(M0930-01-01) Brain-Targeted Oxytocin Nanoparticles Formulated via Microfluidics for Epilepsy

Purpose: Epilepsy is a central nervous system disorder that causes a sudden, uncontrolled electrical disturbance in the brain characterized by a change in behavior, feeling, and state of consciousness. The encapsulation of peptides in polymeric nanoparticles has become a crucial component of pharmaceutical research and development because of their biocompatibility, biodegradability, and sustained release properties. Oxytocin, a neuropeptide, has been identified to control seizures. However, its therapeutic potential has been limited by its large size, hydrophilicity, and short half-life that demands frequent dosing. To overcome these challenges, we optimized the synthesis of Bovine Serum Albumin (BSA) nanoparticles encapsulating Oxytocin using a microfluidics-assisted nanoprecipitation process. Additionally, these nanoparticles are conjugated to rabies virus glycoprotein (RVG) as targeting ligands for targeted brain delivery of the oxytocin nanoparticles.

Methods: In the optimization process, we used an albumin matrix to encapsulate Oxytocin into nanoparticles using a 5-input microfluidics chip by the nanoprecipitation method. It was then conjugated to a rabies virus glycoprotein (RVG). The final nanoparticle formulation was lyophilized, and the formulation parameters such as the effect of flow rate ratio, total flow rate, and polymer concentration on the size and polydispersity index of these particles were evaluated by Zetasizer based on the principle of dynamic light scattering (DLS). Scanning Electron Microscopy (SEM) was used to characterize the shape of the particles. Additionally, the effect of the binary organic solvent mixture polarity index was investigated by manipulating the ratio of acetone and ethanol in the organic phase. The peptide encapsulation efficiency and release profile were assessed using an oxytocin ELISA kit. Further, the cytotoxicity of the nanoparticles was investigated using a cell-based assay.

Results: The results show that the flow rate ratio, total flow rate, and polymer concentration affect these nanoparticles' size and polydispersity index. More importantly, we found that the sizes of the nanoparticles synthesized by this microfluidics system were between 57 nm-75 nm with a PDI less than 0.3. The optimum flow rate ratio of the aqueous phase to the organic phase was found to be 1:1.5. The smaller the flow rate ratio, the smaller the size. Moreover, the encapsulation efficiency of the Oxytocin in the BSA particles was improved > 80%. Interestingly, approximately 58% of Oxytocin was released from the nanoparticles within the first six hours, showing a very significant initial burst ideal for symptom control in epilepsy and exhibiting the Korsmeyer-Peppas release kinetics model. Furthermore,78% of Oxytocin was released over 7 days. By varying the percentage of acetone in the organic phase, there was no significant improvement in the size of the nanoparticles. The nanoparticles were also found to be non-cytotoxic in the cell-based assay.

Conclusion: The advantage of using the automated microfluidics-assisted nanoprecipitation is that it allows precision control over the process parameters in a reliable and reproducible manner. Additionally, using the microfluidics system, we achieved particles of sizes less than 100 nm, which are highly desirable for brain delivery. Overall, we successfully demonstrated the synthesis of less than 100 nm polymeric nanoparticles using the microfluidics system in a reliable and reproducible manner. This final optimized formulation would be investigated for its biological activities in well-characterized animal models.

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Acknowledgements:

Funding: The project is funded by an NIH grant 1R01NS2067601A1

Figure1-5. The effect of process parameters of the size of nanoparticles and cell-based non-cytotoxicity study

Figure 6-7. The in-vitro release profile of Oxytocin from the BSA nanoparticles

Table 1: Analysis of the R-square values and rate constants of release kinetics of BSA Nanoparticles containing Oxytocin fabricated by the microfluidics system.