Poster W13 - Development of poly(ethylene) glycol hydrogel drug delivery device to study intramuscular adipose tissue signaling

Introduction: Intramuscular adipose tissue (IMAT) can potentially be stimulated to secrete pro-myogenic adipokines, emulating brown adipose tissue and promoting muscle regeneration. We have previously shown that β-3 adrenergic receptor agonists can successfully brown white auxiliary adipose depots with intraperitoneal (IP) injection. However, systemic delivery of β-3 adrenergic receptor agonists is associated with undesirable off-target effects. Thus, this project aims to develop a sustained-release, biodegradable system for the local delivery of Mirabegron, a β-3 adrenergic agonist. Here we developed a tunable biodegradable polyethylene glycol (PEG) hydrogel with embedded net negatively charged nanosilicates (NS) for the delivery of the net positively charged Mirabegron. Electrostatic complexation between Mirabegron and NS particles refines drug release kinetics, mitigating burst release.

Materials and

Methods: The drug delivery device (DDS) of 8-arm PEG-vinyl sulfone and PEG-dithiol crosslinker were combined in Michael-type addition with an NS-drug solution, where the NS were pre-incubated with the drug to form electrostatic complexes. The PEG, crosslinker, and complexes were mixed and dotted onto a parafilm-covered glass slide for a bulk DDS. Parafilm strip spacers (0.2 mm) were placed on the edges of the slide and a second covered slide was placed on top. Depot DDS were fabricated in the same manner but were subcutaneously injected into mice immediately before crosslinking.
To test the drug binding affinity to the nanosilicates, the NS was combined with deionized water, incubated on a rocker for 1 hour, spun down, the supernatant retrieved, and the absorbance measured via UV-vis spectrophotometry. The pellet of complexes was resuspended in deionized water and the particle size(s) were measured through dynamic light scattering (DLS). Sustained release of drug from bulk DDS was quantified via bulk release experiments (230 - 350 nm, 5 nm step absorbance of releasate vis spectrophotometry). The mechanical properties of the DDS were evaluated through rheological measurements.
For the evaluation of in vivo degradation and host response, pre-formed bulk hydrogels were cut and implanted in an intramuscular pocket of the rotator cuff and subcutaneously in 12-week-old mice. The hydrogels were recovered after 4, 7, or 14 days, cryo-sectioned, and stained for histology.

Results, Conclusions, and Discussions: DLS measurements of dispersed nanosilicates and complexed nanosilicates [0 mg/mL, 0.01 mg/mL, 0.1 mg/mL] with Mirabegron [1 mg/mL] showed a shift in size between the nanosilicates complexes. This confirmed that the drug did bind to the nanosilicates, forming complexes. The known IMAT stimulatory small molecule Mirabegron has been shown to electrostatically complex with dual-charge nanosilicates, the foundation to our sustained-release DDS. The hydrogel and nanosilicates have been evaluated in vivo for host response and degradation. Upon histological analysis of intramuscularly implanted bulk PEG hydrogels of different volume fractions (20% w/v, 30% w/v, 40% w/v) that were recovered, 20% w/v and 30% w/v 8-arm PEG-VS hydrogels showed the least degradation in the complex, dynamic environment and the most regeneration of the surrounding tissue. The inflammation caused by the implantation of the hydrogel was within acceptable ranges. Histology of the 8-arm PEG-VS gels with nanosilicates did not show any additional inflammation or increased rate of degradation. Bulk release experiments were performed to show the effect of NS-drug complexation on slowing down Mirabegron release and minimizing burst release. The sustained drug release from PEG-NS gels was quantified via UV-Vis spectrophotometry and confirmed with capillary electrophoresis. These results indicate that this DDS will be suitable to evaluate the effect of local β-3 adrenergic stimulation on intramuscular adipose tissue.
Understanding the mechanisms and effects of the browning of white adipose tissue will play a key role in improving muscle regeneration for rehabilitation. To study those mechanisms, the development of a drug delivery device for the local, sustained release of β-3 adrenergic receptor agonists is paramount. Here, we have developed a slow, sustained-release matrix drug delivery device that is present and stable in vivo for a clinically relevant amount of time.

Acknowledgements (Optional): Dr. Samuel Stealey