Method for preparing stem cell exosome composition and application thereof in male sexual dysfunction

Abstract

The application provides a preparation method of a stem cell exosome composition and application of the stem cell exosome composition in male sexual dysfunction, and the preparation method comprises the following steps: extracting stem cell exosomes; combining the stem cell exosomes with a stabilizing peptide; and preparing a stem cell exosome hydrogel; the stabilizing peptide comprises an amino acid sequence as shown in SEQ ID NO: 1. The stem cell exosome composition obtained by the method can improve the stability of the exosomes, improve the in-vivo bioavailability, prevent and relieve testicular injury, and inhibit inflammatory reactions.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology research and development, and specifically provides a method for preparing a stem cell exosome composition and its application in male sexual dysfunction. Background Technology

[0002] Infertility refers to the inability to conceive or carry a pregnancy to full term, and it has gradually become one of the most important issues affecting individual health and social life. Male infertility is influenced by many factors, including age, congenital defects, infections, environmental problems, genetic diseases, and autoimmune diseases. These factors can lead to sperm production failure, reduced sperm concentration, and fertilization failure, thus causing infertility.

[0003] Currently, several effective treatments for infertility have been developed, including hormone therapy, drug therapy targeting specific causes, surgical removal, fertility preservation, and assisted reproductive technologies (ART). However, these therapies have encountered varying degrees of difficulties in their use. For example, hormone therapy is effective in some cases of infertility, such as low sperm count, but overuse of hormone drugs may lead to other diseases (see Vermeulen R., Korse C., Kenter G., et al. Safety of hormone replacement therapy following risk-reducing salpingo-oophorectomy: Systematic review of literature and guidelines. Climacteric. 2019;22:352–360). Artificial insemination (ART) can solve nearly 80% of infertility problems, but it is highly invasive and poses some health risks (see Gómez R., Soares SR, Busso C., et al. Physiology and pathology of ovarian hyperstimulation syndrome. Semin. Reprod. Med. 2010;28:448–457). To overcome the shortcomings of traditional therapies, researchers have begun to shift their attention to stem cell therapy.

[0004] Stem cells are undifferentiated cells with the ability to self-renew and differentiate into various cell types, and are an important component of the body's internal repair system. Researchers have found that stem cell therapy can help infertile individuals obtain offspring. For example, stem cell transplantation has been found to effectively restore fertility in mouse models (see Brinster RL Malegermline stem cells: From mice to men. Science. 2007;316:404–405); and in rhesus monkeys, functional sperm were generated through stem cell transplantation after chemically induced infertility (see Hermann BP, Sukhwani M., Winkler F., et al. Spermatogonial stem cell transplantation into rhesustestes regenerates spermatogenesis producing functional sperm. Cell StemCell. 2012;11:715–726).

[0005] Stem cells have shown promising results in the treatment of male infertility. Reports indicate that stem cells and stem cell-derived vesicles can protect and restore sperm fertility during freeze-thaw cycles (see Qamar AY, Fang X., et al. Improved post-thaw quality of canine semen after treatment with exosomes from conditioned medium of adipose-derived mesenchymal stem cells. Animals. 2019;9:865). In vivo generation of spermatogonial stem cells from bone marrow-derived MSCs (BM-MSCs) can significantly promote male fertility recovery (see Karimaghai N., Tamadon A., Rahmanifar F., et al. Spermatogenesis after transplantation of adipose tissue-derived mesenchymal stem cells in busulfan-induced azoospermichamster. Iran. J. Basic Med. Sci. 2018;21:660–667). In mice, BM-MSCs... Immunosuppression of anti-sperm antibodies (ASA) produced after traumatic testicular rupture caused by infusion (see Mohammad S., Aghamir K., Salavati A., et al. Does Bone Marrow–derived Mesenchymal Stem Cell Transfusion Prevent Antisperm Antibody Production After Traumatic Testis Rupture? Urology. 2014;86:82–86).

[0006] However, many authorities have raised controversies and ethical issues regarding stem cell therapy, and pluripotent stem cell therapy faces some drawbacks, such as low homing efficiency, tumor formation, non-functionality, or immune rejection (Yin L., Liu X., Shi Y., et al. Therapeutic Advances of Stem Cell-Derived Extracellular Vesicles in Regenerative Medicine. Cells. 2020;9:707). Of particular interest is that stem cells can secrete extracellular vesicles (EVs) with the same therapeutic and regenerative effects as maternal stem cells. EVs are nanovesicles containing proteins, lipids, RNA (mRNA, miRNA, and lncRNA), and other biomolecules that play important roles in paracrine signaling (see Mathieu M., Martin-Jaular L., Lavieu G., et al. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nat. Cell Biol. 2019;21:9–17). EVs, such as exosomes (exo) and microvesicles, are continuously released from all living cells. Exosomes, formed from multivesicular endosomes or multivesicular bodies, are easier to isolate and apply. EVs contain the secretome of the source cells and, due to their ability to transfer different molecules, can be used to treat various reproductive diseases. As a cell-free therapy, they can overcome the limitations of traditional stem cell therapies.

[0007] This invention is based on stem cell exosome therapy. It uses a hydrogel containing special stable peptides to encapsulate exosomes, which can improve their in vivo stability and bioavailability, promote testicular repair after injury, and thus benefit the treatment and prevention of male infertility. Summary of the Invention

[0008] A first aspect of the present invention provides a method for preparing a stem cell exosome composition, characterized in that the method comprises:

[0009] (1) Extraction of stem cell exosomes;

[0010] (2) Stem cell exosomes bind to stable peptides;

[0011] (3) Preparation of stem cell exosome hydrogel;

[0012] The stable fusion peptide comprises the amino acid sequence shown in SEQ ID NO: 1.

[0013] Utilizing exosomes for stem cell therapy remains challenging, as the in vivo bioavailability of exosomes is low when used directly, making it difficult to meet therapeutic needs. This invention utilizes an exosome hydrogel composition that effectively delivers exosomes and enhances their viability. Currently, the common method for modifying exosomes into hydrogels is direct mixing, but this can have drawbacks such as low loading efficiency, exosome structural damage, and inhibition of exosome osteogenic potential (see Wang J., Li W., Zhang L., et al. Chemically edited exosomes with dual ligand purified by microfluidic device for active targeted drug delivery to tumor cells. ACS Appl. Mater. Interfaces. 2017;9:27441–27452).To overcome these limitations, multifunctional fusion peptides composed of two or more peptide chains have shown excellent drug delivery capabilities (see Yousefpour P., Jonathan M., Prasad L. V., et al. Genetically encoding albumin binding into chemotherapeutic-loaded polypeptide nanoparticles enhances their antitumor efficacy. NanoLett. 2018;18:7784–7793) and are considered a promising exosome delivery method. For example, the polypeptide sequence CP05 (CRHSQMTVTSRL) can specifically recognize and capture the exosome marker CD63 (see Vicencio JM, Yellon DM, Sivaraman V., et al. Plasma exosomes protect the myocardium from ischemia-reperfusion injury. J. Am. Coll. Cardiol. 2015;65:1525–1536). TKKTLRT and KELNLVY are collagen-binding domains that specifically bind to SIS, respectively. Type I and Type III collagen in hydrogels (see Mcmasters J., Panitch A. Collagen-binding nanoparticles for extracellular anti-inflammatory peptide delivery decrease platelet activation, promote endothelial migration, and suppress inflammation. Acta Biomater. 2016;49:78–88). Furthermore, adding peptide linkers during the construction of fusion peptides can prevent misfolding or impaired biological activity. EAAAK is a rigid linker containing an α-helix structure composed of multiple hydrogen bonds, conferring high structural stability to the fusion peptide (see Chen X., Zaro JL, Shen WC Fusionprotein linkers: property, design and functionality. Adv. Drug Deliv. Rev.2013;65:1357–1369).This invention provides a novel stable peptide with the amino acid sequence shown in SEQ ID NO: 1. When this stable peptide is linked with EAAAK, a fusion peptide is formed, which further enhances the stability of stem cell exosomes.

[0014] Furthermore, the stem cells are mesenchymal stem cells, including at least one of bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord mesenchymal stem cells, dental pulp mesenchymal stem cells, and induced stromal stem cells. Existing technologies have shown that stem cells and their exosomes from various sources have positive therapeutic effects on male infertility; therefore, the method provided by this invention is not limited to stem cells from a single source.

[0015] Furthermore, the stem cells are bone marrow mesenchymal stem cells.

[0016] Furthermore, the step of extracting stem cell exosomes includes: culturing bone marrow mesenchymal stem cells to the logarithmic growth phase, collecting cell supernatant, centrifuging at 3000 g for 10 minutes to collect the supernatant; centrifuging at 10000 g for 30 minutes, collecting the supernatant and filtering it through a 0.22 μm filter membrane; precipitating exosomes by ultracentrifugation at 100000 g for 1 hour, washing the precipitate with sterile PBS, and finally recovering the exosomes by centrifugation at 100000 g for 30 minutes.

[0017] Furthermore, the amino acid sequence of the stable peptide is shown in SEQ ID NO: 2, and the stable fusion peptide is the peptide shown in SEQ ID NO: 1 linked with EAAAK to form a stable fusion peptide.

[0018] Furthermore, the step of binding the stem cell exosomes to the stable peptide includes: dissolving the stable peptide in deionized water at room temperature to prepare a concentration of 2×10⁻⁶. -3 mM peptide solution; take 200 μL of stem cell exosomes with a concentration of 1 μg / μL and mix them with 100 μL of peptide solution. Incubate at room temperature for 30-60 minutes; centrifuge at 100000g for 30 minutes, discard the supernatant, and resuspend the precipitate to obtain a stable peptide-exosome complex.

[0019] Furthermore, the step of preparing stem cell exosome hydrogel includes: adding the stable peptide and exosome complex to a 20% methacrylate gelatin solution, mixing evenly, then adding to liquid paraffin and stirring to emulsify; stirring in an ice-water bath at 4°C and adding a photoinitiator for photocrosslinking; after crosslinking is completed, adding isopropanol to the mixed solution and filtering, then washing three times with isopropanol to obtain stem cell exosome hydrogel.

[0020] A second aspect of the present invention provides a stem cell exosome composition prepared by the method described above.

[0021] A third aspect of the present invention provides the use of the aforementioned stem cell exosome composition in the preparation of a medicament for treating male sexual dysfunction.

[0022] Furthermore, the male sexual dysfunction includes testicular injury.

[0023] Beneficial effects

[0024] This invention provides a method for preparing a stem cell exosome composition and its application in male sexual dysfunction, as detailed below.

[0025] 1. A stem cell exosome composition is provided, wherein the composition is a hydrogel encapsulating stem cell exosomes, which can effectively deliver the stem cell exosomes and maintain their stability;

[0026] 2. The stem cell exosome composition includes cell-stabilizing peptides, which can further improve the stability of the hydrogel complex;

[0027] 3. The stem cell exosome composition can effectively inhibit and prevent testicular damage, maintain sperm count, and combat inflammatory responses. Attached Figure Description

[0028] Figure 1 : Degradation rate of exosome hydrogel;

[0029] Figure 2 : Testicular coefficient;

[0030] Figure 3 Sperm count;

[0031] Figure 4 IL-6 expression level;

[0032] Figure 5 TNF-α expression level. Detailed Implementation

[0033] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents, biological materials, and detection kits are all commercially available.

[0034] The term "stem cell" refers to an undifferentiated cell. Such cells can undergo differentiation when exposed to one or more stimuli. These stimuli can be physical, mechanical, electrical, chemical, biochemical, biological, or any combination of the latter. Here, "stem cell" should be understood as a pluripotent or multipotent cell, as well as an induced cell.

[0035] The term "mesenchymal stem cells" refers to a type of adult stem cells with multi-directional differentiation potential, mainly derived from tissues such as bone marrow, adipose tissue, umbilical cord blood, and placenta.

[0036] The term "exosome" refers to a nanoscale vesicle with a membrane structure that is secreted or released from a cell into the extracellular space, and is also known as an extracellular vesicle or microvesicle.

[0037] The term "mesenchymal stem cell exosomes" refers to all exosomes isolated from, for example, conditioned medium of human umbilical mesenchymal stem cells, blood, human umbilical cord tissue, or equivalent biological products.

[0038] The term "gel," also known as "hydrogel" or "gel solution," refers to a dispersion system formed by the dissolution of hydrophilic polymeric materials (such as gelatin, hyaluronic acid, etc.) in water. This dispersion system may contain one or more hydrophilic polymeric materials, and may also include other non-polymeric materials. Gels can exhibit good cell affinity, which is beneficial for cell preservation or proliferation.

[0039] Example 1: Preparation of stem cell exosomes

[0040] Evidence has shown that bone marrow mesenchymal stem cells (HMSCs) can effectively treat male infertility, and their secreted exosomes contain various active ingredients with reproductive-promoting effects. Therefore, in this invention, human bone marrow mesenchymal stem cells (h-BMSCs) are used to extract exosomes. The h-BMSCs are extracted from the bone marrow of healthy volunteers, cultured in primary culture, and preserved in our laboratory. The mesenchymal stem cells are isolated and extracted using known methods, such as those disclosed in the literature (Yang et al., Effects of HMGB1 on the proliferation and cytokine secretion of bone marrow mesenchymal stem cells in healthy individuals, Chinese Journal of Experimental Hematology, 2021; 29(5):1631-1636).

[0041] Human bone marrow mesenchymal stem cells (h-BMSCs) were seeded in fresh DMEM containing 10% FBS and 1% penicillin / streptomycin and cultured at 37°C and 5% CO2 until the logarithmic growth phase. Cell supernatant was then collected by centrifugation at 3000 g for 10 min, followed by centrifugation at 10000 g for 30 min. The supernatant was then filtered through a 0.22 μm filter. Exosomes were then precipitated by ultracentrifugation at 100000 g for 1 h, washed with sterile PBS, and finally recovered by centrifugation at 100000 g for 30 min. Transmission electron microscopy revealed that the exosomes were round or oval in shape, with intact membrane structures, and approximately 100 nm in size. Western blot analysis identified exosome markers, showing normal expression of markers such as CD9 and TSG101.

[0042] Example 2: Preparation of exosome hydrogel (GelMA-EXO)

[0043] 2.1 Preparation of stable fusion peptides

[0044] This invention provides a novel stable peptide with the amino acid sequence KTKVMSTAKQ as shown in SEQ ID NO: 1, which is then combined with an EAAAK linker to form a stable fusion peptide, further enhancing its stability. The amino acid sequence of the stable fusion peptide is shown in SEQ ID NO: 2. Both the stable peptide and the stable fusion peptide are prepared using a solid-phase synthesis method.

[0045] 2.2 Preparation of stable fusion peptide-exosome complex

[0046] The stable fusion peptide was dissolved in deionized water at room temperature to prepare a concentration of 2×10⁻⁶. -3 mM peptide solution. Take 200 μL of stem cell exosomes with a concentration of 1 μg / μL and mix them with 100 μL of peptide solution. Incubate at room temperature for 30-60 minutes. Centrifuge at 100,000g for 30 minutes, discard the supernatant, and resuspend the precipitate to obtain a stable fusion peptide-exosome complex (EXO-FP).

[0047] 2.3 Preparation of exosome hydrogels

[0048] A 20% methacrylate gelatin (GelMA) solution (purchased from Sigma) was prepared. 500 μg of the stable fusion peptide and exosome complex was added to 5 mL of the GelMA solution and mixed thoroughly at 37°C. Liquid paraffin Span-80 (10:1) (purchased from Sigma) was added to a round-bottom flask and stirred at 350 r / min for 10 minutes to obtain a homogeneous emulsion, which was used as the oil phase. The GelMA and exosome mixture was slowly added to the oil phase and emulsified by stirring at a certain speed for 10 minutes. The mixture was then stirred in an ice-water bath at 4°C, and 200 μL of LAP photoinitiator (purchased from Sigma) was added for photocrosslinking. After crosslinking, isopropanol was added to the mixture to generate flocculants, which were further filtered and washed three times with isopropanol to remove unencapsulated exosomes, yielding exosomes encapsulated with GelMA microspheres (GelMA-EXO-FP).

[0049] 2.4 Stability testing of exosome hydrogels

[0050] To investigate the stability of exosome hydrogels, we used an enzymatic hydrolysis method to detect their in vitro degradation rate. A 100 U / mL type I collagenase solution was prepared, and 1 mL of hydrogel was immersed in 10 mL of the enzyme solution and incubated at 37°C for 1 hour. The remaining hydrogel was washed with PBS, and its wet weight was recorded on days 1, 3, 5, 7, and 10, denoted as (wd). The weight of the remaining sample (wd) was compared with the weight of the original hydrogel (wo) to calculate the degradation rate. Degradation rate = [(wo-wd) / wo] × 100%. Simultaneously, an exosome hydrogel without fused stable peptides (GelMA-EXO) was used as a control, prepared using the same method as in Section 2.3. The results are as follows: Figure 1 As shown, the stability of exosome hydrogels linked with fusion-stabilized peptides is significantly improved, and some activity is still present after 10 days of storage.

[0051] Example 3: Exosome hydrogel promotes testicular repair in mice after injury

[0052] 3.1 Preparation and administration of a mouse testicular injury model

[0053] This invention uses lipopolysaccharide (LPS) to construct a mouse injury model. LPS is a major component of Gram-negative bacteria and is most commonly used as an effective inducer of inflammation in mouse experimental models. It has been reported that LPS-induced orchitis disrupts spermatogenesis and steroid production (see HEDGER MP Immunophysiology and pathology of inflammation in the testis and epididymis. J Androl. 2011;32(6):625-640). LPS has been widely used in the preparation of animal testicular injury models in domestic and international research.

[0054] Eight-week-old male ICR mice were selected and housed in an environment with a temperature of (22±2)℃ and a photoperiod of 12 h. Mice had free access to food and water and were acclimatized for one week. Thirty mice were randomly divided into three groups: the GelMA-EXO-FP group (10 mg / kg GelMA-EXO-FP was injected locally into the testis); the GelMA-EXO group (10 mg / kg GelMA-EXO was injected locally into the testis); and the control group (an equal volume of physiological saline was injected locally into the testis). One hour after treatment, mice in all three experimental groups were intraperitoneally injected with 10 mg / kg LPS. Ten mice served as a normal control group and were injected with an equal volume of physiological saline. After model establishment, all groups of mice continued to be fed for 24 hours.

[0055] 3.2 Determination of the testicular coefficient in mice

[0056] After the experiment, the mice were euthanized, and testicular tissue was collected. The tissue was cleaned with sterile PBS, excess water was removed with absorbent paper, and the testicular mass was weighed. The testicular coefficient was calculated as follows: Testicular coefficient = Testicular weight (g) / Mouse body weight (g) × 100%.

[0057] The results are as follows Figure 2 As shown, LPS treatment reduced the weight of mice, resulting in a significant increase in the testicular index. However, pretreatment with stem cell exosome hydrogel complex, especially GelMA-EXO-FP pretreatment, inhibited the weight loss in animals, causing the testicular index to return to normal levels.

[0058] 3.3 Mouse sperm count determination

[0059] The mouse epididymis was removed, and the distal end of the vas deferens was grasped with forceps. Three incisions were made on the epididymal tail, and the sperm was quickly placed in 200 μL of preheated PBS at 37 ℃ for 10 min to allow for full sperm motility. The sperm suspension was filtered through a 200-mesh nylon mesh, diluted 1:4, and 20 μL of the sperm suspension was transferred to a hemocytometer to count the sperm count. The results are as follows: Figure 3 As shown, compared with the normal group, the number of sperm was significantly reduced after modeling. However, after treatment with stem cell hydrogel, the number of sperm recovered to some extent, and the GelMA-EXO-FP group was closer to the normal level.

[0060] 3.4 Detection of inflammatory factor expression in mouse testicular tissue (RT-PCR or ELISA)

[0061] Testicular tissues from mice in each group were collected, homogenized at 4°C, and the supernatant was centrifuged at 3000 rpm for 10 minutes to remove tissue residue. The expression levels of IL-6 and TNF-α in the supernatant were detected using an ELISA kit (purchased from R&D Company, USA). The specific procedure was performed according to the kit instructions. Results are as follows: Figure 4 , Figure 5 As shown, after LPS modeling, the expression levels of inflammatory factors such as IL-6 and TNF-α in mice were significantly increased. After treatment with exosome hydrogel, the release level of inflammatory factors was inhibited, indicating that this therapy can effectively alleviate the inflammatory response.

[0062] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A stable fusion peptide, characterized in that, The sequence of the stable fusion peptide is shown in SEQ ID NO:

2.

2. A method for preparing a stem cell exosome composition, characterized in that, The method includes: (1) Extraction of stem cell exosomes; (2) Stem cell exosomes bind to the stable fusion peptide described in claim 1; (3) Preparation of stem cell exosome hydrogel; The stem cells mentioned are human bone marrow mesenchymal stem cells; The steps for preparing stem cell exosome hydrogel include: adding a stable fusion peptide and exosome complex to a 20% methacrylate gelatin solution, mixing thoroughly, adding to liquid paraffin, and stirring to emulsify; stirring in a 4 ℃ ice-water bath and adding a photoinitiator for photocrosslinking; after crosslinking is completed, adding isopropanol to the mixed solution and filtering, then washing three times with isopropanol to obtain stem cell exosome hydrogel.

3. The preparation method according to claim 2, characterized in that, The steps for extracting stem cell exosomes include: culturing bone marrow mesenchymal stem cells to the logarithmic growth phase, collecting cell supernatant, centrifuging at 3000 g for 10 minutes to collect the supernatant; centrifuging at 10000 g for 30 minutes, collecting the supernatant and filtering it through a 0.22 μm filter membrane; ultracentrifuging at 100000 g for 1 hour to precipitate the exosomes, washing the precipitate with sterile PBS, and finally centrifuging at 100000 g for 30 minutes to recover the exosomes.

4. The preparation method according to claim 3, characterized in that, The step of binding stem cell exosomes to the stable fusion peptide includes: dissolving the stable fusion peptide in deionized water at room temperature to prepare a concentration of 2×10⁻⁶. -3 mM peptide solution; take 200 μL of stem cell exosomes with a concentration of 1 μg / μL and mix them with 100 μL of peptide solution. Incubate at room temperature for 30-60 minutes; centrifuge at 100000g for 30 minutes, discard the supernatant, and resuspend the precipitate to obtain a stable fusion peptide and exosome complex.

5. A stem cell exosome composition, said composition being prepared by the method described in any one of claims 2-4.

6. The use of the stem cell exosome composition of claim 5 in the preparation of a medicament for treating male sexual dysfunction caused by testicular injury.

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