A biologically active delivery material with dried-rehydrated vesicles containing the anti-inflammatory diclofenac for potential wound healing

Chronic wounds usually remain in the inflammatory phase of the healing process during several months or even years. Hence, a continuous research has been resulting in the development of wound dressings with improved performance. Herein, we report a delivery system for cutaneous wound healing, consisting of a textile material (non-woven gauzes) covered with lipidic vesicles containing diclofenac, a non-steroidal anti-inflammatory drug (NSAID). This study also aims to compare the entrapment efficiency data with previous works and confirm that this parameter and drug amount are not directly correlated. A method of dehydration–rehydration of the liposomes presenting different sizes and lamellarities was used to assess the best conditions to attain the highest drug entrapment efficiency. Optimum conditions for the NSAID release were achieved with high phospholipid concentrations and dried-rehydrated vesicles (DRVs) prepared from multilamellar liposomes (MLVs). A chemical activation of the gauzes was performed to enhance the vesicles attachment, also contributing to a higher drug amount in the surrounding media. In spite of the entrapment efficiency being lower comparatively with other values presented by us previously, the diclofenac concentration was considerably higher in this formulation. Entrapment efficiency is, therefore, not sufficient per se to define the real amount of drug contained in the formulation. The cytocompatibility assessment in human skin fibroblasts showed that DRVs from MLVs and DRVs from large unilamellar liposomes (LUVs) with less than 750?μM of egg-yolk phosphatidylcholine (EPC), containing diclofenac, were not cytotoxic after 72?h of contact, greatly implying potential for their application in the chronic wounds healing.     

A novel form of epithelial wound healing of the embryonic epidermis

The embryonic epidermis of amniotes is a two-cell layer sheet with a periderm positioned superficial to the basal cell layer which, itself, attaches apically to the basal surface of the periderm and basally to the basal lamina. The presence of the periderm is essential to maintain the basal layer as a two-dimensional monolayer. Wounds to the epidermis that remove selectively just the periderm are healed by a stacking of the basal layer cells that constitute the wound bed. Basal cell stacking involves the desertion of the basal lamina by many of the cells so as to increase their contact area with other basal layer cells. This suggests that a preferential adhesion to the planar basal lamina is not important for the monolayered organization of the basal layer but, instead, association with inner surface of the planar periderm is the principal process that maintains the basal layer as a monolayer. The conversion of the basal layer from monolayer to multilayer during wound healing diminishes its planar area, resulting in movement of the wound borders toward the center of the wound. This is a novel scenario for wound healing.     

The use of adipose-derived stem cells as sheets for wound healing

Cellular therapies have shown immense promise in the treatment of nonhealing wounds. Cell sheets are an emerging strategy in tissue engineering, and these cell sheets are promising as a delivery method of mesenchymal stem cells to the wound bed. Cell sheet technology utilizes temperature dependent polymers to allow for lifting of cultured cells and extracellular matrix without the use of digestive enzymes. While mesenchymal stem cells (MSCs) have shown success in cell sheets for myocardial repair, examination of cell sheets in the field of wound healing has been limited. We previously developed a novel cell sheet composed of human adipose-derived stem cells (ASCs). Both single and triple layer cell sheets were examined in a full-thickness murine wound model. The treatment cell sheets were compared with untreated controls and analyzed at timepoints of 7, 14, 18 and 21 d. The ASC cell sheets showed increased healing at 7, 14 and 18 d, and this effect was increased in the triple layer cell sheet group. Future development of these cell sheets will focus on increasing angiogenesis in the wound bed, utilizing multiple cell types, and examining allogeneic cell sheets. Here we review our experiment, expand upon our future directions and discuss the potential of an off-the-shelf cell sheet. In the field of wound healing, such a cell sheet is both clinically and scientifically exciting.     

Solvent tolerant Pseudomonads as a source of novel lipases for applications in non-aqueous systems

Abstract

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are ubiquitous biocatalysts known to catalyze the hydrolysis of water insoluble triglycerides in aqueous medium and carry out the reverse reaction (synthesis) under organic solvent rich medium. Microbial lipases have received a great deal of attention in the field of food technology, pharmaceutical sciences, chemical and detergent industries due to their stability, selectivity, mild operation conditions and broad substrate specificity. Despite these advantages, low activity and stability displayed in organic medium has restricted their commercial application in organic synthesis. Researchers have explored alternative ways to modify the enzymes making them suitable for use in non-conventional media. In this context, harvesting lipases from “Solvent Tolerant Microbes” has recently become an attractive approach. These microbes are able to grow in the presence of high concentrations of organic solvents, generally known to have detrimental effect on microorganisms. Such microbes survive through novel adaptation mechanisms and secretion of solvent stable enzymes having efficient functionality in solvent-rich media. These enzymes could be useful for bioconversion in non-conventional media. In the current review, this approach is described with an emphasis on characteristics, applications and genetic aspect of lipases from the genus Pseudomonas.     

Curcumin nanofibers for the purpose of wound healing

Poor wound healing is a highly prevalent clinical problem with, as yet, no entirely satisfactory solution. A new technique, termed electrospinning, may provide a solution to improve wound healing. Due to their large surface area to volume ratio and porosity, the nanofibers created by electrospinning are able to deliver sustained drug release and oxygen to the wound. Using different types of polymers with varying properties helps strengthening nanofiber and exudates absorption. The nanofibers appear to have an ideal structure applicable for wound healing and, in combination with curcumin, can blend the anti-inflammatory and antioxidant properties of curcumin into a highly effective wound dressing. The use of suitable curcumin solvents and the slow release of curcumin from the nanofiber help in overcoming the known limitations of curcumin, specifically its low stability and limited bioavailability. Here, we review the studies which have been done on synthesized nanofibers containing curcumin, produced by the electrospinning technique, for the purpose of wound healing.     

Nanobiotechnology as a novel paradigm for enzyme immobilisation and stabilisation with potential applications in biodiesel production

Nanobiotechnology is emerging as a new frontier of biotechnology. The potential applications of nanobiotechnology in bioenergy and biosensors have encouraged researchers in recent years to investigate new novel nanoscaffolds to build robust nanobiocatalytic systems. Enzymes, mainly hydrolytic class of enzyme, have been extensively immobilised on nanoscaffold support for long-term stabilisation by enhancing thermal, operational and storage catalytic potential. In the present report, novel nanoscaffold variants employed in the recent past for enzyme immobilisation, namely nanoparticles, nanofibres, nanotubes, nanopores, nanosheets and nanocomposites, are discussed in the context of lipase-mediated nanobiocatalysis. These nanocarriers have an inherently large surface area that leads to high enzyme loading and consequently high volumetric enzyme activity. Due to their high tensile strengths, nanoscale materials are often robust and resistant to breakage through mechanical shear in the running reactor making them suitable for multiple reuses. The optimisation of various nanosupports process parameters, such as the enzyme type and selection of suitable immobilisation method may help lead to the development of an efficient enzyme reactor. This might in turn offer a potential platform for exploring other enzymes for the development of stable nanobiocatalytic systems, which could help to address global environmental issues by facilitating the production of green energy. The successful validation of the feasibility of nanobiocatalysis for biodiesel production represents the beginning of a new field of research. The economic hurdles inherent in viably scaling nanobiocatalysts from a lab-scale to industrial biodiesel production are also discussed.     

Development of gallic acid formazans as novel enoyl acyl carrier protein reductase inhibitors for the treatment of tuberculosis

The enoyl acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis (MTB) is an attractive target for developing novel antitubercular agents. A series of gallic acid formazans, were computationally designed and docked into the active site of InhA to understand their binding mode and potential to inhibit InhA. Nine compounds from the designed series were identified as potential InhA inhibitors, on the basis of good Glide score. These compounds were synthesized in the laboratory and evaluated for in vitro antitubercular activity against drug-sensitive and multi-drug resistant strains of MTB. Out of nine compounds, three compounds exhibited the most promising MIC of <2 μM against the sensitive strain of MTB, H37Rv. The compounds were evaluated against five resistant strains of MTB. Most of the compounds exhibited activity superior to the standard, linezolid, against all these resistant strains. The mechanism of action of these compounds was concluded to be InhA inhibition, through InhA enzyme inhibition study. Insignificant cytotoxicity of these compounds was observed on RAW 264.7 cell line. Inactivity of all these compounds against gram positive and gram negative bacteria indicated their specificity against MTB. The compounds were further analyzed for ADME properties and showed potential as good oral drug candidates. The results clearly identified some novel, selective and specific InhA inhibitors against sensitive and resistant strains of MTB.     

Nucleotide receptors as targets in the pharmacological enhancement of dermal wound healing

With a growing interest of the involvement of extracellular nucleotides in both normal physiology and pathology, it has become evident that P2 receptor agonists and antagonists may have therapeutic potential. The P2Y2 receptor agonists (diquafosol tetrasodium and denufosol tetrasodium) are in the phase 3 of clinical trials for dry eye and cystic fibrosis, respectively. The thienopyridine derivatives clopidogrel and ticlopidine (antagonists of the platelet P2Y12 receptor) have been used in cardiovascular medicine for nearly a decade. Purines and pyrimidines may be of therapeutic potential also in wound healing since ATP and UTP have been shown to have many hallmarks of wound healing factors. Recent studies have demonstrated that extracellular nucleotides take part in all phases of wound repair: hemostasis, inflammation, tissue formation, and tissue remodeling. This review is focused on the potent purines and pyrimidines which regulate many physiological processes important for wound healing.     

The healing skin wound: a novel site of action of the chemokine C10

To gain insight into the molecular mechanisms underlying the wound repair process, we searched for genes that are regulated by skin injury. For this purpose we generated a subtractive cDNA library from normal mouse back skin and 1-day full-thickness excisional wounds. One of the differentially expressed genes encodes the chemokine C10. Using Northern blotting, RNase protection assay and Western blotting, we confirmed the injury-induced expression of C10 at the mRNA and protein level. Maximal levels of C10 mRNA and protein were seen at day 1 after wounding, and expression levels subsequently declined. In situ hybridization and immunohistochemistry revealed expression of C10 in macrophages of the clot and the granulation tissue as well as in keratinocytes of the epidermis and the hair follicles at the wound edge. Since C10 is a potent chemoattractant for macrophages, our results suggest that this chemokine contributes to the strong macrophage influx observed in the healing skin wound.     

Mesenchymal stem cell-conditioned media: A novel alternative of stem cell therapy for quality wound healing

Mesenchymal stem cells-conditioned media (MSCs-CM) contains several growth factors and cytokines, thus may be used as a better alternative to stem cell therapy, which needs to be elucidated. The present study was conducted to evaluate the therapeutic potential of caprine, canine, and guinea pig bone marrow-derived MSCs-CM in excision wound healing in a guinea pig model. MSCs were obtained from bone marrow, expanded ex vivo and characterized as per ISCT criteria. CM was collected assayed by western blot to ascertain the presence of important secretory biomolecules. Quantitative estimation by enzyme-linked immunosorbent assay was done for a vascular epidermal growth factor (VEGF) and interleukin-6 (IL-6) in caprine MSCs-CM and optimum time for collection of CM was decided as 72 hr. CM from all the species was lyophilized by freeze-drying method. Full-thickness (2 × 2 cm²) excision skin wounds were created in guinea pigs (six animals in each group) and respective lyophilized CM mixed with laminin gel was applied topically at weekly interval. On Day 28, histopathological examinations of healed skin were done by hemotoxylin and eosin staining. MSCs were found to secrete important growth factors and cytokines (i.e., VEGF, transforming growth factor-β1, fibroblast growth factor-2, insulin-like growth factor-1, stem cell factor, and IL-6) as demonstrated by immunohistochemistry and western blot assay. It was found that allogenic and xenogenic application of CM significantly improved quality wound healing with minimal scar formation. Thus, MSCs-CM can be used allogenically as well as xenogenically for quality wound healing.     

Development of low-cost microfluidic systems for lab-on-a-chip biosensor applications

In this work, we develop low-cost microfluidic systems based on polydimethylsiloxane (PDMS) for lab-on-a-chip applications. PDMS microfluidic structures have been fabricated by micromolding, PDMS casting, and plasma bonding processes. The micromolding technique is used to fabricate PDMS slabs with micro-sized grooves, and the complete microchannel is formed by bonding PDMS slab with glass or PDMS substrate. The molding procedure using SU-8 photoresist patterning on silicon wafer, PDMS microchannel fabrication, and PDMS surface treatment using oxygen plasma and TiO₂ coating, are discussed. The various parameters for oxygen plasma treatment including RF power and treatment time are studied in order to obtain conditions for good bonding with the glass substrate. The best condition for plasma treatment is found to be the low RF power (8 W) with 5 min treatment time. In addition, TiO₂ coating with oxygen plasma treatment has been applied to make PDMS surface more hydrophilic to improve aqueous solution compatilbility. The microfluidic channels for various applications, including sample injection cross channel, micropump channel, T and Y sample mixers, PCR thermocyling chamber and channel, capillary electrophoresis flow channel, and conductimetric systems have been fabricated. Finally, a typical application of the PDMS chip in a flow injection conductimetric system for sodium chloride detection has been demonstrated.     

Influence of extremely low-frequency magnetic field on the activity of antioxidant enzymes during skin wound healing in rats

The aim of this study was to evaluate the activity of the antioxidant enzymes mitochondrial and cytosolic superoxide dismutase (EC 1.15.1.1), glutathione peroxidase (POX, EC 1.11.1.9) and glutathione S-transferase (EC 3.1.2.7), as well as the concentration of malone dialdehyde (MDA), as an indicator of lipid peroxidation rate in the liver tissue homogenates and blood serum of male rats exposed to extremely low-frequency magnetic field (ELF-MF) in order to improve the healing process of an experimental cut wound on the back of each animal. The exposure to ELF-MF with frequency 40 Hz and magnetic flux density 10 mT induced an increase in POX serum activity and a decrease in MDA contents in the liver tissue, which suggests the inhibition of phospholipid peroxidation and subsequent stabilization of cellular membranes, as a result of ELF-MF action. Based on the results obtained, it seems that ELF-MF could be a useful supplement in the complex treatment of prolonged wound healing, due to the activation of endogenous enzymatic antioxidant system.     

The antioxidant systems in Toxoplasma gondii and the role of cytosolic catalase in defence against oxidative injury

Superoxide dismutase, catalase, glutathione peroxidase and peroxiredoxins form an antioxidant network protecting cells against reactive oxygen species (ROS). Catalase is a potent H2O2-detoxifying enzyme, which is unexpectedly absent in some members of the Kinetoplastida and Apicomplexa, but present in Toxoplasma gondii. In T. gondii, catalase appears to be cytosolic. In addition, T. gondii also possesses genes coding for other types of peroxidases, including glutathione/thioredoxin-like peroxidases and peroxiredoxins. This study presents a detailed analysis of the role of catalase in the parasite and reports the existence of antioxidant enzymes localized in the cytosol and the mitochondrion of T. gondii. The catalase gene was disrupted and, in addition, T. gondii cell lines overexpressing either catalase or a cytosolic 1-cys peroxiredoxin, TgPrx2, under the control of a strong promoter were created. Analysis of these mutants confirmed that the catalase activity is cytosolic and is encoded by a unique gene in T. gondii. Furthermore, the catalase confers protection against H2O2 exposure and contributes to virulence in mice. The overexpression of Prx2 also increases protection against H2O2 treatment, suggesting that catalase and other peroxidases function as a defence mechanism against endogenously produced reactive oxygen intermediates and the oxidative stress imposed by the host.     

Sus Scrofa immune tissues as a new source of bioactive substances for skin wound healing

Influence of a new protein-peptide complex on promoting skin wound healing in male BALB/c mice was studied. Protein-peptide complex, extracted from Sus scrofa immune organs, was percutaneously administered using two methods: by lecithin gel-like liquid crystals and by liquid microemulsion. On the fifth day, wound closure in mice with a linear wound model become faster in group (less 2 days comparison to other ones), which was treated with lecithin liquid crystals carrying the protein-peptide complex. This promoting healing can be caused by resorption of bioactive high-molecular compounds the animal skin. In mice with the linear wound model, the tensile strength of the scars were respectively higher both in mice, treated using lecithin liquid crystals with protein-peptide complex, and in mice, treated using microemulsion containing protein-peptide complex, by 215.4% and 161.5% relative to the animals, which did not receive bioactive substances for wound treatment. It was associated with the regeneratory effects of tissue- and species-specific protein-peptide complexes, including α-thymosin Sus scrofa (C3VVV8_PIG, m/z 3802.8) and other factors, which were described as parts of the new extracted complex. This reveals that percutaneous administration of the complex reliably activates local regenerative processes in animals.     

An analysis of the adequacy of the asymmetric carrier model for sugar transport

In 1972, Lieb, W. R.; Stein, W. D. (Biochim. Biophys. Acta 265, 187–207) in their review of sugar transport in human erythrocytes concluded that the conventional two-state carrier model was inconsistent with the experimental data available at that time. Since then, other papers have appeared which question the validity of the model. In this paper, we give a brief derivation of the equations describing the two-state carrier model, and analyze the predictions of the model in the classical experiments, i.e. zero-trans, infinite-cis, and equilibrium exchange. We show that the estimate of the half saturatiion constant of 2.8 mM for glucose at the inner face of the human red cell membrane for the infinite-cis procedure reported by Hankin, B. L., Lieb, W. R. and Stein, W. D ((1972) Biochim. Biophys. Acta 288, 114–126) is unreliable. We note that all of the other experimental findings are consistent with the asymmetric carrier model.