(T1330-07-44) Brain Targeted Delivery of siRNA Incorporated Polymeric Nanoparticles with PEG and Polysorbate 80 Surface Modification for Global Cerebral Ischemia

Purpose: Global cerebral ischemia is a cerebrovascular disease caused by cardiac arrest or cardiac surgery, which affects 200,000 Americans annually. Chronic and excessive neuroinflammation is a major contributor to cell death in the pathophysiology of global ischemia, likely having a detrimental effect on cognitive function. Bromodomain-containing protein 4 (BRD4) plays a pivotal role in the transcriptional regulation of inflammation in this event. Therefore, inhibiting BRD4 may be a novel therapeutic strategy for decreasing neuroinflammation and delaying cognitive deficits. We aim to suppress BRD4 using siRNA, thereby protecting neurons from neuroinflammation. However, the blood-brain barrier (BBB) causes a significant challenge in delivering siRNA to the brain. To address this issue and suppress BRD4 expression, this study developed PLGA nanoparticles surface modified with Polysorbate-80 (PS-80) and Polyethylene glycol (PEG) to incorporate siRNA and effectively deliver 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: Nanoparticles were prepared using a modified nanoprecipitation method. Briefly, 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) (2.5mg/mL) was mixed with 1nmol of BRD4 siRNA to form a lipoplex. Lipoplex was combined with the organic phase consisting of PLGA (5mg/mL) in acetone. In case of PEGylated nanoparticles, the organic phase also consisted of PEG (MW 1450) (2.5%). The organic phase was slowly added to the aqueous phase, consisting of 1% Polysorbate-80 or 1% PVA, under magnetic agitation for 1 hour. The dispersion was left undisturbed for 30 minutes, then centrifuged (3100 rpm, 40 minutes), washed, and resuspended in PBS. The resulting nanoparticles were physically characterized using a Zetasizer. Morphology was confirmed by Transmission Electron Microscopy (TEM). The encapsulation efficiency was analyzed via the Ribogreen Assay. BBB permeability of nanoparticles was carried out using Parallel Artificial Membrane Permeability (PAMPA) assay. Cellular Uptake of nanoparticles was observed via confocal microscopy in Neuro-2-a cells using Rhodamine 123 loaded nanoparticles. Hemolysis and protein adsorption were performed via UV-visible spectroscopy and BCA Assay. Furthermore, age-matched adult male Sprague Dawley rats weighing 150-200 g were subjected to tail vein injection or stereotaxic injection to analyze the uptake of nanoparticles in brain tissues.

Results: The study engineered siRNA-incorporated PLGA nanoparticles with PS-80 and PEG coating via a modified nanoprecipitation method. A change in the zeta potential from 14.66±1.15 mV to -24.27±0.99 mV indicated the formation of a lipoplex. The particle size of all the nano-formulations was observed to be less than 250 nm with a negative charge and a PDI less than 0.3. Encapsulation efficiency of PS-80, PEG+PS-80 and PVA nanoparticles are 52.36±6.9%, 79.25±1.39%, 74.95±24.67% respectively. TEM image of siRNA incorporated particles shows spherical morphology and variation in sizes. Furthermore, PEG+PS-80 coated nanoparticles showed higher BBB permeability compared to the high control of the PAMPA assay. PS-80 coated nanoparticles showed moderate BBB Permeability. Cellular uptake studies revealed a higher uptake of PEG+PS-80 coated nanoparticles in Neuro-2-a cells as compared to other nano-formulations. The nanoparticles showed low hemolysis compared to the positive control, Triton X-100. All the nano-formulations showed hemolysis of less than 10%. Low serum protein adsorption was observed for PEG+PS-80 coated nanoparticles owing to the presence of PEG on the surface.

Conclusion: In conclusion, this study developed a novel delivery system for siRNA by PEG and PS-80 surface modifications on PLGA nanoparticles using a modified nanoprecipitation method. These nanoparticles were characterized and evaluated for their efficacy and safety profiles. The formed nanoparticle size lies below 250nm, indicating its potential to cross the BBB. In-vitro data suggests its potential to cross the BBB, showing high permeability and cellular uptake. Blood compatibility data indicates its safety as a delivery system to be administered intravenously. With the ongoing in vivo studies to evaluate the uptake of these nanoparticles, we can further validate the formation of a successful delivery system to deliver BRD4 siRNA to the brain.

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Acknowledgements: We would like to acknowledge the School of Pharmacy and Health Professions, Department of Pharmacy Sciences, Creighton University and 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.


Physical Characterization. A) Lipoplex formation B) Particle size C) PDI D) Zeta Potential E) TEM image of PEG+PS-80 siRNA-NP F) Encapsulation Efficiency using Ribogreen Assay


A) BBB Permeability via PAMPA Assay B) Quantification of the Cellular Uptake in N2A cells using confocal Microscopy. C) Confocal Microscopy of the cellular Uptake in N2A cells of all the Rhodamine 123 loaded nano-formulations. a: control b: PS-80 NP c: PEG+PS-80 NP d: PVA NP


A) Hemolysis Study B) Serum Protein Adsorption Studies