Abstract 4404: A novel chitosan-based hydrogel for intratumoral release of immunotherapeutic cytokines
Authors: Lowry Ethan D. , Wallace Christopher , Koppolu Bhanu prasanth , Smith Sean , Zaharoff David 
Published: August 2015

Controlled delivery of IL-12 has been shown to stimulate the immune system to eliminate primary tumors and elicit tumor-specific, protective immunity. Recently, we have developed a novel in situ gelling chitosan-based hydrogel that appears capable of further improving cytokine delivery within the tumor microenvironment while preventing systemic dissemination of potentially toxic, pro-inflammatory cytokines.

Change in viscosity during gelation was measured using a Brookfield DV-III Ultra Rheometer, revealing a 300-fold increase in viscosity. Manipulation of chitosan's chemical properties such as molecular weight, concentration, and degree of acetylation was found to control the time of gelation, which ranged from ten seconds to one minute.

A protein release study was conducted using fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) as a surrogate for IL-12. Dialysis tubes (with a molecular weight cutoff of 100kDa) were loaded with FITC-BSA co-formulated with hydrogel and FITC-BSA in PBS alone. The tubes were placed in a PBS bath and samples were taken at 48, 96, and 168 hours. The degree of release from the tubes was determined by measuring the fluorescence of protein in samples using a spectrofluorimeter. In vitro drug release studies demonstrated zero-order release of FITC-BSA from the hydrogel over a one-week period. Comparison to FITC-BSA in PBS indicates that the hydrogel exhibits a controlled kinetics profile.

Bioactivity of IL-12 when co-formulated with the hydrogel was determined by quantifying the production of interferon gamma (IFNγ) by NK-92MI cells, a line of human Natural Killer immune cells. NK-92MI cells were grown in the presence of human IL-12 with and without the hydrogel, and IFNγ production was measured via ELISA. The bioactivity assay indicated that IL-12 bioactivity was not compromised when mixed with the hydrogel. Specifically, after a 24 hour incubation period, the amount of IFNγ produced by the NK-92MI cells was found to be similar between groups treated with IL-12 alone and IL-12/hydrogel co-formulation.

After bioactivity was confirmed, mice (n = 3 per group) were subcutaneously injected with the mouse urothelial carcinoma cell line, MB49 (∼300,000 cells per mouse). After sufficient (approximately 50mm3) tumor growth, the mice were injected with either a hydrogel/IL-12 formulation or saline, which served as a control. Tumor volume was measured on days 7, 9, 14 and 19 after tumor implantation. In the anti-tumor study, mice treated with the IL-12/hydrogel co-formulation experience reduction in tumor size, while those treated with saline experience expected tumor growth.

These studies demonstrate that a novel chitosan-based hydrogel allows for sustained delivery of antitumor cytokines without altering bioactivity.

Citation Format: Ethan D. Lowry, Christopher Wallace, Bhanu prasanth Koppolu, Sean Smith, David Zaharoff. A novel chitosan-based hydrogel for intratumoral release of immunotherapeutic cytokines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4404. doi:10.1158/1538-7445.AM2015-4404