N26 - Development of poly(ethylene) glycol hydrogel drug delivery device to study intramuscular adipose tissue signaling

Introduction: : Intramuscular adipose tissue (IMAT) can 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) to deliver the net positively charged small molecule drug Mirabegron (MIR). Electrostatic complexation between Mirabegron and NS particles refines drug release kinetics, mitigating burst release.

Materials and

Methods: : The drug delivery device (DDS) was created by combining an 8-arm PEG-vinyl sulfone with a PEG-dithiol crosslinker through a Michael-type addition with an NS-MIR solution. The NS were pre-incubated with the drug to form electrostatic complexes. The polymer solutions and complexes were mixed and dotted onto a glass slide covered with parafilm to form a bulk DDS. Parafilm spacers (0.2 mm) were placed along the edges of the slide, and a second covered slide was placed on top. Depot DDS were produced similarly but were subcutaneously injected into mice immediately after crosslinking.
To evaluate in vivo host response, pre-swollen bulk hydrogels were implanted in the intramuscular pocket of the rotator cuff in 12-week-old mice. The hydrogels were recovered after 4, 7, or 14 days, cryo-sectioned, and stained for histological analysis.
To assess the DDS properties, DMSO (9% v/v) and NS (0.1 mg/mL) were incorporated into a 20% w/v gel. The control group excluded NS. To calculate swelling ratio and mesh size, the gels were weighed immediately after crosslinking, after swelling in PBS overnight, and after incubating overnight at 60°C. The storage modulus (G') of the swollen gels was measured at a strain of 0.1%, which was within the linear viscoelastic region, over a frequency range of 0.1 to 10 rad/s. The diffusion coefficient (De) of Alexa 647 through the gels was measured using fluorescence correlation spectroscopy (FCS). The gels were prepared in glass chamber slides and incubated in a 0.2 nM solution of Alexa 647 at 4°C overnight before measurement.

Results, Conclusions, and Discussions:: MIR was shown to electrostatically complex with dual-charge NS, the foundation of our sustained-release DDS. The hydrogel and nanosilicates were 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 greatest stability in the complex, dynamic environment and encouraged the most regeneration of the surrounding tissue (Figure 1). The inflammation caused by the gel implantation was within acceptable ranges. Histology of the 8-arm PEG-VS gels with NS did not show additional inflammation or increased degradation.
Mechanical and chemical properties of the DDS are summarized in Table 1. Laponite addition slightly reduced the gel stiffness but did not affect mesh size. We used FCS to measure the diffusion of Alexa 647 in the gels. Alexa 647, a small molecule dye with a similar (+1) charge and size to MIR, was used as a surrogate for MIR, which is non-fluorescent. We noted a 5-fold decrease in De in the presence of LAP, confirming that the electrostatic interactions between the negatively charged NS and the positively charged small molecule slowed the diffusion to allow for sustained release. Additional mass spectrometry results confirmed that the drug was released over at least 30 days of incubation in buffer at 37 °C and complexation experiments confirmed that we can achieve up to 60% MIR binding to LAP by tuning the LAP and MIR concentrations (data not shown). Together, these results indicate that this DDS will be suitable for the sustained release of MIR.
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.

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