(M1430-02-12) Microfluidics Generated Aspirin Loaded Albumin Nanoparticles for Targeted Brain Delivery in the Treatment of Ischemic Stroke

Purpose: Stroke is one of the leading causes of death and disability. In the United States alone, more than 795,000 people experience a stroke every year, of which 87% are ischemic strokes, and 25% of the cases are due to the recurrence of stroke cases. Aspirin, at low doses, alone or in combination, is approved for the secondary prevention of acute non-cardioembolic ischemic stroke and transient ischemic stroke conditions due to its ability to induce anti-inflammatory and antiplatelet aggregation effects soon after an acute event. Additionally, it is recommended for patients who have already had an ischemic stroke or transient ischemic attack (TIA), as they are at high risk for stroke recurrence. Currently, Aspirin is being delivered via oral and intravenous (IV) routes, which come with several shortcomings including the lessened 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 comotose patients. Therefore, there is a need to develop a brain-targeted drug delivery system for Aspirin to increase its penetrance into the brain tissue, further increasing its local potency and reducing systemic side effects. The goal is to develop a brain-targeting intranasal nanoparticle drug delivery system for Aspirin using a microfluidics-assisted nanoprecipitation technique. Further, we aim to characterize and quantitatively determine in vitro and in vivo properties of the formulated NPs using a dynamic Multiple Reaction Monitoring (dMRM) method on a Liquid chromatography - triple quadrupole Mass spectrometry (LC-MS/MS) technique.

Methods: Using an automated microfluidics system (Dolomite, Roystone, UK) with a 5-input 3D chip and employing the nanoprecipitation method, we formulated Aspirin nanoparticles. The formed NPs upon solvent evaporation, are conjugated with brain-targeting Rabies Virus Glycoprotein (RVG) ligands. We lyophilized the final preparation and determined the % yield. We characterized the formulation for 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 as % cumulative release and in vivo analysis. We evaluated a single dose in-vivo pharmacokinetic profiles of the Aspirin NPs by administering a dose 50 mg/kg dose of the NPs intranasally to the mice. At 1 hour, 3 hours, and 6 hours, we collected blood samples and brain tissues from the mice and evaluated the pharmacokinetics of the NPs. We homogenized the tissue samples in the presence of trypsin, followed by precipitation and separation of the protein content from the samples. We analyzed the samples for quantitative estimation of the drug using LC-MS/MS.

Results: The percent recovery yield was more than 85% w/w. The characterization results revealed the size to be 149.3 nm and 114.1 nm; PDI of 0.222 and 0.238 and zeta potential to be 17.8 +/- 3.58 mV and -22.6 +/- 2.3 mV for Aspirin NPs and blank NPs respectively. From the LC-MS/MS analysis, % Encapsulation Efficiency was determined to be 74.7% and a release pattern suggesting 82.3% cumulative release was achieved at 72 hours and 84.2% at 120 hours. We observed 50% of the release of the drug around 2.6 hours. The single dose pharmacokinetics study revealed that Aspirin detected in the blood samples at 1 hour, 3 hours, and 6 hours are 190 ng, 240 ng, and 170 ng, respectively. In the brain samples, Aspirin at 1 hour, 3 hours and 6 hours was 0.074 ug, 19.56 ug, and 0.17 ug, respectively. Additionally, we observed that the aspirin concentration is at its maximum at 3 hours and cleared out > 90% by 6 hours of administration.

Conclusion: In conclusion, we successfully formulated brain-targeted Aspirin NPs using microfluidics. We were successful in delivering Aspirin NPs using intranasal administration. Effective concentrations of Aspirin were detected in the mice's brain tissue and blood serum using LC-MS/MS. Based on the data, since the amount of drug detected in the brain tissue was high at all time points compared to the blood samples, the brain-targeted intranasal delivery of the NPs reached and sustained in the brain tissue with minimal concentrations of the drug in the systemic circulation, with a >90% clearance by 6 hours of administration.

References: 1. 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.
2. Pu L, Wang L, Zhang R, Zhao T, Jiang Y, Han L. Projected Global Trends in Ischemic Stroke Incidence, Deaths and Disability-Adjusted Life Years From 2020 to 2030: Stroke 2023;54:1330–9. https://doi.org/10.1161/STROKEAHA.122.040073.

Figure 1: Graphical Abstract. Aspirin drug was encapsulated in BSA NPs using microfluidics assisted nanoprecipitation method. The NPs were characterized for size, PDI, zeta potential, %EE and release pattern. The NPs were administered intranasally into mice and single dose pharmacokinetic analysis was performed using dMRM method on LC-MS/MS as a quantitative analytical tool.

Table 1: Characterization results for Aspirin-loaded BSA NPs and blank BSA NPs illustrating size (nm), PDI and zeta potentials (mV).