The Development of Alginate-Based Microspheres for Endovascular Applications

This work presents findings on the potential of calcium alginate microspheres for endovascular applications. Unique ionic cross-linking mechanism of calcium alginate microsphere were explored and utilized for development of specially designed biomaterials for endovascular therapies. First, the studies for developing the alginate-based microspheres were conducted for breaking through the drawbacks of currently developed permanent and temporary embolic agents for embolotherapies, which were caused by non-degradability of permanent embolic agent and unpredictable degradation rate together with their degradation behaviors of temporary embolics. Calcium alginate microspheres embedded with iron-oxide nanoparticle that can provide MRI-visibility were synthesized by spraying-coagulation method. The synthetic optimization was conducted for controlling the synthesis of alginate microspheres with desired size range and uniform size distribution. On-demand degradability of calcium alginate microspheres by treatment of EDTA was demonstrated in in vitro systems. Addition of EDTA in the microsphere suspension showed the instant degradation of calcium alginate microspheres, and the complete degradation time was measured with various concentration of EDTA. This result also presented the minimum concentration of EDTA to degrade the alginate microspheres. Also, the on-demand degradable embolic microsphere system did not show severe cytotoxicity. Plus, to mimic the real blood vessel, custom-made capillary was prepared and tested with occlusion of with alginate microspheres and infusion of EDTA. To check the safety profile after the degradation occurred, the solution passed through the capillary was observed with optical microscope, and there was no visible debris that may cause side effects such as distal migration. In vivo studies showed the embolization performance of calcium alginate microspheres and demonstrated the instant restoration of blood flow observed by naked eyes, DSA and MRI in mice and rabbit model. Finally, the histology of liver section that were the downstream of embolization site showed there was no visible tissue toxicity and residual embolization in the liver. Next, the studies for the development of new type of bacterial delivery system were conducted to enhance the tumor eradication performance of anaerobic C. novyi bacteria by increasing hypoxic region in the tumor. Calcium alginate microspheres embedded with C. novyi spores were synthesized using spraying-coagulation method. As well as the confirmation of successful encapsulation of C. novyi spores in the microspheres, the germination process and the release of living bacteria was successfully observed in hypoxic conditions. The microspheres incubated in normoxia showed minimal leakage of spores loaded in the matrix. Furthermore, it was found out that the germination process of C. novyi spores triggered the swelling and degradation of calcium alginate microspheres. Hence, the possible mechanisms of degradation during germination process of spores were explored. The calcium quantification assays, and OD loss assays suggested the calcium ions could act as co-germinants of C. novyi spores. Also, DPA accumulated in the core of C. novyi spores was released during germination of C. novyi spores. Therefore, the effect of DPA on the morphology of alginate-based microspheres was explored using calcium or barium alginate microspheres. DPA showed selective chelation of calcium ions showing that the degradation of calcium alginate microspheres while the barium alginate microspheres did not show morphological changes. The uptake of calcium ion and subsequent release of DPA contributed to the degradation of calcium alginate microspheres. in vivo studies showed the synergistic effect of therapeutic bacteria and embolization therapy, shown in tumor responses. Also, administrated C. novyi bacteria via alginate-based bacterial delivery system were found in not only necrotic tumor region but also vascularized tumor rim. In the meantime, the immune modulation of alginate-based bacterial delivery system was characterized. Maturation of dendritic cell was triggered, and the higher accumulation of CD4+ T lymphocytes was observed in tumor. Surprisingly Immunosuppressive regulatory T cells were strongly suppressed among CD4+ T cell population. The possibility of the involvement of T-helper 17 lymphocytes was observed by flow cytometry analysis. Overall, this work may contribute to the advance of alginate-based material system for endovascular applications where the choice of materials may be extremely conservative and limited. The alginate microspheres showed unique advantages showing stimuli responsive behavior by their special ionic cross-linking mechanism.