pH-Responsive Hexa-Histidine Metal Assembly (HmA) with Enhanced Biocatalytic Cascades as the Vehicle for Glucose-Mediated Long-Acting Insulin Delivery

Abstract

Diabetes has been listed as one of the three major diseases that endanger human health. Accurately injecting insulin (Ins) depending on the level of blood glucose (LBG) is the standard treatment, especially controlling LBG in the long-term by a single injection. Herein, the pH-responsive hexa-histidine metal assembly (HmA) encapsulated with enzymes (GOx and CAT) and Ins (HmA@GCI) is engineered as the vehicle for glucose-mediated insulin delivery. HmA not only shows high proteins loading efficiency, but also well retained proteins activity and protect proteins from protease damage. Within HmA, the biocatalytic activities of enzymes and the efficiency of the cascade reaction between GOx and CAT are enhanced, leading to a super response to the change of LBG with insulin release and efficient clearance of harmful byproducts of GOx (H₂ O₂ ). In the treatment of diabetic mice, HmA@GCI reduces LBG to normal in half an hour and maintains for more than 5 days by a single subcutaneous injection, and nearly 24 days with four consecutive injections. During the test period, no symptoms of hypoglycemia and toxicity to tissues and organs are observed. These results indicate that HmA@GCI is a safe and long-acting hypoglycemic agent with prospective clinical application.

Keywords: diabetes; glucose-response; hexahistidine-metal-assembly; insulin delivery; pH sensitive.

Scheme 1

Schematically illustration of a) co‐encapsulating GOx, CAT, and Ins into HmA and b) rapid response to glucose and extended release of Ins in vivo for T1D treatment.

Figure 1

a) Hydrodynamic size distribution, and b) TEM images of HmA, HmA@Ins, and HmA@GCI. Scale bar, 200 nm. c) The entrapment efficiency (EE%) of Ins, GOx, and CAT into HmA.

Figure 2

a) pH changes over time of HmA@GCI solution incubated with various concentrations of glucose. b) Disintegration over incubated time of HmA@Ins (top) and HmA@GCI (bottom) in glucose (4 mg mL⁻¹). c) Hydrodynamic size distribution of HmA@GCI incubated with glucose solution (4 mg mL⁻¹) at various time. Cumulative insulin release from HmA@GCI (d) and HmA@Ins (e) at various concentrations of glucose. f) Pulsatile release profile of FITC‐Ins when exposed between a hyperglycemic and normal state alternatively.

Figure 3

Bioactivity assays of a) HmA@Ins, b) HmA@CAT, and c) HmA@GC and their native counterparts under protease K treatment or control conditions (no treatment of protease K). d) The scheme of biocatalytic cascades of GOx and CAT. e) The biocatalytic cascades of free GCI and HmA@GCI.

Figure 4

Hemolysis test of red blood cells at various concentrations of a) HmA@Ins and b) HmA@GCI for 4 h. c) Cell viability of dendritic cells (DCs) and d) NIH3T3 cells after co‐incubation with HmA at various concentrations for 24 h.

Figure 5

a) The retention of F‐Ins and HmA@GCI in vivo. b) Corresponding fluorescent content of injection site over time after injection. c) Level of blood glucose (LBG) in diabetic mice within the first 8 h. LBG in d) diabetic mice and e) healthy mice after subcutaneous injection with NS, free Ins, HmA@Ins, and HmA@GCI. f) Hypoglycemia index in different groups. g) Glucose tolerance test in healthy and diabetic mice. h) The responsiveness to intraperitoneal glucose tolerance test (IPGTT) was calculated based on the area under the curve from 0 to 2 h. i) LBG in diabetic mice after continuous injection with HmA@GCI.

Figure 6

a) Pictures of mice dorsum treated with subcutaneous injection of NS, HmA@GI, or HmA@GCI after different days. Red circles refer to injection sites. b) H&E stained sections of the subcutaneous injection site with surrounding tissues. Scale bar, 200 µm. c) The weight change of healthy mice and diabetic mice injected with NS and HmA@GCI. d) The water consumption of healthy mice and diabetic mice injected with NS and HmA@GCI. e–g) The blood routine examination of RBC, WBC, and PLT of healthy mice and diabetic mice injected with NS and HmA@GCI at the 24^th day. Blood biochemistry levels including h) AST, i) BUN after treatment at the 24^th day. j) Serum levels of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) in healthy mice and diabetic mice after injection with NS and HmA@GCI. l) H&E stained section of major organs after injected with NS, Ins, HmA@Ins, and HmA@GCI at the 24^th day, respectively. Scale bar, 100 µm.