Nasal delivery PF4 mRNA delivery system and preparation method and application thereof

Abstract

The invention discloses a nasal delivery PF4 mRNA delivery system, the nasal delivery PF4 mRNA delivery system comprises a mesenchymal stem cell exosome encapsulated with PF4 mRNA and a pharmaceutically acceptable auxiliary material, and the nucleotide sequence of the PF4 mRNA is as shown in SEQ NO: 1. According to the invention, the MSC-Exo is utilized to deliver the PF4 mRNA, and the administration is carried out in a nasal administration manner, so that the MSC-Exo encapsulated with the PF4 mRNA can bypass BBB to efficiently enter the brain through olfactory bulbs, and the PF4 mRNA and the MSC-Exo can play a synergistic effect, so that the inflammatory reaction induced by A beta is obviously alleviated, and the cognitive level of AD mice is improved. The nasal delivery PF4 mRNA delivery system provided by the invention can be applied to preparation of drugs for treating neurodegenerative diseases, and preferably can be applied to preparation of drugs for treating Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a nasal administration PF4 mRNA delivery system, a preparation method and application thereof. BACKGROUND

[0002] mRNA gene therapy is one of the frontiers of the current biomedical field, and its prospects in clinical application are constantly improving. For example, mRNA vaccines have shown remarkable effects and good safety in preventing viral infections; mRNA tumor vaccines can activate the body's immune system by encoding tumor-specific antigens or immune-stimulating factors, and have shown certain efficacy in melanoma, glioblastoma and other tumors; meanwhile, mRNA can guide cells to synthesize growth factors, cytokines and the like, and promote tissue regeneration and repair, thus having broad prospects in the fields of tissue regeneration and organ repair. However, the success of mRNA therapy largely depends on whether a suitable delivery system can be found, which should be able to safely and efficiently and stably convert genetic material into functional proteins. Among the existing various delivery methods, lipid nanoparticles (LNPs) are currently the preferred mRNA delivery system, but as one of the main components of LNPs, ionizable cationic lipids are considered to be the main factor causing cytotoxicity, and the polyethylene glycol (PEG) lipids in LNPs are prone to trigger immune responses in the body. In addition, LNPs also have the defects of limited blood-brain barrier (BBB) penetration ability, low cellular uptake efficiency, and insufficient in-vivo stability and pharmacokinetics.

[0003] Mesenchymal stem cell exosomes (MSC-Exo) are considered to be a promising alternative to stem cell transplantation therapy, which can reduce inflammatory responses by inhibiting microglial activation, reduce dendritic spine loss and promote neuron regeneration, and improve cognitive function, which makes MSC-Exo a potential candidate for the treatment of neurodegenerative diseases, but the single therapy of using MSC-Exo alone is limited in clinical application due to its limited efficacy, so how to improve the efficacy of MSC-Exo needs to be further explored.

[0004] The study found that the circulating level of platelet-derived chemokine platelet factor 4 (PF4) decreased in the plasma preparations of old mice and humans compared with young mice and humans, and administration of exogenous PF4 could reduce age-related hippocampal neuroinflammation in old mice, trigger synaptic plasticity-related molecular changes and improve cognition. Chinese patent CN117384857B discloses a PF4 gene modified MSC-Exo, which uses an adeno-associated virus to transfect the optimized PF4 gene into MSC, and then the MSC secretes PF4 gene modified MSC-Exo, which can improve the cognitive function of aging mice by inhibiting the inflammatory response of microglia. However, this method of preparing PF4 gene modified MSC-Exo by endogenous loading has the following defects: (1) the entire preparation process is complex and time-consuming; (2) there is a risk of random insertion of the adeno-associated virus genome into the MSC genome, which may interfere with the normal function of MSC, and even pose a long-term potential mutation risk, thereby indirectly affecting the quality of Exo secreted by MSC, and there are certain safety problems; (3) the amount of PF4 protein carried by Exo is affected by the state of MSC itself (such as proliferation and metabolism), and it is difficult to precisely control. At the same time, Exo carries PF4 protein synthesized in advance by MSC, and these proteins will be gradually degraded by host cell metabolism after entering the host cell, with a short action time, and continuous delivery of exosomes is needed to maintain the effect, which belongs to "one-time supply", ultimately resulting in limited efficacy of the prepared PF4 gene modified mesenchymal stem cell exosomes.

[0005] Currently, the administration methods for nervous system diseases mainly include intravenous injection, intrathecal injection and brain stereotactic injection, but all have the defects of invasiveness and high operation difficulty, and the accumulation concentration of drugs in the brain is limited due to the presence of BBB. SUMMARY

[0006] The main purpose of the present application is to provide a nasal administration PF4 mRNA delivery system to solve at least one of the above technical problems.

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

[0008] The application delivers PF4 mRNA by MSC-Exo and administers by intranasal administration, so that the mesenchymal stem cell exosome encapsulating PF4 mRNA (hereinafter referred to as mPF4@Exo) can bypass the BBB and enter the brain through the olfactory bulb efficiently, and then reach the hippocampus and cortex regions. The mPF4@Exo provided by the application can make the hippocampus and cortex highly express functional PF4 protein, and play a synergistic effect with MSC-Exo, significantly improve the symptoms of neurodegenerative diseases by reducing inflammatory response, promoting neuron regeneration and inducing synaptic plasticity-related molecular changes. The intranasal administration PF4 mRNA delivery system provided by the application can be applied to the preparation of a drug for treating neurodegenerative diseases, preferably, to the preparation of a drug for treating Alzheimer's disease.

[0009] In some embodiments, the mesenchymal stem cell exosome encapsulating PF4 mRNA is prepared mainly by transfecting PF4 mRNA into mesenchymal stem cell exosomes.

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

[0011] In some embodiments, the dosage form of the PF4 mRNA delivery system can be any intranasal administration preparation acceptable in medicine, preferably, nasal drops. The related dosage form can be prepared by conventional methods.

[0012] In some embodiments, the pharmaceutically acceptable adjuvant is physiological saline.

[0013] In some embodiments, the pharmaceutically acceptable adjuvant can also include one or more of pH adjusters, preservatives, stabilizers, antioxidants, etc. as needed.

[0014] According to another aspect of the application, a method for preparing an intranasal administration PF4 mRNA delivery system is provided, comprising the following steps: Isolating and extracting mesenchymal stem cell exosomes; Transfecting PF4 mRNA into mesenchymal stem cell exosomes to obtain mesenchymal stem cell exosomes encapsulating PF4 mRNA; Preparing an intranasal administration preparation by mixing the mesenchymal stem cell exosomes encapsulating PF4 mRNA and the pharmaceutically acceptable adjuvant.

[0015] In some embodiments, the method for isolating and extracting mesenchymal stem cell exosomes can use any method known in the art that can isolate and extract cell exosomes, including but not limited to at least one of ultracentrifugation, ultrafiltration, immunocapture, polymer precipitation, density gradient centrifugation, tangential flow filtration, microfluidics, affinity chromatography, molecular exclusion.

[0016] In some embodiments, the mesenchymal stem cell exosome can be separated by ultracentrifugation, comprising the following steps: (1) Collecting the supernatant of the mesenchymal stem cell culture medium, and obtaining the supernatant 2,000xg g , 4℃ centrifugation 10 min to remove dead cells, and taking the supernatant; (2) The supernatant obtained in step (1) is centrifuged at 10,000xg g , 4℃ centrifugation 30 min to remove apoptotic bodies, and taking the supernatant; (3) The supernatant obtained in step (2) is centrifuged at 100,000xg g , 4℃ centrifugation 70 min, and taking the precipitate; thus obtained.

[0017] In some embodiments, the following steps can also be included: resuspending the precipitate obtained in step (3) with PBS, 100,000xg g , 4℃ centrifugation 70 min to purify the exosome, and taking the precipitate.

[0018] The beneficial effects of the present application include: (1) The present application utilizes MSC-Exo to deliver PF4 mRNA, and MSC-Exo has excellent biocompatibility and low immunogenicity, good pharmacokinetic characteristics and high cell uptake capacity, which can efficiently deliver PF4 mRNA into cells and stably express PF4 protein to exert efficacy.

[0019] (2) The mPF4@Exo provided by the present application, MSC-Exo and PF4 mRNA can exert synergistic effect, significantly improve the cognitive function of AD patients by reducing inflammatory response, inducing synaptic plasticity-related molecular changes and promoting neuron generation.

[0020] (3) The PF4 mRNA delivery system provided by the present application can administer mPF4@Exo through nasal administration, which can greatly improve patient compliance, bypass the BBB and efficiently enter the brain through the olfactory bulb to exert efficacy, and provide therapeutic effect.

[0021] (4) The present application directly transfects PF4 mRNA into MSC-Exo to prepare mPF4@Exo, the whole synthesis process is simple and convenient, time-consuming is short, and there is no need to genetically modify MSC, which reduces the safety hazard; the prepared mPF4@Exo can be used as a template after entering the host cell with MSC-Exo, guiding the host cell to continuously synthesize PF4 protein, continuously producing effect, belonging to "long-acting supply", which can significantly improve the efficacy. BRIEF DESCRIPTION OF DRAWINGS

[0022] 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. Figure 2 Expression of exosome markers CD9, CD63, CD81, and Calnexin (negative) proteins in MSCs, MSC-Exo, and mPF4@Exo; 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. 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. 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). 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. 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. Figure 9 The 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. 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

[0023] 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.

[0024] Example 1 (1) Extraction of MSC-Exo 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). S2. Take the supernatant obtained in S1, 2,000 × g Centrifuge at 4℃ for 10 min and collect the supernatant (to remove dead cells). S3. Take the supernatant obtained in S2, 10,000 × g Centrifuge at 4℃ for 30 min and collect the supernatant (to remove apoptotic bodies). 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). 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.

[0025] (2) PF4 mRNA transfection 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: Take 10 μL Exo-Effect Reagent solution, 5 μL PF4 mRNA (concentration 2 μg / μL), 100 μL MSC-Exo (102 μg / mL), 35 μL PBS, and constitute a total transfection reaction system of 150 μL, mix the components well by flicking or overturning (do not vortex), and incubate the exosome transfection solution in a shaking bed at 37°C for 10 min. Then immediately stop the reaction by placing the test tube on ice. Add 30 μL ExoQuick-TC solution to the transfection exosome sample suspension, mix well by overturning (do not vortex), and then place the transfection exosome sample on ice (or at 4°C) for 30 min. After 30 min, centrifuge the exosome transfection sample at 13,000-14,000 rpm for 3 min, and finally remove the supernatant to obtain mPF4@Exo.

[0026] Resuspend mPF4@Exo in a small amount of PBS and store at -80°C for standby use.

[0027] Test Example 1, Characterization of mPF4@Exo (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 potential instrument.

[0028] The results are shown in Figure 1 From the A and D graphs of Figure 1 , it can be seen that the morphology of MSC-Exo and mPF4@Exo is cup-shaped, and the morphology and integrity of mPF4@Exo remain unchanged after transfection of PF4 mRNA. From the B and C graphs of the particle size Figure 1 and the E and F graphs of the potential Figure 1 of MSC-Exo and mPF4@Exo, it can be seen that compared with MSC-Exo, the particle size of mPF4@Exo increases and the potential also changes significantly after transfection of PF4 mRNA, which indirectly proves that PF4 mRNA is successfully loaded into MSC-Exo.

[0029] (2) Western blotting detection of exosome marker proteins MSC, MSC-Exo and mPF4@Exo were lysed with RIPA buffer (Solarbio, R0010), and the protein concentration in the extract was quantified using a BCA protein detection kit (KGB2101). About 25 μg of protein was electrophoresed on 10% SDS-PAGE, then transferred to PVDF membrane for 2 h, blocked with 5% (w / v) skim milk solution at room temperature for 2 h, and incubated with different primary antibodies at 4°C overnight. Then, the membrane was incubated with the appropriate secondary antibody for 1 h, and the expression of exosome markers CD9, CD63, CD81 and Calnexin (negative) proteins was shown using an electrochemiluminescence (ECL) detection kit (Merck millipore, WBKLS0500).

[0030] 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), horseradish peroxidase (HRP) labeled anti-mouse IgG antibody (CST, 7076S, 1:2000).

[0031] The results are shown in Figure 2 From Figure 2 It can be seen that MSC-Exo and mPF4@Exo both express exosome marker proteins CD9, CD63 and CD81, and negative protein Calnexin has no expression, which indirectly proves the successful extraction and synthesis of MSC-Exo and mPF4@Exo.

[0032] Test Example 2, Western Blotting Detection of PF4 Protein Expression HT22 cells (neuronal cells) or BV2 cells (microglial cells) were cultured according to conventional techniques, and HT22 cells or BV2 cells were inoculated in a 6-well plate at 2×10 5When 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 into the culture medium, respectively, and co-incubated with HT22 cells or BV2 cells for 48 h, then the cells were collected and lysed with RIPA buffer (Solebo, R0010), and the protein concentration in the extract was quantified using BCA protein detection kit (Kaiji, KGB2101). About 25 μg of protein was electrophoresed on 10% SDS-PAGE, then transferred to PVDF membrane for 1 h, blocked with 5% (w / v) skim milk solution at room temperature for 2 h, and incubated with different primary antibodies at 4°C overnight. Then, the membrane was incubated with the appropriate secondary antibody for 1 h, and the expression of PF4 protein in HT22 cells or BV2 cells after different treatments was shown using an electrochemiluminescence (ECL) detection kit (Merck millipore, WBKLS0500). β-actin was used as an internal control for total protein.

[0033] Among them, anti-PF4 rabbit monoclonal antibody (Abeam, ab303494, 1:2000), anti-β-actin rabbit monoclonal antibody (Proteintech, 20536-1-AP, 1:4000), horseradish peroxidase (HRP) labeled anti-rabbit IgG antibody (CST, 7074S, 1:2000).

[0034] The results are shown in Figures 3-4

[0035] Compared with the Control and MSC-Exo groups, the expression of PF4 protein in HT22 and BV2 cells after mPF4@Exo treatment was significantly increased, among which the expression of PF4 protein in HT22 cells increased by about 7 times, and the expression of PF4 protein in BV2 cells increased by about 5 times, indicating that MSC-Exo can efficiently deliver PF4 mRNA into cells and stably express it as PF4 protein.

[0036] Test Example 3, Evaluation of Nasal Administration Effect (1) In vitro brain slice fluorescence imaging experiment ​PBS and PKH26 (UR52302) labeled MSC-Exo (PKH26@Exo) (containing MSC-Exo 300 μg / mL, PKH26 50 μM) were used to nose drop 5x FAD mice at 6 months old, the nose drop volume was 30 μL per mouse, 3 mice in each group, and the brain tissue was taken at 12 h after nose drop, cut into 20 μm tissue sections, stained with DAPI, and then observed with a fluorescence confocal laser microscope.

[0037] The results are shown in Figures 5-6

[0038] Compared with the PBS group, the olfactory bulb, hippocampus and cortex of the PKH26@Exo group mice had obvious red PKH26 infiltration, indicating that PKH26@Exo could bypass the BBB by nose drop administration, and MSC-Exo successfully delivered PKH26 to the hippocampus and cortex of mice through the olfactory bulb.

[0039] (2) Fluorescence imaging of brain tissue in vitro PBS and DiR (UR21017) labeled MSC-Exo (DiR@Exo) (containing MSC-Exo 300 μg / mL, DiR 20 μM) were used to nose drop 5x FAD mice at 6 months old, the nose drop volume was 30 μL per mouse, 3 mice in each group, and the brain tissue was taken at 12 h after nose drop, and the in vitro imaging system (IVIS Lumina S5) was used to image the mouse brain tissue in vitro, and the fluorescence imaging results and average radioactivity intensity were reported.

[0040] The results are shown in Figure 7

[0041] Compared with the PBS group, the brain tissue of the DiR@Exo group mice had obvious red DiR fluorescent dye infiltration, indicating that DiR@Exo could bypass the BBB by nose drop administration, and MSC-Exo successfully delivered DiR to the brain tissue of mice through the olfactory bulb.

[0042] Test Example 4, Effect of mPF4@Exo on the expression level of inflammatory factors of BV2 cells induced by Aβ According to the conventional technique, BV2 cells were cultured, and 2x10 5 ​​When 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, refer 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 inflammatory factor detection.

[0043] The contents of IL-1β and TNF-α in the cell supernatant were detected according to the instructions of the mouse interleukin 1β ELISA kit (Solabio Biotech Co., Ltd., SEKM-0002) and the mouse tumor necrosis factor α ELISA kit (Solabio Biotech Co., Ltd., SEKM-0034). The test sample or standard solution of different concentration gradients was added to the 96-well ELISA plate, and incubated at 37°C for 90 min. After washing to remove other unbound substances, the primary antibody solution was added and incubated at 37°C for 60 min, and then the solution was discarded. The HRP-labeled secondary antibody was added and incubated at 37°C for 30 min, and then the unbound enzyme-labeled antibody was washed thoroughly. The substrate was added for color development, and the absorbance value was measured at 450 nm. The inflammatory factor IL-1β and TNF-α levels in the sample were calculated according to the standard curve.

[0044] The results are shown in Figure 8

[0045] Compared with the Aβ model group, MSC-Exo can reduce the levels of IL-1β and TNF-α in the cell supernatant to a certain extent. After transfection of PF4 mRNA, the levels of IL-1β and TNF-α in the cell supernatant of the mPF4@Exo group were further significantly reduced. Compared with the control drug Exo-PF4-optimized, mPF4@Exo can 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 inflammatory factors IL-1β and TNF-α induced by Aβ in BV2 cells to a certain extent, and MSC-Exo and PF4 mRNA can exert a synergistic effect, and the anti-inflammatory effect is further enhanced after administration of mPF4@Exo. Compared with the Exo-PF4-optimized group, the anti-inflammatory effect of the mPF4@Exo administration group is better.

[0046] Test Example 5: Evaluation of the therapeutic effect of mPF4@Exo on AD (1) Water maze experiment ​The purchased 6-month-old C57BL / 6 mice and 5xFAD mice were divided into 5 groups, 8 in each group, namely: WT group (PBS nose drop normal C57BL / 6 mice, volume: 30 μL), AD group (PBS nose drop 5xFAD mice, volume: 30 μL), AD+MSC-Exo group (300 μg / mL MSC-Exo nose drop 5xFAD mice, volume: 30 μL), AD+mPF4@Exo group (mPF4@Exo containing MSC-Exo 300 μg / mL and PF4 mRNA 80 μg / mL nose drop 5xFAD mice, volume: 30 μL), AD+Exo-PF4-optimized group (300 μg / mL Exo-PF4-optimized nose drop 5xFAD mice, volume: 30 μL), 3 times a week, for 3 weeks.

[0047] After the completion of nose drop administration, each mouse was placed in a circular swimming pool with a diameter of 120 cm, and a platform about 1 cm from the water surface was provided in the pool (temperature: 22°C). The first 5 days, each mouse was trained to swim onto the platform within 60 s. On the 6th day, the mice were trained to swim freely for 60 s without the platform, and the swimming path was recorded using a video tracking system.

[0048] The results are shown in Figure 9 .

[0049] As can be seen from B (number of crossings of the platform) and C (platform quadrant residence time) in Figure 9 , MSC-Exo nose drop can increase the number of crossings of the platform and the platform quadrant residence time of AD mice, and after mPF4@Exo nose drop, the number of crossings of the platform and the platform quadrant residence time of AD mice are further increased, basically close to the level of WT mice. Compared with the control drug Exo-PF4-optimized, the number of crossings of the platform and the platform quadrant residence time of AD mice are significantly increased after mPF4@Exo nose drop. These results show that MSC-Exo nose drop can improve the cognitive function of AD mice to a certain extent, and after mPF4@Exo nose drop, MSC-Exo and PF4 mRNA can play a synergistic role, further improving the cognitive level of AD mice, basically close to the cognitive level of WT mice. And compared with the Exo-PF4-optimized group, the ability to improve the cognitive function of AD mice is stronger after mPF4@Exo nose drop.

[0050] (2) Y maze novel arm exploration experiment The experimental grouping and administration scheme are the same as those in "(1) water maze experiment".

[0051] The Y maze was cleaned with alcohol, and the three arms of the Y maze were randomly set as the start arm, the novel arm and the other arm, the middle triangular area was blank, and the baffle was closed in the novel arm. After the nasal administration was completed, the head of each mouse was gently faced to the center of the Y maze and placed in the start arm. After 5 min of adaptation, the experimental animals were placed back into the cage. After 1 h, the baffle of the novel arm was removed, the head of each mouse was gently faced to the center of the Y maze and placed in the same position of the start arm, and the test was performed for 5 min. The activity information of the mouse was recorded by software.

[0052] The results are shown in Figure 10

[0053] As can be seen from the C graph (the number of times of entering the novel arm), the D graph (the time of staying in the novel arm) and the E graph (the distance of the novel arm) in Figure 10 , the MSC-Exo nasal administration can increase the number of times of entering the novel arm, the time of staying in the novel arm and the distance of the novel arm of the AD mice to a certain extent. After the mPF4@Exo nasal administration, the number of times of entering the novel arm, the time of staying in the novel arm and the distance of the novel arm of the AD mice are further increased and basically close to the level of the WT mice. Compared with the Exo-PF4-optimized group, the number of times of entering the novel arm, the time of staying in the novel arm and the distance of the novel arm of the AD mice are significantly increased after the mPF4@Exo nasal administration. These results show that the MSC-Exo nasal administration can improve the cognitive function of the AD mice to a certain extent. Moreover, the MSC-Exo and the PF4 mRNA can play a synergistic role after the mPF4@Exo nasal administration, further improve the cognitive level of the AD mice and basically close to the cognitive level of the WT mice. Furthermore, compared with the Exo-PF4-optimized group, the ability to improve the cognitive function of the AD mice is stronger after the mPF4@Exo nasal administration.

[0054] The above only describes some embodiments of the present application. For those skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the protection scope of the present application.​

Claims

1. A PF4 mRNA delivery system for intranasal administration, characterized in that, The PF4 mRNA delivery system comprises mesenchymal stem cell exosome encapsulating PF4 mRNA and a pharmaceutically acceptable adjuvant, wherein the nucleotide sequence of the PF4 mRNA is shown as SEQ NO: 1, the mesenchymal stem cell exosome encapsulating PF4 mRNA is prepared by transfecting PF4 mRNA into mesenchymal stem cell exosome, and the dosage form of the PF4 mRNA delivery system is nasal drops.

2. The PF4 mRNA delivery system of claim 1, wherein, The transfection method is at least one selected from chemical reagent transfection, electroporation, ultrasound, and freeze-thaw cycle.

3. The PF4 mRNA delivery system of claim 2, wherein, The pharmaceutically acceptable adjuvant is physiological saline.

4. The method of claim 1-3, wherein the PF4 mRNA delivery system is prepared by, The method comprises the following steps: isolating and extracting mesenchymal stem cell exosome; transfecting PF4 mRNA into mesenchymal stem cell exosome to obtain mesenchymal stem cell exosome encapsulating PF4 mRNA; preparing a preparation for intranasal administration by mixing the mesenchymal stem cell exosome encapsulating PF4 mRNA and the pharmaceutically acceptable adjuvant.

5. The preparation method according to claim 4, characterized in that, The method for isolating and extracting mesenchymal stem cell exosome comprises at least one selected from ultracentrifugation, ultrafiltration, immunocapture, polymer precipitation, density gradient centrifugation, tangential flow filtration, microfluidic, affinity chromatography, and molecular exclusion.

6. The production method according to claim 5, wherein The method for isolating and extracting mesenchymal stem cell exosome comprises the following steps: (1) Collect mesenchymal stem cell culture medium supernatant, and obtain supernatant 2,000 x g g , 4°C centrifugation 10 min, take supernatant; (2) The supernatant obtained in step (1) was centrifuged at 10,000 x g at 4°C for 30 min, and the supernatant was collected. g , 4°C for 30 min, and the supernatant was collected. (3) The supernatant obtained in step (2) was centrifuged at 100,000 x g for 70 min at 4°C, and the precipitate was collected. Thus, the product was obtained. g , 4°C for 70 min, and the precipitate was collected. Thus, the product was obtained.

7. Use of the PF4 mRNA delivery system according to any one of claims 6 in the preparation of a medicament for treating neurodegenerative diseases.

8. Use according to claim 7, characterized in that, The neurodegenerative disease is Alzheimer's disease.

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