(M1030-02-14) A Novel Self-Crosslinkable Hydrogel-Based Matrix Microneedle Patch for Smart Glucagon Delivery

Purpose: Currently, around 10% of people living with diabetes have type 1 diabetes (T1D). The primary treatment of T1D relies on frequent insulin administration, which often carries the risk of hypoglycemia, a state of abnormally low blood glucose (≤ 70 mg/dL) that can be life-threatening if left untreated, which causes significant anxiety to patients and caregivers. Severe cases of hypoglycemia require immediate intervention by using emergency kits of glucagon injection or nasal powder spray. However, hypoglycemia unawareness, hypoglycemia during sleep, or other circumstances could prevent the patient or caregiver from administering glucagon on time, putting patients at significant risk. To reduce the risk of insulin overdose-induced hypoglycemia, glucose-responsive insulin analogs and glucose-responsive insulin delivery strategies have been employed. However, these approaches cannot address other factors caused hypoglycemia. Our group has thus developed a microneedle patch for glucose-responsive delivery of glucagon.1,2 Herein, we present a novel catechol-based microneedle (MN) patch made from self-crosslinkable polymers using a simple fabrication method. This patch enables high drug-loading efficiency via zinc-glucagon and zinc-polymer complexation and releases glucagon under hypoglycemic conditions.

Methods: The biocompatible hyaluronic acid polymer was functionalized by conjugating catechol and boronic acid-containing moieties to its backbone. The conjugation was verified using proton nuclear magnetic resonance spectroscopy (¹H-NMR). The dual-crosslinking method was confirmed via Fourier transform infrared spectroscopy (FTIR). The complexation between zinc and glucagon was achieved via metal-ligand complexation, which is based on their intrinsic binding affinity. The zinc-glucagon-loaded matrix MN was prepared via self-crosslinking and a secondary dynamic-crosslinking process under physiological pH in a one-step procedure. The mechanical strength of the MN tips was measured using a compression testing machine and evaluated on shaven rat skin. In vitro glucose-dependent glucagon release was determined using the Bradford protein assay. The secondary structure of released glucagon was determined using circular dichroism (CD) spectropolarimetry. The in vivo efficacy of the matrix MN patch was evaluated in a streptozotocin-induced T1D rat model and the plasma glucagon delivered by the patch was determined using an ELISA kit.

Results: ¹H-NMR confirmed the successful attachment of catechol and boronic acid conjugates onto the polymer backbone. The FTIR analysis determined the successful crosslinking between catechol with boronic acid moieties and its secondary dynamic crosslink with boronic acid moieties. The matrix MN was able to penetrate the first layer of rat skin, which was evaluated by delivering trypan blue dye into the rat skin. In vivo release studies showed that the matrix MN patch released glucagon in a glucose-dependent manner, with faster release at low glucose compared to high glucose. CD spectropolarimetry verified that the released glucagon exhibited a native secondary structure, which demonstrated that the bioactivity of loaded zinc-glucagon was preserved after using the mild crosslink method. The efficacy of the MN patch was further demonstrated in vivo by its glucose-responsive delivery of glucagon, which prevented hypoglycemia for up to 6 hours in T1D rat model against two consecutive challenges of insulin overdoses.

Conclusion: A prototype “smart” matrix MN patch has been successfully designed using a simple and peptide-friendly catechol-based chemistry approach. This method eliminated chemical crosslinkers, photoinitiators and other harsh crosslinking methods allowing the maintenance of the native structure and bioactivity of glucagon. This new MN patch technology is also useful for delivering other biologics.

References: [1] GhavamiNejad, A., et al. Adv Mater (2019) 31(3) e1901051
[2] Lu, B., et al. ACS Appl Mater Interf. (2022) 10.1021/acsami.1c24955

Acknowledgements:The work was supported through funding from The Leona M. & Harry B. Helmsley Charitable Trust Grant #2109-04810, Natural Sciences and Engineering Research Council of Canada (NSERC) Equipment grants (EQPEQ 374799-09; EQPEQ 440689-13) to X.Y.W. The authors would also like to thank Ontario Graduate Scholarship to BL, and University of Toronto Open scholarships to BL and JFL, the staff of the Analytical Laboratory for Environmental Science Research (ANALEST), Centre for Research and Applications in Fluidic Technologies (CRAFT), and Microscopy Imaging Laboratory (MIL) facilities at the University of Toronto for helping with analytical experiments. Conflict of Interest: The authors declare no conflict of interest.