(W1030-07-38) Development of Targeted Nanovehicles to Deliver Biomolecules to Cerebrovascular Endothelium

Purpose: Alzheimer’s disease (AD) constitutes an array of pathological hallmarks, with aggregated amyloid beta plaques and phosphorylated tau tangles being the central disease-modifying targets (Serrano-Pozo et al., 2011). Ameliorating the toxic effects of these harmful proteins is as critical as intervening in the preceding pathogenic molecular pathways. Alterations in molecular mechanisms of endothelial cells within the neurovascular unit significantly contribute to blood-brain barrier degeneration (BBB) (Yamazaki & Kanekiyo, 2017). These endothelial cells express genes that may enhance susceptibility to AD (Tsartsalis et al., 2024). Ribonucleic acid interference (RNAi) is a promising tool to silence the causative genes by degrading messenger RNA (mRNA) in molecular pathways that are not targetable by traditional therapeutics (Kim, 2020). However, the in vivo stability and transport of these RNA-based drugs to the BBB endothelium pose significant challenges. We have previously demonstrated that nano vehicles formulated from chitosan- a cationic, bioadhesive, and biocompatible polymer- can deliver therapeutic and diagnostic agents to the cerebrovasculature for the detection and treatment of cerebral amyloid angiopathy (Agyare et al., 2008). This study aims to develop chitosan-polycarbophil nanoparticles for the delivery of RNA to BBB endothelial cells in vitro model constructed from human microvascular endothelial cells (hCMEC/D3). The nanoparticles are crosslinked with bovine serum albumin (BSA) or anti-amyloid antibody IgG 4.1 to facilitate endocytosis and specifically target amyloid-beta peptides.

Methods: Chitosan-polycarbophil nanoparticles were prepared using the ionic gelation technique. A solution of low and medium-molecular-weight chitosan was prepared in cell culture water containing 1% glacial acetic acid. To prepare fluorescent nanoparticles, medium molecular weight FITC-chitosan was used in combination with low molecular weight chitosan. Polycarbophil-RNA solution was prepared separately and then gradually added to the chitosan solution. The nanoparticles were allowed to form and equilibrate at room temperature for 20 minutes. Nanoparticles were then conjugated with either BSA or IgG 4.1 using carbodiimide modification. The BSA was also adsorbed on the nanoparticle surface by co-incubation of nanoparticles and BSA solution (referred to as mixed nanoparticles). After surface modification, they were purified with spin filtration using 1000 KMWCO filters to remove unconjugated proteins and undergo buffer exchange, as shown in Figure 1 A. The size and zeta potential of the nanoparticles were characterized using Malvern Zetasizer and NanoSight instruments. The encapsulation efficiency of RNA nanoparticles was determined using Ribogreen assay. A bicinchoninic acid (BCA) assay was used to calculate the amount of conjugated proteins on the nanoparticles. In vitro studies were carried out in hCMEC/D3 monolayers. The particles were first assessed for any cytotoxicity using live/dead cell assay, where cells were treated with nanoparticles for 24 hours and analyzed using flow cytometry. Then, the cellular uptake of nanoparticles was assessed using flow cytometry and confocal microscopy. The uptake of BSA or IgG 4.1 conjugated nanoparticles and mixed nanoparticles was compared to the chitosan-polycarbophil core (control). The impact of amyloid beta 42 (Abeta42) pretreatment on the cellular uptake of these particles was also investigated. Furthermore, the uptake mechanisms of various nanoparticles were investigated upon pretreatment with multiple inhibitors. Specifically, 4 and 37 ^°C preincubation was used to differentiate between active and passive uptake mechanisms; dynasore pretreatment was employed to assess dynamin mediated endocytosis, and cytochalasin-A, an inhibitor of actin polymerization, was used to examine the nanoparticle uptake by macropinocytosis.

Results: Average particle size and zeta potential for control nanoparticles, BSA/ IgG 4.1 conjugated, and mixed nanoparticles are presented in Figure 1 B. Using the Ribogreen assay, we detected encapsulated RNA through heparin displacement. These studies have shown an insignificant amount of RNA in the external buffer, suggesting an encapsulation efficiency between 97-100%. This could be due to the assay's detection limit (20 base pairs) and the scrambled sequence of the model RNA used in the study (10 to 120 base pairs). The Live/Dead cell assay indicated over 92 % cell viability for both concentrations. Hence, the nanoparticles were deemed to show low cellular toxicity. The cellular uptake of BSA or IgG4.1 conjugated nanoparticles was significantly higher than the control nanoparticles- with IgG 4.1 conjugated nanoparticles showing higher uptake. Mixed nanoparticles, however, showed lower uptake than the control. Pretreatment with Abeta42 increased the uptake of IgG 4.1 conjugated nanoparticles and decreased the uptake of mixed nanoparticles, but no significant change was observed with the uptake of BSA conjugated nanoparticles. Z-stacks were used to confirm the internalization of nanoparticles. A substantial reduction in the nanoparticle uptake by BBB endothelial monolayers was observed at 4 ^°C and upon cytochalasin A pretreatment, which indicates a possible energy-dependent and micropinocytosis-mediated internalization of nanoparticles.

Conclusion: Cationically charged chitosan-polycarbophil nanoparticles effectively encapsulated RNA. Surface cross-linking with IgG 4.1 significantly enhanced intracellular uptake, which was further enhanced in the presence of Abeta42. These findings indicate that the chitosan-polycarbophil nanoparticles improved targetability of RNA-based therapeutics to BBB endothelial cells, especially in the presence of Abeta42.

References: Serrano-Pozo, A., Frosch, M. P., Masliah, E., & Hyman, B. T. (2011). Neuropathological Alterations in Alzheimer Disease. Cold Spring Harbor Perspectives in Medicine:, 1(1), a006189.
Yamazaki, Y., & Kanekiyo, T. (2017). Blood-Brain Barrier Dysfunction and the Pathogenesis of Alzheimer’s Disease. International Journal of Molecular Sciences, 18(9), 1965.
Tsartsalis, S., Sleven, H., Fancy, N., Wessely, F., Smith, A. M., Willumsen, N., Cheung, T. K. D., Rokicki, M. J., Chau, V., Ifie, E., Khozoie, C., Ansorge, O., Yang, X., Jenkyns, M. H., Davey, K., McGarry, A., Muirhead, R. C. J., Debette, S., Jackson, J. S., … Matthews, P. M. (2024). A single nuclear transcriptomic characterisation of mechanisms responsible for impaired angiogenesis and blood-brain barrier function in Alzheimer’s disease. Nature Communications, 15(1), 2243.
Kim, Y.-K. (2020). RNA Therapy: Current Status and Future Potential. Chonnam Medical Journal, 56(2), 87–93. https://doi.org/10.4068/cmj.2020.56.2.87
Serrano-Pozo, A., Frosch, M. P., Masliah, E., & Hyman, B. T. (2011). Neuropathological Alterations in Alzheimer Disease. Cold Spring Harbor Perspectives in Medicine:, 1(1), a006189.
Agyare, E. K., Curran, G. L., Ramakrishnan, M., Yu, C. C., Poduslo, J. F., & Kandimalla, K. K. (2008). Development of a Smart Nano-vehicle to Target Cerebrovascular Amyloid Deposits and Brain Parenchymal Plaques Observed in Alzheimer’s Disease and Cerebral Amyloid Angiopathy. Pharmaceutical Research, 25(11), 2674–2684.

Acknowledgements: This work was supported by the Minnesota Partnership for Biotechnology and Medical Genomics (MNP#15.31) and National Institutes of Health/National Institute of Neurological Disorders and Stroke (R01NS125437).

Figure 1 Formulation and characterization of nanoparticles: A- Formulation steps for nanoparticle preparation and surface modification followed by purification and buffer exchange. B- Particle size and zeta potential were measured by a zeta sizer for control and conjugated nanoparticles. C- Snapshot of the video of conjugated RNA nanoparticles from nanosight.

Figure 2 Cellular uptake in hCMEC/D3 cells: A-Uptake in D3 cells assessed by flow cytometry, bar chart represents intracellular median FITC fluorescence intensity, data is expressed as mean ± standard deviation. Significance levels: *p < 0.05, ***p < 0.001,****p < 0.0001 determined by one-way ANOVA.

B-E Confocal micrographs illustrating nanoparticle uptake for different samples. FITC chitosan is visualized in green, while nuclei are stained blue (DAPI). Scale bar 50 µm. B- Control nanoparticles, C- BSA conjugated nanoparticles, D - IgG 4.1 conjugated nanoparticles, E- Mixed nanoparticles



Figure 3 Effect of Abeta 42 pretreatment on cellular uptake of nanoparticles: A-Uptake in D3 cells assessed by flow cytometry, bar chart represents intracellular median FITC fluorescence intensity, data is expressed as mean ± standard deviation. Significance levels: **p < 0.01, ***p < 0.001,****p < 0.0001 determined by one-way ANOVA.

B-C Confocal z-stacks illustrating nanoparticle internalization for IgG4.1 conjugated nanoparticles. FITC chitosan is visualized in green, while nuclei are stained blue (DAPI). Scale bar 50 µm. B- IgG 4.1 conjugated nanoparticles treated cells, C- Abeta 42 pre-treated cells followed by treatment with IgG4.1 conjugated nanoparticles.