Novel Injectable Thermoresponsive Hydrogels For Sustained Release Vaccines

Purpose: Current vaccine development has focussed on the use of subunit vaccines that utilise highly purified protein and peptide antigens. Subunit antigens are safe to use, however they lack immunogenicity and need to be administered multiple times along with potent adjuvants in order to stimulate effective immune responses. The aim of this study was to develop a single-shot injectable thermoresponsive hydrogel based delivery system that was a free-flowing liquid at ambient temperature and could sustain the release of antigen over time with trend resembling that of an acute infection, thereby reducing the possibility of immune exhaustion. The thermogelling vaccine formulations were made using pentablock copolymers and Pluronic® grafted chitosan (CP). The hydrogels were loaded with poly (lactic-co-glycolic acid) nanoparticles (PLGA-NP) to provide synchronous release of vaccine components. Rheological characteristics of these formulations were then investigated as was the in vitro release of vaccine components and immune responses stimulated in mice. Methods: Pentablock copolymer based sol-gels were prepared using two novel pentablock copolymers consisting of polyethyleneglycol-polycaprolactone-polylactide-polycaprolactone-polyethyeneglycol (PEG-PCL-PLA-PCL-PEG) and using Pluronic® grafted chitosan copolymer, synthesised using EDC/NHS chemistry. PLGA-NP containing the antigen ovalbumin (OVA), and monophosphoryl lipid A (MPL) and Quil A (QA) as adjuvants were prepared using the W/O/W double emulsion method. The polymer solutions were loaded with PLGA-NP or free antigen and adjuvant at 4 °C and stirred for 24 hours. The sol-gel transitions were characterised with an oscillatory rheometer using a cone and plate geometry. In vitro gel stability and release studies were performed at 37 °C for 30 days. C57BL/6 mice were immunised subcutaneously and antigen specific CD4+ and CD8+ T cells in lymph nodes and the production of antigen specific IgG in serum was quantified. B16 OVA melanoma cells (1x105) were injected s.c. into mice 7 weeks after immunisation and tumor development was assessed. Results: The polymers examined were freely flowable solutions at ambient temperature and transformed rapidly into gels at body temperature. Rheological analysis revealed that addition of PLGA-NP (size range of 325-345 nm) to the polymer solutions resulted in a decrease in gelation temperature and time, and an increase in the complex viscosity of the gel. Pentablock hydrogel formulations were stable even after 30 days and showed incomplete release of vaccine components whereas CP hydrogels were stable up to 18 days and shown complete release of vaccine components. Pentablock hydrogels formed gel depots that persisted for several weeks at the site of immunisation resulting in a more chronic release of antigen while CP hydrogels depots demonstrated antigen release more resembling an acute infection. Both pentablock and CP hydrogel formulations stimulated cellular and humoral responses in vivo for up to 49 days. PLGA-NP loaded pentablock hydrogels did not induce early immune responses whereas CP hydrogels loaded with PLGA-NP were able to induce strong cellular and humoral responses at early time points. No significant difference was observed in immune responses in mice immunised with hydrogels directly loaded with antigen and adjuvant or with hydrogels loaded with vaccine in PLGA-NP, which is consistent with the synchronous release of antigen and adjuvant measured in vitro. Immunisation of mice with CP hydrogel formulations resulted in higher survival of mice than did immunisation with pentablock hydrogels in the prophylactic tumor challenge studies. Conclusion: Both CP and pentablock copolymer sol-gel formulations met the criteria for thermoresponsive hydrogel delivery systems. CP hydrogels appear to be an ideal sustained release vaccine delivery system and incorporation of PLGA-NP resulted in higher cellular and humoral responses and the development of a stronger prophylactic anti-tumor effect