(T1230-01-05) Polymeric Micelles for Nose-to-Brain Delivery of Rapamycin

Purpose: Alzheimer’s disease is a complex disorder that significantly impairs the patient's memory, behavior, and emotions. The effect of the conventional therapies for treating Alzheimer’s disease is limited due to the difficulties in penetrating the Blood Brain Barrier (BBB). Rapamycin is an mTOR inhibitor used to treat renal transplant patients. In the last few years, it has been reported that rapamycin can function as an anti-aging agent and/or a drug candidate for improving cognitive functions for patients with Alzheimer’s diseases. Yet, it is controversial as long-term treatment with rapamycin appears to cause off-target consequences such as fatal thrombocytopenia and hyperlipidemia. To overcome the aforementioned limitations, the present study aimed to formulate and characterize brain-targeted polymeric micelles for nose-to-brain delivery of rapamycin.

Methods: Rapamycin-loaded polymeric micelle was formulated using a solvent evaporation technique. Briefly, N-hydroxysuccinimide ester-poly (ethylene glycol-block-poly(DL-lactide) (NHS ester PEG-b-PLA) and rapamycin were dissolved in an organic solvent and an aqueous buffer at pH 8.4 was added dropwise with continuous stirring.  The organic solvent was evaporated, and fibronectin CS1 peptide was incoporated to develop a tumor-targeted polymeric micelles carrying rapamycin Optimized formulations were then evaluated on particle size distribution, peptide conjugation, % entrapment efficiency, in-vitro drug release, cell viability, nasal cell permeability, and neuroinflammation.

Results: Optimized formulation were composed of a 1:10 ratio of drug:polymer, which showed c.a. 96.40% of entrapment efficiency and c.a. 35.40% of drug loading.The average particle size of brain-targeted micelles carrying rapamycin was 81±0.32 nm with 0.31±0.01 PDI. The NMR result demonstrated that fibronectin CS1 peptide was successfully conjugated to PEG-b-PLA micelles via NHS ester-amine reaction.   (Fig. 1A). An in vitro drug release study demonstrated c.a. 82% of rapamycin was release from the micelle in 24h. There was a burst release in the first 30 min, which released c.a. 35% of the loaded rapamycin. (Fig 1B). Polymeric micelle (12.5 and 25 µM) treated on microglia and astrocyte cells did not cause any notable cytotoxic effect as compared to free rapamycin and negative control, after 48 h post-treatment (Figs. 1C and D). Nasal permeability study demonstrated that brain-targeted micelles improved the permeability of rapamymycin (19.78 ± 0.22µg/cm²) compared to the permeability of free rapamycin (11.27 ± 0.12 µg/cm²) (Figs. 2A-F). A neuroinflammation study showed that microglia cells had low levels of CD11b antibodies compared to the lipopolysaccharide (LPS) treated microglia cells. Treatment with micelles formulation (12.5 and 25µM) on LPS-treated cells resulted in a notable decrease in CD11b expression compared to the LPS-treated cells, which is indicative of the reduction of neuroinflammation. (Fig. 2G)

Conclusion: We successfully formulated polymeric micelle for effective intranasal delivery of rapamycin for potentially treating Alzheimer’s disease. Future studies involve behavior studies for testing cognitive functions in vivo and the evaluation of molecular markers expressed on brain tissues ex vivo.


Figure 1. Physicochemical characterizations A) NMR, B) Rapamycin release profile, C) Cell viability on microglia cells, and D) Cell viability on astrocyte cells.


Figure 2.
A) Transepithelial electrical resistance (TEER) value, B) Cumulative permeation of Rapamycin (11 g/mL), C) Cumulative permeation of rapamycin (23 g/mL), D) Apparent permeability of rapamycin (12.5 g/mL), E) Apparent permeability of Rapamycin (25 g/mL), F) Summary of rapamycin permeation across the nasal epithelial cells, G) Neuroinflammation in microglia cells (LPS-treated cells vs. LPS-treated cells incubated with polymeric micelles carrying rapamycin).