Design of Experiment, Preparation, and in vitro Biological Assessment of Human Amniotic Membrane Extract Loaded Nanoparticles.

BACKGROUND: Human amniotic membrane grafting could be potentially useful in ocular surface complications due to tissue similarity and the presence of factors that reduce inflammation, vascularization, and scarring. However, considerations like donor-derived infectious risk and the requirement of an invasive surgery limit the clinical application of such treatments. Moreover, the quick depletion of bioactive factors after grafting reduces the efficacy of treatments. Therefore, in the current study, the possibility of nano delivery of the bioactive factors extracted from the human amniotic membrane to the ocular surface was investigated. MATERIALS AND METHODS: Nanoparticles were prepared using polyelectrolyte complexation from chitosan and dextran sulfate. The effect of polymer ratio, pH, and the amount of extract on particle size and encapsulation efficacy were studied using Box-Behnken response surface methodology. RESULTS: The optimum condition was obtained as follows: 4.9:1 ratio of dextran sulfate to chitosan, 600 µL amniotic membrane extract, and pH of 6. The prepared nanoparticles had an average size of 213 nm with 77% encapsulation efficacy. In the release test, after 10 days, approximately 50% of entrapped bioactive proteins were released from the nanocarriers in a controlled manner. Biological activity assessment on endothelial cells revealed amniotic membrane extract loaded nanoparticles had a longer and significant increase in anti-angiogenic effect when compared to the control. CONCLUSION: Our data elucidate the ability of nanotechnology in ocular targeted nano delivery of bioactive compounds.

Design, modeling, and expression of erythropoietin cysteine analogs in Pichia pastoris: improvement of mean residence times and in vivo activities through cysteine-specific PEGylation.

In this study, the low-cost production of recombinant human erythropoietin cysteine analogs (Cys-rhEPOs) from Pichia pastoris and the potential to increase their serum residency and in vivo activity through cysteine-specific PEGylation were investigated. Three-dimensional structures of several Cys-rhEPOs were generated using homology modeling, and three stable Cys-rhEPOs were selected on the basis of model stability in molecular dynamics simulation and surface accessibility of the inserted cysteine. cDNAs encoding Cys-rhEPOs were constructed by site-directed mutagenesis and expressed as secreted proteins in flask cultures of P. pastoris. The selection of highly expressing clones and the optimization of certain culture parameters resulted in protein expression levels of 100-170 mg/l. Purified Cys-rhEPOs were cysteine-specifically PEGylated using 20 kDa and 30 kDa mPEG-maleimides (methoxy polyethylene glycol-maleimides). The E89CEPO analog with the highest (96.6%) cysteine accessibility was conjugated to PEG-polymers with the largest yields (about 80%). In comparison with rhEPO, 30 kDa PEG-E89CEPO demonstrated a significant (approximately 30%) increase in the mean residence time. Whereas the in vitro activities of 30 kDa PEG-E89CEPO were comparable to those of rhEPO, the in vivo activity of this conjugate was more prolonged compared to rhEPO (12 days vs. 7 days). Our results demonstrate that the site-specific PEGylation of Pichia-expressed EPO analogs may be considered as a promising approach for generating cost-effective and long-acting erythropoiesis-stimulating agents.