Sustained release thermoresponsive hydrogels for vaccine delivery

Abstract

Purpose: Modern vaccine development has concentrated on the use of highly purified peptides and proteins as target antigens. While these subunit antigens exhibit good safety profiles, they are generally not potent in activating immune responses. Therefore for effective vaccination subunit vaccines require the addition of adjuvants and the use of potent delivery systems in order to eliminate the requirement for multiple administrations. The aim of this study was to develop a hydrogel-based injectable thermoresponsive system which was a free-flowing liquid at room temperature and could sustain the release of antigen over time. The hydrogel-based delivery systems investigated were poloxamer 407 (P407) and chitosan loaded with cubosomes. Optimised formulations were investigated in vivo for their potential to sustain antigen release and their capacity to stimulate immunological responses. Methods: Modified P407 solutions were prepared through the addition of titrated amounts of methyl cellulose (MC), dextran, carrageenan and reversed poloxamer (25R4). Chitosan solutions modified with either dextran, MC, carrageenan or carbopol were prepared by dissolving the polymers in dilute acidic solution followed by the addition of a polyol salt. Cubosomes were prepared using the liquid precursor method and were loaded with the model antigen ovalbulmin (OVA) and the adjuvants Quil A and monophosphoryl lipid A (MPL). The rheological characteristics of the systems and in vitro gel stability in an aqueous environment were investigated. Particulate systems and thermoresponsive gel based systems containing soluble antigen and adjuvants or particulate antigen and adjuvants were investigated in vivo for their ability to sustain antigen release, to stimulate proliferation of CD4+ and CD8+ T cells and to stimulate the secretion of cytokines and antibody. Results: Initial experiments involving the addition of various polymers to overcome the lack of P407 gel stability in an aqueous environment found that P407-25R4 showed the most promising results. Rheological studies and in vitro erosion studies showed that this gel existed as a free-flowing solution at < 17 °C and formed gels upon heating. P407-25R4 sol-gels and gels containing particles were found to have a minimal ability to sustain antigen release in vivo. Mice immunised with P407-25R4 showed a significantly greater ability to stimulate CD4+ and CD8+ T cell response as compared to mice immunised with the immediate release formulation however the inclusion of cubosomes had no beneficial effect which reflected cubosomes instability in the gel as shown by small-angle X-ray scattering (SAXS). Chitosan modified with MC in the presence or absence of cubosomes demonstrated the desired rheological characteristics. Antigen release appeared to be sustained up to at least 14 days after vaccine administration. The immunogenicity of chitosan sol-gels containing soluble antigen and adjuvants was very promising with this formulation inducing T cells and cytokine responses. Again the inclusion of cubosomes into the formulation did not improve immune responses which may require further study. Conclusion: Thermoresponsive sustained release chitosan gel appears to promote greater immune responses than chitosan sol-gel containing cubosomes and immediate release systems. Further investigation is required on methods to enhance the stability of poloxamer-based gels. Overall, the inclusion of cubosomes into the gel did not significantly enhance immune responses. This highlights the importance of selecting a particulate carrier to match the properties of the hydrogel system. Taken together, 1% chitosan-MC sol-gels loaded with soluble antigen and adjuvants show promise as a potential candidate for sustained release vaccine delivery systems.