Long-term glycemic control and prevention of diabetes complications in vivo using oleic acid-grafted-chitosan‑zinc-insulin complexes incorporated in thermosensitive copolymer

Abstract

Daily injections for basal insulin therapy are far from ideal resulting in hypo/hyperglycemic episodes associated with fatal complications in type-1 diabetes patients. Here we report a delivery system that provides controlled release of insulin closely mimicking physiological basal insulin requirement for an extended period following a single subcutaneous injection. Stability of insulin was significantly improved by formation of zinc-insulin hexamers, further stabilized by electrostatic complex formation with chitosan polymer. Insulin complexes were homogenously incorporated into PLA-PEG-PLA, a biodegradable thermogel copolymer, that instantaneously forms a subcutaneous gel-depot following injection. Chitosan polymer was hydrophobically modified using oleic acid prior to complex formation with insulin to enable distribution of oleic acid-grafted-chitosan‑zinc-insulin complexes into the hydrophobic core of PLA-PEG-PLA thermogel-copolymer micelles. In vivo, daily administration of marketed long-acting insulin, glargine, resulted in fluctuating blood glucose levels between 91 and 443 mg/dL in type 1 diabetic rats. However, single administration of thermogel copolymeric formulation successfully demonstrated slow diffusion of insulin complexes maintaining peak-free basal insulin level of 21 mU/L for 91 days. Sustained release of basal insulin also correlated with efficient glycemic control (blood glucose <120 mg/dL), prevention of diabetic ketoacidosis and absence of cataract development, unlike other treatment groups. Moreover, there was no sign of inflammation, tissue damage, or collagen deposition around depot site, suggesting exceptional biocompatibility of the formulation for long-term use.

Keywords: Basal insulin; Controlled release; Glycemic control; Hydrophobic modification; Prevention of diabetes complications; Protein stability; Thermosensitive copolymer.

Figure 1.

Differential scanning calorimetry thermogram of insulin, zinc-insulin hexamers and chitosan-zinc-insulin complexes prepared using oleic acid-grafted chitosan polymers. [Insulin: 1 mg/mL; Zinc ions: insulin hexamer (1:5); CSO monomer unit: insulin monomer (5:1); in phosphate buffered saline (10 mM, pH 7.4)]

Figure 2.

Integrated heats of interaction from calorimetric titrations of oleic acid grafted chitosan polymer (titrant) into zinc-insulin hexamers (titrand). [2 mg/mL, ~0.057 mM zinc-insulin hexamers; 1.2 mg/mL, CSO polymer solution; in phosphate buffered saline (10 mM, pH 7.4)] Data are expressed as mean ± S.D, n=3.

Figure 3.

Effect of hydrophobic modification on chitosan oligosaccharide (CSO, 5 kDa) on in vitro release of insulin from 35% (w/v) PLA₁₅₀₀-PEG₁₅₀₀-PLA₁₅₀₀ copolymer, drug loading: 0.15% (w/v). Data is expressed as mean ± SD, n=4. [Key: (●) free insulin, (◆) zinc-insulin hexamers, (▲) CSO-zinc-insulin complex, (■) oleic acid_(45%)-grafted-CSO-zinc-insulin complex; *: significantly lower compared to zinc-insulin hexamers; #: significantly lower compared to CSO-zinc-insulin complex; at p < 0.05]

Figure 4.

(A) Near-UV circular dichroism spectrum, (B) Far-UV circular dichroism spectrum, (C) Nano-differential scanning calorimetry fitted thermogram, and (D) Reversed phase high performance liquid chromatography, of insulin released in vitro at 1, 30, 60 and 90 days from OA-g-CSO-zinc-insulin complexes incorporated in thermosensitive copolymer formulation at 37 °C. [Key: green - standard solution, pink - one day, red - 30 days, blue - 60 days, and purple - 90 days, on stability analysis at 37 °C.]

Figure 5.

(A) Near-UV circular dichroism spectrum, (B) Far-UV circular dichroism spectrum, (C) Nano-differential scanning calorimetry fitted thermogram, and (D) Reversed phase high performance liquid chromatography, of insulin extracted from OA-g-CSO-zinc-insulin complexes incorporated in thermosensitive copolymer formulation after 1, 3, 6 and 9 months of storage at 4 °C. [Key: green - standard solution, pink - one month, red - 3 months, blue - 6 months, and purple - 9 months, on stability analysis at 4 °C.]

Figure 6.

Graphical representation of percent relative cell viability after 24, 48 and 72 h at different dilutions of copolymer degradation products incubated with (A) 3T3–L1 fibroblast cell line (B) HEK 293 cell line. Thermosensitive copolymer PLA-PEG-PLA incorporating oleic acid-g-CSO polymer was incubated with PBS for 10 days at 37 °C to extract the copolymer, CSO polymer and their degradation products. Biocompatibility was evaluated using MTT assay. Positive control (formalin) showed ≤ 1.0 ± 0.2 % relative cell viability at the highest dilution (1:16) in cell culture medium, and negligible cell survival at lowest dilution (1:1) in cell culture medium for both 3T3–L1 and HEK 293 cell lines. Data expressed as mean ± SD, n = 4.

Figure 7.

Bright field micrographs of H & E stained rat injection site skin tissue after (A) no injection (negative control), (B) 5% (v/v) Formalin (positive control), (C – F) 1, 7, 30 and 90 days’ post administration of Saline, (G – J) 1, 7, 30 and 90 days’ post administration of OA-g-CSO incorporated in thermosensitive copolymer formulation. Bright field micrographs of Gomori’s trichome stained rat injection site skin tissue after (K) no injection, and (L) 30 days, (M) 90 days, post administration of OA-g-CSO incorporated in thermosensitive copolymer formulation.

Figure 8.

Serum insulin concentration and blood glucose level of STZ-induced diabetic rats upon treatment with (A) single administration of recombinant human insulin and (B) daily administration of insulin glargine (Lantus® U-100). [Data are expressed as mean ± S.D, n=6; arrows mark administration of glargine; insulin dose: 0.5 IU/kg/day]

Figure 9.

Serum insulin concentration and blood glucose level of STZ-induced diabetic rats upon insulin treatment. Key: Treatment with single administration of: (●) free insulin in thermosensitive copolymer, dose 45 IU/kg; (▲) CSO-zinc-insulin complex in thermosensitive copolymer, dose 45 IU/kg; or (■) oleic acid_(45%)-grafted-CSO-zinc-insulin complex in thermosensitive copolymer, dose 45 IU/kg; (■) untreated STZ-induced diabetic control; (●) healthy non-diabetic control. [Data are expressed as mean ± S.D, n=6; *: significantly different compared to glargine treated control; #: significantly different compared to CSO-zinc-insulin complex; at p < 0.05]

Figure 10.

Body weight of rats following STZ and insulin treatment. Key: (●) healthy non-diabetic control; (■) untreated STZ-induced diabetic control; STZ-induced diabetic rats upon treatment with single administration of: (●) insulin solution, dose 0.5 IU/kg; (●) free insulin in thermosensitive copolymer, dose 45 IU/kg; (▲) CSO-zinc-insulin complex in thermosensitive copolymer, dose 45 IU/kg; and (■) oleic acid_(45%)-grafted-CSO-zinc-insulin complex in thermosensitive copolymer, dose 45 IU/kg; or (◆) daily administration of glargine (Lantus® U-100), dose 0.5 IU/kg/day. [Data are expressed as mean ± S.D, n=6; *: significantly higher compared to untreated STZ-induced diabetic control; at p < 0.01]

Figure 11.

(A) Blood ketone level in rats following STZ and insulin treatment; (B) Clear eye lens following treatment with oleic acid_(45%)-grafted-CSO-zinc-insulin complex in thermosensitive copolymer; (C) Partial, or (D) total, cataract formation in untreated STZ-induced diabetic control; (E) Detection of anti-insulin antibodies following insulin treatment. Key: (●) healthy non-diabetic control; (■) untreated STZ-induced diabetic control; STZ-induced diabetic rats upon treatment with single administration of: (●) insulin solution, dose 0.5 IU/kg; (●) free insulin in thermosensitive copolymer, dose 45 IU/kg; (■) CSO-zinc-insulin complex in thermosensitive copolymer, dose 45 IU/kg; and (▲) oleic acid_(45%)-grafted-CSO-zinc-insulin complex in thermosensitive copolymer, dose 45 IU/kg; or (◆) daily administration of glargine (Lantus® U-100), dose 0.5 IU/kg/day. [Data are expressed as mean ± S.D, n=6; *: significantly higher compared to healthy non-diabetic control; at p < 0.05]