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Formulation and Delivery - Biomolecular

(T1230-09-53) Development of siRNA Incorporated PEGylated and Polysorbate 80 Coated Polymeric Nanoparticles for Global Cerebral Ischemia

Tuesday, October 22, 2024
12:30 PM – 1:30 PM MT
Purpose: Global cerebral ischemia is a cerebrovascular disease caused by cardiac arrest or cardiac surgery and affects approximately 200,000 Americans each year (1). Global cerebral ischemia events that occur in humans or induced experimentally in animals result in selective and delayed neuronal death that causes the onset of neurological deficits (2). Chronic and excessive neuroinflammation is a major contributor to cell death in the pathophysiology of global ischemia, which likely has a detrimental effect on cognitive function post-global ischemia. Bromodomain-containing protein 4 (BRD4) acts as an epigenetic reader and plays a pivotal role in the transcriptional regulation of inflammation in cancer, ischemic stroke, and central nervous system (CNS) diseases (3, 4). Therefore, inhibiting BRD4 may be a novel therapeutic strategy for inhibiting neuroinflammation and delaying cognitive deficits following global cerebral ischemia. Our goal is to target BRD4 with siRNA which is known for suppressing genes in a sequence specific way, to suppress its expression and protect neurons from neuroinflammation, thereby improving cognitive deficits following global ischemia. However, transferring siRNA to the brain is a challenge due to the presence of the blood-brain barrier (BBB), which restricts the delivery of substances and chemicals in. Therefore, the aim of this study is to develop PLGA nanoparticles coated with polysorbate-80 and PEG was created to aid siRNA in effective delivery across the BBB. We hypothesize that these particles will cross the BBB successfully and show neuroprotection by suppression of BRD4 in the affected neuronal cells.

Methods: PLGA nanoparticles were prepared using a modified nanoprecipitation method. Briefly, PLGA (50:50) was used as a biodegradable polymer. The organic phase consisted of PLGA (5mg/mL) in acetone. In case of PEGylated nanoparticles, organic phase also consisted of poly-ethylene glycol (PEG 1450) (1 mg/mL). The aqueous phase contained 0.1% of Polysorbate-80 or 0.1% PVA. The organic phase was slowly added into the aqueous phase under magnetic agitation for 1 hour. The dispersion was left undisturbed for 1 hour and centrifuged at 3100 rpm for 30 minutes. It was washed with PBS and resuspended in water/PBS. For siRNA incorporated nanoparticles, 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) (2.5 mg/mL) was mixed with 2 nmol of BRD4 siRNA, then combined with PLGA in acetone, and added to the aqueous phase with 0.1% Polysorbate-80, centrifuged and washed. The resulting nanoparticles were characterized for their particle size, zeta potential, electrophoretic mobility and polydispersity index (PDI) using a Zetasizer. Formation of the nanoparticles was further confirmed by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The cytotoxicity of blank nanoparticles was tested on Neuro-2-a cells via MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) cytotoxicity assay.

Results: The particle size of PLGA nanoparticles coated with either Polysorbate 80, PEG or PVA is below 150 nm (Table 1). The particle size of siRNA incorporated particles was 228.23±7.15nm. The zeta potential of Polysorbate 80 and PVA-coated nanoparticles was -17.24± 2.00 mV and -10.81 ±0.72mV, respectively. The zeta potential of PEGylated nanoparticle was towards the neutral side -9.17 ±1.89 mV indicating PEGylation of these particles. The zeta potential of siRNA incorporated PLGA-Polysorbate 80 particles was -17.52 ± 2.23mV like the blank Polysorbate 80 nanoparticles. The PDI value varies from 0.067 to 0.08 for the blank nanoparticles and it was 0.65±0.36 for siRNA incorporated nanoparticles. The electrophoretic mobility of the nanoparticles ranged from -0.72 to -1.36 with PEGylated nanoparticles having the lowest number indicating the presence of PEG layer (Table 1). SEM images of polysorbate 80 coated and PVA coated nanoparticles (Figure 1A, Figure 1B) indicate formation of spherical nanoparticles and uniformity in size and shape. TEM images of PEGylated nanoparticle confirm nanoparticle formation with PEG layer and morphological uniformity (Figure 1C). SEM image of siRNA incorporated particles show spherical morphology and variation in sizes consistent with the PDI data (Figure 1D). Cell viability of polysorbate 80 and PVA blank PLGA nanoparticle was 85.84% and 96.46%, respectively which is higher than the working standard solutions of 0.1% Polysorbate 80 and PVA after 72 hrs of exposure (Figure 2).

Conclusion: These studies demonstrated successful formulation and characterization of blank PLGA nanoparticles coated with Polysorbate 80, PEG, PVA and siRNA incorporated PLGA-polysorbate 80 nanoparticles. All the particle sizes lie in nano ranges indicating good potential to cross the BBB. The zeta potential of all the particles is negative and PDI indicates polydisperse particles. PEGylation of particles increased the particle size and decreased the zeta potential and electrophoretic mobility as compared to non-PEGylated particles indicating successful PEGylation of nanoparticles. An increased particle size of siRNA PLGA-Polysorbate 80 nanoparticles indicates successful incorporation of siRNA. Both Polysorbate 80 and PVA coated blank nanoparticles have less cytotoxicity on the Neuro-2-a cells indicating its further use for siRNA incorporation and BBB targeting.

References: 1. Ofengeim D, Miyawaki T, RS Z. Molecular and Cellular Mechanisms of Ischemia-induced Neuronal Death. In: Mohr JP, Wolf P, Grotta JC, Moskowitz MA, Mayberg M, von Kummer R, editors. Stroke: Pathophysiology, Diagnosis and Management. 5th ed. Philadelphia: Churchill Livingstone Elsevier; 2011. p. 1-39.
2. Zukin RS, Jover T, Yokota H, Calderone A, Simionescu M, C.G. L. Molecular and Cellular Mechanisms of Ischemia-Induced Neuronal Death. In: Mohr JP, Choi DW, Grotta JC, Weir B, Wolf PA, editors. Stroke: Pathophysiology, Diagnosis, and Management. 4 ed. Philadelphia: Churchill Livingstone; 2004. p. 829-54.
3. Dhalluin C, Carlson JE, Zeng L, He C, Aggarwal AK, Zhou MM. Structure and ligand of a histone acetyltransferase bromodomain. Nature. 1999;399(6735):491-6.
4. Liu L, Yang C, Candelario-Jalil E. Role of BET Proteins in Inflammation and CNS Diseases. Front Mol Biosci. 2021;8:748449.

Acknowledgements: Acknowledgements: We would like to acknowledge the School of Pharmacy and Health Professions, Department of Pharmacy Sciences, Creighton University, and George F. Haddix President’s Faculty Research fund for funding and the University of Nebraska Medical Center for assistance in TEM.

Funding: The authors gratefully thank the Department of Pharmacy Sciences, Creighton University, for the financial support.
Conflict of interest statement: We confirm that there are no conflicts of interest associated with this work, and there has been no significant financial support for this work that could have influenced its outcome.

Table 1: Particle characterization of PLGA Nanoparticles with Polysorbate 80, PVA and Polysorbate 80 and PEG.

Figure 1. A) SEM images of Polysorbate 80-PLGA Nanoparticles B) SEM images of PVA-PLGA Nanoparticles C) TEM image of Polysorbate 80-PEG-PLGA Nanoparticles D) SEM image of siRNA-PLGA-Polysorbate 80

Figure 2. Cell viability of blank nanoparticles treated for 72 hours