(T1230-10-56) Nanosac Formulation for Nucleic Acid Delivery to the Brain

Purpose: Alzheimer’s disease (AD) is one of the most significant neurodegenerative diseases. Currently, AD is treated with acetylcholinesterase inhibitors, N-methyl-d-aspartate receptor antagonists, or anti-amyloid antibodies. They modestly improve cognitive outcomes and behaviors of AD patients in the early stage but do not stop or reverse the disease progression [1]. Therefore, there is a critical unmet need for new AD therapy. We propose to develop a nanoparticle for the brain delivery of nucleic acid therapeutics. Nanosac is a polyphenol capsule, soft and negatively charged, co-opts albumin corona in serum, and enters cells bypassing endosomal trafficking [2]. These features bode well for systemic delivery of nucleic acids to the brain. In this study, we aim to optimize the Nanosac for systemic delivery of nucleic acids to the brain. We hypothesize that optimal surface and size of Nanosac will enhance its interaction with the brain endothelium and transport across the blood-brain barrier, promoting siRNA delivery to neurons.

Methods: Preparation of Nanosac: Nanosac was produced by sequential coating of mesoporous silica nanoparticles (MSNs) with siRNA and polydopamine, followed by removing the sacrificial MSN core. Surface modification of Nanosac for brain delivery: The surface of Nanosac was modified with albumin to improve colloidal stability of Nanosac (Alb-Nanosac). Alternatively, Nanosac was modified with polyvinyl alcohol (PVA) to reduce aggregation (PVA-Nanosac). Cytotoxicity of Nanosac: Cytotoxicity of Nanosac was tested with human cortical microvesselendothelial cells (hCMEC/d3) and human neuroblastoma cells (SH-SY5Y) by MTT assay. Transport of Nanosac through endothelial layer: hCMEC/d3 were seeded in a Transwell insert. When the Transepithelial electrical resistance (TEER) value reached a plateau (indicating confluency), the hCMEC/d3 layer was treated with Nanosac containing a fluorescent dye (Alexa Fluor™ 594 NHS Ester, AF594) for 6 h. The fluorescence intensity of Nanosac suspension before incubation and those in the apical and basolateral sides of a Transwell insert after the incubation were measured by spectrofluorometry. The Transwell membrane was cut and imaged by confocal microscopy. Cell uptake: Nanosac containing AF594 was visualized by confocal microscopy to trace Nanosac delivery. Cy3-siRNA or Cy5-siRNA was loaded in Nanosac to trace siRNA delivery by Nanosac to SH-SY5Y cells.

Results: The Z-average of Nanosac was 155.90 ± 10.3 nm in diameter. Zeta potential of Nanosac was -9.3 ± 1.1 mV (Fig. 1a). The cytotoxicity of MSN, MSNa (amine-modified MSN, precursor to Nanosac), and Nanosac were examined with hCMEC/d3 (Fig. 1b) and SH-SY5Y cells (Fig. 1c). After 24 h incubation with cells, MSN, MSNa, and Nanosac did not show significant toxicity on cell viability at a concentration of up to 1000 μg/mL. Nanosac was aggregated in the complete DEME media containing 10% FBS, but the intensity peak of pre-albuminylated Nanosac (Alb-Nanosac) shifted from about 400 nm to about 150 nm, indicating that albumin surface prevented the aggregation of Nanosac (Fig. 1d). To further reduce the aggregation of Nanosac in water, PVA was added during polydopamine coating. The Z-average of PVA-stabilized Nanosac (PVA-Nanosac) was around 78 nm (Fig. 1e). The percentage of Nanosac transported across the hCMEC/d3 endothelial monolayer was tested using AF594-labeled Nanosac after 6 h incubation (Fig. 2a, b). 47.7 ± 6.2% of the Nanosac was detected in the lower chamber. We also evaluated the Nanosac uptake by neurons. AF594-labeled Nanosac was seen inside the induced pluripotent stem cells (iPSC)-derived neurons, rat primary cortical neurons, and SH-SY5Y cells in 1 h (Fig. 2c). Nanosac uptake by the neurons increased with time. Nanosac entered the cell body as well as projections of iPSC-derived neurons and rat primary cortical neurons (Fig. 2d). Cy3-siRNA was delivered by Nanosac to SH-SY5Y cells (Fig. 2e). These results indicate that Nanosac can pass the confluent endothelial barrier and enter neuron cells, including iPSC-derived neurons, rat primary cortical neurons, and SH-SY5Y cells, to deliver siRNA. To evaluate intracellular trafficking, Nanosac and Alb-Nanosac were located in SH-SY5Y cells relative to the lysosome marker, LysoTracker™ Green DND-26 (LysoTracker) (Fig. 3). For comparison, Cy5-siRNA loaded with Lipofectamine 3000 (L3K), a commercial transfection reagent known for entering cells through endocytosis [3], was tested in the same way. L3K was colocalized with LysoTracker, indicating that most of the internalized L3K was transported to lysosomes, consistent with the literature. No colocalization was observed between Nanosac or Alb-Nanosac and LysoTracker in either serum-free or serum-containing medium, suggesting that Cy5-siRNA-loaded Nanosac entered cells bypassing the lysosomes. These results indicate that Nanosac can deliver siRNA to neuron cells avoiding the destructive lysosomal sequestration.

Conclusion: Pre-albuminylated Nanosac maintained colloidal stability in serum-containing medium. Aggregation of Nanosac was further reduced by introducing PVA during the Nanosac formation. Additionally, Nanosac traversed the endothelial cell layer and was taken up by neuron cells, including the iPSC-derived neurons, rat primary cortical neurons, and SH-SY5Y neuroblastoma cells in 1 h. siRNA delivered by Nanosac avoided lysosomal sequestration. These results support the potential of Nanosac to deliver siRNA to the neurons in the brain by systemic administration.

References: [1] J. Versijpt, Effectiveness and cost-effectiveness of the pharmacological treatment of Alzheimer's disease and vascular dementia, J Alzheimers Dis 42 Suppl 3 (2014) S19-25.
[2] H. Kim, S.A. Yuk, A.M. Dieterly, S. Kwon, J. Park, F. Meng, H.H. Gadalla, M.J. Cadena, L.T. Lyle, Y. Yeo, Nanosac, a Noncationic and Soft Polyphenol Nanocapsule, Enables Systemic Delivery of siRNA to Solid Tumors, ACS Nano 15(3) (2021) 4576-4593.
[3] C. Del Toro Runzer, S. Anand, C. Mota, L. Moroni, C. Plank, M. van Griensven, E.R. Balmayor, Cellular uptake of modified mRNA occurs via caveolae-mediated endocytosis, yielding high protein expression in slow-dividing cells, Mol Ther Nucleic Acids 32 (2023) 960-979.

Acknowledgements: This work was supported by NIH R01 CA258737, Ross-Lynn Research Scholar Fund, PIDD Institutional Programmatic Areas grant, and Yanchick Geriatric Research Award.

Fig.1. In vitro characterization and optimization of Nanosac. (a) Z-average and zeta potential of Nanosac. (b) Cytotoxicity of Nanosac on hCMEC/d3, and (c) SH-SY5Y cells. (d) Effect of pre-albuminylation on Nanosac colloidal stability. (e) Effect of PVA on the particle size of Nanosac.

Fig. 2. Nanosac transport across the endothelial layer and cell uptake by neurons. (a) The percentage of Nanosac transport across the confluent hCMEC/d3 endothelial layer. (b) Neurons on Transwell membrane. Blue: DAPI (Nuclei); Green: Wheat Germ Agglutinin (WGA)-AF488 (Cell membrane); Red: AF594 (Nanosac) (c) Nanosac cell uptake by iPSC-derived neurons, rat primary cortical neurons, and SH-SY5Y cell in 1, 2, and 6 h. Blue: DAPI (Nuclei); Red: AF594 (Nanosac) (d) Nanosac in the cell body and projections after incubation for 6 h. Blue: DAPI (Nuclei); Green: WGA-AF488 (Cell membrane); Red: AF594 (Nanosac) (e) Cy3-siRNA delivered by Nanosac to SH-SY5Y cells after incubation for 6 h. Blue: DAPI (Nuclei); Red: Cy3-siRNA.

Fig. 3. Confocal microscope images locating Cy5-siRNA loaded Nanosac, Alb-Nanosac, and L3K relative to lysosomes in SH-SY5Y cells. Blue: Hoechst 33342 (nuclei); Green: Lysotracker (lysosome); red: Cy5-siRNA. Incubation time: 6 h. Scale bars: 10 μm.