(M1430-08-52) Development of an Advanced Hydrogel-Based Drug Delivery System for Enhanced Neuroprotection in Traumatic Brain Injury

Purpose: Traumatic brain injury (TBI) presents a significant global health burden. Particularly in military and emergency settings open skull injuries are more concerning, since despite their frequent occurrence, advanced neurosurgical interventions is delayed or unavailable. Current treatment approaches are limited by the need for repeated dosing, systemic side effects, and poor bioavailability of neuroprotective agents at the injury site. To address these challenges, we aimed to develop an advanced hydrogel-based drug delivery system designed for sustained, localized, and multimodal therapy in open skull TBI. The hydrogel system integrates antioxidant, anti-inflammatory, and neuroprotective agents with antimicrobial functionality into a single thermosensitive platform. Our goal in this study was to establish the feasibility of this modular hydrogel for injectable in situ gelation, prolonged drug release, cytocompatibility, and broad-spectrum antimicrobial activity—supporting its potential for use in prolonged field care and austere clinical environments.

Methods: Preliminary Glutathione and Minocycline hydrogels were formulated from Chitosan and beta glycerophosphate. Hydrogels were investigated for sol-to-gel transition, gelation time, viscosity, Conductivity and Glutathione loading. Hemoglobin loaded liposomes were synthesized using the thin film hydration technique. Methyl prednisolone was incorporated into the system by initially incorporating it into solid lipid nanoparticles synthesized via the solvent emulsification-evaporation technique. To promote controlled and sustained Glutathione and Minocycline release, these drugs were further incorporated into microparticles. In addition to loading glutathione and minocycline into microparticles, both hemoglobin loaded liposomes and Methyl prednisolone-loaded Solid lipid nanoparticles were introduced into the microparticles. Microparticles were synthesized using the air atomization technique and characterized for size and surface zeta potential using a master sizer. Finally, drug and nanoparticle loaded microparticles were incorporated into the drug loaded hydrogel system. The final system was characterized for gelation time, pH, conductivity, drug loading, cumulative glutathione release and biocompatibility with U87 glioblastoma cells.

Results: Study efforts centered on the integration of multiple delivery technologies—hydrogels, solid lipid nanoparticles (SLNs), functionalized liposomes, and microparticles—into a multifunctional therapeutic system. Liposomes functionalized with a cell-penetrating lycotoxin peptide successfully encapsulated hemoglobin, a model protein structurally comparable to erythropoietin. The incorporation of hemoglobin increased liposome size from 102 nm (empty) to 148.6 nm, while the polydispersity index (PDI) improved from 0.387 to 0.256, indicating a more uniform particle distribution post-loading. Proton NMR confirmed successful conjugation of the lycotoxin peptide to DSPE-PEG. Solid lipid nanoparticles loaded with methylprednisolone were optimized through a Design of Experiments approach, identifying Trimyristin and Tristearin as key lipids for improved encapsulation. The final SLNs exhibited an average particle size of ~218 nm with a PDI of 0.2378, reflecting good stability and uniformity. Microparticles co-loaded with glutathione, minocycline, SLNs, and liposomes demonstrated favorable physicochemical properties. The particles exhibited an average diameter of 367 ± 7.08 µm and a surface charge of +32.67 ± 6.32 mV, suggesting strong electrostatic stability and potential for enhanced cellular interaction. High zeta potential values further supported the physical stability of the formulation. Hydrogels formed from chitosan and β-glycerophosphate showed temperature-sensitive sol-to-gel transition properties. Gelation time decreased with increasing minocycline concentration and remained below 120 seconds at physiological temperature. Hydrogels co-loaded with microparticles and nanoparticles retained near-neutral pH (7.02 ± 0.06), exhibited conductivity values of 23.8 ± 1.56 µS/cm, and transitioned to a gel state in 76.71 ± 30.96 seconds at 37°C. Controlled release studies indicated sustained glutathione release, with up to 50% released over a 7-day period.

Conclusion: The integration of liposomes, solid lipid nanoparticles, microparticles, and thermosensitive hydrogels led to the successful development of a multifunctional drug delivery system with favorable physicochemical characteristics and controlled release capabilities. The formulations demonstrated efficient encapsulation of model therapeutic agents, improved particle uniformity, and strong indicators of stability, while the hydrogel matrix enabled sustained drug release and temperature-triggered gelation under physiological conditions. These results collectively support the potential of this system for delivering combination therapies in a stable, biocompatible, and controlled manner, providing a strong foundation for future in vivo evaluation and clinical translation.

Acknowledgements: This study has been developed in collaboration with the U.S. Department of Defense (DoD) and is supported by the Defense Health Agency (DHA), Walter Reed Army Institute of research, US Army Medical Research and Development Command (USAMRDC), and the US Army Medical Research Acquisition Activity (USAMRAA).

Hydrogel formulations of MedTBI solutions. The release kinetics of glutathione alone in hydrogel, glutathione in microparticles entrapped in hydrogel, and co-loaded microparticles entrapped in hydrogel are compared with nanoparticle-loaded formulations.