A stem cell exosome composition for treating erectile dysfunction and its uses

Abstract

The present invention provides a stem cell exosome composition for treating erectile dysfunction and its uses, including stem cell exosomes and vasoactive intestinal peptide, and the amino acid sequence of the vasoactive intestinal peptide is as shown in SEQ ID NO:2. The present invention modifies the amino acid structure of vasoactive intestinal peptide to improve its biological stability; the combined use of the vasoactive intestinal peptide and adipose mesenchymal stem cell exosomes can relieve ED symptoms; the combined use of the vasoactive intestinal peptide and adipose mesenchymal stem cell exosomes can regulate the expression level of cytokines and improve the penile tissue microenvironment.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research and development, and specifically provides a stem cell exosome composition for treating erectile dysfunction and its uses. Background Art

[0002] Erectile dysfunction (ED) refers to the inability to achieve or maintain an erection sufficient for satisfactory sexual performance, thereby affecting the sexual life status of men. ED is a prevalent medical problem that seriously affects male health. Over 150 million men worldwide suffer from ED to varying degrees, and the prevalence of ED is higher in men over 40 years old. It is estimated that there will be more than 322 million people globally by 2025 (see Burnett AL, Nehra A, Breau RH, et al.. Erectile dysfunction: AUA guideline. J Urol. (2018) 200(3): 633–41). In the past, mental illness was commonly considered an important factor in the development of ED. However, many studies have shown that organic etiologies account for more than 80% of ED cases. Cardiovascular diseases, diabetes, dyslipidemia, hypogonadism, and nerve damage are independent risk factors for ED. More importantly, ED is no longer solely associated with sexual dysfunction; it may also indicate potential vascular endothelial dysfunction and is an early indicator of cardiovascular diseases (see Gandaglia G, Briganti A, Jackson G, et al. A systematic review of the association between erectile dysfunction and cardiovascular disease. Eur Urol. (2014) 65(5): 968–78). Selective phosphodiesterase type 5 inhibitors (PDE5i), such as sildenafil citrate, have been widely used as first-line therapeutic drugs for ED because they can enhance the erectile function of 63% of patients and act based on the role of nitric oxide (NO) in the relaxation of cavernous smooth muscle (see Xu W, Jiang H, Liu J, et al. Non-coding RNAs: New dawn for diabetes mellitus induced erectile dysfunction. Front Mol Biosci (2022) 9: 888624). However, due to the complexity of the pathways regulating penile erection, up to 35% of patients do not respond to drug treatment. Since current treatment methods cannot bring the greatest benefits to patients, it is crucial to study new strategies for treating ED.

[0003] Extracellular vesicles are cell-derived membrane structures with diameters ranging from 40 nm to 1000 nm. Exosomes are nanosized particles with diameters of approximately 40 - 160 nm secreted by various cells under physiological or pathological conditions. Exosomes contain many components, such as metabolites, proteins, lipids, and nucleic acids. Exosomes are involved in various physiological and pathological processes in the human body and can serve as molecular and signal carriers for intercellular communication. Exosomes may be involved in cardiovascular and metabolic diseases, play a role in the pathogenesis of neurological diseases, and dynamically affect cancer growth (see Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science (2020) 367(6478):eaau6977).

[0004] The effect of exosomes on the improvement of ED was first analyzed in 2017. Chen et al. established a type 2 diabetes rat model and isolated exosomes from adipose-derived stem cells (ADSCs) by ultracentrifugation. They found that ADSC-derived exosomes promoted the recovery of erectile function by increasing the ratio of maximum intracavernous pressure (ICP) to mean arterial pressure (MAP), increasing endothelial and smooth muscle content, and reducing apoptosis of cavernous endothelial cells and smooth muscle cells (see Chen F, Zhang H, Wang Z, et al.. Adipose-derived stem cell-derived exosomes ameliorate erectile dysfunction in a rat model of type 2 diabetes. J Sex Med (2017) 14(9):1084–94). Exosomes isolated from ADSCs and BMSCs can be used not only to treat diabetic erectile dysfunction but also to treat BCNI-induced erectile dysfunction. Ouyang et al. found that four weeks after injecting exosomes into the corpus cavernosum of rats with BCNI-induced erectile dysfunction, erectile function was significantly improved by inhibiting apoptosis in CCSMCs (see Ouyang X, Han X, Chen Z, et al. MSC-derived exosomes ameliorate erectile dysfunction by alleviation of corpus cavernosum smooth muscle apoptosis in a rat model of cavernous nerve injury. Stem Cell Res Ther (2018) 9(1):246). However, exosomes also showed uncertainty in the efficacy during the treatment of ED, such as the significant difference in the treatment effects of exosomes obtained from different cell sources and different culture conditions.

[0005] Vasoactive intestinal polypeptide (VIP) is a novel peptide with strong vasoactivity isolated by Said and Mutt from porcine intestinal extracts. Subsequently, the biological functions of VIP and its signal transduction pathways have been extensively studied. Various in vitro and in vivo studies have shown that VIP expressed in intrinsic non-adrenergic non-cholinergic (NANC) neurons is a potent regulator of gastrointestinal (GI) motility, water absorption and ion flux, mucus secretion, and immune homeostasis. These VIP actions are thought to be mainly mediated by interaction with highly expressed VPAC1 receptors and the production of nitric oxide (NO) (see Abad C, Martinez C, Juarranz MG, et al. Therapeutic effects of vasoactive intestinal peptide in the trinitrobenzene sulfonic acid mice model of Crohn’s disease. Gastroenterology. (2003) 124:961–71). And studies have shown that the combination of VIP and phentolamine mesylate can treat moderate to severe ED. VIP has a strong effect on the veno-occlusive mechanism and can also reduce the incidence of penile pain (see Dinsmore W, Wyllie M. Vasoactive intestinal polypeptide / phentolamine for intracavernosal injection in erectile dysfunction, BJU Int, (2008) 102(8):933-7). Although VIP shows good effects in inhibiting the occurrence of ED, its biological stability is poor, which is not conducive to clinical application.

[0006] In order to overcome the defects of the above-mentioned ED treatment drugs, the present invention provides a stem cell exosome composition for treating erectile dysfunction and its use. The amino acid structure of VIP is modified to improve stability and bioavailability. When used in combination with adipose mesenchymal stem cell exosomes, it can significantly relieve ED symptoms, thus providing an effective means for treating ED. Summary of the Invention

[0007] The first aspect of the present invention provides a stem cell exosome composition for treating erectile dysfunction, comprising stem cell exosomes and vasoactive intestinal peptide, and the amino acid sequence of the vasoactive intestinal peptide is as shown in SEQ ID NO:2.

[0008] In the present invention, the amino acid sequence of vasoactive intestinal peptide is modified to significantly increase its stability, improve its bioavailability in vivo, and make it more suitable for the slightly alkaline environment in vivo; and it is combined with stem cell exosomes to treat erectile dysfunction, which can improve the penile tissue microenvironment, regulate cytokine secretion, and effectively treat erectile dysfunction.

[0009] Further, the stem cells are mesenchymal stem cells.

[0010] Further, the mesenchymal stem cells are selected from at least one of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, and adipose mesenchymal stem cells.

[0011] Further, the mesenchymal stem cells are adipose mesenchymal stem cells, and the preparation method thereof includes: cutting adipose tissue into pieces, filtering through a 100-mesh filter, adding type IV collagenase and mixing evenly for digestion for 1 h; centrifuging at 1000 rpm for 5 min to collect cells; washing the cells again with PBS and centrifuging at 1000 rpm for 5 min to collect cells; resuspending in complete medium, culturing under the conditions of 37 °C and 5% CO2, changing the medium once every 2 days, and when the density reaches 80%, digesting the cells with trypsin and collecting the cells.

[0012] Further, the preparation method of the stem cell exosomes includes: culturing adipose mesenchymal stem cells in complete medium with exosome-depleted serum for 48 h to collect cell supernatant; centrifuging at 4 °C and 2000 g for 20 min, collecting the supernatant, and discarding the precipitate; centrifuging the supernatant at 4 °C and 10000 g for 40 min, collecting the supernatant, and discarding the precipitate; filtering the supernatant through a 0.22-μm filter and collecting the filtrate; ultracentrifuging the filtrate at 4 °C and 100000 g for 70 min, discarding the supernatant, and retaining the precipitate; resuspending the precipitate with PBS, and after ultracentrifuging the resuspended solution at 4 °C and 100000 g for 70 min, discarding the supernatant, and retaining the exosome precipitate; resuspending the exosome precipitate with an appropriate amount of PBS and storing at -80 °C.

[0013] Further, the mass ratio of the stem cell exosomes to vasoactive intestinal peptide is 1:5 to 5:1.

[0014] Further, the mass ratio of the stem cell exosomes to vasoactive intestinal peptide is 2:1.

[0015] The second aspect of the present invention provides an application of the composition as described above in the preparation of drugs for erectile dysfunction.

[0016] The erectile dysfunction includes vascular erectile dysfunction, diabetic erectile dysfunction, organic lesion erectile dysfunction, neurogenic erectile dysfunction, and so on.

[0017] Beneficial effects

[0018] The present invention provides a stem cell exosome composition for treating erectile dysfunction and its uses, and the specific beneficial effects are as follows:

[0019] (1) Provide a novel vasoactive intestinal peptide, modify its amino acid structure, and improve its biological stability;

[0020] (2) The combined use of the vasoactive intestinal peptide and adipose mesenchymal stem cell exosomes can relieve ED symptoms;

[0021] (3) The combined use of the vasoactive intestinal peptide and adipose mesenchymal stem cell exosomes can regulate the expression level of cytokines and improve the penile tissue microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : ICP / MAP detection in rats;

[0023] Figure 2 : Expression level of eNOS in penile tissues of rats;

[0024] Figure 3 : Expression level of NGF in penile tissues of rats;

[0025] Figure 4 : Expression level of VEGF in penile tissues of rats. DETAILED DESCRIPTION OF THE INVENTION

[0026] In the following examples, the experimental methods are all conventional methods unless otherwise specified; the reagent biomaterials and detection kits can all be obtained from commercial channels unless otherwise specified.

[0027] Example 1 Preparation of muVIP

[0028] Natural VIP is an endogenous neuropeptide composed of 28 amino acids, and its amino acid sequence is shown in SEQ ID NO: 1. It has various physiological activities and has a good therapeutic effect on ED, but its in vivo stability is poor, its half-life is short, and it is not conducive to in vitro preservation, preparation and transportation. Some studies have shown that VIP is stable in a slightly acidic environment and slowly degrades in a basic environment, and will degrade rapidly when the pH value > 11 (see Cui Xu, Han Xuan, Wang Zhimin, etc. Study on the chemical and biological stability of vasoactive intestinal peptide, Chinese Journal of New Drugs. (2011) 20(19): 1922-1925).

[0029] Therefore, the present invention has modified its amino acid sequence, replacing some amino acids with basic amino acids, thereby providing its stability. The amino acid sequence of the modified muVIP is shown in SEQ ID NO:2. Using the solid-phase synthesis method, MBHA resin was selected, and through steps such as resin swelling, deprotection, condensation reaction, polypeptide cleavage, and extraction, muVIP was obtained according to the order of the amino acid sequence.

[0030] Natural VIP and muVIP were respectively treated in environments with pH values of 7, 8, 9, 10, 11, and 12 for 1 h, and then HPLC was used to detect the characteristic peaks of the target protein, as shown in Table 1. The pH stability of muVIP was significantly increased, and it could still maintain partial activity under the conditions of pH 11 or 12.

[0031] Table 1 VIP Stability Detection

[0032]

[0033] Note: +++ indicates that there are obvious and clear characteristic peaks of the target protein and no obvious impurity peaks; ++ indicates that there are obvious characteristic peaks, but there is partial degradation and a small amount of impurity peaks appear; + indicates that there are characteristic peaks of the target protein, but the degradation increases and the impurity peaks are obvious; - indicates that there are no obvious characteristic peaks of the target protein.

[0034] Example 2 Preparation of Adipose Mesenchymal Stem Cell Exosomes

[0035] Healthy volunteer adipose tissue was taken, rinsed 3 times with PBS containing penicillin and streptomycin, the adipose tissue was minced, filtered through a 100-mesh sieve, and 1 mg / mL type IV collagenase was added and mixed for digestion for 1 h; the cells were collected by centrifugation at 1000 rpm for 5 min; the cells were washed again with PBS and collected by centrifugation at 1000 rpm for 5 min; the cells were resuspended in α-MEM medium containing 10% fetal bovine serum and cultured under the conditions of 37 °C and 5% CO2, and the medium was changed once every 2 days. When the density reached 80%, the adipose-derived stem cells (ADSCs) were digested with trypsin and collected for subsequent experiments.

[0036] The ADSCs were cultured in complete medium with exosome-depleted serum for 48 h, and the cell supernatant was collected. Exosomes were extracted from the collected stem cell culture medium by ultra-high speed centrifugation. The culture medium was centrifuged at 2,000 g for 20 min at 4 °C, and the supernatant was collected, discarding the precipitate. The supernatant was centrifuged at 10,000 g for 40 min at 4 °C, and the supernatant was collected, discarding the precipitate. The supernatant was filtered through a 0.22-μm filter and the filtrate was collected. The filtrate was ultra-centrifuged at 100,000 g for 70 min at 4 °C, the supernatant was discarded, and the precipitate was retained. The precipitate was resuspended with a large amount of PBS, and after ultra-centrifugation at 100,000 g for 70 min at 4 °C, the supernatant was discarded, and the exosome precipitate was retained. The exosome precipitate was resuspended with an appropriate amount of PBS and stored at -80 °C for subsequent experiments. Transmission electron microscopy was used to observe the microscopic morphology of exosomes, and the size distribution of exosome particles was measured. It was detected that the exosomes were spherical vesicles with a diameter of about 100 nm.

[0037] Example 3 Treatment of rat ED model with muVIP and stem cell exosomes

[0038] 3.1 Preparation of rat ED model and administration

[0039] SPF-grade healthy male SD rats weighing 200 - 220 g were taken and raised in an environment with a room temperature of (21 ± 2) °C, a relative humidity of 50% - 60%, and a light-dark cycle of 12 h / 12 h, and the rats were allowed to eat and drink freely. The rats were adaptively raised for 1 week. An ED model was established by bilateral internal iliac artery ligation. The SD rats were anesthetized with 3% sodium pentobarbital, a midline abdominal incision was made, the abdominal wall muscle tissue was separated, the abdominal aorta and common iliac artery were exposed, and the internal iliac artery was carefully separated along the common iliac artery under a surgical magnifying glass until the internal iliac artery was reached, and the bilateral internal iliac arteries were ligated with surgical thread. The rats in the sham operation group received sham operation, with the same incision and separation as in the modeling step, but without ligation, and then each layer of tissue was sutured in turn. The APO experiment was used to screen the rats with successful modeling as the subjects for subsequent experiments.

[0040] Two days after modeling, 40 ED rats were selected and randomly divided into 4 groups, with 10 rats in each group, namely: the muVIP group, in which 50 mg / kg of muVIP was injected into the corpus cavernosum of the penis twice a week (on the 1st and 4th days); the exosome group (EXO group), in which 100 mg / kg of stem cell exosomes was injected into the corpus cavernosum of the penis twice a week (on the 1st and 4th days); the combined group, in which 50 mg / kg of muVIP and 100 mg / kg of stem cell exosomes were injected into the corpus cavernosum of the penis twice a week (on the 1st and 4th days); the control group, in which an equal volume of normal saline was injected into the corpus cavernosum of the penis twice a week (on the 1st and 4th days). Another 10 rats in the sham operation group were injected with an equal volume of normal saline into the corpus cavernosum of the penis twice a week (on the 1st and 4th days). Each group was treated for 8 weeks.

[0041] 3.2 ICP / MAP Detection

[0042] After anesthesia, the right common carotid artery and corpus cavernosum of the rats were exposed. A PE-50 tube filled with heparin solution was inserted into the proximal end of the right common carotid artery along the incision to record the mean arterial pressure (MAP) level, and another PE-50 tube filled with heparin solution was inserted into the corpus cavernosum. The cavernous nerve of the corpus cavernosum was electrically stimulated (voltage 5V, frequency 20Hz, pulse 1.2ms, duration 60s), and the value of maximal intracavernosal pressure (ICPmax) in the erectile state was recorded. The results are as Figure 1 shown. After modeling, the maxICP / MAP level of the rats decreased significantly, and it recovered after treatment. Among them, the recovery degree of maxICP / MAP in the combined group was the largest and was close to the normal level.

[0043] 3.4 Detection of eNOS Expression Level

[0044] After the above experiments were completed in each group of rats, they were sacrificed. After taking part of the corpus cavernosum tissue to make tissue homogenate, it was centrifuged at 3000 rpm for 15 min, and the supernatant was taken and stored for later use. According to the operation steps of the ELISA kit (purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.), the erectile-related bioactive factor eNOS in the corpus cavernosum tissue of each group of rats was measured respectively.

[0045] As Figure 2 shown, compared with the sham operation group, the eNOS expression level in the control group decreased significantly, and the eNOS expression increased to varying degrees after treatment. Among them, the muVIP group was higher than the EXO group but lower than the combined group, indicating that the combined use of muVIP and EXO had a better therapeutic effect.

[0046] 3.5 Detection of Cytokine Expression Level

[0047] The occurrence and development of ED are related to abnormal expression of multiple cytokines. In the present invention, an ELISA kit (purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.) was used to detect the expression levels of NGF and VEGF in the corpus cavernosum tissue homogenate, and the specific steps were carried out according to the kit instructions.

[0048] As Figure 3 , Figure 4 shown, compared with the normal level, the expression levels of NGF and VEGF in the model group decreased significantly, indicating that the secretion of the above two factors was significantly inhibited, and the expression levels of NGF and VEGF recovered after treatment, and this trend was more obvious in the combined group.

[0049] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements 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 vasoactive intestinal peptide, characterized in that: The amino acid sequence of the vasoactive intestinal peptide is shown in SEQ ID NO:

2.

2. A stem cell exosome composition for treating erectile dysfunction, comprising stem cell exosomes and vasoactive intestinal peptide, wherein the amino acid sequence of the vasoactive intestinal peptide is as shown in SEQ ID NO:2, the stem cells are mesenchymal stem cells, and the mesenchymal stem cells are adipose mesenchymal stem cells. Its preparation method includes: The adipose tissue was minced, filtered through a 100-mesh sieve, and mixed with type IV collagenase for digestion for 1 h; the cells were collected by centrifugation at 1000 rpm for 5 min; The cells were washed again with PBS and collected by centrifugation at 1000 rpm for 5 min; resuspended in complete medium, cultured at 37 °C and 5% CO2, and the medium was changed every 2 days. When the density reached 80%, the cells were digested with trypsin and collected.

3. The composition according to claim 2, characterized in that, The preparation method includes: culturing adipose mesenchymal stem cells in complete medium with exosome-depleted serum for 48 h to collect the cell supernatant; centrifuging at 4 °C and 2000 g for 20 min, collecting the supernatant, and discarding the precipitate; centrifuging the supernatant at 4 °C and 10,000 g for 40 min, collecting the supernatant, and discarding the precipitate; filtering the supernatant with a 0.22-μm filter and collecting the filtrate; centrifuging the filtrate at 4 °C at a condition of 100,000 g for 70 min, discarding the supernatant, and retaining the precipitate; resuspending the precipitate with PBS, and after centrifuging the resuspended solution at 4 °C and 100,000 g for 70 min, discarding the supernatant, and retaining the exosome precipitate; resuspending the exosome precipitate with an appropriate amount of PBS and storing it at -80 °C.

4. The composition according to claim 2, wherein The mass ratio of the stem cell exosomes to the vasoactive intestinal peptide is 1:5 to 5:

1.

5. The composition according to claim 2, wherein The mass ratio of the stem cell exosomes to the vasoactive intestinal peptide is 2:

1.

6. Use of the composition according to any one of claims 2-5 in the preparation of a drug for erectile dysfunction.

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