(L-447) Novel Chitosan-Glycerol Injectable Gel for Intratumoral Delivery of Immunotherapeutics

Introduction::

 

Materials and Methods:: Chitosan-glycerol hydrogels were formed via simple addition. Briefly, chitosan was dissolved into a 0.1M HCl dilute acid to form a chitosan solution. Glycerol was then incorporated into the solution via positive displacement pipette and mixed thoroughly using a vortex. Next, PBS 1x was incorporated to buffer the solution before 1M NaCl was added to neutralize the mixture, resulting in gelation. The solution containing the gel was then condensed via centrifugation at 10,000 rpm for 5 minutes, allowing for separation of the hydrogel. The influence of chitosan solution concentration (1%,1.5%,2%), chitosan:glycerol volume ratio (5:95 to 95:5 by steps of 5), and chitosan type (degree of acetylation and molecular weight) on gelation was then examined. To evaluate local retention, tumor phantoms comprised of solid agar were injected with saline solutions, chitosan solutions, or chitosan-glycerol hydrogel. All injected samples were loaded with a red dye to visualize injection retention over a 24-hour period (Figure 1). To assess biocompatibility, a cell viability test was then performed utilizing 3T3 mouse embryonic cells. Cells were seeded in a T24 transmembrane well plate for 12 hours before the created hydrogel was loaded into the wells. After 48 hours, cells were counted utilizing trypan blue cell counting, with both live:dead ratio and cell count analyzed.

Results, Conclusions, and Discussions::

The formation of chitosan-glycerol hydrogels was found to be independent of chitosan concentration as long as glycerol comprised between 70 and 85% of the total volume. Increasing chitosan solution concentration and chitosan molecular weight both led to an increase in gelation occurrences outside of this glycerol range. Increasing chitosan degree of deacetylation led to a shift in gelation parameters to favor glycerol percentages as low as 60% of total volume. Hydrogels were easily injected through a 25-gauge needle into agar-based tumor phantoms and displayed increased retention of co-formulated dye compared to saline and chitosan-based vehicles solutions over a 24-hour period (Figure 2). Chitosan-glycerol hydrogels did not significantly impact cell viability in a contact test implying suitability for in-vivo use (Figure 3).  

In conclusion , a set of parameters was defined for the formation of chitosan-glycerol hydrogels. These hydrogels displayed positive qualities for localized delivery of cancer therapeutics, including formation at physiological pH, injectability through a 25-gauge needle, and non-cytotoxicity. Most importantly, chitosan-glycerol hydrogels demonstrated vast improvements in local retention following an injection compared to less viscous solutions such as saline and chitosan solutions which rapidly leak from an injection site. Ongoing studies are focused on quantifying this increase in retention in vitro as well as rheological shifts brought about by varying gel composition. Planned in vivo studies will evaluate biocompatibility and local retention in murine tumor models. In the long term, chitosan-glycerol hydrogels may offer a way to maximize the delivery of large molecule immunotherapeutics to the tumor microenvironment while minimizing adverse events associated with their systemic administration.

Acknowledgements (Optional): : I would like to thank the Joint Department of Biomedical Engineering and Office of Undergraduate Research at North Carolina State University for funding this research.

References (Optional): : Eccleston DS et al. Rationale for local drug delivery. Semin Interv Cardiol. 1996 Mar;1(1):8-16. PMID: 9552480

Nilsen-Nygaard J et al. Chitosan: Gels and Interfacial Properties. Polymers. 2015; 7(3):552-579. https://doi.org/10.3390/polym7030552