Fig. 4. In vivo evaluation of insulin complex for type 1 diabetic mice treatment.

(A) BGLs of type 1 diabetic mice treated with subcutaneously injected native insulin, F-insulin, or B-insulin. PBS was used as a control. The insulin-equivalent dose was 80 U/kg. Data points are means ± SD (n = 5). Statistical significance between groups treated with F-insulin and native insulin was calculated. (B) Normoglycemic time of mice treated with subcutaneously injected F-insulin, B-insulin, and native insulin. Statistical significance was calculated. Data points are means ± SD (n = 5). (C) Live imaging of subcutaneously injected F-insulin and native insulin labeled with sulfo-Cy5. (D) IPGTT in diabetic mice at 3 hours after treatment of F-insulin or native insulin. Glucose dose was set to 1.5 g/kg. Healthy mice were used as control. Data points are means ± SD (n = 5). Statistical significance between F-insulin– and native insulin–treated groups was calculated. (E) Responsiveness to IPGTT in terms of the area under the curve (AUC) in 150 min, with the baseline set at the 0-min blood glucose reading. Data points are means ± SD (n = 5). Statistical significance between groups was calculated. (F) In vivo glucose-responsive insulin release triggered by intraperitoneal glucose injection at 4 hours after treatment of F-insulin at a dose of 80 U/kg. Glucose dose was set to 2 g/kg. Data points are means ± SD (n = 4 to 5). Statistical significance between the plasma insulin levels at various time points after glucose challenge and that at 0 min was calculated. Inset: Representative image of the resultant light yellow solution associated with enzyme-linked immunosorbent assay (ELISA) measurement. Plasma collected at 0, 10, 20, 30, 40, 60, 90, and 120 min after treatment of F-insulin was added to each well one by one from left to right. 5,5′-Tetramethylbenzidine (TMB) was used as the substrate of horseradish peroxidase, and the addition of sulfuric acid (0.6 N) turned the blue product of TMB to light yellow. (G) Representative scanning electron microscopy image of the MN arrays. Inset: Representative fluorescence image of the MNs loaded with F-insulin. Insulin was labeled with rhodamine B. Scale bar, 300 μm. (H) BGLs of type 1 diabetic mice treated with MN array patches loaded with insulin (MN-insulin) or F-insulin (MN–F-insulin) (2 mg of insulin per patch). Blank MN loaded with PBS was used as a negative control. One patch was applied to one mouse. Statistical significance between the groups treated with MN-insulin and MN–F-insulin was calculated. Data points are means ± SD (n = 5). (I) In vivo glucose-responsive insulin release triggered by intraperitoneal glucose injection at 3 hours after treatment of MN array patch load with F-insulin. Each mouse was treated with one patch containing 2 mg of insulin encapsulated in the complex. Glucose was given at 2 g/kg. Data points are means ± SD. (n = 4). Statistical significance between the plasma insulin levels at various time points after glucose challenge and that at 0 min was calculated. All the statistical analyses were performed by one-way analysis of variance (ANOVA) with a Tukey post hoc test or Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.