Vitamin B12 functionalized layer by layer calcium phosphate nanoparticles: A mucoadhesive and pH responsive carrier for improved oral delivery of insulin.

The present study investigates the potential of layer by layer coated calcium phosphate nanoparticles - for oral delivery of insulin where Vitamin B12 grafted chitosan and sodium alginate have been used as cationic and anionic polyelectrolyte respectively. The major emphasis has been given on the role Vitamin B12 conjugated chitosan as cationic polyelectrolyte (VitB12-Chi) in the delivery system. VitB12-Chi conjugate was prepared by carbodiimide reaction. The formulated VirB12-Chi-CPNPs were tested for in vitro and in vivo efficacy studies carried out in Caco-2 monolayers and diabetic rats. VitB12-Chi-CPNPs with particle size <250nm and zeta potential+32.56(±2.34) exhibited pH responsive insulin release at simulated gastric fluid and simulated intestinal fluid. Fluorescence microscopy and flow cytometry studies revealed higher uptake of VitB12-Chi-CPNPs in Caco-2 monolayer in comparison to Chi-CPNPs. Further reduction in TEER supported paracellular transport of insulin because of opening of tight epithelial junctions. In vivo intestinal uptake of FITC tagged Vit-B12-Chi-CPNPs from different intestinal segments supported paracellular and receptor mediated uptake of VitB12-Chi-CPNPs. Plasma insulin and blood glucose levels were measured in diabetic rats and showed about four fold increases in insulin bioavailability and sustained hypoglycemic effects up to 12h of administration with VitB12-Chi-CPNPs in comparison to Chi-CPNPs. Results of the study revealed the potential of layer by layer nanoparticles for oral insulin delivery. The study also specifically highlighted the role of VitB12 as a pH sensitive and targeting ligand which significantly participated in enhancing insulin oral bioavailability. STATEMENT OF SIGNIFICANCE: Oral delivery of insulin is always the most desirable approach for diabetic patients however it's also the most challenging in respect to formulation development due to harsh gastrointestinal conditions. Several groups have been working from decades for oral delivery of insulin. However the beauty of this prototype formulation is that it exhibits the pH responsive behavior in natural condition of gastrointestinal tract. It resists the release of insulin at gastric condition however stimulate the release at intestinal conditions. Apart from pH responsive behavior it utilizes multiple pathways to improve the overall bioavailability of insulin including paracellular transport and receptor mediated endocytosis.