Development and characterization of mucoadhesive chitosan films for ophthalmic delivery of cyclosporine A.

Ocular chitosan films were prepared in order to prolong ocular delivery of cyclosporine A. The mucoadhesive films were prepared using the solvent casting evaporation method. A 2(4) full factorial design was used to evaluate the effect of 4 preparation parameters on the film thickness, swelling index and mechanical properties. Moreover, uniformity of content and in vitro drug release were investigated. Possible interactions between the film excipients were studied by FTIR analysis. In vitro experiments were performed in order to evaluate the cytotoxicity and anti-inflammatory activity of the chitosan films. Film thickness, water uptake, mechanical properties and in vitro release of cyclosporine A were dependent on film composition, especially on the amount of plasticizer. Lower drug release was measured from chitosan films containing a higher amount of plasticizer as glycerol decreased the swelling of chitosan films. FTIR spectra suggest a reorganization of hydrogen bonds between chitosan chains in the presence of glycerol and cyclodextrins. None of the film formulations showed significant cytotoxicity as compared to the negative control using human epithelial cells (HaCaT). Cyclosporine A dispersed in the various film formulations remained anti-inflammatorily active as significant suppression of interleukin-2 secretion in concanavalin A stimulated Jurkat T cells was measured.

Full factorial design, physicochemical characterisation and biological assessment of cyclosporine A loaded cationic nanoparticles.

Cyclosporine A loaded poly(lactide-co-glycolide) nanoparticles coated with chitosan were prepared using the o/w emulsification solvent evaporation method. A 2(3) full factorial design was used to investigate the effect of 3 preparation parameters on the particle size, polydispersity index, zeta potential and drug release. In vitro experiments were performed in order to evaluate the cytotoxicity and anti-inflammatory activity of the developed nanoparticles. Particle sizes varied from 156 nm to 314 nm, and polydispersity index values of 0.07-0.56 were obtained depending on the different preparation parameters. All nanoparticles showed positive zeta potential values. Nanoparticles prepared with the highest concentration chitosan retained a positive zeta potential after dispersion in simulated lachrymal fluid, which supports the possibility of an electrostatic interaction between these particles and the negatively charged mucus layer at the eye. The in vitro release profile of cyclosporine A from the chitosan-coated nanoparticles was strongly dependent on the release medium used. None of the cationic nanoparticle formulations showed significant cytotoxicity compared to the negative control using human epithelial cells (HaCaT). Cyclosporine A encapsulated in the various nanoparticle formulations remained anti-inflammatory active as significant suppression of interleukine-2 secretion in concanavalin A stimulated Jurkat T cells was observed.

Pre-clinical Evaluation of a New Antimicrobial Enzyme for the Control of Wound Bioburden.

 A new, optimized, antimicrobial enzyme system was developed for the control of wound bioburden. This Glucose oxidase-Lactoperoxidase-Guaiacol (GLG) system was analyzed for antimicrobial activity and cytotoxicity. The susceptibility of a wide range of antibiotic-resistant bacterial strains to the GLG-enzyme system was analyzed using minimum inhibitory concentration (MIC90), minimum bactericidal concentration (MBC) determination, and growth kinetics analysis. Additionally, challenge tests and cytotoxicity tests were performed with a new hydroactive alginate gel dressing with antimicrobial activity obtained by the presence of the GLG-enzyme system (Flaminal® Forte, Flen Pharma, Kontich, Belgium). All bacterial strains were susceptible to the GLG-enzyme system at low concentrations. The exact concentration required for growth arrest and cell death was dependent on the experimental design. Further, a 20% (w/v) GLG dilution showed no cytotoxicity toward fibroblasts and keratinocytes. Conversely, other antimicrobial wound-care products applied with the same dilution showed a high degree of cytotoxicity. With increasing concerns about bacterial resistance to antibiotics, this study shows that low concentrations of the GLG-enzyme system are successful in killing antibiotic-resistant bacterial strains. Furthermore, results show that GLG-enzyme system combines strong antimicrobial activity with non-cytotoxicity and promotes optimal wound healing.

Treatment of recalcitrant wounds of diverse etiology with a new hydroactive gel.

Our knowledge about wound care has progressed considerably in recent years. Nonetheless there is a continuing need for new topical hydroactive gels in this field. Flamigel® (Flen Pharma NV, Kontich, Belgium) has recently been used on a limited number of burn patients. The hydroactive colloid gel maintains the wound in an ideal moist environment. The polymer in the tube is present in two physical forms-active and inactive. In a dry wound, the active polymer will release part of its moisture to hydrate the wound and to create and maintain a moist wound environment ("hydrogel" effect). The inactive polymer remains inactive. In an exuding wound, the inactive polymer is activated by the wound exudate and starts absorbing wound exudate ("hydrocolloid" effect). The absorption ceases when the entire polymer is activated and saturated with exudate. The wound itself decides whether the hydroactive gel absorbs or hydrates; hydration in case of a dry wound, absorption in case of an exuding wound. As a result, the wound is kept in a moist environment, which optimizes wound-healing speed and reduces the likelihood of scarring. This study investigated the wound-healing capacities of the hydroactive gel in wounds that have failed to respond to other treatments. .

Differential cell death programmes induced by silver dressings in vitro.

In past decades the gold standard for topical burn treatment was the use of silver sulfadiazine. Due to toxicity caused by the silver, the cream base itself, or a combination of both negatively influencing the wound healing process, the healthcare industry searched for alternatives. In recent years, various dressings containing silver have become available to wound professionals. Although these have been reported to be a significant improvement, the dressings still show residual cytotoxicity. Given the ongoing debate about whether and how these dressings influence cell survival, this article endeavours to clarify some of the mystique surrounding the subject. Various commercially available silver-type dressings were analysed in vitro and attention was paid to the cell death stage induced by these dressings on different cell lines. The results show that within 2 hours, for all dressings tested, cells undergo cell death and further analysis suggests that the death stage induced is dependent on the cell line and type of dressing investigated. Further, the antimicrobial activity of all dressings was analysed. The silver dressings tested have potent antimicrobial activity. Our results showed, however, that silver dressings induce rapid cell death of cells involved in wound healing. We therefore recommend the use of silver dressings only on critically contaminated wounds rather than use on a de facto basis.

Cytotoxicity of submicron emulsions and solid lipid nanoparticles for dermal application.

The cytotoxicity and physical properties of various submicron O/W emulsions and solid lipid nanoparticles for dermal applications were investigated. Droplet size and zetapotential of submicron emulsions depended on the composition of the cosurfactant blend used. The viability of J774 macrophages, mouse 3T3 fibroblasts and HaCaT keratinocytes was significantly reduced in the presence of stearylamine. Nanoparticles consisting of stearic acid or different kinds of adeps solidus could be manufactured when formulated with lecithin, sodium taurocholate, polysorbate 80 and stearylamine. Survival of macrophages was highly affected by stearic acid and stearylamine. In general a viability of more than 90% was observed when semi-synthetic glycerides or hard fat was employed to formulate nanoparticles.

In vitro studies on Itm2a reveal its involvement in early stages of the chondrogenic differentiation pathway.

The Integral membrane protein 2A (Itm2A) is a transmembrane protein belonging to a family composed of at least two other members, Itm2B and Itm2C, all of them having a different expression pattern. The Itm2a gene serves as a marker for early stages in endochondral ossification. In order to understand the role of Itm2A in this process, expression of the gene was investigated in different cell systems. In C3H10T1/2 cells, the gene was upregulated early on when the cells were induced to the chondrogenic lineage but less to the osteogenic lineage. In MCT cells, expression was upregulated at permissive temperatures but not at non-permissive temperatures. When induced with insulin, ATDC5 cells expressed Itm2a in early stages but not at late stages. Furthermore, PTH treatment seems to upregulate Itm2a transcription. In order to understand the role of Itm2a in the chondrogenic differentiation process in more detail, we constitutively overexpressed exogenous Itm2A in mouse ATDC5 cells. Two clones expressing high levels of Itm2a were isolated and characterized. Gene expression analysis of the overexpresser clones demonstrated that expression of collagen type X was delayed. These results demonstrate that overexpression of Itm2a in mouse ATDC5 cells impede the transition to hypertrophic cells. Taken together, our observation supports the involvement of Itm2a in the early stages of chondrogenesis in vitro.

Constitutive overexpression of the integral membrane protein Itm2A enhances myogenic differentiation of C2C12 cells.

Integral membrane protein 2A (Itm2A) is a transmembrane protein belonging to a family composed of at least two other members, Itm2B and Itm2C, all of them having a different expression pattern. The protein serves as a marker for early stages in chondrogenesis and T-cell development. Itm2A is also highly expressed in skeletal muscle. In order to understand the role of Itm2A in muscle development, we constitutively overexpressed exogenous Itm2A in C2C12 myoblast cells. Several clones expressing high levels of Itm2a were isolated and characterized. Overexpression was associated with enhanced tube formation and the appearance of multinuclear cells. Gene expression analysis demonstrated that muscle creatin kinase was upregulated in the presence of exogenous Itm2A. Interestingly, proliferation rates were not altered in the undifferentiated myoblast C2C12 cells. These results demonstrate that overexpression of Itm2a in C2C12 enhances myogenic differentiation in vitro.

Efficient removal of LoxP-flanked genes by electroporation of Cre-recombinase mRNA.

Introduction of Cre-recombinase in target cells is currently achieved by transfection of plasmid DNA or by viral-mediated transduction. However, efficiency of non-viral DNA transfection is often low in many cell types, and the use of viral vectors for transduction implies a more complex and laborious manipulation associated with safety issues. We have developed a non-viral non-DNA technique for rapid and highly efficient excision of LoxP-flanked DNA sequences based on electroporation of in vitro transcribed mRNA encoding Cre-recombinase. A K562-DSRed[EGFP] cell line was developed in order to measure Cre-mediated recombination by flow cytometric analysis. These cells have a stable integrated DSRed reporter gene flanked by two LoxP sites, and an EGFP reporter gene, which could only be transcribed when the coding sequence for DSRed was removed. The presented data show recombination efficiencies, as measured by appearance of EGFP-fluorescence, of up to 85% in Cre-recombinase mRNA-electroporated K562-DSRed[EGFP] cells. In conclusion, mRNA electroporation of Cre-recombinase is a powerful, safe, and clinically applicable alternative to current technologies used for excision of stably integrated LoxP-flanked DNA sequences.