Synthesis and in vitro characterization of poly(ethylene glycol)-albumin hydrogel microparticles.

High water content hydrogel microparticles based on the cross-linking of albumin with activated poly(ethylene glycol) were synthesized. The influence of different synthesis parameters on the physicochemical characteristics of the microparticles, such as the type of oil and of albumin, and the molecular weight of PEG, was evaluated. The water content of the microparticles ranged from 95 to 98%, increasing with an increase of the molecular weight of PEG. At optimal conditions, microparticles with sizes ranging from 3 to 50 µm were prepared. These microparticles showed a negatively charged surface. They were freely dispersed in PBS buffer and they were stable at 4°C for times varying from 0.5 to 10 months. Initial stirring speed and molecular weight of PEG were the 2 main factors that significantly affected microparticle size. High hydrophilicity, good stability and modulable size make this hydrogel an attractive matrix for protein or cell immobilization for biomedical applications.

Mechanism of cell death induced by spermine and amine oxidase in mouse melanoma cells.

Polyamines such as spermine, spermidine and putrescine are necessary for cell proliferation and are detected at higher concentrations in most tumor tissues, compared to normal tissues. The amine oxidase enzymes can generate cytotoxic products such as hydrogen peroxide and aldehydes from these polyamines. This study investigates the mechanisms of cell death in B16-F0 mouse melanoma tumor cells exposed to bovine serum amine oxidase and exogenous spermine. The bovine serum amine oxidase/spermine enzymatic system induced inhibition of cell proliferation in B16-F0 melanoma cells and cell death by both apoptotic and necrotic processes. Bovine serum amine oxidase or spermine, alone, did not induce cytotoxicity or cell death by apoptosis, indicating that the enzymatic reaction products were responsible. Catalase and NAD-dependent aldehyde dehydrogenase, inhibitors of hydrogen peroxide and aldehydes, respectively, decreased cell death by apoptosis and necrosis. This further confirms that the cytotoxic products are responsible for causing cell death. Use of inhibitors of different caspases showed that melanoma cells were sensitive to processes involving caspase-3 and -9, but were insensitive to caspase-6. Bovine serum amine oxidase in the presence of spermine could be useful as a promising new tool for anticancer treatment by the selective generation of toxic compounds from polyamines in tumors.

Anti-tumoral effect of native and immobilized bovine serum amine oxidase in a mouse melanoma model.

Bovine serum amine oxidase (BSAO) oxidatively deaminates polyamines containing primary amine groups, spermidine and spermine, to form the cytotoxic products hydrogen peroxide and aldehyde(s). Polyamines are present at elevated levels in many tumor tissues. The aims of the study were to evaluate the anti-tumoral activities of native and immobilized BSAO in mouse melanoma and also to determine the mechanism of tumor cell death. C57BL mice received a subcutaneous injection of B16 melanoma cells to induce formation of tumors, prior to antitumor treatments with native and immobilized BSAO. The enzyme was immobilized in a poly(ethylene glycol) (PEG) biocompatible matrix. Antitumor treatments consisted of a single injection of enzyme into the tumor. When immobilized BSAO (2.5mU) was injected into the tumor, there was a marked decrease of 70% of the tumor growth. This was compared with a decrease of only 32% of tumor size when the same amount of native BSAO was administered. The type of cell death was analysed in tumors that were treated with native or immobilized BSAO. When tumors were treated with immobilized BSAO, there was induction of a high level of apoptosis (around 70%), compared to less than 10% with the native enzyme. Apoptotic cell death was assessed by nuclear chromatin condensation using Hoechst staining and labelling of externalized phosphatidylserine using Annexin V. However, native BSAO, probably due to a burst of cytotoxic products, induced a high level of necrosis of about 40%, compared to less than 10% with immobilized BSAO. In conclusion, the advantage is that immobilized BSAO can act by allowing the slow release of cytotoxic products, which induces tumor cell death by apoptosis rather than necrosis.