Enzymatic Microneedle Patches: A Promising Approach to Address Nocturnal Hypoglycemia in Diabetes Care

Introduction:: Insulin therapy revolutionized the care of patients with diabetes starting ~100 years ago, yet insulin-induced hypoglycemia remains a serious life-threatening complication of insulin therapy. Glucagon is highly effective for the treatment of hypoglycemia; however current delivery methods require persistent glucose monitoring and manual intervention. This is challenging for patients during sleep. Automated hypoglycemia-triggered glucagon delivery systems have the potential to mitigate the risk of hypoglycemia and eliminate the burden of patient glucose self-monitoring and manual glucagon administration. However, recent efforts to develop counter-regulatory artificial pancreas systems have been hampered by the unstable and incompatible nature of current glucagon formulations. Chemically driven glucose-responsive systems show promise, however systems previously developed by our group and others^1–3 share critical design architectures that may impede clinical translation including (1) the use of a non-specific hypoglycemia-triggering mechanism based on phenylboronic acid, which is known to interact with interfering sugars and compounds commonly found in the body (e.g., fructose, galactose, mannose, sialic acid)⁴, and (2) glucagon loading strategies that are prone to continuous drug elution at euglycemic and even hyperglycemic conditions which may exacerbate hyperglycemia and lead to hyperglucagonemia. To overcome these challenges, we report an enzyme-driven wearable transdermal microneedle patch that provides automated glucagon delivery during episodes of nocturnal hypoglycemia (Fig. 1A). Critically, the proposed system exhibits no drug release prior to hypoglycemia triggering and employs a glucose-responsive mechanism based on the highly specific enzyme glucose oxidase.


Materials and Methods:: Glucose-responsive microneedle patches were constructed by a sequential solution casting and spray deposition process yielding a microneedle structure with four distinct functional layers: (1) an enzyme-functionalized xerogel microneedle shell, (2) an pH-responsive enteric polymer film, (3) a high-density, thermostable and readily-soluble solid glucagon formulation, and (4) a rigid epoxy backing. Confocal laser scanning microscopy was used to characterize the resulting structure (Fig. 1B).

The ability of the microneedles to penetrate the stratum corneum was assessed by manual patch application to ex vivo porcine skin, followed by visualization using methylene blue dye and histology (Fig. 1C) and histologically in rats (Fig. 1H). Glucagon stability at different stages of device fabrication and following long-term storage at 40˚C was assessed using RP-HPLC (Fig. 1D). In vitro glucagon release kinetics from the patches was evaluated by incubation in pH 7.4 phosphate buffer solution supplemented with 50 mg/dL or 200 mg/dL of glucose to simulate hypoglycemic and euglycemic conditions, respectively. Glucagon levels in the release medium was quantified using RP-HPLC (Fig. 1E, F).

In vivo glucagon release was characterized in streptozotocin-induced diabetic rats (Fig. 1G). All animal experiments were performed after MIT CAC approval. Animals were randomized into two experimental groups and treated with Insulin Degludec to induce stable euglycemia or hypoglycemia that persisted for over 9 hours. Transdermal patches were then applied to the skin and secured in place using Tegaderm bandage. Blood glucose levels (BGL) and plasma glucagon levels (PGL) were measured over the course of 6 hours (Fig. 1I-L).


Results, Conclusions, and Discussions:: We report the first enzymatically driven mechanism to achieve hypoglycemia-responsive delivery of glucagon (Fig 1A). The proposed system is driven by a pair of enzymatic reactions which catalyzes the conversion of interstitial glucose molecules to gluconic acid leading to an acidic pH microenvironment. An abundance of glucose during periods of euglycemia and hyperglycemia drives this pair of enzymatic reactions forward resulting in ample production of gluconic acid and maintenance of an acidic microenvironment within the hydrogel matrix. This low pH environment inhibits the dissolution of the enteric polymer film, thereby preventing glucagon release from the microneedle patch. Conversely, the diminished interstitial glucose concentration during periods of hypoglycemia decreases the production of gluconic acid leading to pH neutralization within the hydrogel, rapid dissolution of the enteric polymer film, and release of the glucagon from the microneedle patch.

The proposed mechanism was implemented in a 1 cm x 1 cm microneedle patch for painless self-administration (Fig. 1B). The microneedles can pierce the stratum corneum to access the underlying interstitial fluid (Fig. 1C, H). Glucagon remained stable (~95% purity) following device loading and storage at elevated temperatures (Fig. 1D). In vitro release studies showed significantly faster glucagon release in low glucose conditions compared to high glucose conditions (Fig. 1E). The relative glucagon release times can be tuned by changing the thickness of the enteric polymer film (Fig. 1F). Pharmacokinetic and pharmacodynamic studies in diabetic rats showed that application of the patches to euglycemic rats resulted in no significant change in BGL or PGL compared to euglycemic control animals (Fig 1I, J). In contrast, patch application to hypoglycemic rats induced an immediate and sustained increase in BGL and PGL resulting in a return to euglycemia within ~1-2 h (Fig. 1K, L). Critically, we observed no glucagon release prior to hypoglycemic triggering.

Our approach overcomes two challenges that impede clinical translation of chemically driven closed-loop glucagon delivery systems, namely (1) achieving hypoglycemia-responsive drug release via a glucose-specific transduction moiety, and (2) undesired glucagon elution under euglycemic conditions. We believe that this system shows promise for the prophylactic treatment of nocturnal hypoglycemia in patients with diabetes.


Acknowledgements (Optional): : We thank the MIT Koch Institute Swanson Biotechnology Center histology and high throughput cores for technical support. Pharmaceutical grade 280 kDa hyaluronic acid was gifted from Bloomage Freda Biopharm Co. (Jinan, China). Concept illustrations by Virginia E. Fulford, Alar Illustrations. Work funded in part by: The Natural Sciences and Engineering Research Council of Canada (NSERC) postdoctoral fellowship (J.L.), the National Science Foundation (Grant #2127309 to the Computing Research Association for the CIFellows 2021 Project) (C.B.), Novo Nordisk Grant (R.L., G.T.), Karl Van Tassel (1925) Career Development Professorship, Department of Mechanical Engineering, Massachusetts Institute of Technology (G.T.).




References (Optional): : 1. GhavamiNejad, A. et al. Glucose-Responsive Composite Microneedle Patch for Hypoglycemia-Triggered Delivery of Native Glucagon. Adv. Mater. Deerfield Beach Fla 31, e1901051 (2019).

2. Wang, Z. et al. Dual self-regulated delivery of insulin and glucagon by a hybrid patch. Proc. Natl. Acad. Sci. 117, 29512–29517 (2020).

3. Glucose-responsive microneedle patch for closed-loop dual-hormone delivery in mice and pigs | Science Advances. https://www.science.org/doi/full/10.1126/sciadv.add3197.

4. Wu, X. et al. Selective sensing of saccharides using simple boronic acids and their aggregates. Chem. Soc. Rev. 42, 8032–8048 (2013).