Rapid in situ gelling PEG hydrogels for mucosal drug delivery

Introduction: : Mucosal layers amount to ~400 m2 in total surface area in the human body. They keep bodily tissues hydrated and function as a protective layer by blocking foreign substances and pathogens. Thus, mucosal tissues such as the eye, stomach, nose, lung, and vagina are often sites of inflammatory and infectious diseases making them a desirable site for therapeutic delivery. Moreover, the mucosa may also be useful as a non-invasive route for drug delivery to local and distal sites as these tissues possess a large surface area that is highly vascularized and immunologically active. However, mucosal tissues possess natural defense mechanisms to facilitate the rapid clearance of potentially infectious or irritating materials from these sites which can lead to reduced drug bioavailability when locally administered. To address this challenge, we developed a mucoadhesive hydrogel material which forms rapidly through thiol-pyridyl disulfide exchange. This material is composed of polyethylene glycol (PEG), a widely used polymer, and forms nearly instantaneously upon mixing under physiological conditions. Since the prepared hydrogels have thiol and pyridyl sulfides, we hypothesized adhesion of the PEG hydrogel to mucosal tissues may be favored through direct reaction with thiol groups on mucin biopolymers. To determine if potentially useful for drug delivery applications, we assessed the release rate of different types of cargo encapsulated within the PEG gels and evaluated their biocompatibility. This study establishes a new simple approach to formulate rapid in situ forming PEG gels which could be used in mucosal drug delivery applications.

Materials and

Methods: : To synthesize rapid forming PEG gels, 4-arm thiol terminated polyethylene glycol (PEG-4SH) and 4-arm Ortho pyridyl disulfide terminated PEG (PEG-4OPSS) solutions in phosphate buffered saline are mixed in equal volumes. Tube inversion method was used to measure gelation time at body temperature of 37℃. Bulk rheological measurements were performed using parallel plate on ARES G2 rheometer to confirm successful formation of PEG hydrogels and to understand polymer weight percentages on mechanical properties. PEG hydrogel degradation was measured in PBS, PBS+ dithiothreitol (DTT) and PBS+ glutathione by placing 100 µL cylindrical gels in respective buffers and weighing before and at different time points after immersion in PBS. Functional resilience of the rapidly forming PEG hydrogels was examined in shear induced failure and recovery experiments using oscillatory time sweeps at a frequency of 10 rad/sec. TRITC-BSA, Cy5-IgG, and fluorescent nanoparticles were encapsulated in gels to study release kinetics using fluorescence spectroscopy. Mucoadhesive strength of the gels was measured using a pull-apart adhesion test with porcine intestine tissue using dynamic mechanical analyzer.5 HEK 293T cells were used to evaluate polymer and gel biocompatibility using resazurin assay and live/dead staining. To evaluate hydrogel retention in vivo, mice were intranasally administered with hydrogel loaded with near-infrared-labeled 100 nm polystyrene nanoparticles and compared against control group of mice receiving nanoparticles suspended in PBS under identical conditions. Fluorescence signals from the hydrogel-encapsulated nanoparticles were measured over 15-days post-administration using IVIS Spectrum Imaging System.

Results, Conclusions, and Discussions:: In this study, we developed a new method for rapid-forming PEG hydrogels at physiological conditions (37°C, pH 7.4). An overview of crosslinking mechanism for 4-arm PEG-SH and 4-arm PEG-OPSS is depicted in Figure 1A. We identified 6 lead formulations (Fig. 1B), which rapidly form disulfide-linked PEG hydrogels in ≤30 seconds and retain stability in PBS for ≥ 24 hours. Disulfide reducing agents DTT and glutathione degraded gels in < 10 minutes and 1-3 days, respectively, opposed to more than 30 days in PBS alone suggesting faster in vivo degradation (Fig. 1B, 1D). Fig. 1C shows effect of Polymer weight % and MW on hydrogel strength. Pull-apart tests showed mucoadhesive strengths >600 Pa. Scanning electron microscope images (Fig. 1F) of freeze-dried hydrogels made with 20kD polymers reveals porous structure of the hydrogel network. The self-healing behavior assessed via cyclic time sweep test with alternating low/high strains using formulation F6 showed near-complete recovery, regaining a substantial portion of their original mechanical strength (Fig. 1E). This self-healing capability further highlights their potential for use in dynamic physiological environments where repeated deformation and recovery are critical for sustained functionality. Polymer weight % and swelling ratio minimally affected protein release but significantly impacted nanoparticle release (Fig. 1G). Adhesion likely occurs through PEG-mucin entanglement and hydrogen bonding, rather than disulfide bonds with mucin cysteines, as evidenced by comparable adhesion of PEG gels without thiol end groups (Fig. 2A-2D). HEK 293T cells showed >80% viability when treated with gel precursor components and PEG gel, except for 10 kDa 4-arm PEG OPSS at 4% w/v concentration (Fig. 1E). In vivo cargo retention study showed NPs administered in PBS (without hydrogel) were cleared from the nasal cavity within 5 hours and completely undetectable by 48 hours. In contrast, NPs delivered using the hydrogel vehicle exhibited near-complete retention up to 48 hours (Fig. 3A-3B). These results demonstrate that the rapid in situ forming PEG hydrogel significantly enhances cargo retention in the nasal cavity and provides the ideal formulation strategy for sustained drug release at mucosal sites. Future work aims to develop sprayable and/or injectable PEG gels for mucosal therapeutic delivery.

Acknowledgements and/or References (Optional):: This study was supported by the NIH (R21 EB030834 to GAD, R01 GM141132 to PK) and UM Ventures Medical Device Development Fund grant.