Helical‐Like Assembly of Nateglinide as Coating for Oral Delivery of Insulin and Their Synergistic Prevention of Diabetes Mellitus

Abstract Oral delivery of antidiabetic active components promises to free millions of people from daily suffering who require routine injections. However, oral insulin (Ins) and other short‐acting compounds such as nateglinide (NG) in harsh gastrointestinal tract still face great challenging, including low bioavailability, and rapid elimination. In this study, inspired by the self‐assembly of phenylalanine‐based peptides in nature, it is showed that NG a small phenylalanine derivative, assembles into left‐handed helical nanofibers in the presence of Ca2+. These helical NG nanofibers functioned as a coating layer on the surface of Ca2+‐linked alginate (Alg) microgels for the effective encapsulation of Ins. As expected, the sustained release and prolonged circulation of Ins and NG from the Ins‐loading Alg/NG microgels (Ins@Alg/NG) in the intestinal tract synergistically maintain a relatively normal blood glucose level in streptozotocin‐induced diabetic mice after oral administration of Ins@Alg/NG. This further confirms that Ins@Alg/NG ameliorated Ins resistance mainly through activating Insreceptor substrate 1 (IRS1), protein kinase B (AKT), and AMP‐activated protein kinase (AMPK), as well as by repressing glycogen synthase kinase‐3β (GSK‐3β). The strategy of using the assembly of NG as a coating achieves the oral delivery of insulin and showcases a potential for the treatment of diabetes.

crushed and then dissolved in methanol, 0.45 μm microporous membrane was used for ultrasonic filtration.the content of Ins and NG were determined by 1260 high performance liquid chromatography (HPLC) (Agilent Technologies Inc., California, USA).For Ins, the detection wavelength was 214 nm, the mobile phase consisted of a mixture of acetonitrile and ultrapure water (containing 0.1% phosphate) (30: 70, v/v), flow rate was 0.8 mL/min and retention time was 15 min.For NG, the detection wavelength was 210 nm, the mobile phase consisted of a mixture of acetonitrile and ultrapure water (containing 0.1% phosphate) (54: 46, v/v), flow rate was 0.8 mL/min and retention time was 15 min.Based on the standard concentration and peak area, the sample content was calculated.Encapsulation ratio (EE) and drug loading (DL) were calculated as follows: [1]

DL (%) =
The weight of Ins/NG in microspheres The weight of microspheres × 100% (2) Rheological testing: The rheological properties of each sample were measured by the rheometer (DHR-1, TA, USA).The prepared gel was equilibrated at room temperature for 2 hours.PP25 plate was selected with a gap of 1 mm, and rheological measurement was performed at 25 ℃.To test the oscillatory shear rheology, appropriate amount of NG gel was used and the dynamic sweep frequency was measured at 0.01% strain, and the frequency was between 0.01 Hz and 100 Hz.The rheological behavior of NG gel was studied by temperature scanning test between 25 ℃ to 65 ℃, at 10 Hz.Dynamic frequency scanning was used to further detect the viscoelastic properties of the gel, and the linear viscoelastic region was selected from the angular frequency range of 0.1 to Hemolysis analysis: Blood samples from mice were added to NaCl solution and were centrifuged at 1500 rpm for 10 min to obtain red blood cells (RBCs).After washed with NaCl solution, the RBCs were diluted to 1/50 of their volume with PBS solution (pH 7.2).The samples were divided into following groups: (1) Positive control group: purified water; (2) Negative control group: PBS; (3) Ins@Alg/NG: 0.5 mg/mL; (4) Ins@Alg/NG: 1.0 mg/mL; (5) Ins@Alg/NG: 1.5 mg/mL.The diluted red blood cell suspension was mixed evenly with the above samples and incubated at 37 ℃ for 3 hours.
After centrifugation (1500 rpm for 10 min) at room temperature, the absorbance of the supernatant at 541 nm was determined by microplate analyzer.
Particle size analysis of Ins@Alg and Ins@Alg/NG: Microspheres were observed by light microscopy (Leica DM2500, Leica Microsystems GmbH, Wetzlar, Germany).
Particle size analysis of microspheres (n>100) were performed using Image J software and standard deviation calculated.
Cytotoxicity testing of Ins@Alg and Ins@Alg/NG: The cytotoxicity of Ins@Alg and Ins@Alg/NG to human normal liver cells (L-02) was determined.L-02 cells were inoculated in 96-well plates (density 10 5 μL/ well) and cultured in an incubator for 24 hours.After cell adhesion, the cells were treated with different concentrations of Ins@Alg and Ins@Alg/NG (0, 5, 10 and 20 μmol/mL).After treated for 24 hours, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added and the mixtures were incubated at 37 ℃ for 4 hours.The culture medium was removed and 200 μL DMSO was added into each well, and the absorbance was determined at 490 nm using a microplate reader.
Cytotoxicity testing of PA: The cytotoxicity of palmitic acid (PA) to L-02 cells was determined.L-02 cells were inoculated in 96-well plates (density 10 5 μL/ well) and cultured in an incubator for 24 hours.After cell adhesion, the cells were treated with different concentrations of PA (0, 5, 10 ,15, 20, 25, 30, 35 and 40 μmol/mL) for 24 hours.After treated for 24 hours, MTT was added and the mixtures were incubated at 37 ℃ for 4 hours.The culture medium was removed and 200 μL DMSO was added into each well, and the absorbance was determined at 490 nm using a microplate reader.
Ins@Alg/NG increases glucose consumption by L-02 cells: L-02 cells were inoculated in 12-well plates and cultured in incubators for 24 hours，then, cells were treated with PA (concentration of 30 μmol/mL).After cultured for 24 hours, Ins@Alg and Ins@Alg/NG (concentration of 20 μmol/mL) were added into the culture.At 24 and 48 hours, MTT was added and the mixtures were incubated at 37 ℃ for 4 hours.The culture medium was removed and 200 μL DMSO was added into each well, and the absorbance was determined at 490 nm using a microplate reader.
Apoptosis analysis of Ins@Alg/NG: The protective effect of Ins@Alg/NG on apoptosis of L-02 cells induced by PA was determined.L-02 cells were inoculated into 6-well plates (density 10 5 μL/ well) and cultured in an incubator for 24 hours.After cell adhesion, Ins@Alg and Ins@Alg/NG (20 μmol/mL) were added and incubated for 24 hours.After 24 hours, PA (30 mg/mL) was added, and the culture continued for 24 hours.
The fluorescence staining was continued according to the instructions of the Caspase-3 kit, and incubated at 37 ℃ for 30 min.The expression of apoptotic cells was observed under inverted fluorescence.

Figure S5 .
Figure S5.Overlap the plot of AA and CG Connolly surfaces of deprotonated NGs (AA

Figure S7 .Figure S8 .Figure S9 .
Figure S7.Preparation and characterization of Alg and Alg/NG.a,b) Particle size distribution of Alg and Alg/NG.c) Microscopic images of Alg and Alg/NG.

Figure S14 .
Figure S14.Biocompatibility test of Ins@Alg/NG.Data were presented as mean ±

Figure S15 .
Figure S15.H&E staining of main organs after treated with Ins@Alg/NG in diabetic

Figure S16 .
Figure S16.Masson staining of main organs after treated with Ins@Alg/NG in diabetic mice.