臺灣博碩士論文加值系統

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.

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.

TABLE OF CONTENT

Abstract..........................................................................................iii
Acknowledgement................................................................................v
Table of Content................................................................................vi
List of Abbreviations........................................................................viii
List of Figures..................................................................................ix
Chapter 1. Introduction........................................................................1
1.1. The Voyage of Life: Inevitable Human Aging.......................................1
1.2. The Success of Brain-Targeted Pharmaceutics......................................3
1.2.1. The Incredible Brain’s Proxy Firewalls: BBB and BCSFB.......................3
1.2.2. Breaking The Brain Proxy Firewalls is Worth the Risk.........................5
1.2.3. Intranasal Administration.............................................................7
1.2. The Science Behind Vampires: How Blood May Keep You Young?.................7
1.3. Platelet-Derived Secretomes: The “Game Changers” in Brain
Rejuvenation?..............................................................................................................8
1.3.1. Human Platelet Pellet Lysate (HPPL)..............................................8
1.3.2. Profile of Extracellular Vesicle: Recent Paradigm in Therapeutic Potential
1.3.3. Platelet-derived EV (p-EV).........................................................12
1.4. Labeling of HPPL and EV: Methods and Pitfall to Conquer..................13
1.5. Size Exclusion Chromatography (SEC).............................................16
1.6. Objective of The Research.............................................................18
Chapter 2. Material and Methods...........................................................19
2.1. Experimental Materials and Equipments..........................................19
2.2. Overall Experimental Design...........................................................23
2.3.1. PC Collection...........................................................................26
2.3.2. HPPL and p-EV Preparation.......................................................26
2.3.3. HPPL Labeling & Validation......................................................26
2.3.4. p-EV (NPMP) Labeling & Validation.............................................31
2.4. Intranasal administration of ATTO 488 NHS Ester-Labeled HPPL and p-EV
to the Mice........................................................................................31
2.5 Perfusion and Brain Isolation..........................................................32
2.6. Brain slicing and Fluorescence Detection...........................................32
Chapter 3. Results.............................................................................34
3.1. HPPL Labeling Validation.............................................................34
3.2. P-EV Labeling Validation..............................................................37
3.3. Fluorescence detected in Hippocampus.............................................44
Chapter 4. Discussion........................................................................47
4.1. HPPL Labeling Validation............................................................48
4.2. p-EV Labeling Validation..............................................................49
Chapter 5. Conclusion........................................................................55
References.......................................................................................56

List of Figures

Figure 1.1. Multifaceted deteriorations due to aging in the hippocampus……………2
Figure 1.2. The Anatomy of BBB and BCSFB……………………………………….5
Figure 1.3. Current Approaches of Brain-Targeted Drug Delivery…………………..6
Figure 1.4. Composition of The Whole Blood…………………………………….….9
Figure 1.5. Human Platelet Pellet Lysate (HPPL) Preparation………………………10
Figure 1.6. Platelet-derived EV (p-EV) Preparation…………………………………12
Figure 1.7. ATTO 488 NHS Ester……………………………………………………15
Figure 1.8. NHS Ester’s chemical bond to amine……………………………………15
Figure 1.9. Sephadex G-25 (5 mL)…………………………………………………..16
Figure 1.10. Size Exclusion Chromatography as Free-dye Removal Method………17
Figure 2.2.1. HPPL Labeling and Validation Procedure……………………………23
Figure 2.2.2. p-EV Labeling and Validation Procedure…………………………….24
Figure 2.2.3. In vivo study summary………………………………………………..25
Figure 3.1.1. HPPL Protein Concentration Performed by BCA Assay after
Free-dye Removal………………………………………………...........34
Figure 3.1.2. HPPL Fluorescence Intensity after Free-dye Removal…………...……35
Figure 3.1.3. The Morphological Difference of Fractions composed of the
HPPL-ATTO 488 NHS Ester (6th ,7th ,8th) and The Washing Materials (33th)……...35
Figure 3.1.4. SDS-PAGE of HPPL after Free-dye Removal…………………………36
Figure 3.2.1. p-EV Protein Concentration Performed by BCA Assay after
Free-dye Removal……………………………………………………...37
Figure 3.2.2. p-EV Fluorescence Intensity after Free-dye Removal…………………38
Figure 3.2.3. p-EV NTA Result Before pH Adjustment……………………………..39
Figure 3.2.4. p-EV NTA Result After pH Adjustment………………………...……..39
Figure 3.2.5. p-EV DLS Result Before pH Adjustment……………………………..40
Figure 3.2.6. p-EV DLS Result After pH Adjustment………………………………40
Figure 3.2.7. Fraction 6th of p-EV NTA Result After Free-dye Removal………….41
Figure 3.2.8. Fraction 7th of p-EV NTA Result After Free-dye Removal………….41
Figure 3.2.9. Fraction 88h of p-EV NTA Result After Free-dye Removal…………42
Figure 3.2.10. Fraction 6th of p-EV DLS Result After Free-dye Removal………….42
Figure 3.2.11. Fraction 7th of p-EV DLS Result After Free-dye Removal………….42
Figure 3.2.12. Fraction 8th of p-EV DLS Result After Free-dye Removal………….43
Figure 3.1.13. SDS-PAGE of p-EV after Free-dye Removal……………………….43
Figure 3.3.1. Hippocampus Area- Control ATTO 488 NHS Ester…………………45
Figure 3.3.2. Hippocampus Area- Labeled p-EV Administered……………………45
Figure 3.3.3. Hippocampus Area- Labeled HPPL Administered…………………...46
Figure 4.1. The Structure of Neuron Cell…………………………………………48
Figure 4.2 Human anatomy in intranasal drug delivery………………………….53
Figure 4.3. Intranasal drug delivery route sites…………………………………...54

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