Purpose: Insulin is a vital hormone which mediates glucose transport from bloodstream into the cells for energy production. Type 1 diabetes is a disorder where autoimmune mechanisms cause slow and progressive destruction of body’s own healthy insulin-producing pancreatic β-cells. Consequently, people suffering from this disorder need exogenous insulin therapy for maintaining their blood glucose levels either by taking multiple insulin injections per day or through an insulin pump whilst checking their blood glucose multiple times a day. In a healthy human, other than large amount of insulin secretion following meals, insulin is secreted at basal level (0.5 – 1 U/h) between meals and throughout the night. This slow and constant release of insulin at basal level ensures a controlled glucose output from liver maintaining a steady energy source to cells. None of the currently available strategies of insulin delivery are able to deliver basal level insulin at a controlled rate throughout the day. Repeated administration of exogenous insulin expose the body to chronic high blood glucose which causes serious damage to nerves, blood vessels and organs resulting in macrovascular and microvascular complications concomitant to diabetes. In this study, triblock thermosensitive copolymer poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PLA) was explored to deliver insulin at basal rate up to 2 months following a single subcutaneous injection. Release of drug incorporated in thermosensitive copolymeric matrix is effected by a combination of diffusion from the copolymer matrix and slow hydrolytic degradation of the copolymer. Several parameters such as copolymer concentration, concentration of drug, size of drug molecule, and hydrophobicity of drug/copolymer may affect release rate from such controlled drug delivery systems. In this study we evaluated effect of different copolymer concentration and varied drug loading on the release profile of insulin. Distinctive properties of insulin to self-associate in the presence of zinc and to form polyelectrolyte complex with cationic chitosan were also explored.
Methods: Thermosensitive copolymer PLA-PEG-PLA was synthesized by ring opening polymerization method. 1H and 13C nuclear magnetic resonance (NMR) spectrometry and gel permeation chromatography (GPC) were used to determine the structure, number average molecular weight, and molecular weight distribution (polydispersity index, PDI) of the copolymer. Increasing concentration of aqueous copolymer solution were examined for their phase transition temperature and injectability by tube inversion method and passing through 25G needle, respectively. Insulin was modified using zinc to form zinc-insulin hexamers. At physiological pH zinc-insulin hexamers are negatively charged which form polyelectrolyte complex with positively charged chitosan polymers. Release profile of chitosan-zinc-insulin complex incorporated in thermosensitive copolymer was evaluated at increasing copolymer concentration (20, 25, and 30% w/v). Different loading doses of insulin selected to meet the basal insulin requirement (0.5—1 U/h corresponding to 0.5—1 mg/day) for ~2 months were also tested. Kinetics of drug release were determined by calculating correlation coefficients for zero and first order release rate.
Results: Thermosensitive copolymer PLA-PEG-PLA was successfully synthesized. Structural composition of the synthesized copolymer was confirmed using 1H and 13C NMR. Number average molecular weight was calculated to be 5107.77 Da and molecular weight distribution of copolymer was found to be uniform with PDI ~1.14. Sol-gel transition temperature was found to be in the range 18 — 26 °C, increasing with decreasing copolymer concentration. Copolymer concentrations were found to be injectable through a 25G needle in the concentration range 10 — 35% (w/v). Rate of insulin release was modulated by changing thermosensitive copolymer concentration. Slower release rate was observed with higher initial copolymer concentration, owing to tighter copolymer-copolymer contacts among the gel matrix at higher concentration of the copolymer (Figure 1). Furthermore, rate of release of insulin increased with increase in initial loading dose of insulin corresponding to meet basal insulin requirement at 0.5 or 1.0 U/h for ~2 months (Figure 2). All formulations followed zero order kinetics with correlation coefficient (R2) value ranging between 0.93—0.97, implying constant release rate of insulin over a period of time (Table 1).
Conclusion: Copolymer concentration and loading dose of drug are both essential parameters in controlling the rate of release of incorporated therapeutic from a thermosensitive copolymeric depot based delivery system. Studying effect of formulation parameters on the release profile of incorporated drug provides a deeper understanding of the delivery system. Results from this study demonstrate that insulin delivery at a controlled rate can potentially be modulated by modifying essential formulation parameters such as copolymer concentration and amount of insulin loaded into the copolymer. Furthermore, the results exemplify a chitosan-zinc-insulin complex incorporated into a thermosensitive copolymeric system as a superior and cheaper alternative to the conventional daily basal insulin therapy. This research was supported by the National Institutes of Health (NIH) grant# R15GM114701.