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Formulation and Delivery - Biomolecular

Pioneering the Targeted Brain Delivery of Aspirin to Elicit COX-1&2 Inhibition, Protecting from Ischemic Stroke and Neurodegeneration

(M1230-03-17) Pioneering the Targeted Brain Delivery of Aspirin to Elicit COX-1&2 Inhibition, Protecting from Ischemic Stroke and Neurodegeneration

Monday, October 21, 2024
12:30 PM – 1:30 PM MT
Purpose: Owing to its property to inhibit cyclooxygenases in the arachnoid acid and prostaglandin pathway, Aspirin, a small molecule NSAID, exhibits anti-inflammatory properties both centrally and peripherally. Inhibiting the production of prostaglandins reduces their hyperalgesia modulation activities in the nociceptive pathways, thus regulating pain sensitivity. Currently, Aspirin is being delivered via oral and intravenous (IV) routes, which has several shortcomings, including the lower ability of Aspirin to cross the blood-brain barrier (BBB) and severe systemic side effects like gastric bleeding, thus increasing the risk of comorbidities in critically ill and comatose patients. Therefore, there is a need to develop a brain-targeted drug delivery system and an effective route of administration for Aspirin to increase its penetrance into the brain tissue to elicit COX-1&2 inhibition, protecting from ischemic stroke and neurodegeneration. This will help to further increase its local potency and reduce systemic side effects. Here we developed a brain-targeting intranasal nanoparticle drug delivery system for Aspirin using an automated microfluidics system (Dolomite, Roystone, UK) with a 5-input 3D chip and employing the nanoprecipitation method and Rabies Virus Glycoprotein (RVG29) ligand conjugation. We conducted an in vivo timeline study where we observed that the aspirin concentration was at its maximum at 3 hours and cleared out > 90% by 6 hours after intranasal administration. The goal is to determine the in vivo drug deposition profile of Aspirin in mice blood serum and various organs—brain, kidneys, spleen, liver, lungs and heart—at 3 hours of intranasal administration of brain-targeted Aspirin NPs using the highly sensitive and reliable dMRM method on LC-MS/MS. We also aim to establish the pharmacokinetic parameters of the drug Aspirin when administered as Aspirin NPs based on the concentrations obtained from the quantitative determination in the biological samples.

Methods: Using the nanoprecipitation method, we formulated Aspirin NPs using a Dolomite microfluidics system with a 5-input 3D chip. The formed NPs upon solvent evaporation were conjugated with brain-targeting RVG ligands and lyophilized. We characterized the formulation for % yield and physicochemical properties like size (nm), zeta potential (mV), and polydispersity index (PDI) using Malvern Zetasizer. Additionally, we developed a Dynamic Multiple Reaction Monitoring (dMRM) method using Liquid chromatography – triple quadrupole Mass spectrometry (LC-MS/MS) for all quantitative characterization of the NPs, including percent encapsulation efficiency (%EE) and drug release profile from the NPs and in vivo analysis of drug deposition in various organs and blood serum samples. We established using a single-dose pharmacokinetic study that at 3 hours upon administration of a 50mg/kg dose of the NPs intranasally, the drug deposition in the brain tissue was at its highest: 159.4 ng/mg. Therefore, to establish the drug deposition profile of Aspirin, we administered the Aspirin NPs intranasally and orally at a dose of 50mg/kg to each animal. At 3 hours after administration, the mice were sacrificed, and blood serum samples along with the selected organs were carefully removed and processed for LC-MS/MS analysis. Extensive sample preparation was conducted for analysis using the dMRM method on LC-MS/MS by homogenizing the tissue samples in the presence of trypsin, followed by precipitation and separation of the drug from the protein content of the samples.

Results: The characterization results of the NPs are mentioned in Table 1. The timeline study revealed that upon intranasal administration, Aspirin was detected in the brain samples at 1 hr, 3 hrs, and 6 hrs at 8.93 ng/mg, 159.4 ng/mg, and 15.5 ng/mg, respectively, and in blood serum at 1 ng/uL, 3.2 ng/uL, and 2 ng/uL, respectively. Upon intranasal and oral administration, at 3 hours, the concentration of Aspirin in brain tissues was 159.4 ng/mg and 12.6 ng/mg, respectively. Upon intranasal administration, Aspirin was detected in the liver, heart, and lungs at 2.253 ng/mg, 26.4 ng/mg, and 16.2 ng/mg, respectively, and upon oral administration, Aspirin in the liver, kidney, spleen, and heart was at 3 ng/mg, 77.5 ng/mg, 363.8 ng/mg, and 176.4 ng/mg, respectively.

Conclusion: In conclusion, we successfully formulated microfluidics-assisted brain-targeted Aspirin NPs via the 5-chip nanoprecipitation method and characterized them. Effective concentrations of Aspirin were detected in the mice's brain tissue and blood serum using LC-MS/MS. We established that the highest Aspirin deposition occurred at 3 hours when administered intranasally. The study's findings confirm that intranasal delivery, in conjunction with the targeted nanoparticle formulation of aspirin, leads to increased drug deposition in the brain tissue. This approach also demonstrates sustained release characteristics within the brain while minimizing concentrations in systemic circulation and non-targeted organs when compared to oral administration. These findings imply promising advancements in both the safety and effectiveness of the aspirin formulation. Such a targeted strategy holds significant promise, particularly for conditions necessitating direct central nervous system impact, such as ischemic stroke or neurodegenerative diseases.

References: 1] Ajmone-Cat MA, Bernardo A, Greco A, Minghetti L. Non-Steroidal Anti-Inflammatory Drugs and Brain Inflammation: Effects on Microglial Functions. Pharmaceuticals 2010;3:1949–65. https://doi.org/10.3390/ph3061949.
[2] Ansara AJ, Nisly SA, Arif SA, Koehler JM, Nordmeyer ST. Aspirin Dosing for the Prevention and Treatment of Ischemic Stroke: An Indication-Specific Review of the Literature. Ann Pharmacother 2010;44:851–62. https://doi.org/10.1345/aph.1M346.
[3] Strauss KI. Antiinflammatory and neuroprotective actions of COX2 inhibitors in the injured brain. Brain, Behavior, and Immunity 2008;22:285–98. https://doi.org/10.1016/j.bbi.2007.09.011.
[4] Zaman RU, Mulla NS, Braz Gomes K, D’Souza C, Murnane KS, D’Souza MJ. Nanoparticle formulations that allow for sustained delivery and brain targeting of the neuropeptide oxytocin. International Journal of Pharmaceutics 2018;548:698–706. https://doi.org/10.1016/j.ijpharm.2018.07.043.

Acknowledgements: Graphics are created using Biorender.com


Representative image of formulation and in vivo studies of the microfluidics generated brain targeted Aspirin loaded nano particles


Quantitative determination of biodistribution of Aspirin upon oral and intranasal administration in mice using LC-MS/MS


Physiochemical characterization of RVG conjugated Aspirin loaded BSA NPs