Drug-loaded exosomes for improving the ability of leydig cells to secrete testosterone, and preparation method and application thereof

Abstract

The application belongs to the field of regenerative medicine and nano drug delivery system, and particularly relates to a drug-loaded exosome for improving the testosterone secretion capacity of testicular interstitial cells and a preparation method and application thereof. The application links a targeting FATE1 polypeptide to the surface of MSC-derived exosomes by a chemical coupling method, and loads a small molecule compound in the exosomes by electroporation, so as to obtain modified MSC-derived exosomes. The obtained exosomes can obtain the targeting capacity in testicular interstitial cells, and the natural repair capacity of MSC-derived exosomes and the release of small molecule drugs promote the repair of damaged cells, improve the testosterone secretion capacity of testicular interstitial cells, and relieve male erectile dysfunction caused by diseases or aging.

Description

Technical Field

[0001] This invention belongs to the fields of regenerative medicine and nanomedicine technology, specifically relating to a drug-loaded exosome that enhances the ability of testicular interstitial cells to secrete testosterone, its preparation method, and its application. Background Technology

[0002] Leydig cells are the core functional cells for testosterone synthesis and secretion in men. Their functional decline or damage is a key pathological basis for disease / aging-related erectile dysfunction in men. Under physiological conditions, testosterone maintains normal erectile function by regulating the relaxation of the smooth muscle of the corpus cavernosum, vascular endothelial function, and nerve conduction. However, in chronic diseases (such as diabetes and chronic inflammation) or during the aging process, a decrease in the number of Leydig cells and a decline in the activity of steroid synthases directly lead to a decrease in serum testosterone levels, thereby causing erectile dysfunction, accompanied by decreased libido and reproductive decline.

[0003] FATE1 (Fetal and adult testis-expressed gene 1), a membrane protein specifically highly expressed in testicular interstitial cells, plays a crucial role in regulating testosterone synthesis and maintaining cellular homeostasis. On one hand, FATE1 participates in testosterone biosynthesis by regulating mitochondrial function and steroid hormone synthesis pathways; on the other hand, its membrane localization characteristics in testicular interstitial cells make it an ideal target for targeted drug delivery to this cell population. Currently, clinical treatment for erectile dysfunction related to testosterone deficiency mainly relies on exogenous testosterone replacement therapy (such as oral testosterone preparations and testosterone patches), but this has limitations such as increased liver metabolic burden, cardiovascular risks, and the inability to fundamentally repair damaged testicular interstitial cells. While traditional exosome or stem cell therapies possess tissue repair potential, they lack precise targeting of testicular interstitial cells, easily leading to systemic drug distribution and insufficient local treatment efficiency.

[0004] MSC-derived exosomes possess immunomodulatory effects, promoting tissue repair and regeneration, exhibiting anti-fibrotic and neuroprotective repair properties. They carry the repair instructions of MSCs and inherit their therapeutic potential in tissue repair and regeneration. As a "cell-free therapy" product, they offer higher safety, more delivery methods, and easier standardization in production and quality control. More importantly, MSC-derived exosomes do not induce immunogenicity. While natural exosomes possess therapeutic potential, their limited targeting, low drug loading efficiency, and functional singularity restrict their application.

[0005] Currently, methods for modifying exosomes include cell engineering and direct modification. Cell engineering requires higher technical expertise and can easily alter the state of the parent cell. In contrast, direct modification of exosomes has a shorter production cycle and a lower probability of heterogeneity, making it more suitable for small molecule or surface modification.

[0006] Therefore, in order to obtain exosomes with targeting capabilities in testicular interstitial cells, further promote the repair of damaged cells and enhance the ability of testicular interstitial cells to secrete testosterone, it is urgent to provide a drug-loaded exosome that can target FATE1. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention utilizes a chemical coupling method to link a FATE1-targeting peptide to the surface of MSC-derived exosomes, and then encapsulates small molecule compounds within the exosomes via electroporation, thereby obtaining modified MSC-derived exosomes. The modified exosomes acquire targeting capabilities within testicular interstitial cells, leveraging the natural repair capabilities of MSC-derived exosomes and the release of small molecule drugs to promote the repair of damaged cells, enhance the testosterone secretion capacity of testicular interstitial cells, and alleviate erectile dysfunction in men caused by disease or aging.

[0008] In a first aspect, the present invention provides a drug-loaded exosome targeting FATE1, the exosome being modified with a peptide targeting the FATE1 protein.

[0009] Furthermore, the polypeptide targeting the FATE1 protein is selected from one or more polypeptides with amino acid sequences as shown in SEQ ID NO. 1-5.

[0010] Furthermore, the modification of FATE1 by targeting the peptide specifically involves conjugating a peptide targeting the FATE1 protein with DSPE-PEG-NHS to form a conjugate that is then linked to the exosome.

[0011] Further, the exosomes are exosomes derived from mesenchymal stem cells; optionally, the source of the exosomes may be bone marrow mesenchymal stem cells, adipose tissue mesenchymal stem cells, umbilical cord or umbilical cord blood mesenchymal stem cells, skin mesenchymal stem cells or synovial mesenchymal stem cells; preferably, the exosomes are derived from bone marrow mesenchymal stem cells.

[0012] Furthermore, the peptides targeting the FATE1 protein include five peptides with amino acid sequences as shown in SEQ ID NO. 1-5.

[0013] Secondly, the present invention also provides a method for preparing drug-loaded exosomes targeting FATE1 as described in the first aspect, the method comprising the following steps: Step 1) Dissolve DSPE-PEG-NHS, the FATE1 protein-targeting peptide, and triethylamine in N,N-dimethylformamide (DMF) solution to form a reaction mixture. Incubate at room temperature for 10-15 hours. Then, place the reaction mixture in a dialysis bag with a molecular weight cutoff of less than 1000 Da, dialyze with deionized water for 22-25 hours, and freeze-dry to obtain the conjugate DSPE-PEG-targeting FATE1 peptide. The FATE1 protein-targeting peptide is selected from one or more peptides with amino acid sequences as shown in SEQ ID NO. 1-5. Step 2) Take the purified exosome suspension, resuspend it with PBS to a concentration of 0.5-1.5 mg / mL, weigh a certain amount of the lyophilized conjugate DSPE-PEG-targeting FATE1 peptide, dissolve it in PBS, and add it dropwise to the resuspended exosomes. Shake and incubate for 1.5-3 h. Step 3) After incubation, centrifuge the mixture and wash with PBS to obtain exosomes targeting FATE1.

[0014] Furthermore, the mass concentration ratio of the DSPE-PEG-NHS to the peptide targeting the FATE1 protein is 100:1-1.5.

[0015] Further, in step 2), the molar ratio of the exosomes to the conjugate DSPE-PEG-targeting FATE1 peptide is 1:4-6.

[0016] Furthermore, the peptides targeting the FATE1 protein include five peptides with amino acid sequences as shown in SEQ ID NO. 1-5.

[0017] Furthermore, the mass ratio of the five polypeptides with amino acid sequences as shown in SEQ ID NO.1-5 is 1:1:1:1:1, and the total concentration after mixing is 1.1 mM.

[0018] Thirdly, the present invention also provides the application of the FATE1-targeting exosomes described in the first aspect in the preparation of drug delivery vectors.

[0019] Furthermore, the drug is a small molecule drug.

[0020] Furthermore, the small molecule chemical drug is selected from one or more of β-nicotinamide mononucleotide (NMN), nicotinamide nucleoside (NR), and β-hydroxybutyric acid (BHB).

[0021] Fourthly, the present invention also provides a drug delivery carrier containing exosomes targeting FATE1 as described in the first aspect.

[0022] Furthermore, the drug is a small molecule drug.

[0023] Furthermore, the small molecule chemical drug is selected from one or more of β-nicotinamide mononucleotide, nicotinamide nucleoside, and β-hydroxybutyric acid.

[0024] Fifthly, the present invention also provides a medicament for the prevention and / or treatment of erectile dysfunction in men, the medicament comprising an active ingredient and an exosome targeting FATE1 as described in the first aspect or a drug delivery carrier as described in the fourth aspect.

[0025] Furthermore, the active ingredient is a small molecule drug, which can enhance testosterone synthesis and secretion levels.

[0026] Furthermore, the small molecule chemical drug is selected from one or more of β-nicotinamide mononucleotide (NMN), nicotinamide nucleoside (NR), and β-hydroxybutyric acid (BHB).

[0027] Compared with the prior art, the present invention has the following technical effects.

[0028] 1) This invention obtains peptides with high specificity targeting the FATE1 receptor through screening. These peptides can improve the retention of exosome particles in testicular tissue and have higher tissue specificity than ordinary exosome particles.

[0029] 2) In this invention, the targeted peptides obtained through screening are coupled to the surface of exosomes via DSPE-PEG-NHS, preserving the proteins on the exosome surface and making them less susceptible to endocytosis by the exosomes. Therefore, the drug-loaded exosomes of this invention can precisely target testicular interstitial cells.

[0030] 3) This invention significantly improves the mitochondrial function and steroid synthesis pathway of damaged testicular interstitial cells by using a loaded small molecule drug (NMN) in conjunction with the repair effect of MSC exosomes, thereby increasing the synthesis and secretion levels of testosterone and effectively alleviating male erectile dysfunction caused by disease or aging. At the same time, it reduces the toxic side effects of systemic administration and has higher therapeutic safety and targeted efficacy. Attached Figure Description

[0031] Figure 1 The results of screening FATE1 as a testicular interstitial cell-specific gene.

[0032] Figure 2 The experiment verified the specific high expression of FATE1 in testicular interstitial cells and its intracellular membrane localization.

[0033] Figure 3 The results show the morphological characteristics and flow cytometry identification of primary cells.

[0034] Figure 4The particle size distribution and morphological characteristics of exosomes.

[0035] Figure 5 This is the standard curve for NMN encapsulation.

[0036] Figure 6 These are nanoflow cytometry results.

[0037] Figure 7 The results are from immunofluorescence.

[0038] Figure 8 The results of detecting testosterone concentration in the supernatant of primary testicular interstitial cells in each group using ELISA were obtained.

[0039] Figure 9 The results of immunofluorescence staining in different tissues.

[0040] Figure 10 To detect the distribution and temporal dynamics of DiR-labeled FATE1-Exo in various mouse tissues.

[0041] Figure 11 The serum testosterone concentration in mice was detected by ELISA. Detailed Implementation

[0042] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer.

[0043] Example 1: Screening of testicular interstitial cell-specific membrane proteins Transcriptome TPM expression matrices of 49 normal human tissues were obtained from the GTEx v8 database. After filtering out low-expression genes, the expression ratio of each gene in the testis tissue and other tissues and the tissue specificity index (TSI) were calculated. A set of genes with high expression in the testis tissue and TSI ≥ 0.8 was screened out. Combined with Uniprot database annotation, proteins with transmembrane domains or membrane localization signals were further enriched to obtain a candidate library of high-expression membrane proteins in the testis.

[0044] like Figure 1 As shown in Figure A, based on the GTEx database analysis, membrane proteins with high expression in testis tissue were identified in various human tissues. To further elucidate their cellular distribution characteristics within the testis, single-cell transcriptome sequencing results of testis tissue were analyzed. Unsupervised clustering successfully identified the main cell subpopulations, including interstitial cells. Figure 1(B) Combined analysis of GTEx tissue expression profiles and testicular single-cell data revealed that the FATE1 gene was specifically enriched and highly expressed in testicular interstitial cells, and its expression signal was almost entirely confined to this cell subpopulation. Figure 1 (C)

[0045] Subsequently, a publicly available human testicular tissue single-cell sequencing dataset was downloaded, and the Seurat tool was used to perform data quality control, standardization, dimensionality reduction, and cell clustering to annotate testicular interstitial cell subpopulations. Differential expression analysis was performed on Leydig cells and other testicular cells to screen differentially expressed genes with log2FC≥1.5 and corrected p-value<0.01. The intersection of these differentially expressed genes with the aforementioned membrane protein candidate library was used to obtain a preliminary candidate list of Leydig cell-specific membrane proteins.

[0046] Finally, the expression specificity of the candidate genes was verified by qRT-PCR in purified Leydig cells, supporting cells and germ cells, and the expression localization of the candidate membrane proteins on the Leydig cell membrane was confirmed by immunofluorescence colocalization assay to obtain high-confidence testicular interstitial cell-specific membrane protein targets.

[0047] like Figure 2 As shown, the qRT-PCR results indicate that ( Figure 2 (Figure A) FATE1 showed significant and specific high expression in purified testicular interstitial cells, with a relative mRNA level much higher than that in Sertoli cells and germ cells; while in Sertoli cells and germ cells, FATE1 expression was almost undetectable, with extremely low expression levels. This result further confirms the specificity of FATE1 expression in testicular interstitial cells. Immunofluorescence staining results showed ( Figure 2 (Figure B) In primary cultured testicular interstitial cells, FATE1 (red fluorescence) exhibits a dense filamentous / reticular cytoplasmic distribution, with the signal extending outward from the cell nucleus. This is a typical morphological feature of FATE1 as a mitochondrial / endoplasmic reticulum-associated intracellular membrane protein. Further protein-level evidence confirms that FATE1 is highly expressed in testicular interstitial cells and is mainly located in the intracellular membrane system region, rather than on the cell membrane surface.

[0048] Table 1. Peptides designed based on the FATE1 protein target.

[0049] Example 2: MSC Cell Culture Isolation and culture of bone marrow mesenchymal stem cells (BMSCs): C57BL / 6J (male, 6 weeks old) were euthanized and sterilized with 75% alcohol. The femur and tibia were dissected and dissected distally. Bone marrow was washed with PBS, and the washed bone marrow cells were collected and resuspended in mouse bone marrow mesenchymal stem cell culture medium (MUXMX-90011, OriCell, Cyagen Bioscience). When the cells reached 80% confluence, they were isolated using Solase cell dissociation buffer (RP01021, Xiuren Biotechnology Co., Ltd.). Cells were cultured to passage 3 for subsequent experiments.

[0050] Positive surface markers [CD90 (bsm-30163A-FITC, CD105 (A25377, ABclonal Technology) and CD73 (E-AB-F1089D, ElbScience)] and negative markers for BMSCs [CD34 (A23108, ABclonal Technology and CD45 (E-AB-F1136UF, ElbScience)] were used for flow cytometry (FCM) (FongCyte) TM Cell identification (Beijing Cenglang Biotechnology Co., Ltd.)

[0051] like Figure 3 As shown in Figure A, primary cells exhibit a typical elongated spindle-shaped, whorled arrangement during in vitro culture, consistent with the morphological characteristics of MSCs. To further identify the cell type, the expression of MSC-related surface markers was detected by flow cytometry. Figure 3 (Figure B in the middle). The results showed that this cell population highly expressed CD105, CD90, and CD73, with a significantly higher positive rate than the isotype control; simultaneously, it expressed low or no hematopoietic markers CD34 and CD45, meeting the phenotypic criteria for mesenchymal stem cells defined by the International Society for Cell Therapy (ISCT). These results confirm that the present invention successfully isolated and purified mesenchymal stem cells, providing a reliable cell model basis for subsequent experiments.

[0052] Example 3: Preparation and characterization of MSC-derived exosomes 1) Collect MSC cell culture supernatant and filter the supernatant through a 0.45μm filter membrane to remove cell debris; 2) The exosome particles in the MSC cell culture supernatant were solvent-displaced and enriched by tangential flow filtration; 3) The concentration and particle size of exosomes were detected using an NTA nanoparticle tracking analyzer. The density of the prepared exosome solution was approximately 1*10⁻⁶. 10 / mL, store at -80℃.

[0053] 4) The morphology of RCNVs was observed using a transmission electron microscope (JEM-1200EX, JEOL Ltd.).

[0054] like Figure 4 As shown in Figure A, the NTA results indicate that the extracted particles are mainly distributed in the range of 50–300 nm, with a main peak particle size of 97 nm, consistent with the typical particle size distribution characteristics of exosomes (30–200 nm), suggesting that the sample is enriched with extracellular vesicles, mainly exosomes. Transmission electron microscopy (TEM) further confirmed the morphological characteristics of the exosomes. Figure 4 (See the middle image). As can be seen, the sample contains numerous round or near-round membrane vesicles with a clear double-membrane structure. The particle size is consistent with the NTA results, conforming to the typical morphology of exosomes. These results confirm that this invention successfully extracted exosomes with intact morphology and uniform particle size.

[0055] Example 4: Preparation of MSC-derived exosomes targeting FATE1 by chemical coupling method.

[0056] 100 mg of DSPE-PEG-NHS, 1.1 mM of the targeting peptide (a mixture of 5 peptides of equal mass), and 3 mM triethylamine were dissolved in 3 mL of DMF solution and incubated at room temperature for 12 h. The reaction mixture was placed in a dialysis bag with a molecular weight cutoff of less than 1000 Da and dialyzed with deionized water for 24 h. The dialyzed solution was then lyophilized to obtain the DSPE-PEG-targeting FATE1 peptide.

[0057] Purified MSC exosomes were resuspended in PBS to a concentration of 1 mg / mL. The lyophilized conjugate was weighed at a molar ratio of 5:1 (DSPE-PEG-targeting FATE1 peptide: exosomes), dissolved in a small amount of PBS, and added dropwise to the exosome suspension. The mixture was incubated at 37°C with gentle shaking for 2 h to allow the DSPE-PEG-FATE1 peptide to insert into the exosome phospholipid bilayer.

[0058] After incubation, the mixture was transferred to a 100 kDa ultrafiltration centrifuge tube, centrifuged at 14000 g and 4℃ for 30 min, and washed three times with PBS to remove uninserted conjugates, thus obtaining MSC exosomes targeting FATE1.

[0059] Example 5: Electroporation preparation of drug-loaded exosomes Weigh 20 mg of β-nicotinamide mononucleotide (NMN), dissolve it in 1 mL of 5×PBS to prepare a 60 mM solution, and filter it for sterilization at 0.22 μM. Mix the above-mentioned targeted modified exosomes with the NMN solution (30 mM concentration), and perform electroporation according to the set electroporation parameters. After electroporation, incubate the mixture at 37°C for 1 h, dilute it with 5×PBS, concentrate it using a 100 kDa MWCO ultrafiltration centrifuge tube at 14000 g for 30 min, and wash away the free NMN.

[0060] The filtrate inside the ultrafiltration centrifuge tube was collected, and the concentration of NMN in the filtrate was measured; the encapsulation rate of exosomes was then calculated.

[0061] NMN loading rate = (total NMN - filtrate NMN) / total NMN × 100%. Electroporation parameters are shown in Table 2.

[0062] Table 2 Electrical Transfer Parameters

[0063] The method for detecting the loading rate is as follows: NMN standard samples with concentrations of 0, 5, 10, 20, 70, 80, and 100 μmol / L were prepared using 5×PBS buffer. The samples were dissolved in appropriate solvents to ensure the concentration was within the detection range. The absorbance was measured at 235 nm using a UV spectrophotometer. The absorbance values ​​of the NMN standard samples at different concentrations were recorded. The data were processed using software, and a standard curve was plotted with NMN concentration on the x-axis and the corresponding absorbance on the y-axis. The standard curve is shown below. Figure 5 As shown, the loading rate is 86.52%.

[0064] Example 6: Nanoflow cytometry detection of FATE1 peptide-MSC exosome conjugation The chemically conjugated FATE1 peptide-MSC exosome complex (modified group), unconjugated naked MSC exosomes (negative control group), a mixture of free fluorescently labeled FATE1 peptide and naked exosomes (non-specific adsorption control group), and MSC exosomes with only the conjugating reagent (reagent control group) were washed three times with PBS buffer (pH 7.4), centrifuged at 8000×g, 4℃ for 30 min to remove free peptides, unbound conjugating reagents, and impurities. The exosomes were then resuspended in 100 μL PBS buffer and the concentration was adjusted to 1×10⁻⁶. 9 / mL. Take each of the above groups of samples, add 5 μL of CD81 (A27605, ABclonal Technology), and incubate at 37℃ in the dark for 20 min to label exosomes and delineate exosome populations; simultaneously, the FATE1 peptide was pre-labeled with a FITC fluorescent group during the chemical synthesis stage (or after coupling, added with a FITC-labeled FATE1 peptide-specific antibody, and incubated at 37℃ in the dark for 30 min) to detect the presence of the peptide. After labeling, add 10% fetal bovine serum to stop staining, and centrifuge and wash twice to remove free dye and antibody. Detect using nanoflow cytometry.

[0065] The results are as follows Figure 6 As shown, with modification group ( Figure 6 D) Obvious PKH67 / FITC double positive signals were observed, with a significantly higher positive rate than that of naked MSC exosomes without FATE1 peptide (negative control group). Figure 6 A) MSC exosomes with only the coupling reagent added (reagent control group) Figure 6 B) A mixture of free fluorescently labeled FATE1 peptide and naked exosomes (non-specific adsorption control group) Figure 6 C) serves as the criterion for successful chemical coupling of the FATE1 peptide to the surface of MSC exosomes.

[0066] Example 7: In vitro targeting verification Testicular interstitial cells with high FATE1 expression were selected and seeded in confocal culture dishes at a density of 2 × 10⁶ cells / year. 5 Cells / wells were cultured in DMEM / F12 medium containing 10% FBS at 37°C and 5% CO2 until cell confluence reached 70%–80%. Control groups were set up simultaneously: unmodified exosome group (Exo group) and blank control group (medium only).

[0067] FATE1-targeted exosomes and unmodified exosomes were resuspended in PBS to the same protein concentration (e.g., 100 μg / mL). Lipophilic fluorescent dye DiO (final concentration 5 μM) was added, and the mixture was incubated at 37°C in the dark for 30 min. The cells were then centrifuged at 14000 g at 4°C for 30 min using a 100 kDa MWCO ultrafiltration centrifuge tube. The cells were washed three times with PBS to remove free dye, yielding DiD-labeled exosomes. The cell supernatant was discarded, and the cells were washed twice with PBS. Serum-free medium containing DiD-labeled exosomes (final exosome concentration 50 μg / mL) was added, and the cells were incubated at 37°C for 2 h. After incubation, the supernatant was discarded, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, stained with DAPI for 5 min, and mounted with anti-fluorescence quenching. Observation under laser confocal microscopy: excitation wavelength 644 nm, emission wavelength 665 nm, and DiO fluorescence signal acquisition; verification by flow cytometry: cells were digested and collected, and the proportion of DiO positive cells was detected by flow cytometry to quantitatively compare the uptake efficiency of the two groups.

[0068] like Figure 7 Immunofluorescence results showed that Dio-labeled exosomes (green) and FATE1 protein (red) exhibited significant co-localization. Specifically, only a small number of exosomes in the Exo group showed co-localization with FATE1 protein, while the co-localization signal was significantly enhanced in the FATE1-Exo group, suggesting that FATE1-targeting exosomes prepared via chemical coupling can efficiently enter target cells.

[0069] Example 8: Detection of testosterone concentration in testicular interstitial cell supernatant Testicular interstitial cells were seeded into 6-well plates and cultured in groups for 24 h. The cell culture supernatant was collected, centrifuged at 3000 rpm for 10 min to remove impurities, and the supernatant was then transferred to a microplate reader. The absorbance (OD value) of the testosterone ELISA kit (PT872, Beyotime) was measured at 450 nm using a microplate reader. The testosterone concentration in each group was calculated based on the standard curve.

[0070] Figure 8 To investigate the effect of exosomes on testosterone synthesis and secretion at the cellular level, the ELISA method was used to detect testosterone concentration in the supernatant of primary testicular interstitial cells in each group. Results showed that the testosterone concentration in the Exo group was significantly higher than that in the control group, suggesting that exosomes can significantly enhance testosterone synthesis and secretion in testicular interstitial cells. The testosterone secretion level in the FATE1-Exo group was significantly higher than that in the exosome-only group. These results confirm that FATE1-Exo can further promote testosterone synthesis and secretion in testicular interstitial cells at the cellular level.

[0071] Example 9: In vivo tissue specificity verification of FATE1 protein Eight to ten-week-old SPF-grade male C57BL / 6 mice were selected and acclimatized for one week. The main organs, such as testes, heart, liver, lungs, and kidneys, were isolated, paraffin sections were prepared, and FATE1 protein was immunofluorescently stained. The distribution of FATE1 protein in various tissues was observed under a confocal microscope.

[0072] like Figure 9 Immunofluorescence staining results showed that FATE1 protein exhibited a highly tissue-specific expression pattern in different mouse tissues. FATE1 (green fluorescence) was mainly enriched in testicular tissue; while in heart, lung, liver, and kidney tissues, the fluorescence signal of FATE1 was extremely weak, with almost no specific expression and only background fluorescence signal. This result further validated the testicular-specific expression characteristics of FATE1 at the protein level, which is completely consistent with the conclusions of previous GTEx database and single-cell transcriptome analysis.

[0073] Example 10: In vivo targeting validation Eight to ten-week-old SPF-grade male C57BL / 6 mice were randomly divided into: a FATE1-targeted exosome group; an unmodified exosome group; and a blank control group (injected with PBS only).

[0074] Six mice were placed in each group and allowed to acclimatize for three days before the experiment began. DiD-labeled exosomes were injected via tail vein at a dose of 10 mg / kg, with an injection volume of 100 μL per mouse. Mice were sacrificed at 30 min, 1 d, 3 d, 7 d, and 15 d after injection. Major organs such as the testes, heart, liver, lungs, and kidneys were isolated, frozen sections were prepared, nuclei were stained with DAPI, and the distribution of DiO fluorescence in the testicular interstitial region was observed under a confocal microscope.

[0075] like Figure 10 As shown, 30 minutes after administration, exosomes were mainly enriched in lung tissue, while a significant fluorescent signal was detected in testicular tissue. By day 1, the fluorescent signal in the testes reached its peak, while the signal in the lung tissue began to weaken. At day 3, the fluorescent signal in the testes was still clearly discernible but gradually decreased, while the signal in tissues such as the lungs and liver was significantly reduced. Except for the testes, the fluorescent signals in other tissues were close to background levels, with only very weak signals remaining. At day 15, the fluorescent signals in all tissues had essentially disappeared, indicating that the exosomes had been completely metabolized and cleared. These results indicate that FATE1-Exo has testicular-targeting enrichment characteristics in vivo, can remain in testicular tissue for a relatively long time, and is rapidly metabolized and cleared in other tissues, providing morphological evidence for its targeted therapeutic application in testicular-related diseases.

[0076] Example 11 Testosterone Concentration Detection After grouping and treatment in mice, blood was collected from the orbital sinus, and serum was separated by centrifugation at 3500 rpm for 15 min. Serum testosterone levels were detected using the same ELISA kit, strictly following the kit steps for sample addition, incubation, washing, and color development. The OD450 nm value was read, and the testosterone concentration was calculated.

[0077] The results are as follows Figure 11 As shown, serum testosterone concentration in the Exo group was not significantly different from that in the control group, while serum testosterone levels in the FATE1-Exo group were significantly higher than those in the control and Exo groups. This indicates that FATE1-modified exosomes have a significant targeting effect compared to exosomes alone. They can target testicular interstitial cells and significantly increase testosterone secretion.

[0078] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An exosome targeting FATE1 for drug delivery, characterized in that, The exosomes are modified with peptides that target the FATE1 protein.

2. The exosomes according to claim 1, characterized in that, The peptide targeting the FATE1 protein is selected from one or more peptides with amino acid sequences as shown in SEQ ID NO.1-5.

3. The exosomes according to claim 1, characterized in that, Specifically, the modification of FATE1 by targeting the peptide involves conjugating the peptide targeting the FATE1 protein with DSPE-PEG-NHS to form a conjugate that is then linked to the exosome.

4. The exosomes according to claim 1, characterized in that, The exosomes are exosomes derived from mesenchymal stem cells.

5. The method for preparing FATE1-targeting exosomes for drug delivery according to any one of claims 1-4, characterized in that, The preparation method Includes the following steps, Step 1) Dissolve DSPE-PEG-NHS, the FATE1 protein-targeting peptide, and triethylamine in N,N-dimethylformamide solution to form a reaction mixture. Incubate at room temperature for 10-15 hours. Then, place the reaction mixture in a dialysis bag with a molecular weight cutoff of less than 1000 Da, dialyze with deionized water for 22-25 hours, and freeze-dry to obtain the conjugate DSPE-PEG-targeting FATE1 peptide. The FATE1 protein-targeting peptide is selected from one or more peptides with amino acid sequences as shown in SEQ ID NO. 1-5. Step 2) Take the purified exosome suspension, resuspend it with PBS to a concentration of 0.5-1.5 mg / mL, weigh a certain amount of the lyophilized conjugate DSPE-PEG-targeting FATE1 peptide, dissolve it in PBS, and add it dropwise to the resuspended exosomes. Shake and incubate for 1.5-3 h. Step 3) After incubation, centrifuge the mixture and wash with PBS to obtain exosomes targeting FATE1.

6. The preparation method according to claim 5, characterized in that, In step 1), the mass concentration ratio of the DSPE-PEG-NHS to the peptide targeting FATE1 protein is 100:1-1.

5.

7. The preparation method according to claim 5, characterized in that, In step 2), the molar ratio of the exosomes to the conjugate DSPE-PEG-targeting FATE1 peptide is 1:4-6.

8. The use of the FATE1-targeting exosomes according to any one of claims 1-4 in the preparation of drug delivery carriers.

9. A drug delivery carrier comprising exosomes targeting FATE1 as described in any one of claims 1-4.

10. A medicine for the prevention and / or treatment of erectile dysfunction in men, characterized in that, The drug comprises an active ingredient and an exosome targeting FATE1 as described in any one of claims 1-4 or a drug delivery carrier as described in claim 9.

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