(M1230-03-15) Biotin-Targeted Pluronic/Bile Salt Mixed Micelles for Enhanced Bioavailability of Therapeutic peptide

Purpose: The primary objective of this study was to synthesize and characterize biotin-targeted pluronic/bile salt mixed micelles aimed at enhancing the bioavailability of insulin. Given the challenges associated with traditional insulin administration methods, particularly in overcoming gastrointestinal barriers, this research focused on leveraging the unique absorption properties of biotin through sodium-dependent multivitamin transporters (SMVT).

Methods: In this study, biotinized pluronic (F127) mixed micelles were synthesized using a thin-film hydration method and optimized under various environmental conditions. The resulting micellar formulations underwent several characterizations, including assessments of particle size, polydispersity index (PDI), and entrapment efficiency (%EE) via dynamic light scattering and zeta potential analysis. Micelles were evaluated for their morphology using Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). In vitro release profiles were measured under gastrointestinal conditions, and the ex vivo permeability of insulin across Caco-2 cell monolayers was assessed. Cell uptake studies were performed using FITC-labeled insulin to evaluate the internalization efficacy of micelles. In addition, in vivo pharmacokinetic & pharmacodynamic studies were conducted to evaluate the therapeutic potential of these formulations.

Results: The synthesis of biotin-targeted micelles resulted in optimal particle size and polydispersity, with results indicating that the formulations exhibited favorable dimensions for intestinal uptake. The particle size of the developed micelles ranged between 200 to 300 nm, with a PDI below 0.2, demonstrating their uniformity and stability. Importantly, the %EE of insulin significantly improved when formulated as a phospholipid complex, addressing the inherent challenges of loading hydrophilic molecules within the micelle structure. In in vitro release studies, biotinized formulations demonstrated a controlled and sustained release profile. After 2 hours in simulated gastric fluid, ~10% of insulin was released, while ~20% was released after 6 hours in intestinal fluid. In stark contrast, free insulin exhibited a complete release (~100%) within 6 hours. The slower release kinetics from the biotin-targeted micelles suggest a potential for prolonged therapeutic action, contributing to more stable blood glucose levels. Ex vivo permeability studies revealed that incorporating insulin into the biotin-targeted nanocarriers resulted in a remarkable fourfold increase in intestinal permeability compared to native insulin. This substantial enhancement underscores the efficacy of using biotin to facilitate transport via SMVT. Moreover, cell uptake studies utilizing FITC-labeled insulin indicated a significant increase in fluorescence intensity within Caco-2 cells following 2 hours of incubation with the biotin-targeted micelles. This increase in cellular uptake was approximately 4.5 times greater than that of free insulin. The stability of the formulations was also assessed through storage experiments at various temperatures, revealing that both biotinized mixed micelles remained stable for over six months when stored at 4 °C. Dynamic light scattering measurements showed minimal changes in size and PDI under different storage conditions, suggesting that these micellar systems possess promising stability characteristics for long-term therapeutic use. Furthermore, morphologically, the micelles adopted a spherical structure, as evidenced by SEM and TEM imaging, supporting their feasibility as drug carriers. The rough surface morphology of the insulin-phospholipid complexes indicated their ability to interact effectively with surrounding media, enhancing the potential for drug loading and release. In vivo studies were conducted using a diabetic animal model. Serum insulin levels were monitored following the oral administration of the biotin formulations versus traditional insulin injections. The areas under the curve (AUC) calculations showed a notable increase in the bioavailability of insulin when delivered via the biotin micelles, suggesting a sustained pharmacological effect over time.The targeted mixed micelles revealed ~1.5 folds increment in AUC compared to plain oral insulin solution and ~1.2 folds increment in comparison with subcutaneous injection. Thus, the better hypoglycemic response observed in case of prepared formulations could be ascribed to the combined effect of better protection of IN in micellar systems and enhanced uptake though SMV transporters.

Conclusion: The study demonstrates that biotin-targeted pluronic/bile salt mixed micelles offer a promising new strategy for the oral delivery of insulin, significantly improving its bioavailability and absorption efficiency. The optimization of particle size, enhanced entrapment efficiency, and sustained release profiles all contribute to the enhanced therapeutic efficacy observed in this formulation. The specific interaction with SMVT transporters identified in Caco-2 cell studies indicates a targeted approach that could lead to better management of diabetes.

Acknowledgements: Authors are thankful to NIPER, SAS Nagar, Mohali for providing necessary infrastructure. Authors would thankful to Rahul Mahajan and Mahesh Kashyap for technical assistance. Madhunika Agrawal and Bhargavi Nallamothu are thankful to DHR, India and Lady TM Trust, India for fellowship. Conflict of Interest Statement: All authors declare that there is no conflict of interest. Institutional Animal Ethics Committee duly approved all animal protocols (Protocol number: IAEC/19/56)


Fig 1. Aims and Objectives


Fig 2. Stability studies and Kinetic release


Fig 3. invitro and invivo studies