Enhancing Nose-to-Brain Transport of HEX via Nebulized PEG-b-PLA Polymersomes

Introduction: :

Introduction: The blood-brain barrier (BBB) plays a key role in maintaining homeostasis and preventing foreign pathogens from entering the brain. However, certain pathogens have developed mechanisms to bypass the BBB and infect the central nervous system (CNS) through the olfactory and trigeminal nerves. The BBB is a substantial hurdle to treat CNS infections due to it preventing ~98% of small molecule drugs from entering the brain1. One such pathogen is Naegleria fowleri (Nf) which is the causative agent of pathogenic amoeba-associated meningoencephalitis (PAM). PAM poses a dire threat with a mortality rate exceeding 97%. Recent research highlights enolase, an enzyme involved in glycolysis, as a potential treatment target. Enolase inhibitors, such as HEX, have demonstrated promise in ENO-1 deleted cancers2 and due to the enolase homology, also show promise as a therapeutic agent against Nf3. However, HEX suffers from a short half-life, complicating its therapeutic potential. Our laboratory aims to extend half-life by using polyethylene glycol-b-poly lactic acid (PEG-b-PLA) polymersomes to encapsulate HEX and facilitate transport across the Nose-to-Brain (N2B) pathway. N2B can increase on-target delivery to the brain but is plagued by small absorption areas and difficult-to-penetrate nasal mucosa. To facilitate N2B targeting, we are examining the viability of delivering HEX-loaded PEG-b-PLA polymersomes (HEX-PPs) via nebulization, enabling consistent and direct delivery to the olfactory tract. Using nebulization, we are examining the characteristics and viability of intranasal delivery of HEX-PPs compared to intranasal droplets as a potential therapy for PAM.

Materials and

Methods: : PEG-b-PLA polymersomes have been explored by our laboratory with great success for applications in drug delivery. HEX was synthesized by and received from Dr. Dan Whitehead (Clemson University). Polymersomes were synthesized using standard techniques4. HEX was neutralized by progressively adding 1M NaOH until reaching a pH of 7. HEX-PPs were loaded by vortexing PEG-b-PLA polymersomes with neutralized HEX. Nf was cultured under sterile conditions, then incubated with varying concentration of HEX and HEX-PPs for 48 hours. Unloaded and HEX-PPs underwent nebulization using both standard and small Aeroneb lab nebulizers. Diameter changes before and after nebulization and loading were measured using dynamic light scattering (DLS). Relative humidity was evaluated using data reported from local weather services. Encapsulation efficiency was calculated by loading polymersomes with Cy5. Free Cy5 was removed from the samples by 100kDa dialysis tubes in MilliQ water. To quantify encapsulated Cy5, a sample of each wash was measured for fluorescence using a plate reader at 645 nm. Cell viability was quantified by treating SHSY5Y cells with free HEX and HEX-PPs then measuring viability using a CellTiter 96 aqueous one solution cell proliferation assay.

Results, Conclusions, and Discussions:: The polydispersity index (PDI) for non-nebulized unloaded polymersomes and nebulized polymersomes was 0.405 ± 0.192 and 0.476 ± 0.158 respectively which suggests that both groups were relatively monodispersed. However, size varied between the two groups with sizes with average sizes of 136.5 ± 36.1 nm and 412.25 ± 214.8 nm, respectively, indicating that size was affected by the nebulization process. Similarly, non-nebulized and loaded polymersomes had PDIs of 0.436 ± 0.172 and 0.442 ± 0.143 respectively, indicating that their populations were monodispersed. However, size greatly differed between non-nebulized and nebulized HEX-PPs with sizes of 138.9 ± 28.8 and 1040.7 ± 2478.9 nm respectively. Additionally, humidity did not affect particle size or PDI before or after nebulization whether loaded or unloaded (Figures 1 & 2). Cy5 dye was encapsulated at an efficiency of 90.2 % ± 0.301 pre-nebulization and 88.5% ± 0.426 post nebulization indicating minimal drug loss during the process despite the increase in size. HEX-PPs also inhibit Nf growth as effectively as free HEX with EC50 values of 0.278 ± 0.022 µM and 0.855 ± 0.076 µM respectively (Figure 3). PEG-b-PLA polymersomes had a limited impact on SHSY5Y cell viability, indicating that they are biocompatible for intranasal administration.

PEG-PLA polymersomes are a promising vehicle for enabling the treatment of PAM. Polymersomes also remain monodispersed following nebulization. Additionally, polymersomes were not impacted by humidity during nebulization. The ability to nebulize HEX-PPs with minimal drug loss and maintained stability supports the feasibility of developing intranasal therapies for treating Nf infections leading to PAM.

Acknowledgements and/or References (Optional):: 1. Larsen, J. M., Martin, D. R. & Byrne, M. E. Recent Advances in Delivery through the Blood-Brain Barrier. Curr Top Med Chem 1148–1160 (2014).
2. Lin, Y. H., Satani, N., Hammoudi, N., Yan, V. C., Barekatain, Y., Khadka, S., Ackroyd, J. J., Georgiou, D. K., Pham, C. D., Arthur, K., Maxwell, D., Peng, Z., Leonard, P. G., Czako, B., Pisaneschi, F., Mandal, P., Sun, Y., Zielinski, R., Pando, S. C., Wang, X., Tran, T., Xu, Q., Wu, Q., Jiang, Y., Kang, Z., Asara, J. M., Priebe, W., Bornmann, W., Marszalek, J. R., DePinho, R. A. & Muller, F. L. An enolase inhibitor for the targeted treatment of ENO1-deleted cancers. Nat Metab 2, 1413–1426 (2020).
3. Milanes, J. E., Yan, V. C., Pham, C.-D., Muller, F., Kwain, S., Rees, K. C., Dominy, B. N., Whitehead, D. C., Millward, S. W., Bolejack, M., Shek, R., Tillery, L., Phan, I. Q., Staker, B., Moseman, E. A., Zhang, X., Ma, X., Jebet, A., Yin, X. & Morris, J. C. Enolase inhibitors as therapeutic leads for Naegleria fowleri infection. PLoS Pathog 20, e1012412 (2024).
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