(T0930-06-36) Development of Injectable Chitosan-Based Hydrogels for Sustained Bladder Delivery of Therapeutic Proteins

Purpose: Bladder drug delivery via intravesical administration remains a challenge due to rapid therapeutic clearance and limited mucosal adhesion of injected payloads [1]. This study aim to develop and evaluate thermosensitive hydrogels composed of chitosan and β-glycerophosphate (β-GP) [2] for intravesical administration, with the goal of achieving localized and sustained delivery of therapeutic proteins to the bladder. Ovalbumin was used as a model protein to evaluate the hydrogel’s delivery performance.

Methods: Thermosensitive hydrogels were formulated using small and medium molecular weight (MW) chitosan derivatives. Formulations were optimized using gelation kinetics assays, leading to the selection of 1.2% chitosan and 12% β-GP as the final composition. These were designated as S12 (small MW) and M12 (medium MW). Hydrogels incorporating ovalbumin were labeled as S12-OVA and M12-OVA. Microstructures were characterized using scanning electron microscopy (SEM), and hydrogel stability was assessed over a 7-day period by measuring residual gel volume. Ovalbumin release kinetics were evaluated over 7 days. Cytotoxicity was assessed using LDH assays in T24 bladder cancer cells and RAW 264.7 macrophages. Cellular uptake was examined in T24 cells using fluorescence microscopy. In vivo gelation studies were conducted by intravesical injection of pre-hydrogel solution into the bladders of 8–9-week-old C57BL/6 mice. Gel formation was confirmed, and gelation yield was calculated based on the mass of formed gel.

Results: S12 and M12 hydrogels successfully transitioned from sol to gel state at 37°C within 15 minutes, displaying rapid and stable gelation. Ovalbumin loading did not interfere with gelation, and the resulting S12-OVA and M12-OVA hydrogels maintained structural integrity. SEM imaging revealed distinct porous architectures. Ovalbumin was released gradually and continuously over 7 days. Stability assays confirmed consistent residual gel volume throughout the 7-day period. LDH assays indicated minimal cytotoxicity in both T24 and RAW 264.7 cells, comparable to untreated controls, confirming high biocompatibility. Fluorescence microscopy demonstrated efficient cellular uptake of ovalbumin from both S12-OVA and M12-OVA in T24 cells. In vivo experiments further validated successful gel formation in the bladder, with quantifiable gelation yield supporting the practical applicability of the formulation.

Conclusion: These findings highlight the potential of chitosan-based thermosensitive hydrogels for intravesical delivery of therapeutic proteins. The optimized formulations enable effective gelation, sustained release, cellular uptake, and biocompatibility, supporting their application as injectable biomaterials for bladder-targeted therapy.

References: 1. Liu, X., Luo, Z., Zhang, L., & Huang, Y. (2020). Intravesical drug delivery: Challenges, current status, and future trends. Journal of Controlled Release, 329, 645–668. https://doi.org/10.1016/j.jconrel.2020.10.040
2. Kim, M. H., Park, H., Nam, K. T., & Lee, D. S. (2021). Injectable hydrogels based on chitosan and glycerophosphate as local drug delivery systems. Pharmaceutics, 13(4), 530. https://doi.org/10.3390/pharmaceutics13040530

Acknowledgements: This work was supported in part by a grant from CoBRE-Chemical Biology of Infectious Disease (NIH award number P20GM113117) (H.K). Conflict of Interest Statement: The authors declare no conflicts of interest related to this study.


Figure 1. Schematic Illustration of Hydrogel Administration and Gelation in the Mouse Bladder


Figure 2. LDH Cytotoxicity and Cellular Uptake Assay Using Alexa Fluor 647-Labeled Ovalbumin in T24 Cells.
(A) LDH assay results in RAW 264.7 and (B) T24 cells treated with S12, S12-OVA, M12, and M12-OVA.
(C) Fluorescence images of T24 cells after 24-hour incubation with Alexa Fluor 647-labeled ovalbumin, S12-OVA, and M12-OVA hydrogels, showing effective cellular uptake.



Figure 3. Hydrogel Stability and Ovalbumin Release Profile Over 168 Hours
(A) Residual hydrogel mass over 144 hours, showing physical stability of S12 and M12 formulations.
(B) Cumulative ovalbumin release from S12-OVA and M12-OVA hydrogels over 144 hours, demonstrating sustained protein release.