Application and preparation method of exosome EGF-FGF-EXO containing double growth factors

Abstract

The invention discloses application and a preparation method of exosome EGF-FGF-EXO containing double growth factors, the exosome EGF-FGF-EXO is uniform in particle size, high in loading efficiency and good in biological activity, the double growth factors comprise an epidermal growth factor EGF and a fibroblast growth factor FGF, and the epidermal growth factor EGF and the fibroblast growth factor FGF are added into the exosome EGF-FGF. The preparation method of the exosome EGF-FGF-EXO comprises preparation of an induced pluripotent stem cell (iPSC) exosome and loading of EGF-FGF, a recombinant epidermal growth factor (EGF) and a fibroblast growth factor (FGF) are loaded on the exosome from iPS-clone 12c, and the exosome product which has high biological activity and is used for treating nervous system diseases of neuroinflammation is prepared. The invention develops a brand new technical path for treatment of nervous system diseases, and in the fields of epilepsy, neurodegenerative diseases, brain injury and the like, the life quality of patients can be improved and important theoretical basis and technical support can be provided for precise medical treatment of nervous system diseases by precisely regulating and controlling neuroinflammation.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosomes, and specifically relates to an application of exosomes containing double growth factors EGF-FGF-EXO and a preparation method thereof. Background Art

[0002] Neuroinflammation is one of the core difficulties in modern neuroscience research and plays a vital role in a variety of neurological diseases. The occurrence and development of neurodegenerative diseases such as epilepsy, Alzheimer's disease, and Parkinson's disease are closely related to neuroinflammation. Traditional neuroinflammation treatment methods are limited by insufficient targeting and limited therapeutic effects. There is an urgent need to develop new and efficient neuroinflammation regulation strategies. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) show unique therapeutic effects in the treatment of neurological diseases. EGF has significant neuroregeneration and synaptic plasticity regulation capabilities, can promote neuronal proliferation and differentiation, and effectively reduce nerve damage; FGF plays a key role in the survival and repair of nerve cells, and significantly improves nerve function by inhibiting the inflammatory cascade. The synergistic effect of these two growth factors provides new possibilities for the treatment of neurological diseases. As an emerging intercellular communication carrier, extracellular vesicles (Exosomes) have shown unprecedented potential in the treatment of neurological diseases. Compared with traditional drug delivery systems, exosomes have unique advantages such as natural cross-biological barriers, low immunogenicity, and multifunctional carriers, becoming an important breakthrough in the precision treatment of neurological diseases. Their natural membrane structure and precise targeted delivery capabilities make them ideal biological treatment carriers.

[0003] Based on the patented technology of human induced pluripotent stem cell iPS-clone 12c, the present invention proposes an innovative therapy, which uses iPS-clone 12c-derived exosomes to load two growth factors, EGF and FGF, and presents a multi-dimensional mechanism of action in the regulation of neuroinflammation. By inhibiting the NF-κB signaling pathway, regulating microglial polarization, and reducing the release of pro-inflammatory factors, it can not only effectively alleviate neuroinflammatory reactions, but also directly protect neuronal function, showing a significant neuroprotective effect. The synergistic effect of EGF and FGF further enhances the therapeutic efficacy of exosomes in neurological diseases, especially the application of exosome delivery systems combined with EGF and FGF in the regulation of neuroinflammation, achieving precise biological modification of exosomes, significantly improving their targeting and functionality in the treatment of neurological diseases; by precisely combining growth factors, optimizing the biological characteristics of exosomes, and constructing a multifunctional treatment platform. Summary of the invention

[0004] The present invention discloses an application of exosomes containing double growth factors EGF-FGF-EXO and a preparation method thereof, wherein recombinant epidermal growth factor (EGF) and fibroblast growth factor (FGF) are loaded into exosomes derived from pluripotent stem cells iPS-clone 12c to prepare an exosome product with high biological activity for treating neuroinflammatory nervous system diseases. The technical scheme is as follows: An application of dual growth factor exosomes EGF-FGF-EXO in the application of nervous system diseases, which plays the role of disease modulator by inhibiting NF-κB signaling pathway, regulating microglial activation and polarization phenotype, and reducing the release of pro-inflammatory factors, including inhibiting the polarization of M1 microglia and promoting their polarization to M2 phenotype, anti-inflammation in glial cells, inhibiting neuronal epileptiform activity, inhibiting epileptic seizures and maintaining normal excitation-inhibition balance.

[0005] The invention discloses an exosome containing dual growth factors, EGF-FGF-EXO, which has uniform particle size, high loading efficiency and good biological activity. The average particle size of the exosome EGF-FGF-EXO is 138±15nm, the double-layer membrane structure is complete, and the exosome markers CD9, CD63, CD81 and TSG101 are all positive. The biological activities of EGF and FGF in the exosome EGF-FGF-EXO are significantly improved compared with those of EXO exosomes, wherein the activity of EGF is increased by about 7.2 times, and the activity of FGF is increased by about 10.8 times. The dual growth factors include epidermal growth factor EGF and fibroblast growth factor FGF.

[0006] A method for preparing exosomes containing dual growth factors EGF-FGF-EXO includes preparing induced pluripotent stem cell iPSC exosomes and EGF-FGF loading, and the specific steps are as follows: (1) After culturing iPS-clone 12c cells for 36-72 hours, the culture supernatant was collected, and exosomes were purified by multi-step differential centrifugation. The iPS-clone 12c cell clone was classified and named as human induced pluripotent stem cell iPS-clone 12cHomo sapiens, and was deposited in the China Center for Type Culture Collection on July 23, 2024, with the deposit number of CCTCC NO: C2024238; (2) The exosome pellet was resuspended in sterile PBS precooled at 4°C, ultracentrifuged again at 80,000-120,000 g for 60-90 min to obtain high-purity exosomes, and filtered; (3) Dilute the purified iPS-clone 12c exosomes with PBS to 0.5-1.0 mg / mL, add EGF and FGF to a final concentration of 8-15 μg / mL, and use an ultrasonic probe to perform ultrasonic treatment in an ice bath at 4-8°C to allow the growth factors to fully bind to the exosomes; (4) After treatment, the samples were immediately placed on ice, and the loaded exosomes EGF-FGF-EXO were collected by ultracentrifugation and resuspended in pre-chilled PBS.

[0007] Furthermore, the multi-step differential centrifugation method includes centrifugation at 2,000-3,000 g for 10-15 min to remove cells, centrifugation at 8,000-12,000 g for 20-40 min to remove cell debris, and ultracentrifugation at 80,000-120,000 g for 60-90 min to obtain exosome precipitation.

[0008] Furthermore, the multi-step differential centrifugation method is sequentially 2,000g centrifugation for 10 min, 10,000g centrifugation for 30 min, and 100,000g centrifugation for 70 min.

[0009] Furthermore, the ultracentrifugation in step (2) and step (4) is performed at 100,000 g for 70 min.

[0010] Furthermore, in step (2), the iPS-clone 12c exosomes were sterilized and filtered through a 0.22 μm filter membrane.

[0011] Furthermore, after obtaining the purified iPS-clone 12c exosomes in step (3), the exosomes were diluted to 0.8 mg / mL with PBS, and recombinant human EGF and recombinant human FGF were added to adjust the final concentrations to 12 μg / mL respectively.

[0012] Furthermore, the ultrasonic parameters of step (3) are: power 25-35W, working time 3-5s, intermittent time 6-10s, and cycle 4-6 times.

[0013] Furthermore, the ultrasonic parameters of step (3) are: power 30W, working time 4s, rest time 8s, and 5 cycles.

[0014] The above technical solution can achieve the following beneficial effects: Compared with the existing technology, the method for preparing engineered exosomes of the present invention, with its unique biological modification strategy, precise inflammation regulation mechanism, and safe delivery system, will provide a new solution for the treatment of neurological diseases, and has important scientific value and application prospects. In particular, the deep fusion of EGF, FGF and iPS-clone 12c exosomes has opened up a new technical path for the treatment of neurological diseases.

[0015] The technical route of the present invention has broad application prospects. In the fields of epilepsy, neurodegenerative diseases, brain damage, etc., engineered exosomes are expected to become a revolutionary treatment method. By precisely regulating neuroinflammation, this technology can not only improve the quality of life of patients, but also provide important theoretical basis and technical support for precision medicine of neurological diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the main characterization of exosomal EGF-FGF-EXO; From left to right are nanoparticle tracking analysis and transmission electron microscopy of exosomes EGF-FGF-EXO.

[0017] Figure 2 This is a schematic diagram of ELISA detection of growth factor activity.

[0018] Figure 3 This is a schematic diagram of EGF-FGF-EXO inhibiting neuronal epileptiform activity; Among them: (A) Experimental design of BV2 cells induced by LPS; (BD) mRNA expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α in BV-2 cells after 4 hours of LPS treatment; (E) Experimental design of primary cultured hippocampal neurons induced by CTZ; (F) Sample traces showing that EGF-FGF-EXO treatment inhibited CTZ-induced epileptic burst activity in cultured hippocampal neurons; (GI) Bar graph display; (J) Restoration effect of EGF-FGF-EXO on CTZ-induced inhibitory transmission in hippocampal neurons; (KL) Patch clamp recording display; ∗ P<0.05, ∗∗ P<0.01 and ∗∗∗ P<0.001 compared with PBS group; # P<0.05, ## P<0.01 and #### P<0.001 compared with LPS group or CTZ group.

[0019] Figure 4 It is a schematic diagram of EGF-FGF-EXO inhibiting the seizure activity of PTZ-induced mice; Among them: (A) Animal experiment design diagram; (B) Scatter plot showing the Racine Score of mice in different groups; (C) Bar graph showing the proportion of mice with the maximum epilepsy score, R3, R4, and R5 represent the epilepsy seizure scores of Racine3, Racine4, and Racine5, respectively; (D) Histogram showing the latency of each epileptic seizure stage in mice; (E) Histogram showing the duration of each epileptic seizure stage in mice; (F) Representative EEG of each group of mice and representative EEG energy spectrum; PTZ, pentazobacterium tetrazolium; # P<0.05, ## P<0.01 and ### P<0.001 compared with the PTZ group.

[0020] Figure 5 Schematic diagram of EGF-FGF-EXO treatment reversing PTZ-induced hippocampal neuronal loss and inflammatory cytokine release in epileptic mice; Among them: (A) Representative schematic diagram of neuronal loss in the CA1 region of the hippocampus of each group of mice 24 hours after PTZ injection; (B) Quantification of the number of neurons in the CA1 region of the hippocampus of each group of mice; (C) Representative schematic diagram of neuronal loss in the CA3 region of the hippocampus of each group of mice 24 hours after PTZ injection; (D) Quantification of the number of neurons in the CA3 region of the hippocampus of each group of mice; (EG) Quantification of mRNA levels in the hippocampus within 30 minutes showed that EGF-FGF-EXO could reduce the expression levels of IL-1β, IL-6 and TNF-α after epilepsy. ∗ P<0.05, ∗∗ P<0.01 and ∗∗∗ P<0.001 compared with the saline group; # P<0.05, ## P<0.01 and ### P<0.001 compared with the PTZ group.

[0021] Figure 6 Schematic diagram of the inhibitory effect of EGF-FGF-EXO pretreatment on microglial activation and M1 polarization in the CA1 region of the hippocampus; Among them: (A) Representative immunofluorescence images showed that EGF-FGF-EXO could inhibit the PTZ-induced increase in Iba1 (green) and CD16 / 32 (red) areas in the CA1 region of the hippocampus; (BC) Quantitative analysis showed that EGF-FGF-EXO significantly reduced the number of Iba1 (green) cells and the percentage of CD16 / 32 (red) positive cells in the CA1 region of the hippocampus induced by PTZ, compared with PTZ mice; (D) Representative immunofluorescence images showed that EGF-FGF-EXO alleviated the PTZ-induced decrease in the percentage of CD206 (red) positive cells in the CA1 region of the hippocampus; (E) Quantitative analysis showed that EGF-FGF-EXO significantly increased the percentage of CD206 (red) positive cells in the CA1 region of the hippocampus induced by PTZ, compared with PTZ mice; (F) Representative immunofluorescence images showed the effect of EGF-FGF-EXO on the PTZ-induced GFAP (green) area in the CA1 region of the hippocampus. Quantitative analysis showed that the number of GFAP (green)-positive astrocytes did not change significantly after PTZ induction for 30 min. ∗ P<0.05, ∗∗ P<0.01 and ∗∗∗ P<0.001 compared with the saline group; # P<0.05, ## P<0.01 and ### P<0.001 compared with the PTZ group.

[0022] Figure 7Schematic diagram showing that EGF-FGF-EXO reduces the severity of epilepsy and the release of inflammatory cytokines after epilepsy in PTZ-induced mice, and achieves the above effects by inhibiting NF-κB activity; Among them, (A) Western blotting results showed the changes in p-P65 and P65 expression in the hippocampus of rats in the saline, PTZ, PTZ+Con.Exos, or PTZ+EGF-FGF-EXO groups, and the relative protein levels were normalized to the average value of the saline group; (B) The bar graph showed that EGF-FGF-EXO reduced the increase in the p-P65 / P65 ratio after PTZ induction; (C) Animal experiment design diagram after PMA injection (PMA: 12-octadecenoyl-13-acetic acid phosphate, NF-κB activator); (D-E) PMA pretreatment reversed the effect of EGF-FGF-EXO on reducing the percentage of epileptic seizures in PTZ-induced epileptic mice (D) and reduced animal scores (E); (F) PMA reversed the inhibitory effect of EGF-FGF-EXO on NF-κB activation; (G) The bar graph showed that PMA increased the p-P65 / P65 ratio in the PTZ+EGF-FGF-EXO group of mice; (HJ) After PMA treatment, the mRNA expression levels of proinflammatory cytokines including IL-1β, IL-6 and TNF-α in the PTZ+EGF-FGF-EXO group of mice were decreased; (K) *P<0.05, **P<0.01 and ***P<0.001 compared with the saline group; (L) #P<0.05, ##P<0.01 and ###P<0.001 compared with the PTZ group.

[0023] Figure 8 Schematic diagram of EGF-FGF-EXO inhibiting post-epileptic microglial M1 polarization and promoting M2 polarization by inhibiting NF-κB inactivation; Wherein: (A) Representative immunofluorescence images showed that PMA upregulated the Iba1 (green) and CD16 / 32 (red) areas in the CA1 region of the hippocampus reduced by EGF-FGF-EXO; (BC) Quantitative analysis showed that PMA significantly increased the number of Iba1 (green) cells and the percentage of CD16 / 32 (red) positive cells in the CA1 region of the hippocampus reduced by EGF-FGF-EXO; (D) Representative immunofluorescence images showed that PMA reduced the CD206 positive (red) area in the CA1 region of the hippocampus restored by EGF-FGF-EXO; (E) Quantitative analysis showed that PMA significantly reduced the percentage of CD206 (red) positive cells in the CA1 region of the hippocampus compared with the PTZ+EGF-FGF-EXO group. ∗ P<0.05, ∗∗ P<0.01 and ∗∗∗ P<0.001 compared with the saline group; # P<0.05, ## P<0.01 and ### P<0.001 compared with the PTZ group. DETAILED DESCRIPTION

[0024] The following is combined with Figure 1-8 The present invention is further described as follows: Example 1: Preparation of exosomes EGF-FGF-EXO (1) Preparation of exosomes: iPS-clone 12c cells (the cell clone classification is named human induced pluripotent stem cell iPS-clone 12cHomo sapiens, deposited in the China Center for Type Culture Collection on July 23, 2024, with the deposit number CCTCC NO: C2024238) were cultured in mTeSR™ 1 complete medium (STEMCELL Technologies, 85850) for 48 h, and exosomes were collected and purified by differential centrifugation: first, centrifuge at 2,000g for 10 min at 4°C to remove cells and collect the supernatant; centrifuge at 10,000g for 30 min to remove cell debris and other subcellular components, and collect the supernatant again; finally, centrifuge at 100,000g for 70 min to obtain the exosome precipitate. The pellet was resuspended in sterile PBS precooled at 4°C and ultracentrifuged again at 100,000 g for 70 min to obtain highly pure exosomes, and the iPSC exosomes resuspended in PBS were filtered through a 0.22 μm filter membrane (Millipore).

[0025] (2) Loading of EGF-FGF: After obtaining the purified iPS-clone 12c exosomes, dilute them with PBS to 0.8 mg / mL, add recombinant human EGF (PeproTech, AF-100-15) and recombinant human FGF (PeproTech, AF-100-18B), and adjust the final concentration to 12 μg / mL. Use an ultrasonic probe to treat under ice bath conditions, with the specific parameters of: power 30 W, working time 4 s, rest time 8 s, and 5 cycles. During the treatment, the temperature was monitored in real time and maintained in the range of 4-8 °C. After the treatment, the sample was immediately placed on ice, and the modified exosomes EGF-FGF-EXO were collected by ultracentrifugation at 100,000g for 70 min and resuspended in pre-cooled PBS.

[0026] (3) Product quality testing and characterization: After testing, the main quality indicators of the final product exosomes EGF-FGF-EXO are as follows Figure 1 , Figure 2 As shown, the prepared dual growth factor exosomes have the characteristics of uniform particle size, high loading efficiency and good biological activity: (1) NTA detection showed that the average particle size was 138±15nm and the Zeta potential was -31.8±2.5 mV; (2) Transmission electron microscopy showed that exosomes maintained a typical double-layer membrane structure intact; (3) Western blot confirmed that the exosome markers CD9, CD63, CD81, and TSG101 were all positive; (4) The ELISA method showed that the biological activities of EGF and FGF in the prepared exosomes EGF-FGF-EXO were significantly improved compared with those in EXO exosomes, with the EGF activity increased by about 7.2 times and the FGF activity increased by about 10.8 times.

[0027] Example 2: EGF-FGF-EXO significantly reduces LPS-induced pro-inflammatory cytokines IL-1β and IL-6 mRNA levels in BV2 microglia In the present invention, BV2 microglia were cultured in LPS (1ug / ml) or PBS (phosphate buffered saline) for 4h, and 293T-EXO or EGF-FGF-EXO (200ug) were added, and then the levels of pro-inflammatory cytokines were examined by RT-qPCR. Compared with the 293T-EXO treatment group: the IL-1β and IL-6 mRNA levels of BV2 cells in the EGF-FGF-EXO treatment group were significantly reduced; Figure 3 : Figure 3 (BD) After 4 hours of LPS treatment, the mRNA expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α in BV-2 cells was significantly upregulated. EGF-FGF-EXO significantly reduced the mRNA expression of IL-1β and IL-6, and attenuated the increase in TNF-α mRNA expression; Figure 3 (GI) Bar graphs show that EGF-FGF-EXO induced a significant decrease in the percentage of neurons showing epileptiform burst discharges (G) and burst discharge frequency (H) compared with the CTZ group and CTZ+Con.Exos group, and alleviated the CTZ-induced increase in the total number of action potentials within 10 minutes (I); Figure 3 (KL) Patch clamp recordings showed that EGF-FGF-EXO incubation significantly increased the frequency of mIPSCs compared with the LPS or CTZ groups, but the amplitude was not affected.

[0028] In summary, Figure 3 BC; LPS+EGF-FGF-EXO: 629.8-fold vs LPS+293T-EXO: 1137.0-fold, IL-1β, P<0.05; LPS+EGF-FGF-EXO: 506.0-fold vs LPS+293T-EXO: 790.0-fold, IL-6, P<0.05; 1 control group; one-way ANOVA multiple comparisons, but no significant changes in TNF-α mRNA levels, such as Figure 3 D. The above results indicate that EGF-FGF-EXO has an anti-inflammatory effect in LPS-treated BV2 microglia.

[0029] Example 3: EGF-FGF-EXO inhibited CTZ-induced epileptiform activity in primary cultured hippocampal neurons and led to an increase in mIPSC frequency In order to study whether the anti-inflammatory effect of EGF-FGF-EXO is also involved in the treatment of epilepsy, the present invention tested the epileptic discharge activity of primary hippocampal neurons at DIV14-16, and these neurons were randomly divided into four groups: PBS, CTZ (40µM, 3h), CTZ+293T-EXO (50ug / ml) and CTZ+EGF-FGF-EXO (50ug / ml), and treated accordingly. Whole-cell patch clamp recordings showed that under CTZ exposure, the abnormal synchronous discharge activity of the EGF-FGF-EXO treatment group was reduced, as shown in the figure. Figure 3 F. About 57.1% (14 / 24) of the neurons recorded in the CTZ group showed discharge activity, which was similar to the 293T-EXO-treated group (70.0%, 10 / 14). However, the EGF-FGF-EXO-treated group showed a very low proportion of spontaneous epileptiform discharge activity (5.9%), with only 1 of 17 neurons showing discharge activity ( Figure 3 G; P<0.01 compared with CTZ; P<0.001 compared with 293T-EXO; Fisher's test). In addition, the high-frequency burst activity of neurons induced by CTZ was also reduced under the incubation of EGF-FGF-EXO ( Figure 3 H; EGF-FGF-EXO: 0.002 ± 0.001 Hz, n = 17, vs. CTZ: 0.013 ± 0.005 Hz, n = 14, P < 0.05; vs. 293T-EXO: 0.010 ± 0.003 Hz, n = 10, P < 0.01; Kruskal-Wallis test with multiple comparisons). During the 10-min recording period, the number of action potentials (APs) observed in neurons treated with EGF-FGF-Exo was significantly reduced compared with that in the CTZ group ( Figure 3 I; EGF-FGF-Exo: 104.5±46.5 vs CTZ: 298.7±99.16; P<0.05, Mann-Whitney test). These findings suggest that EGF-FGF-EXO has the effect of inhibiting neuronal epileptiform activity.

[0030] To determine the role of EGF-FGF-EXO in CTZ-induced inhibitory synaptic imbalance, the present invention studied miniature inhibitory synaptic currents (mIPSCs) in primary cultured hippocampal neurons, such as Figure 3J: Compared with the control group (n=11), the mIPSC frequency in the CTZ (n=16) and 293T-EXO treated groups (n=12) was significantly reduced, see Figure 3 L; PBS: 2.26±0.43Hz vs CTZ: 0.75±0.14Hz, P<0.0001; vs CTZ+293T-EXO: 0.83±0.16Hz, p<0.001; one-way ANOVA multiple comparisons, while this decrease was completely reversed after EGF-FGF-EXO treatment (n=22) (CTZ+EGF-FGF-EXO: 1.59±0.14Hz vs CTZ, P<0.05; one-way ANOVA multiple comparisons. However, there was no significant difference in mIPSC amplitude among the four groups, see Figure 3 K.

[0031] Taken together, these results suggest that EGF-FGF-EXO may play a role in suppressing epileptic seizures and maintaining normal excitation-inhibition balance.

[0032] Example 4: EGF-FGF-EXO inhibits PTZ-induced epileptic seizures in mice In order to further verify the inhibitory effect of EGF-FGF-EXO on epileptic seizures in epileptic animals, the present invention administered EGF-FGF-EXO (100ug, ip) 15min before administering PTZ (50mg / kg). We found that EGF-FGF-EXO reduced the highest epilepsy score of PTZ-induced epileptic mice, see Figure 4 B; PTZ+EGF-FGF-EXO: 3.9±0.3, vs PTZ: 4.8±0.3, P<0.01; vs PTZ+293T-EXO: 4.7±0.3, P<0.05; one-way ANOVA for multiple comparisons. Pre-administration of EGF-FGF-EXO significantly reduced the proportion of animals showing epileptic behavior with a Racine score ≥4 after PTZ injection.

[0033] See also Figure 4 C; EGF-FGF-EXO treatment: 55%, n=20, vs PTZ: 100%, n=14, P<0.01, vs 293T-EXO treatment: 87%, P<0.05; Fisher test. At the same time, compared with the PTZ group, the average latency of stages II, III, and IV in the EGF-FGF-EXO group was significantly prolonged, see Figure 4D; PTZ vs EGF-FGF-EXO treatment; R-II: 1.2±0.1 min vs 1.9±0.3 min, P<0.05; R-III: 2.1±0.1 min vs 3.6±0.5 min, P<0.05; R-IV: 4.5±0.4 min vs 7.2±0.7 min, P<0.05; ordinary one-way ANOVA multiple comparison. In addition, we observed that the duration of Racine-5 behavior in the EGF-FGF-EXO group was shortened, see Figure 4 E; EGF-FGF-EXO: 0.07±0.02 min vs PTZ: 0.2±0.04 min, P<0.05; vs 293T-EXO: 0.2±0.04 min, P<0.05; ordinary one-way ANOVA multiple comparisons. These results indicate that pre-administration of EGF-FGF-EXO in mice can inhibit PTZ-induced epileptic seizures.

[0034] In summary, Figure 4 F shows the representative EEG of each group and its corresponding energy spectrum. Among them, the influence trend of EGF-FGF-EXO on the EEG signals of each group is consistent with its inhibitory effect on the observed convulsive behavior. These results further prove that EGF-FGF-EXO may have an inhibitory effect on epileptic discharges.

[0035] Example 5: EGF-FGF-EXO pretreatment can reduce hippocampal neuronal loss (24h) and the release of inflammatory cytokines (30min) in PTZ-induced epileptic mice The present invention studies the protective effect of EGF-FGF-EXO on hippocampal neurons of PTZ-induced epileptic mice by H&E staining. Figure 5 A. Compared with the saline group, the number of neurons in the CA1 region of the hippocampus in the PTZ group and the 293T-EXO-treated group was significantly reduced, see Figure 5 C; saline group: 58.16, PTZ group: 41.10, P < 0.05; and 293T-EXO (+PTZ) group: 31.20, P < 0.001, one-way ANOVA multiple comparison. In contrast, the EGF-FGF-EXO group showed a significant increase in the number of neurons, see Figure 5 C; EGF-FGF-EXO (+PTZ) group: 52.45, PTZ group, P < 0.05, t test. At the same time, the PTZ group still showed neuronal loss and disordered arrangement in the CA3 area of ​​the hippocampus, such as Figure 5As shown in D and 5B: saline group: 35.97, PTZ group: 28.90, P<0.05; and 293T-EXO (+PTZ) group: 25.70, P<0.001; one-way ANOVA multiple comparison. EGF-FGF-EXO also showed an anti-apoptotic protective effect in neurons in the CA3 region of the hippocampus EGF-FGF-EXO (+PTZ) group: 35.92, PTZ group, P<0.05; one-way ANOVA multiple comparison. Therefore, it was proved that EGF-FGF-EXO can significantly inhibit the loss of hippocampal neurons in PTZ-induced epileptic mice. Exposure to inflammatory factors may cause neuronal damage. Previous cell data showed that EGF-FGF-EXO can inhibit the release of inflammatory factors in BV2 microglia induced by LPS. To further explore whether EGF-FGF-EXO can inhibit inflammation in vivo, we evaluated the mRNA levels of inflammatory cytokines by RT-qPCR after mice were injected with PTZ. The results showed that EGF-FGF-EXO treatment significantly reduced the levels of IL-1β, IL-6, and TNF-α induced by PTZ injection. Figure 5 EG, PTZ vs EGF-FGF-EXO (+PTZ), mRNA: IL-1β: 2.78±0.49 vs 0.88±0.09, P<0.05; IL-6: 2.77±0.65 vs 0.49±0.19, P<0.05; TNF-α: 3.46±1.01 vs 0.50±0.14, P<0.05; one-way ANOVA multiple comparison.

[0036] Example 5: EGF-FGF-EXO regulates microglial activation and polarization phenotype in epileptic brain The activation of glial cells is closely related to neuroinflammation. The present invention examined the activation characteristics of microglia and astrocytes in the hippocampus by immunofluorescence staining to study the condition of glial cells after PTZ (50 mg / kg) treatment. Compared with the PTZ group and the 293T-EXO treatment group, EGF-FGF-EXO significantly reduced the number of activated microglia (Iba1-positive cells) in the CA1 region of the hippocampus, such as Figure 6 AB; EGF-FGF-EXO (+PTZ): 37.95 ± 2.41 vs PTZ: 53.55 ± 1.69, P < 0.01; vs 293T-EXO (+PTZ): 56.52 ± 1.74, P < 0.001; one-way ANOVA multiple comparison. However, there was no significant difference in the number of astrocytes (GFAP-positive cells), see Figure 6E. To evaluate the effect of EGF-FGF-EXO on microglial polarization, we measured the classic M1 marker CD16 / 32 and M2 marker CD206, which co-localize with microglia (Iba1+). Double immunofluorescence staining results showed that EGF-FGF-EXO treatment significantly reduced the polarization of M1 microglia and promoted the polarization of M2 phenotype, see Figure 6 A, 6D. Figure 6 B shows that the proportion of M1 microglia (CD16 / 32+ / Iba1+) in the PTZ and 293T-EXO treatment groups increased significantly: Saline: 30.30%±2.59% vs PTZ: 56.73%±3.07%, P<0.001; vs 293T-EXO (+PTZ): 49.32%±4.17%, P<0.01; multiple comparisons of one-way ANOVA, while this proportion was significantly reduced after EGF-FGF-EXO pretreatment, see Figure 6 C; EGF-FGF-EXO (+PTZ): 30.30%±2.59% vs PTZ, P<0.01; multiple comparisons of one-way ANOVA. At the same time, the proportion of M2 cells in the CA1 region of the hippocampus of PTZ-induced mice was also reduced, see Figure 6 B; CD206+ / Iba1+: Saline (31.90%±2.36%) vs PTZ (8.36±1.55%), P<0.05; multiple comparisons by one-way ANOVA. This downward trend was reversed by EGF-FGF-EXO, such as Figure 6 E: EGF-FGF-EXO (52.23%±1.56%) vs PTZ, P<0.01; one-way ANOVA multiple comparison. Therefore, EGF-FGF-EXO can alleviate the activation of microglia in the hippocampus after PTZ injection. In addition, EGF-FGF-EXO inhibits the polarization of M1 microglia and promotes their polarization to M2 phenotype.

[0037] Example 6: EGF-FGF-EXO acts as a disease modifier by inhibiting the NF-κB signaling pathway, thereby inhibiting neuroinflammatory responses NF-κB is crucial for neuroinflammation and can promote the expression of various inflammatory cytokines. Therefore, the present invention determines the potential molecular mechanism of EGF-FGF-EXO in inhibiting neuroinflammatory response by examining the activation of the transcription factor NF-κB. In this study, the present invention used Western blot to examine the protein levels of key molecules involved in the NF-κB pathway. The results showed that PTZ injection increased the phosphorylation level of NF-κB p65, while EGF-FGF-EXO significantly reduced the increase in the ratio of p-P65 / P65, as shown in Figure 2. Figure 7AC shows: EGF-FGF-EXO (+PTZ): 0.99±0.06 vs PTZ: 1.40±0.10, p<0.05; vs 293T-EXO (+PTZ): 1.47±0.21, p<0.05; 1 control group; one-way ANOVA multiple comparisons. This suggests that EGF-FGF-EXO may inhibit NF-κB pathway activation mainly by reducing the level of P65 phosphorylation in PTZ-induced epileptic mice.

[0038] Furthermore, these changes could be reversed by intracerebroventricular injection of the NF-κB agonist PMA, see Figure 7 DE, 5H-I; DMSO (+PTZ+EGF-FGF-EXO): 1.05±0.06 vs PMA (+PTZ+EGF-FGF-EXO): 1.61±0.11, P<0.01; 1 control group; one-way ANOVA multiple comparisons. Next, the effects of restoring the NF-κB pathway on epileptic behavior, proinflammatory cytokine mRNA levels, and activated microglia polarization phenotype were analyzed. The results showed that the use of PMA increased the proportion of mice reaching stage IV / V, see Figure 7 F; DMSO (+PTZ+EGF-FGF-EXO): 37.5%, n=8, PMA (+PTZ+EGF-FGF-EXO): 86.7%, n=8, P=0.056; Fisher's test, and the highest PTZ-induced epilepsy score in the EGF-FGF-EXO treatment group was improved, see Figure 7 G; DMSO: 3.525±0.32, PMA: 4.500±0.26, P=0.055; t test. In addition, PMA reversed the suppressed TNF-α, IL-6 and IL-1β levels under EGF-FGF-EXO treatment, see Figure 7 DE; DMSO (+PTZ+EGF-FGF-EXO): PMA (+PTZ+EGF-FGF-EXO), IL-1β: 0.7543±0.2793 vs 3.638±1.776, P<0.05; IL-6: 0.881±0.351 vs 4.903±2.189, P<0.05; TNF-α: 0.689±0.109 vs 2.796±1.69, P<0.05; one-way ANOVA. In addition, compared with the DMSO (+PTZ+EGF-FGF-EXO) group, the expression of M1 marker CD16 / 32 increased and the expression of M2 marker CD206 decreased in the PMA (+PTZ+EGF-FGF-EXO) group, see Figure 8AD; DMSO compared with PMA, CD16 / 32+ / Iba1+: 45.03%±7.36% vs 78.44%±4.63%, P<0.05; CD206+ / Iba1+: 51.98%±3.89% vs 37.23%±3.68%, P<0.05; one-way ANOVA multiple comparison. These results indicate that EGF-FGF-EXO inhibits M1 and promotes M2 polarization by inhibiting the NF-κB signaling pathway.

[0039] The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, various equivalent modifications to the present invention belong to the protection scope of the claims attached to this application.

Claims

1. An application of exosomes containing dual growth factors EGF-FGF-EXO, characterized in that: In its application in neurological diseases, it plays the role of a disease modulator by inhibiting the NF-κB signaling pathway, regulating microglial activation and polarization phenotype, and reducing the release of pro-inflammatory factors, including inhibiting the polarization of M1 microglia and promoting their polarization to M2 phenotype, anti-inflammation in glial cells, inhibiting neuronal epileptiform activity, inhibiting epileptic seizures and maintaining normal excitation-inhibition balance.

2. The dual growth factor exosome EGF-FGF-EXO according to claim 1, characterized in that: The exosome EGF-FGF-EXO has uniform particle size, high loading efficiency and good biological activity, and the dual growth factors include epidermal growth factor EGF and fibroblast growth factor FGF.

3. A method for preparing double growth factor exosomes containing EGF-FGF-EXO as claimed in claim 2, characterized in that: The method includes preparing induced pluripotent stem cell iPS-clone 12c exosomes and EGF-FGF loading, and the specific steps are as follows: (1) After culturing iPS-clone 12c cells for 36-72 hours, the culture supernatant was collected, and exosomes were purified by multi-step differential centrifugation. The iPS-clone 12c cell clone was classified and named as human induced pluripotent stem cell iPS-clone 12c Homosapiens, and was deposited in the China Center for Type Culture Collection on July 23, 2024, with the deposit number of CCTCC NO: C2024238; (2) The exosome pellet was resuspended in sterile PBS precooled at 4°C, ultracentrifuged again at 80,000-120,000 g for 60-90 min to obtain high-purity exosomes, and filtered; (3) Dilute the purified iPS-clone 12c exosomes with PBS to 0.5-1.0 mg / mL, add EGF and FGF to a final concentration of 8-15 μg / mL, and use an ultrasonic probe to perform ultrasonic treatment in an ice bath at 4-8°C to allow the growth factors to fully bind to the exosomes; (4) After treatment, the samples were immediately placed on ice, and the loaded exosomes EGF-FGF-EXO were collected by ultracentrifugation and resuspended in pre-chilled PBS.

4. The method for preparing double growth factor exosomes EGF-FGF-EXO according to claim 3, characterized in that A method for preparing exosomes containing dual growth factors EGF-FGF-EXO, characterized in that: the multi-step differential centrifugation method includes centrifugation at 2,000-3,000g for 10-15min to remove cells, centrifugation at 8,000-12,000g for 20-40min to remove cell debris, and ultracentrifugation at 80,000-120,000g for 60-90min to obtain exosome precipitation.

5. The method for preparing double growth factor exosomes containing EGF-FGF-EXO according to claim 4, characterized in that: The multi-step differential centrifugation method is sequentially 2,000g centrifugation for 10 min, 10,000g centrifugation for 30 min, and 100,000g centrifugation for 70 min.

6. The method for preparing double growth factor exosomes containing EGF-FGF-EXO according to claim 3, characterized in that: The ultracentrifugation in step (2) and step (4) is performed at 100,000 g for 70 min.

7. The method for preparing double growth factor exosomes containing EGF-FGF-EXO according to claim 3, characterized in that: In the step (2), the iPS-clone 12c exosomes are sterilized and filtered through a 0.22 μm filter membrane.

8. The method for preparing double growth factor exosomes containing EGF-FGF-EXO according to claim 3, characterized in that: After obtaining the purified iPS-clone 12c exosomes in step (3), the exosomes were diluted to 0.8 mg / mL with PBS, and recombinant human EGF and recombinant human FGF were added to adjust the final concentrations to 12 μg / mL respectively.

9. The method for preparing double growth factor exosomes containing EGF-FGF-EXO according to claim 3, characterized in that: The ultrasonic parameters of step (3) are: power 25-35W, working time 3-5s, intermittent time 6-10s, and cycle 4-6 times.

10. The method for preparing double growth factor exosomes containing EGF-FGF-EXO according to claim 3, characterized in that: The ultrasonic parameters of step (3) are: power 30W, working time 4s, rest time 8s, and 5 cycles.

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