Synthetic β-cells for fusion-mediated dynamic insulin secretion Zhaowei Chen1,2, Jinqiang Wang1,2, Zhen Gu1, 2, 3,* 1Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, NC 27695, USA; 2Division of Pharmacoengineering and Molecular Pharmaceutics and Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; 3Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, NC, 27599, USA

Introduction Importantly, AβC(no insulin) were not associated with The β-cells of the pancreas dynamically regulate insulin obvious cytotoxicity at any of the concentrations studied. secretion to maintain blood glucose homeostasis. Destruction or dysfunction of these cells leads to type 1 Conclusions and type 2 diabetes mellitus, a family of chronic diseases To conclude, we have demonstrated that the synthetic that currently affect over 422 million people in the AβCs could recapitulate the key functions of β-cells, world.1-2 On the other hand, the traditional intensive including sensing glucose levels, internally transducing insulin therapy by periodic injections imperfectly signals and dynamically secreting insulin via vesicle simulates the dynamics of β-cells and can cause fusion. Albeit many gaps remain between biomimetic hypoglycemia, which is always associated with the risk of assemblies and natural cells, the described design behavioral and cognitive disturbance, brain damage, or principle lay the groundwork for developing the next death. generation of artificial cells with the goal of achieving long-term replacement therapy to correct for cell function Methods deficiency. Like other biomolecule-secreting cells, β-cells can export cellular cargos to the outside through a vesicle transport system and a membrane fusion process upon external stimulation. Mimicking this biochemical cascade, here, we describe construction of an artificial β-cell (AβC) with a multicompartmental ‘vesicles-in-vesicle’ superstructure that is spatially equipped with a glucose metabolism system and membrane fusion machinery.3 The inner small liposomal vesicles (ISVs) are loaded with insulin to mimic the storage granules inside mature β-cells while the outer large vesicle (OLV) mimicked the role of the Figure 1. (a) Magnified fractured cryo-SEM of the plasma membrane. To endow the system with the vesicles-in-vesicle superstructures (scale bar in c, 5 µm; capability to both sense glycemic levels and release scale bar in d, 200 nm). As shown, small liposomal insulins via fusing ISV with OLV, the hierarchical vesicles can be clearly seen inside the large liposome. (b) liposomal assembly is equipped with a glucose relevant The blood glucose levels of diabetic mice after metabolic system and pH-controllable membrane fusion ‘transplantation’ with AβCs, or control AβCs which machinery. lacked insulins (AβC(no insulin)), membrane fusion peptide- E/peptide-K (AβC(no PE/PK)), or glucose sensing machinery Results (AβC(no GSM)). Data points represent mean ± SD (n = 5). To implement the AβC design, insulin-loaded ISVs were *P<0.05 for AβCs compared with control AβCs. first prepared via the classic lipid film hydration method. ISVs and glucose oxidase/catalase were encapsulated into Acknowledgement OLV. The resulting vesicles-in-vesicle superstructures This work was supported by the grants from American had an overall size 1~5 µm (Figure 1a). Based on the Diabetes Association (grant no. 1-15-ACE-21), Alfred P. glucose sensing ability of AβCs and the reversible Sloan Foundation (Sloan Research Fellowship) and NC attachment of PEG shield on ISV, this new glucose- TraCS, NIH’s Clinical and Translational Science Awards responsive mechanism allows fusion accessible under (CTSA, NIH grant 1UL1TR001111) at UNC-CH. high glycemic conditions while resheileding the ISVs minimized further fusion at normoglycemic levels for References numerous cycles. In vitro, membrane fusion at high (1) Yu, J. Proc. et al. Natl Acad. Sci. USA. 2015, 112, glucose concentration led to a remarkably rapid insulin 8260-8265. release while minimal release was observed at a normal (2) Back, N. et al. Nat. Chem. 2017, in press. glucose level or in glucose-free buffer solution over 15 h. (3) Chen, ZW. et al. Nat. Chem. Biol. 2017, in press. In in vivo experiments, the blood glucose levels in diabetic mice ‘transplanted’ with AβC quickly declined from hyperglycemia to normoglycemia within 1 h, and after that, the blood glucose level maintained around normoglycemic level for up to five days (Figure 1b).