PF4 mRNA delivery system administered via nasal cavity, its preparation method and application

Abstract

This invention discloses a nasal delivery system for PF4 mRNA, comprising mesenchymal stem cell exosomes encapsulating PF4 mRNA and pharmaceutically acceptable excipients, wherein the nucleotide sequence of the PF4 mRNA is shown in SEQ NO:1. This invention utilizes MSC-Exo to deliver PF4 mRNA via nasal administration. Thus, the MSC-Exo encapsulating PF4 mRNA can bypass the BBB and efficiently enter the brain through the olfactory bulb. Furthermore, PF4 mRNA and MSC-Exo can exert a synergistic effect, significantly reducing Aβ-induced inflammatory responses and improving cognitive function in AD mice. The nasal delivery system for PF4 mRNA provided by this invention can be used to prepare drugs for treating neurodegenerative diseases, preferably, for preparing drugs for treating Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a nasal PF4 mRNA delivery system, its preparation method and application. Background Technology

[0002] mRNA gene therapy is one of the cutting-edge technologies in the current biomedical field, and its prospects for clinical application are constantly improving. For example, mRNA vaccines are effective in preventing viral infections and have good safety profiles; mRNA tumor vaccines, by encoding tumor-specific antigens or immunostimulatory factors, can activate the body's immune system and have shown certain efficacy in various tumors such as melanoma and glioblastoma; at the same time, mRNA can guide cells to synthesize growth factors and cytokines, promoting tissue regeneration and repair, and has broad prospects in the fields of tissue regeneration and organ repair. However, the success of mRNA therapy largely depends on finding a suitable delivery system that can safely, efficiently, and stably convert genetic material into functional proteins. Among the various existing delivery methods, lipid nanoparticles (LNPs) are currently the preferred mRNA delivery system. However, ionizable cationic lipids, one of the main components of LNPs, are considered to be a major factor leading to cytotoxicity, and polyethylene glycol (PEG) lipids in LNPs are prone to triggering immune responses. In addition, LNPs also have drawbacks such as limited blood-brain barrier (BBB) ​​penetration, low cellular uptake efficiency, and insufficient in vivo stability and pharmacokinetics.

[0003] Mesenchymal stem cell exosomes (MSC-Exo) are considered a promising alternative to stem cell transplantation therapy. They can reduce inflammation by inhibiting microglia activation, reduce dendritic spine loss, promote neuronal regeneration, and improve cognitive function. This makes MSC-Exo a potential candidate for the treatment of neurodegenerative diseases. However, the limited efficacy of MSC-Exo monotherapy restricts its clinical application. Therefore, further exploration is needed to improve the efficacy of MSC-Exo.

[0004] Studies have found that the circulating levels of the platelet-derived chemokine platelet factor 4 (PF4) are lower in plasma formulations from aged mice and humans compared to younger mice and humans. Furthermore, administration of exogenous PF4 can alleviate age-related hippocampal neuroinflammation in aging mice, induce molecular changes related to synaptic plasticity, and improve cognition. Chinese patent CN117384857B discloses a PF4-modified MSC-Exo, which utilizes adeno-associated virus to transfect the optimized PF4 gene into MSCs. The MSCs then secrete the PF4-modified MSC-Exo, which can improve cognitive function in aged mice by inhibiting the inflammatory response of microglia. However, this method of preparing PF4 gene-modified MSC-Exo through endogenous loading has the following drawbacks: (1) The entire preparation process is cumbersome and time-consuming; (2) There is a risk that the adeno-associated virus genome may be randomly inserted into the MSC genome, which may interfere with the normal function of MSCs, and there may even be a long-term potential mutation risk, which may indirectly affect the quality of Exo secreted by MSCs and pose certain safety issues; (3) The amount of PF4 protein carried by Exo is affected by the state of MSCs themselves (such as proliferation and metabolism), and it is difficult to control precisely. At the same time, Exo carries PF4 protein pre-synthesized by MSCs. After these proteins enter the host cell, they will be gradually degraded by the host cell metabolism. The effect time is short, and continuous delivery of exosomes is required to maintain the effect. It is a "one-time supply", which ultimately leads to the limited efficacy of the PF4 gene-modified mesenchymal stem cell exosomes.

[0005] Currently, the main methods of drug administration for neurological diseases include intravenous injection, intrathecal injection, and stereotactic brain injection. However, all of these methods have drawbacks such as being invasive and difficult to perform. Furthermore, the presence of the brain barrier (BBB) ​​limits the concentration of drugs that can accumulate in the brain. Summary of the Invention

[0006] The main objective of this invention is to provide a PF4 mRNA delivery system via nasal administration to solve at least one of the aforementioned technical problems.

[0007] According to one aspect of the present invention, a nasal PF4 mRNA delivery system is provided, comprising mesenchymal stem cell exosomes encapsulating PF4 mRNA and pharmaceutically acceptable excipients, wherein the nucleotide sequence of the PF4 mRNA is as shown in SEQ NO:1.

[0008] This invention utilizes MSC-Exo to deliver PF4 mRNA via nasal administration. Thus, mesenchymal stem cell exosomes encapsulating PF4 mRNA (hereinafter referred to as mPF4@Exo) can bypass the BBB, efficiently enter the brain through the olfactory bulb, and reach the hippocampus and cortex. The mPF4@Exo provided by this invention can induce high expression of functional PF4 protein in the hippocampus and cortex, and synergistically work with MSC-Exo to significantly improve the symptoms of neurodegenerative diseases by reducing inflammatory responses, promoting neuronal regeneration, and inducing molecular changes related to synaptic plasticity. The nasal PF4 mRNA delivery system provided by this invention can be used to prepare drugs for treating neurodegenerative diseases, preferably, drugs for treating Alzheimer's disease.

[0009] In some implementations, mesenchymal stem cell exosomes encapsulated with PF4 mRNA are obtained primarily by transfecting PF4 mRNA into mesenchymal stem cell exosomes.

[0010] In some embodiments, the transfection method may be selected from at least one of chemical reagent transfection, electroporation, ultrasound, and freeze-thaw cycles.

[0011] In some embodiments, the dosage form of the PF4 mRNA delivery system can be any medically acceptable nasal administration formulation, preferably a nasal drop. The dosage form can be prepared using conventional methods.

[0012] In some implementations, the pharmaceutically acceptable excipient is physiological saline.

[0013] In some implementations, pharmaceutically acceptable excipients may also include one or more of the following, as needed: pH adjusters, preservatives, stabilizers, antioxidants, etc.

[0014] According to another aspect of the present invention, a method for preparing a PF4 mRNA delivery system for intranasal administration is provided, comprising the following steps:

[0015] Isolate and extract exosomes from mesenchymal stem cells;

[0016] PF4 mRNA was transfected into mesenchymal stem cell exosomes to obtain mesenchymal stem cell exosomes encapsulated with PF4 mRNA.

[0017] Mesenchymal stem cell exosomes encapsulated with PF4 mRNA were formulated into a nasal administration formulation together with pharmaceutically acceptable excipients.

[0018] In some embodiments, the method for separating and extracting mesenchymal stem cell exosomes can employ any method known in the art capable of separating and extracting cell exosomes, including but not limited to at least one of ultracentrifugation, ultrafiltration, immune capture, polymer precipitation, density gradient centrifugation, tangential flow filtration, microfluidics, affinity chromatography, and size exclusion.

[0019] In some implementations, mesenchymal stem cell exosomes can be separated and extracted using ultracentrifugation, including the following steps:

[0020] (1) Collect the supernatant of the mesenchymal stem cell culture medium. The obtained supernatant is 2,000× g Centrifuge at 4℃ for 10 min to remove dead cells and collect the supernatant;

[0021] (2) Take the supernatant obtained in step (1), 10,000 × g Centrifuge at 4℃ for 30 min to remove apoptotic bodies and collect the supernatant;

[0022] (3) Take the supernatant obtained in step (2), 100,000 × g Centrifuge at 4℃ for 70 min, and collect the precipitate; this is the final product.

[0023] In some embodiments, the step may also include: resuspending the precipitate obtained in step (3) with PBS, 100,000 × g Purify the exosomes by centrifuging at 4℃ for 70 min and collect the precipitate.

[0024] The beneficial effects of this invention include:

[0025] (1) This invention utilizes MSC-Exo to deliver PF4 mRNA. MSC-Exo has excellent biocompatibility and low immunogenicity, good pharmacokinetic properties and efficient cellular uptake ability, which can efficiently deliver PF4 mRNA into cells and make it efficiently and stably expressed as PF4 protein to exert its effects.

[0026] (2) The mPF4@Exo, MSC-Exo and PF4 mRNA provided by the present invention can play a synergistic role in reducing inflammatory response, inducing molecular changes related to synaptic plasticity and promoting neuronal regeneration, thereby significantly improving the cognitive function of AD patients.

[0027] (3) The PF4 mRNA delivery system provided by the present invention can administer mPF4@Exo via nasal administration, which can not only greatly improve patient compliance, but also bypass the BBB and efficiently enter the brain through the olfactory bulb to exert therapeutic effects.

[0028] (4) In this invention, PF4 mRNA is directly transfected into MSC-Exo to prepare mPF4@Exo. The entire synthesis process is simple and convenient, takes little time, and does not require genetic modification of MSCs, thus reducing safety risks. The obtained mPF4@Exo, after PF4 mRNA enters the host cell with MSC-Exo, can serve as a template to guide the host cell to continuously synthesize PF4 protein and exert its effects. It is a "long-term supply" and can significantly improve the therapeutic effect. Attached Figure Description

[0029] Figure 1 Figure 1 shows the physical characterization results of MSC-Exo and mPF4@Exo. In this figure, (A) and (D) are transmission electron micrographs of MSC-Exo and mPF4@Exo, respectively; (B) and (C) are particle size diagrams of MSC-Exo and mPF4@Exo; and (E) and (F) are Zeta potential diagrams of MSC-Exo and mPF4@Exo.

[0030] Figure 2 Expression of exosome markers CD9, CD63, CD81, and Calnexin (negative) proteins in MSCs, MSC-Exo, and mPF4@Exo;

[0031] Figure 3 The Western blot analysis and corresponding statistical analysis of PF4 protein expression in BV2 cells after treatment with PBS, MSC-Exo and mPF4@Exo for 48 h are shown in the figure.

[0032] Figure 4 The Western blot analysis and corresponding statistical analysis of PF4 protein expression in HT22 cells after treatment with PBS, MSC-Exo and mPF4@Exo for 48 h are shown in the figure.

[0033] Figure 5 and Figure 6 The images are, in order, horizontal plane images of brain slices from 5×FAD mice 12 h after nasal administration of PBS and PKH26@Exo (blue represents cell nuclei, red represents PKH26 fluorescent dye).

[0034] Figure 7 The fluorescence images and corresponding fluorescence intensity statistical analysis of isolated brain tissue from 5×FAD mice 12 h after nasal instillation of PBS and DiR@Exo.

[0035] Figure 8 The results of ELISA detection of inflammatory factors (IL-1β, TNF-α) in cell supernatant after BV2 cells were treated with different drugs for 24 h.

[0036] Figure 9The results of the water maze experiment for mice treated with different drugs are shown below. (A) is a representative diagram of the water maze experiment for mice treated with different drugs, (B) is the statistical result of the number of times different mice crossed the platform, and (C) is the statistical result of the time different mice spent in the platform quadrant.

[0037] Figure 10 The results of the Y-maze novel arm exploration experiment for mice treated with different drugs are shown below. (A) is a schematic diagram of the Y-maze novel arm exploration experiment, (B) is a representative diagram of the Y-maze novel arm exploration experiment for different mice, (C) is the statistical result of the number of times different mice entered the novel arm, (D) is the statistical result of the time different mice spent in the novel arm, and (E) is the statistical result of the distance traveled in the novel arm for different mice. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the embodiments. The embodiments are for illustrative purposes only and do not limit the invention in any way. Unless otherwise specified, the raw materials and reagents used in the embodiments are conventional products that can be obtained commercially; experimental methods that do not specify specific conditions in the embodiments are generally performed under conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0039] Example 1

[0040] (1) Extraction of MSC-Exo

[0041] S1. Take the culture medium (serum-free MSC medium, Beijing Youkang Biotechnology Co., Ltd., NC0103) after culturing MSCs for 48 h, 300× g Centrifuge at 4℃ for 10 min and collect the supernatant (to remove live cells).

[0042] S2. Take the supernatant obtained in S1, 2,000 × g Centrifuge at 4℃ for 10 min and collect the supernatant (to remove dead cells).

[0043] S3. Take the supernatant obtained in S2, 10,000 × g Centrifuge at 4℃ for 30 min and collect the supernatant (to remove apoptotic bodies).

[0044] S4. Take the supernatant obtained in S3, 100,000 × g Centrifuge at 4℃ for 70 min and collect the precipitate (the precipitate is the exosome).

[0045] S5. Resuspend the exosomes obtained from S4 in PBS, and then 100,000× g Centrifuge at 4℃ for 70 min, collect the precipitate to obtain purified MSC-Exo, resuspend MSC-Exo with a small amount of PBS, and store at -80℃ for later use.

[0046] (2) PF4 mRNA transfection

[0047] The PF4 mRNA (nucleotide sequence shown in SEQ NO:1) was transfected into the MSC-Exo obtained in step (1) using the Exo-Fect exosome transfection kit (System Biosciences (SBI), USA, EXFT20A-1), including the following steps:

[0048] Take 10 μL of Exo-Effect Reagent solution, 5 μL of PF4 mRNA (concentration 2 μg / μL), 100 μL of MSC-Exo (102 μg / mL), and 35 μL of PBS to form a total transfection reaction system of 150 μL. Mix the components thoroughly by gently tapping or flipping (do not vortex). Incubate the exosome transfection solution in a shaker at 37°C for 10 min, and then immediately place the test tube on ice to stop the reaction. Add 30 μL of ExoQuick-TC solution to the transfected exosome sample suspension and mix thoroughly by flipping (do not vortex). Then place the transfected exosome sample on ice (or at 4°C) for 30 min. After 30 min, centrifuge the exosome transfected sample at 13,000~14,000 rpm for 3 min. Finally, remove the supernatant to obtain mPF4@Exo.

[0049] Resuspend mPF4@Exo in a small amount of PBS and store at -80°C for later use.

[0050] Experimental Example 1: Characterization of mPF4@Exo

[0051] (1) The morphology of MSC-Exo and mPF4@Exo was observed by transmission electron microscopy (TEM). The particle size and potential of MSC-Exo and mPF4@Exo were determined by Malvern nanoparticle size potentiometry.

[0052] The results are as follows Figure 1 As shown. From Figure 1 As shown in Figures A and D, both MSC-Exo and mPF4@Exo exhibit a cup-shaped morphology. After transfection with PF4 mRNA, the morphology and integrity of mPF4@Exo remain unchanged. The particle sizes of MSC-Exo and mPF4@Exo (…) are also different. Figure 1 (Figures B and C in the diagram) and potential ( Figure 1 As can be seen from Figures E and F, compared with MSC-Exo, after transfection with PF4 mRNA, the particle size of mPF4@Exo increased and the potential also changed significantly, indirectly proving that PF4 mRNA was successfully loaded into MSC-Exo.

[0053] (2) Detection of exosome marker proteins by Western blotting

[0054] MSCs, MSC-Exo, and mPF4@Exo were lysed with RIPA buffer (Solepro Biosciences, R0010), and the protein concentration in the extract was quantified using a BCA protein assay kit (KGI Biosciences, KGB2101). Approximately 25 μg of protein was electrophoresed on 10% SDS-PAGE, then transferred to a PVDF membrane for 2 h, blocked with 5% (w / v) skim milk powder solution for 2 h at room temperature, and incubated overnight at 4°C with different primary antibodies. The membrane was then incubated with a suitable secondary antibody for 1 h, and the expression of exosome markers CD9, CD63, CD81, and Calnexin (negative) proteins was detected using an electrochemiluminescence (ECL) assay kit (Merck Millipore, WBKLS0500).

[0055] Among them, anti-CD9 rabbit monoclonal antibody (Proteintech, 84142-1-RR, 1:5000), anti-CD63 rabbit monoclonal antibody (Proteintech, 25682-1-AP, 1:1000), anti-CD81 mouse monoclonal antibody (Proteintech, 66866-1-Ig, 1:3000), anti-Calnexin rabbit monoclonal antibody (Proteintech, 10427-2-AP, 1:5000), horseradish peroxidase (HRP)-labeled anti-rabbit IgG antibody (CST, 7074S, 1:2000), and horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody (CST, 7076S, 1:2000).

[0056] The results are as follows Figure 2 As shown. From Figure 2 As can be seen, both MSC-Exo and mPF4@Exo express exosome marker proteins CD9, CD63, and CD81, while the negative protein Calnexin is not expressed, indirectly proving the successful extraction and synthesis of MSC-Exo and mPF4@Exo.

[0057] Experimental Example 2: Detection of PF4 protein expression by Western blotting

[0058] HT22 cells (neurons) or BV2 cells (microglia) were cultured using standard techniques. HT22 cells or BV2 cells were seeded into 6-well plates at a density of 2 × 10⁶ cells per well. 5Cells were collected, and when the cell density reached 70%, PBS (Control, 2 mL), MSC-Exo (final concentration: 60 μg / mL), and mPF4@Exo (final concentration: MSC-Exo 60 μg / mL, PF4 mRNA 10 μg / mL) were added to the culture medium and co-incubated with HT22 or BV2 cells for 48 h. Cells were then collected and lysed with RIPA buffer (Solepro Biosciences, R0010). The protein concentration in the extract was quantified using a BCA protein assay kit (KGI Biosciences, KGB2101). Approximately 25 μg of protein was electrophoresed on 10% SDS-PAGE, then transferred to a PVDF membrane for 1 h, blocked with 5% (w / v) skim milk powder solution for 2 h at room temperature, and incubated overnight at 4°C with different primary antibodies. The membrane was then incubated with a suitable secondary antibody for 1 hour, and the expression of PF4 protein in HT22 or BV2 cells after different treatments was visualized using an electrochemiluminescence (ECL) assay kit (Merck Millipore, WBKLS0500). β-actin was used as an internal control for total protein.

[0059] Among them, anti-PF4 rabbit monoclonal antibody (Abcam, ab303494, 1:2000), anti-β-actin rabbit monoclonal antibody (Proteintech, 20536-1-AP, 1:4000), and horseradish peroxidase (HRP) labeled anti-rabbit IgG antibody (CST, 7074S, 1:2000).

[0060] The results are as follows Figures 3-4 As shown.

[0061] Compared with the Control and MSC-Exo groups, mPF4@Exo treatment significantly increased the expression of PF4 protein in HT22 and BV2 cells. PF4 protein expression increased by about 7-fold in HT22 cells and by about 5-fold in BV2 cells, indicating that MSC-Exo can efficiently deliver PF4 mRNA into cells and make it efficiently and stably expressed as PF4 protein.

[0062] Experimental Example 3: Evaluation of the Efficacy of Nasal Intracavitary Drug Delivery

[0063] (1) Fluorescence imaging experiment of isolated brain slices

[0064] Six-month-old 5×FAD mice were intranasally instilled with PBS and PKH26 (Yumeibo Biotechnology Co., Ltd., UR52302) labeled MSC-Exo (PKH26@Exo) (containing MSC-Exo 300 μg / mL, PKH26 50 μM). The intranasal instillation volume was 30 μL / mouse, and 3 mice were in each group. Brain tissue was collected 12 h after intranasal instillation, cut into 20 μm tissue sections, stained with DAPI, and then observed using a fluorescence confocal laser microscope.

[0065] The results are as follows Figures 5-6 As shown.

[0066] Compared with the PBS group, the PKH26@Exo group showed obvious red PKH26 infiltration in the olfactory bulb, hippocampus and cortex of mice, indicating that PKH26@Exo can bypass the BBB by nasal administration and MSC-Exo can successfully deliver PKH26 to the hippocampus and cortex of mice through the olfactory bulb.

[0067] (2) Fluorescence imaging experiment of isolated brain tissue

[0068] Six-month-old 5×FAD mice were intranasally instilled with PBS and DiR (Yumeibo Biotechnology Co., Ltd., UR21017) labeled MSC-Exo (DiR@Exo) (containing MSC-Exo 300 μg / mL, DiR 20 μM). The intranasal instillation volume was 30 μL / mouse, with 3 mice in each group. Brain tissue was harvested 12 h after intranasal instillation. The isolated mouse brain tissue was imaged using an in vivo imaging system (IVIS Lumina S5), and the fluorescence imaging results and mean radioactivity intensity were reported.

[0069] The results are as follows Figure 7 As shown.

[0070] Compared with the PBS group, the brain tissue of mice in the DiR@Exo group showed obvious red DiR fluorescent dye infiltration, indicating that DiR@Exo, administered via nasal drops, can bypass the BBB, and MSC-Exo successfully delivers DiR to the mouse brain tissue through the olfactory bulb.

[0071] Experiment 4: Effect of mPF4@Exo on the expression levels of inflammatory factors in Aβ-induced BV2 cells.

[0072] BV2 cells were cultured using standard techniques, and then seeded into 6-well plates at a density of 2 × 10⁶ cells per well. 5When the cell density reached 70%, Aβ42 (final concentration: 25 μM) and different preparations (PBS (2 mL), 60 μg / mL (final concentration) MSC-Exo, mPF4@Exo (final concentration: MSC-Exo 60 μg / mL, PF4 mRNA 10 μg / mL) and 60 μg / mL (final concentration) Exo-PF4-optimized exosomes (preparation method according to Chinese patent CN117384857B, hereinafter referred to as "Exo-PF4-optimized")) were co-incubated with BV2 cells for 24 h, and then the cell supernatant was collected for the detection of inflammatory factors.

[0073] The levels of IL-1β and TNF-α in cell supernatants were detected according to the instructions of the mouse interleukin 1β ELISA kit (Soleb Biosciences, SEKM-0002) and the mouse tumor necrosis factor α ELISA kit (Soleb Biosciences, SEKM-0034). The test sample or standard solutions of different concentrations were added to each well of a 96-well ELISA plate and incubated at 37°C for 90 min. Unbound substances were washed away. Primary antibody solution was added and incubated at 37°C for 60 min, then the solution was discarded. HRP-labeled secondary antibody was added and incubated at 37°C for 30 min. Unbound enzyme-labeled antibody was thoroughly washed away. The absorbance was measured at 450 nm after substrate development. The levels of inflammatory factors IL-1β and TNF-α in the samples were calculated based on the standard curve.

[0074] The results are as follows Figure 8 As shown.

[0075] Compared with the Aβ model group, MSC-Exo could reduce the levels of IL-1β and TNF-α in the cell supernatant to a certain extent. After transfection with PF4 mRNA, the levels of IL-1β and TNF-α in the cell supernatant of the mPF4@Exo group were further significantly reduced. Furthermore, compared with the optimized control drug Exo-PF4, mPF4@Exo could further significantly reduce the levels of IL-1β and TNF-α in the cell supernatant. These results indicate that MSC-Exo can inhibit the secretion of Aβ-induced inflammatory factors IL-1β and TNF-α in BV2 cells to a certain extent, and that MSC-Exo and PF4 mRNA can exert a synergistic effect, further enhancing the anti-inflammatory effect after mPF4@Exo administration. Moreover, compared with the optimized Exo-PF4 group, the mPF4@Exo group showed better anti-inflammatory effects.

[0076] Experimental Case 5: Evaluation of the therapeutic effect of mPF4@Exo on AD

[0077] (1) Water maze experiment

[0078] Six-month-old C57BL / 6 mice and 5×FAD mice were randomly divided into five groups of eight mice each: WT group (30 μL of PBS intranasal drops on normal C57BL / 6 mice), AD group (30 μL of PBS intranasal drops on 5×FAD mice), AD+MSC-Exo group (300 μg / mL MSC-Exo intranasal drops on 5×FAD mice), AD+mPF4@Exo group (30 μL of mPF4@Exo intranasal drops containing 300 μg / mL MSC-Exo and 80 μg / mL PF4 mRNA in 5×FAD mice), and AD+Exo-PF4-optimized group (300 μg / mL Exo-PF4-optimized intranasal drops on 5×FAD mice). The treatment was administered three times a week for three weeks.

[0079] After intranasal administration, each mouse was placed in a circular swimming pool with a diameter of 120 cm, containing a platform approximately 1 cm above the water surface (temperature: 22℃). For the first 5 days, each mouse was trained to swim to the platform within 60 seconds. On the 6th day, the mice were trained to swim freely for 60 seconds without the platform, and their swimming path was recorded using a video tracking system.

[0080] The results are as follows Figure 9 As shown.

[0081] from Figure 9 Figures B (number of platform crossings) and C (platform quadrant dwell time) show that MSC-Exo nasal drops increased the number of platform crossings and the dwell time in the platform quadrant in AD mice. Furthermore, after mPF4@Exo nasal drops, the number of platform crossings and the dwell time in the platform quadrant in AD mice further increased, approaching the levels of WT mice. Compared with the optimized control drug Exo-PF4, the number of platform crossings and the dwell time in the platform quadrant in AD mice significantly increased after mPF4@Exo nasal drops. These results indicate that MSC-Exo nasal drops can improve cognitive function in AD mice to a certain extent, and that MSC-Exo and PF4 mRNA can synergistically improve cognitive levels in AD mice after mPF4@Exo nasal drops, approaching the cognitive levels of WT mice. Moreover, compared with the optimized Exo-PF4 group, mPF4@Exo nasal drops showed a stronger ability to improve cognitive function in AD mice.

[0082] (2) Y-maze novel arm exploration experiment

[0083] The experimental grouping and drug administration regimen are the same as "(1) Water Maze Experiment".

[0084] The Y-maze was cleaned with alcohol. The three arms of the Y-maze were randomly assigned as the starting arm, the novel arm, and other arms, with the central triangular area as a blank area. The novel arm was closed with a barrier. After intranasal administration, each mouse was gently placed in the starting arm with its head facing the center of the Y-maze. After acclimatization for 5 minutes, the animals were returned to their cages. One hour later, the barrier on the novel arm was removed, and each mouse was gently placed in the same position on the starting arm with its head facing the center of the Y-maze. The test lasted 5 minutes, and mouse activity was recorded using software.

[0085] The results are as follows Figure 10 As shown.

[0086] from Figure 10 Figures C (number of entries into the neo-paraarm), D (time spent in the neo-paraarm), and E (distance traveled in the neo-paraarm) show that MSC-Exo intranasal drops can increase the number of entries, time spent in the neo-paraarm, and distance traveled in AD mice to a certain extent. Furthermore, after mPF4@Exo intranasal drops, these numbers increased further, approaching the levels of WT mice. Compared to the optimized control drug Exo-PF4, mPF4@Exo intranasal drops significantly increased the number of entries, time spent in the neo-paraarm, and distance traveled in AD mice. These results indicate that MSC-Exo intranasal drops can improve the cognitive function of AD mice to a certain extent, and that MSC-Exo and PF4 mRNA work synergistically after mPF4@Exo intranasal drops, further improving the cognitive level of AD mice, approaching the level of WT mice. Moreover, compared to the optimized Exo-PF4 group, mPF4@Exo intranasal drops showed a stronger ability to improve the cognitive function of AD mice.

[0087] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A PF4 mRNA delivery system administered via nasal cavity, characterized in that, The product comprises mesenchymal stem cell exosomes encapsulated with PF4 mRNA and pharmaceutically acceptable excipients, wherein the nucleotide sequence of the PF4 mRNA is shown in SEQ NO:1, the mesenchymal stem cell exosomes encapsulated with PF4 mRNA are obtained by transfecting the PF4 mRNA into the mesenchymal stem cell exosomes, and the dosage form of the PF4 mRNA delivery system is a nasal drop.

2. The PF4 mRNA delivery system according to claim 1, characterized in that, The transfection method is selected from at least one of chemical reagent transfection, electroporation, ultrasound, and freeze-thaw cycles.

3. The PF4 mRNA delivery system according to claim 2, characterized in that, The pharmaceutically acceptable excipient is physiological saline.

4. The method for preparing the PF4 mRNA delivery system according to any one of claims 1 to 3, characterized in that, Includes the following steps: Isolate and extract exosomes from mesenchymal stem cells; PF4 mRNA was transfected into mesenchymal stem cell exosomes to obtain mesenchymal stem cell exosomes encapsulated with PF4 mRNA. Mesenchymal stem cell exosomes encapsulated with PF4 mRNA were formulated into a nasal administration formulation together with pharmaceutically acceptable excipients.

5. The preparation method according to claim 4, characterized in that, The method for separating and extracting mesenchymal stem cell exosomes is selected from at least one of ultracentrifugation, ultrafiltration, immune capture, polymer precipitation, density gradient centrifugation, tangential flow filtration, microfluidics, affinity chromatography, and size exclusion.

6. The preparation method according to claim 5, characterized in that, The method for isolating and extracting mesenchymal stem cell exosomes includes the following steps: (1) Collect the supernatant of the mesenchymal stem cell culture medium. The obtained supernatant is 2,000× g Centrifuge at 4℃ for 10 min and collect the supernatant; (2) Take the supernatant obtained in step (1), 10,000 × g Centrifuge at 4℃ for 30 min and collect the supernatant; (3) Take the supernatant obtained in step (2), 100,000 × g Centrifuge at 4℃ for 70 min, and collect the precipitate; this is the final product.

7. The use of the PF4 mRNA delivery system according to any one of claims 1 to 3 in the preparation of a medicament for treating neurodegenerative diseases, characterized in that, The neurodegenerative disease mentioned is Alzheimer's disease.

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