldability, and breathability. Although BC has already gained increasing relevance in the treatment of burn wounds, its potential and clinical performance for "chronic wound" indications have not yet been sufficiently investigated. This article focuses on experimental and clinical data regarding the application of BC within the indications of chronic, non-healing wounds, especially venous and diabetic ulcers. A recent clinical observation study in a chronic wound setting clearly demonstrated its wound-cleansing properties and ability to induce healing in stalling wounds. Furthermore, the material parameters of BC dressings obtained through the static cultivation of Komagataeibacter xylinus were investigated for the first time in standardized tests and compared to various advanced wound-care products. Surprisingly, a free swell absorptive capacity of a BC dressing variant containing 97% moisture was found, which was higher than that of alginate or even hydrofiber dressings. We hypothesize that the fine-structured, open porous network and the resulting capillary forces are among the main reasons for this unexpected result.

Controlled Release of the α-Tocopherol-Derived Metabolite α-13'-Carboxychromanol from Bacterial Nanocellulose Wound Cover Improves Wound Healing.

Inflammation is a hallmark of tissue remodeling during wound healing. The inflammatory response to wounds is tightly controlled and well-coordinated; dysregulation compromises wound healing and causes persistent inflammation. Topical application of natural anti-inflammatory products may improve wound healing, in particular under chronic pathological conditions. The long-chain metabolites of vitamin E (LCM) are bioactive molecules that mediate cellular effects via oxidative stress signaling as well as anti-inflammatory pathways. However, the effect of LCM on wound healing has not been investigated. We administered the α-tocopherol-derived LCMs α-13'-hydroxychromanol (α-13'-OH) and α-13'-carboxychromanol (α-13'-COOH) as well as the natural product garcinoic acid, a δ-tocotrienol derivative, in different pharmaceutical formulations directly to wounds using a splinted wound mouse model to investigate their effects on the wounds' proinflammatory microenvironment and wound healing. Garcinoic acid and, in particular, α-13'-COOH accelerated wound healing and quality of the newly formed tissue. We next loaded bacterial nanocellulose (BNC), a valuable nanomaterial used as a wound dressing with high potential for drug delivery, with α-13'-COOH. The controlled release of α-13'-COOH using BNC promoted wound healing and wound closure, mainly when a diabetic condition was induced before the injury. This study highlights the potential of α-13'-COOH combined with BNC as a potential active wound dressing for the advanced therapy of skin injuries.

Overcoming the hydrophilicity of bacterial nanocellulose: Incorporation of the lipophilic coenzyme Q10 using lipid nanocarriers for dermal applications.

Although used in a wide range of medical and pharmaceutical applications, the potential of the natural biopolymer bacterial nanocellulose (BNC) as drug delivery system is by far not fully exploited. Particularly, the incorporation of lipophilic drugs is still considered as an unsolved task. In the present study, the homogeneous incorporation of the lipophilic coenzyme Q10 (CoQ10) into BNC was accomplished by several post-synthesis techniques utilizing different nanoemulsions and liposomes. All colloidal carriers were in the range of about 90-120 nm with negative zeta potentials and storage stabilities up to 30 days. The biphasic drug release profiles of loaded BNC were found to be dependent on the type of colloidal carrier and the loading technique. Favorable characteristics such as high mechanical stability and high loading capacity were retained after the incorporation of the lipophilic components. Penetration studies using excised porcine skin revealed CoQ10 distributions also in deeper skin layers dependent on the type of the colloidal carrier system. In conclusion, hydrophilic BNC could be loaded with water-insoluble drugs as shown for the model drug CoQ10 by the use of lipidic colloidal carriers which offers new possibilities of application in pharmacy and medicine.

Modified Bacterial Cellulose Dressings to Treat Inflammatory Wounds.

Natural products suited for prophylaxis and therapy of inflammatory diseases have gained increasing importance. These compounds could be beneficially integrated into bacterial cellulose (BC), which is a natural hydropolymer applicable as a wound dressing and drug delivery system alike. This study presents experimental outcomes for a natural anti-inflammatory product concept of boswellic acids from frankincense formulated in BC. Using esterification respectively (resp.) oxidation and subsequent coupling with phenylalanine and tryptophan, post-modification of BC was tested to facilitate lipophilic active pharmaceutical ingredient (API) incorporation. Diclofenac sodium and indomethacin were used as anti-inflammatory model drugs before the findings were transferred to boswellic acids. By acetylation of BC fibers, the loading efficiency for the more lipophilic API indomethacin and the release was increased by up to 65.6% and 25%, respectively, while no significant differences in loading could be found for the API diclofenac sodium. Post-modifications could be made while preserving biocompatibility, essential wound dressing properties and anti-inflammatory efficacy. Eventually, in vitro wound closure experiments and evaluations of the effect of secondary dressings completed the study.

Development and characterization of bacterial nanocellulose loaded with Boswellia serrata extract containing nanoemulsions as natural dressing for skin diseases.

The combination of the anti-inflammatory lipophilic Boswellia serrata extract with the natural hydropolymer bacterial nanocellulose (BNC) for the treatment of skin diseases is counteracted by their different hydro/lipophilicity. To overcome the hydrophilicity of the BNC, the water in its network was exchanged by single and double nanoemulsions. Incorporation of the Boswellia serrata extract in the nanoemulsions formed particles of about 115 to 150 nm with negative zeta potential and storage stability over 30 days at temperatures between 4 and 32 °C. Their loading into the BNC did not change the preferential characteristics of the nanocellulose like water absorption and retention, softness, and pressure stability in a relevant way. Loaded BNC could be sterilized by an electron-beam procedure. A biphasic drug release profile of lead compounds was observed by Franz cell diffusion test. The biocompatibility of the loaded BNC was confirmed ex ovo by a shell-less hen's egg test. Tape stripping experiments using porcine skin determined a dependency of the drug penetration into skin on the type of nanoemulsion, single vs. repeated applications and the incubation time. In conclusion, the hydrophilicity of BNC could be overcome using nanoemulsions which offers the possibility for the anti-inflammatory skin treatment with Boswellia serrata extract.

Doxorubicin Loaded Poloxamer Thermosensitive Hydrogels: Chemical, Pharmacological and Biological Evaluation.

(1) Background: doxorubicin is a potent chemotherapeutic agent, but it has limitations regarding its side effects and therapy resistance. Hydrogels potentially deal with these problems, but several characterizations need to be optimized to better understand how hydrogel assisted chemotherapy works. Poloxamer 407 (P407) hydrogels were mixed with doxorubicin and physico-chemical, biological, and pharmacological characterizations were considered. (2) Methods: hydrogels were prepared by mixing P407 in PBS at 4 °C. Doxorubicin was added upon solutions became clear. Time-to-gelation, hydrogel morphology, and micelles were studied first. The effects of P407-doxorubicin were evaluated on MC-38 colon cancer cells. Furthermore, doxorubicin release was assessed and contrasted with non-invasive in vivo whole body fluorescence imaging. (3) Results: 25% P407 had favorable gelation properties with pore sizes of 30-180 µm. P407 micelles were approximately 5 nm in size. Doxorubicin was fully released in vitro from 25% P407 hydrogel within 120 h. Furthermore, P407 micelles strongly enhanced the anti-neoplastic effects of doxorubicin on MC-38 cells. In vivo fluorescence imaging revealed that hydrogels retained fluorescence signals at the injection site for 168 h. (4) Conclusions: non-invasive imaging showed how P407 gels retained drug at the injection site. Doxorubicin P407 micelles strongly enhanced the anti-tumor effects.

Bacterial nanocellulose as novel carrier for intestinal epithelial cells in drug delivery studies.

Synthetic cell carriers (A) represent common scaffold structures for the development of cell-based in vitro models of the human intestine but due to their low porosity or unwanted molecular adhesion effects, synthetic carriers can negatively affect cell function. Alternative scaffolds such as natural extracellular matrices (ECMs) (B) were shown to overcome some of the common drawbacks. However, their fabrication is time-consuming, less well standardized and not entirely conform to the 3R principle (replacement, reduction, refinement). Nowadays, biopolymers such as bacterial nanocellulose (BNC) (C) represent interesting scaffold materials for innovative tissue engineering concepts, as they can be generated in a faster and more standardized process workflow without the need for animal material. In this study, we demonstrate the BNC as suitable carrier for the development of Caco-2-based in vitro models of the human intestine. The BNC-based models exhibit organ-specific properties comprising typical cellular morphologies, characteristic protein expression profiles, representative ultrastructural features and the formation of a tight epithelial barrier. The proof of in vivo-like transport activities further validates the high quality of the BNC-based Caco-2 models. In summary, this illustrates the BNC as alternative bioscaffold of non-animal origin to develop functional organ models in vitro.

Thermosensitive hydrogels as sustained drug delivery system for CTLA-4 checkpoint blocking antibodies.

Thermosensitive poloxamer 407 (P407) hydrogels were evaluated as slow release system for optimizing CTLA-4 therapy. Slow release reduces systemic antibody levels and potentially mitigates the side effects of CTLA-4 therapy. The 25% P407 hydrogel is injectable at room temperature and depots are established quickly after subcutaneous injection. Scanning electron microscopy revealed the porous structure of the hydrogel, average pore surface was 1335 µm2. Release studies were optimized using the human IgG antibody. IgG was easily incorporated in the hydrogel by simple mixing and no antibodies were lost during preparation. In vitro, hydrogels showed low burst release within the first 24 h. Total IgG load was gradually released within 120 h. In vitro cytotoxicity assays showed that P407 is not cytotoxic and induces no immune activation by itself. In vivo, P407 hydrogels significantly reduced serum IgG levels, were biocompatible and were broken down 1 week after injection. Finally, local hydrogel delivery of anti-CTLA-4 antibodies near established tumors effectively slowed down tumor growth, whilst significantly reduced serum anti-CTLA-4 levels. Altogether, P407 hydrogels represent promising delivery systems for the optimization of CTLA-4 blocking therapy.

Process control and scale-up of modified bacterial cellulose production for tailor-made anti-inflammatory drug delivery systems.

Bacterial cellulose (BC) has proven its high potential as active wound dressing and drug delivery system in many scientific studies, but the transferability of the methods to efficient manufacturing still needs to be demonstrated. This study presents a technically feasible, straightforward and efficient approach to modify BC according to specific medical requirements, to scale-up the cultivation and to load the active pharmaceutical ingredient of interest. By means of in situ-modification of the network structure using water-soluble poly(ethylene glycol) 400 and 4000 on pilot-scale, up to 41.5 ±â€¯3.0 % higher transparency of the dressing, 40.6 ±â€¯3.8 % increased loading capacity and 9% increased total release of the anti-inflammatory model drug diclofenac sodium could be obtained. Spray loading was investigated as material efficient alternative to absorption loading allowing a significant reduction in loading time.

Immobilization of plasmids in bacterial nanocellulose as gene activated matrix.

The synergy of the local delivery of nucleic acids using a hydrogel-based gene activated matrix (GAM) might support regenerative processes on a genetic level by concurrently providing a cell-friendly microenvironment. To investigate bacterial nanocellulose (BNC) as GAM, two plasmids (pSV-ß-Gal and pGL3) were incorporated by reswelling and injection techniques forming matrix and core-shell systems as determined by SEM and staining experiments. The release was found to be dependent on the type of BNC, the plasmid and the loading technique, and lasted over at least 20 days. No morphological or mechanical changes of the BNC due to the presence of plasmids were observed. Immobilized plasmids especially in the matrix systems were protected against enzymatic degradation by maintaining the high biocompatibility of BNC and transfection efficacy of the plasmids. These results indicate that BNC can be used as a promising and renewable carrier for the application as local gene delivery system.

Tailor-made material characteristics of bacterial cellulose for drug delivery applications in dentistry.

Bacterial cellulose (BC) has shown high potential as innovative wound dressing and drug delivery system. Bringing both together, drug-loaded BC was investigated for applications in dental therapies such as dental extraction or mucosal transplantation. Both applications would benefit from a material which degrades under physiological conditions, and from an antibiotic environment. Consequently, periodate-oxidation of BC was investigated to facilitate modified degradation behaviour. A periodate concentration of 0.14 mol/L at ϑ = 25 °C and t = 8 h resulted in a material loss of <10%, but at the same time a sufficient degree of degradation. Additionally, native and oxidised BC loaded with doxycycline was tested for prophylaxis against infection. An in vitro-toxicity test (MTT assay) provided a first confirmation of biocompatibility, whereas agar diffusion tests proved antibiotic efficiency against pathogenic oral bacteria. Release studies of the drug from native and oxidised BC confirmed a comparative biphasic release behaviour.

Opportunities of Bacterial Cellulose to Treat Epithelial Tissues.

In this mini-review, we highlight the potential of the biopolymer bacterial cellulose to treat damaged epithelial tissues. Epithelial tissues are cell sheets that delimitate both the external body surfaces and the internal cavities and organs. Epithelia serve as physical protection to underlying organs, regulate the diffusion of molecules and ions, secrete substances and filtrate body fluids, among other vital functions. Because of their continuous exposure to environmental stressors, damage to epithelial tissues is highly prevalent. Here, we first compare the properties of bacterial cellulose to the current gold standard, collagen, and then we examine the use of bacterial cellulose patches to heal specific epithelial tissues; the outer skin, the ocular surface, the oral mucosa and other epithelial surfaces. Special emphasis is made on the dermis since, to date, this is the most widespread medical use of bacterial cellulose. It is important to note that some epithelial tissues represent only the outermost layer of more complex structures such as the skin or the cornea. In these situations, depending on the penetration of the lesion, bacterial cellulose might also be involved in the regeneration of, for instance, inner connective tissue.

Combinatory therapy adopting nanoparticle-based cancer vaccination with immune checkpoint blockade for treatment of post-surgical tumor recurrences.

Cancer immunotherapy is emerging as a candidate treatment modality for treating post-surgical metastasis and recurrences. Despite the great promises with therapeutic cancer vaccines and checkpoint blocking antibodies in pre-clinical studies, response rates in the clinic still remain unsatisfactory. The evaluation of immunotherapy after surgery in patients could confront significant unexpected hurdles. Surgery itself tends to cause immune suppression, while wound healing factors also stimulate tumor cell outgrowth and metastasis. Regarding the marked changes in the post-surgical tumor microenvironment, one can anticipate that better tumor growth control is attainable by combining cancer vaccines with immune checkpoint blockade. However, it is important that vaccines and checkpoint blocking antibodies are delivered efficiently to their target cells, are released sustained and locally and do not induce cytotoxic effects. The generation of effective anti-tumor immunity and durable response rates could largely depend on these parameters. In the last decade, researchers spend tremendous effort in optimizing the delivery of immunotherapeutic compounds with the use of nanomedicine. Biocompatible nanoparticle based delivery systems demonstrated intriguing results with regard to specific immune cell activation, improved drug delivery, cell targeting, limiting off target toxicity and improving treatment outcome. It therefore makes sense, to speculate on the promises of combined cancer vaccination and immune checkpoint blocking immunotherapy with the aid of nanomedicine. A powerful nanoparticle combination immunotherapy conferring durable therapeutic benefit whilst leaving healthy tissue untouched represents the base for more efficient post-surgical cancer treatment.

Bacterial nanocellulose stimulates mesenchymal stem cell expansion and formation of stable collagen-I networks as a novel biomaterial in tissue engineering.

Biomimetic scaffolds are of great interest to tissue engineering (TE) and tissue repair as they support important cell functions. Scaffold coating with soluble collagen-I has been used to achieve better tissue integration in orthopaedy, however, as collagen persistence was only temporary such efforts were limited. Adequate coverage with cell-derived ECM collagen-I would promise great success, in particular for TE of mechanically challenged tissues. Here, we have used label-free, non-invasive multiphoton microscopy (MPM) to characterise bacterial nanocellulose (BNC) - a promising biomaterial for bone TE - and their potency to stimulate collagen-I formation by mesenchymal stem cells (MSCs). BNC fleeces were investigated by Second Harmonic Generation (SHG) imaging and by their characteristic autofluorescence (AF) pattern, here described for the first time. Seeded MSCs adhered fast, tight and very stable, grew to multilayers and formed characteristic, wide-spread and long-lasting collagen-I. MSCs used micron-sized lacunae and cracks on the BNC surface as cell niches. Detailed analysis using a collagen-I specific binding protein revealed a highly ordered collagen network structure at the cell-material interface. In addition, we have evidence that BNC is able to stimulate MSCs towards osteogenic differentiation. These findings offer new options for the development of engineered tissue constructs based on BNC.

Bacterial nanocellulose: the future of controlled drug delivery?

Although bacterial nanocellulose (BNC), a natural nanostructured biopolymer network, offers unique material characteristics, the number of drug-loaded BNC-based carriers in clinical trials or on the market is still low. This report provides an overview of aspects still limiting the broad application of BNC as drug-delivery system and the challenges for its future applications. Continuous large-scale production, storability, the loading and controlled release of critical drugs, for example, with high molar mass or highly lipophilic character as well as the formulation of long-term release systems will be highlighted. Recent achievements toward promoting the application of BNC as drug-delivery system and overcoming these obstacles will be discussed. [Formula: see text].

Antimicrobial functionalization of bacterial nanocellulose by loading with polihexanide and povidone-iodine.

Bacterial nanocellulose (BNC) is chemically identical with plant cellulose but free of byproducts like lignin, pectin, and hemicelluloses, featuring a unique reticulate network of fine fibers. BNC sheets are mostly obtained by static cultivation. Now, a Horizontal Lift Reactor may provide a cost efficient method for mass production. This is of particular interest as BNC features several properties of an ideal wound dressing although it exhibits no bactericidal activity. Therefore, BNC was functionalized with the antiseptics povidone-iodine (PI) and polihexanide (PHMB). Drug loading and release, mechanical characteristics, biocompatibility, and antimicrobial efficacy were investigated. Antiseptics release was based on diffusion and swelling according to Ritger-Peppas equation. PI-loaded BNC demonstrated a delayed release compared to PHMB due to a high molar drug mass and structural changes induced by PI insertion into BNC that also increased the compressive strength of BNC samples. Biological assays demonstrated high biocompatibility of PI-loaded BNC in human keratinocytes but a distinctly lower antimicrobial activity against Staphylococcus aureus compared to PHMB-loaded BNC. Overall, BNC loaded with PHMB demonstrated a better therapeutic window. Moreover, compressive and tensile strength were not changed by incorporation of PHMB into BNC, and solidity during loading and release could be confirmed.