Abstract 3188: Chitosan-glycerol injectable gel for intratumoral retention of immunotherapeutics
Authors: Kobrin Robert L. , Mantooth Siena M. , Zaharoff David A. 
Published: March 2024

Abstract
A novel, injectable chitosan-glycerol hydrogel was developed and characterized. Upon injection with co-formulated therapeutics, the gel vastly improved molecule retention at the injection site, as compared to molecule delivery in less viscous media.
Methods. The hydrogel was formed through a centrifugation process. 70% deacetylated, medium viscosity chitosan was dissolved in 0.1M HCl. Glycerol was then added, followed by vortexing. Next, 1x PBS and 1M NaOH were added to neutralize the mixture, which induced gelation. After hydrogel centrifugation at 10,000 rpm for 5 minutes, a hydrogel was separated from precipitate. Several formulation parameters were tested, including chitosan:glycerol volume ratio, chitosan concentration, chitosan degree of deacetylation, and chitosan molecular weight. The retention of model therapeutics, formulated in hydrogel or saline vehicles, following injection in agar-based tumor phantoms was quantified via absorbance and fluorescence readings. To assess biocompatibility, a non-contact cell viability assay was performed utilizing 3T3 fibroblasts seeded in the bottom of transmembrane wells with hydrogels added to the upper chamber. After 48 hours, cells were collected and counted utilizing trypan blue.
New Data. A hydrogel for intratumoral therapeutic delivery was constructed with several desirable qualities, including biocompatibility and injectability through a 25-gauge needle. Different relative glycerol to chitosan volumes, from 5 to 95 w/v % glycerol, were tested to determine a range of gelation conditions. Glycerol volume most significantly impacted gelation conditions independent of chitosan concentration, with gelation occurring between 70-85% relative volume glycerol. Increasing chitosan solution concentration and chitosan molecular weight increased the range of chitosan:glycerol ratios resulting in gelation. The hydrogel improved load retention at the agar tumor phantom injection site for various sizes of model therapeutics compared to less viscous solutions. Cytotoxicity testing of the hydrogel showed a statistically insignificant difference between the live:dead cell ratios of the experimental and negative control groups.
Conclusion. An injectable, biocompatible hydrogel was created through the neutralization and centrifugation of a chitosan-glycerol solution. The hydrogel displays physiological pH, injectability through a 25-gauge needle, biocompatibility, and vastly improved therapeutic load retention at the injection site compared to less-viscous delivery media. Chitosan-glycerol hydrogels may be useful for intratumoral delivery and retention of immunomodulators within the tumor microenvironment.
Citation Format: Robert L. Kobrin, Siena M. Mantooth, David A. Zaharoff. Chitosan-glycerol injectable gel for intratumoral retention of immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3188.