Intranasal Administration of Fluorophore-labelled Platelet Secretome and Isolated Platelet-derived Extracellular Vesicle to The Brain

Background: Aging is a phenomenon associated with a gradual reduction in the effectiveness of mechanisms involved in the maintenance of homeostasis, which leads to an increased risk of various pathologies and death. Aging and cognitive decline take place in a part of the brain located deep within the medial temporal lobe, named hippocampus. Recently, it was found that trophic factors that are important for neuro-regenerative and cognitive functions are stored in blood platelets. However, the value of biomaterials containing blood-derived trophic factors and extracellular vesicles (EVs) to treat brain aging is still unclear. In this research, two main samples derived from clinical-grade platelet concentrates were isolated, namely Human Platelet Pellet Lysate (HPPL) and nanosized platelet-derived EVs (p-EV). Currently, HPPL and p-EV, administered intranasally, are investigated as potential brain rejuvenating agent. Understanding their biodistribution in the brain is important to establish the therapeutic dose, capacity to time estimation of the drug to reach targeted brain region, including the hippocampus site.
Aim: Understand where the HPPL and p-EVs migrate into the brain upon intranasal administration, and in particular whether they can reach the hippocampus as the main site of loss of memory and cognition occurring during aging.
Material and Methods: The direct labeling of both platelet materials was done using ATTO 488 NHS Ester fluorophore prior to intranasal procedure. The labelling procedure was validated by BCA (Bicinchoninic Acid Assay) assay, fluorescence detection, SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamid Gel Electrophoresis), NTA (Nanoparticle Tracking Analysis), DLS (Dynamic Light Scattering). Furthermore, the Size Exclusion Chromatography (SEC) using 5 mL Sephadex G-25 chromatography was performed to separate the labeled samples from the free dye to prevent any artefacts during brain imaging. The intranasal procedure was done by administration of 60 µL of labeled HPPL and p-EV, as well as the dye-only (control) into 3 mice which were sacrificed 2 hours after the intranasal procedure, perfused, before brain observation using ImageXpress Pico Automated Imaging System.
Results: In this research, both platelet-derived materials can be adjusted to pH 8.3-8.5 to optimize the binding affinity with Atto 488 NHS Ester. The SEC procedure generated fractions 6, 7, and 8 to have the highest protein and fluorescence content from BCA assay and florescence detection methods. The validation procedures followed by the SDS-PAGE results that revealed HPPL and p-EV to possess the same protein profiling with the original or unlabeled condition. Furthermore, the NTA and DLS analysis consecutively unveil the comparison of native and labeled p-EV’s condition to be in the same particle number and particle size distribution profile which highlighting no changes after labeling procedures. Therefore, these labeled HPPL and p-EV fractions were selected and pooled from intranasal administration to the mice. The brain imaging results of intranasally administered labeled HPPL and p-EV have shown detectable fluorescence of both materials in the hippocampus after 2 hours of intranasal administration procedures, compared to the control that has no indication of fluorescence.
Conclusion: The current procedures have validated that 60 µL of HPPL and p-EV can be administered through intranasal pathway to diffuse into the brain (most specifically the hippocampus) after 2 hours. This biolabeling procedure is important to better understand the potential translational success of HPPL and p-EV and to help define therapeutic dose, biodistribution timing, and potential side effects.
Keywords: brain aging, brain rejuvenation, extracellular vesicle, human platelet lysate, biolabeling.