Production, purification and controlled release application of gamma-polyglutamic acid.

摘要

Gamma-polyglutamic acid (gamma-PGA) is a commercially important biopolymer with many applications in biopharmaceutical, food, cosmetic and waste-water treatment industries. It is a biodegradable biopolymer produced by microbial fermentation. This work is focused on two main aspects of gamma-PGA research: downstream processing and drug delivery application. Efficient gamma-PGA recovery, development of controlled drug release formulation and cytotoxic evaluation of the formulation were the main objectives of this research.;A novel method for the selective recovery and purification of gamma-PGA from cell free fermentation broth of Bacillus licheniformis was developed. The cell-free fermentation broth was treated with divalent copper ions, resulting in the precipitation of gamma-PGA, which was collected as a pellet by centrifugation. The pellet was resolubilized and dialyzed against de-ionized water to obtain the purified gamma-PGA biopolymer. The efficiency and selectivity of gamma-PGA recovery was compared with the existing ethanol precipitation method. Since ethanol is a commonly used solvent for protein precipitation, the purity of gamma-PGA precipitate was analyzed by measuring proteins that co-precipitated with gamma-PGA. Of the total proteins present in the broth, 48% proteins were found to be co-precipitated with gamma-PGA by ethanol precipitation, whereas in copper sulfate-induced precipitation, only 3% of proteins were detected in the final purified gamma-PGA, suggesting that copper sulfate-induced precipitation offers better selectivity than ethanol precipitation method. Total metal content analysis of the purified gamma-PGA revealed the undetectable amount of copper ions, whereas other metal ions detected were in low concentration range. The purified gamma-PGA was characterized using infrared spectroscopy.;Owing to its biological properties such as non-toxicity, biocompatibility and non-immunogenicity, gamma-PGA is an important biomaterial in the drug delivery applications. A comprehensive review of literature focused on the development of gamma-PGA nanoparticles as drug delivery carriers for anti-cancer therapeutics is presented. Various techniques for the production and characterization of gamma-PGA nanoparticles and controlled-release strategies are also discussed. A fundamental understanding of tumor physiology that forms the basis for the development of various targeted drug delivery approaches in cancer chemotherapy has been summarized briefly.;Formation of drug/polymer complexes through ionic interactions has proven to be very effective for the controlled release of drugs. The stability of such drug/polymer ionic complexes can be greatly influenced by solution pH and ionic strength. The potential of gamma-polyglutamic acid (gamma-PGA) as a carrier for the anticancer drug, Doxorubicin (DOX) was evaluated by the formation of ionic complexes between gamma-PGA and DOX. DOX was found to specifically interact with gamma-PGA forming random colloidal aggregates and results in almost 100% complexation efficiency. In vitro drug release studies illustrated that these complexes were relatively stable at neutral pH, but dissociates slowly under acidic pH environments, facilitating a pH-triggered release of DOX from the complex. Hydrolytic degradation of gamma-PGA and DOX/gamma-PGA complex was also evaluated in physiological buffer. These studies clearly showed the feasibility of gamma-PGA to associate cationic drug such as DOX, and may serve as a new drug carrier for the controlled release of DOX in malignant tissues.;Batch fermentations were run to produce gamma-PGA using B. licheniformis under pre-defined growth conditions. The PGA concentration in the range of 2-4 g/l was obtained after 48 h fermentation. The weight average molecular weight and polydispersity of gamma-PGA in the fermentation broth was 960 kDa, and 1.7, respectively. Parameters such as bacterial growth, substrate consumption and bi-product formation were also monitored throughout the cultivation time. As the fermentation progressed, a substantial reduction in the molecular weight of gamma-PGA was observed, suggesting the secretion of gamma-PGA-degrading enzymes in the later stage of fermentation. The fermentation broth obtained was further used to recover and purify gamma-PGA.;Doxorubicin-loaded gamma-PGA nanoparticle formulation was developed and its activity against model cancer cell line was investigated. The nanoparticles were prepared using DOX/gamma-PGA ionic complex and chitosan in different weight ratios. Up to 50-55% of complexed-DOX was encapsulated within the nanoparticles. The in vitro release studies showed an initial burst release followed by a very slow release. The evaluation of the activity of DOX-loaded nanoparticles and that of released DOX in cell cultures indicated that both were able to maintain anti-proliferative activity relative to free DOX. These studies showed the feasibility of gamma-PGA nanoparticles to entrap DOX and to release it in its active form.;Keywords: Polyglutamic acid, recovery, purification, metal cation induced precipitation, Bacillus licheniformis, nanoparticles, biopolymer, anticancer drug delivery, doxorubicin; polymer-drug complex; controlled release, ionic interactions, in vitro cytotoxicity.