Cell membrane bionic nano-vesicle loaded with resveratrol carbon dots and application of cell membrane bionic nano-vesicle

Abstract

The invention is applicable to the technical field of biological medicine, and provides a resveratrol carbon dot-loaded cell membrane bionic nano-vesicle and application thereof, and the resveratrol carbon dot-loaded cell membrane bionic nano-vesicle is prepared by the following preparation process: mixing pure trans-resveratrol with ultrapure water, adjusting the pH value until complete dissolution, reacting the solution to obtain a carbon dot solution, centrifuging, filtering with a filter membrane, dialyzing and purifying to obtain the resveratrol carbon dot-loaded cell membrane bionic nano-vesicle. Carrying out rotary evaporation and freeze-drying to obtain RES CDs; and dropwise adding Cy7 into the DSPE-PEG solution, stirring overnight, dialyzing to remove free Cy7 to obtain Cy7-coated PEG, and mixing TM4-M, RES CDs and Cy7-coated PEG to obtain the resveratrol carbon dot-loaded cell membrane bionic nano-vesicles RCD-coated PCy-coated EVs. The invention also discloses a preparation method of the resveratrol carbon dot-loaded cell membrane bionic nano-vesicles RCD-coated PCy-coated EVs and application of the resveratrol carbon dot-loaded cell membrane bionic nano-vesicles. The vesicles based on the supporting cell membrane prepared by the invention can specifically deliver the resveratrol carbon dots to the blood testis barrier and treat spermatogenesis disorder.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a cell membrane biomimetic nanovesicle loaded with resveratrol carbon dots and applications thereof. Background Art

[0002] Cryptorchidism is one of the most common genital malformations in male newborns and a leading cause of male infertility and testicular cancer. It is characterized by marked atrophy of the seminiferous tubules and a decrease in the number of spermatogenic cells, primarily due to oxidative stress induced by slightly elevated testicular temperatures. Specifically, high levels of reactive oxygen species (ROS) can easily disrupt the integrity of the blood-testis barrier (BTB) by inhibiting the expression of tight junction-associated molecules, including β-catenin. This further promotes an acute inflammatory response, induces DNA damage within the sperm nucleus, and leads to decreased sperm motility. Although early diagnosis and surgery are the preferred interventions for mitigating the negative effects of unilateral and bilateral cryptorchidism, this approach is not suitable for children one year of age and younger due to the unavoidable adverse effects of anesthesia, as well as the risks of infection and bleeding associated with invasive surgery. Furthermore, human chorionic gonadotropin has long been used to treat cryptorchidism by increasing testicular weight and vascularity and stimulating testosterone and dihydrotestosterone production, but its safety and efficacy remain controversial. Therefore, there is an urgent need to develop non-invasive strategies to simultaneously eliminate reactive oxygen species and accelerate the repair of the blood-testis barrier to treat cryptorchidism.

[0003] Resveratrol (RES) is a natural polyphenol with remarkable protective effects on the male reproductive system. Recent research advances have shown that RES mitigates oxidative damage by activating the AMPK / SIRT1-FOXO1 signaling pathway, which has positive effects on male fertility. Specifically, RES can improve sperm motility by protecting DNA integrity and chromatin structure caused by cryptorchidism. However, its physicochemical properties severely limit its application in the treatment of spermatogenesis disorders, such as its poor water solubility, low chemical stability, short biological half-life, and toxic side effects with long-term administration. Carbon dots (CDs), a class of carbon-based nanomaterials, have attracted considerable attention due to their excellent biocompatibility, low toxicity, anti-inflammatory, and antioxidant properties. They have been considered a promising alternative strategy for the diagnosis and healthcare management of diseases including bacterial infections and vascular diseases. However, RES has relatively poor ability to penetrate the blood-testis barrier and lacks testicular-targeted delivery. Therefore, there is an urgent need to construct a multifunctional system for the targeted delivery of nanoformulations to the seminiferous tubules. Summary of the Invention

[0004] The purpose of the embodiments of the present invention is to provide a cell membrane biomimetic nanovesicle loaded with resveratrol carbon dots, aiming to solve the problems raised in the above background technology.

[0005] The embodiment of the present invention is achieved by: a cell membrane biomimetic nanovesicle loaded with resveratrol carbon dots is prepared by the following preparation process:

[0006] Pure trans-resveratrol was mixed with ultrapure water, the pH was adjusted to completely dissolve, and the solution was reacted to obtain a carbon dot solution, which was then purified by centrifugation, membrane filtration, and dialysis, followed by rotary evaporation and freeze-drying to obtain RES CDs;

[0007] Cy7 was added dropwise to the DSPE-PEG solution, stirred overnight, and dialyzed to remove free Cy7 to obtain Cy7@PEG. TM4-M was mixed with RES CDs and Cy7@PEG to obtain cell membrane biomimetic nanovesicles RCD@PCy@EVs loaded with resveratrol carbon dots.

[0008] Preferably, NaOH is used for adjusting the pH.

[0009] Preferably, in the step of reacting the solution to obtain the carbon dot solution, the reaction temperature is 180° C. and the reaction time is 8 hours.

[0010] Preferably, the concentrations of Cy7, DSPE-PEG solution and RES CDs are 1 mg / mL respectively, and the volume ratio of TM4-M to RESCDs and Cy7@PEG is 2:1:1.

[0011] Another object of the embodiment of the present invention is to provide an application of cell membrane biomimetic nanovesicles loaded with resveratrol carbon dots in the preparation of drugs for treating cryptorchidism.

[0012] Preferably, the cell membrane biomimetic nanovesicles loaded with resveratrol carbon dots achieve the treatment of cryptorchidism by targeted repair of the blood-testis barrier.

[0013] Another object of an embodiment of the present invention is to provide a drug for treating cryptorchidism, wherein the drug comprises cell membrane biomimetic nanovesicles loaded with resveratrol carbon dots.

[0014] The cell membrane biomimetic nanovesicles RCD@PCy@EVs loaded with resveratrol carbon dots provided in an embodiment of the present invention can improve the spermatogenesis of cryptorchidism in vivo. RCD@PCy@EVs effectively targets the seminiferous tubules through the homing and fusion tendency of the cell membrane of the mouse testicular supporting cell line (TM4), thereby enhancing the ability of resveratrol carbon dots to penetrate the blood-testis barrier. In addition, RCD@PCy@EVs can eliminate the oxidative stress and inflammation induced by cryptorchidism and accelerate the repair of blood-testis barrier damage by activating the SIRT1 / AMPKα / FOXO1 signaling pathway. Through sperm function tests and mating and reproduction experiments, it was found that RCD@PCy@EVs can repair sperm dysfunction in cryptorchidism mice. In summary, the supporting cell membrane-based vesicles prepared in an embodiment of the present invention can specifically deliver resveratrol carbon dots to the blood-testis barrier and treat spermatogenesis disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the RCD@PCy@EVs synthesis and testis-targeted drug delivery system provided by an embodiment of the present invention;

[0016] Figure 2 Characterization results of RES CDs and RCD@PCy@EVs provided in the embodiments of the present invention (A, full scan XPS spectra; high-resolution XPS spectra of BC, C1s, and O1s peaks; DE, particle size and morphology of resveratrol carbon dots and RCD@PCy@EVs; F, UV-visible absorption spectra of resveratrol carbon dots and pure resveratrol precursor; G, Fourier transform infrared (FTIR) spectra of resveratrol carbon dots and pure resveratrol precursor, vertical dashed lines indicate characteristic bands of phenolic residues; H, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein analysis of TM4 cell membrane shells, TM4 cell exosomes, RCD@PCy@EVs, resveratrol carbon dots, and Cy7@PEG);

[0017] Figure 3 The biosafety evaluation results of RCD@PCy@EVs provided in the embodiment of the present invention (AB, RES CDs and RCD@PCy@EVs were incubated with fresh red blood cells for 12 hours at different concentrations (μg mL -1 ) under different treatment groups after intravenous injection of RCD@PCy@EVs; C, Hematoxylin-eosin (H&E) staining results of different organs collected;

[0018] Figure 4 The results of constructing the cryptorchidism mouse model provided in the embodiments of the present invention (A, testicles in the abdominal cavity of mice in the non-cryptorchidism group; B, testicles in the scrotum of mice in the non-cryptorchidism group; C, testicles in the abdominal cavity of mice in the cryptorchidism group);

[0019] Figure 5 Results of targeted therapy for testis and male mouse reproductive capacity testing provided by the embodiments of the present invention (A, in vivo dosing regimens for different treatments; B, in vivo fluorescence imaging results; C, quantitative analysis of Cy7 signal intensity in the testis and epididymis at 0 hours, 12 hours, and 24 hours after injection; D, testicular morphology; E, sperm live / dead staining; F, hematoxylin-eosin (H&E) staining of testicular sections);

[0020] Figure 6 Results of male mouse reproductive capacity testing provided by the present invention (A, metabolic process of RES CDs@PCy@EVs in vivo; B, sperm density; C, sperm motility; D, testicular weight; EF, serum hormone levels: testosterone (T) and inhibin B (INHB). p < 0.05, p < 0.01, p < 0.001);

[0021] Figure 7 Figure 2: Permeability results of the blood-testis barrier (BTB) provided in the examples of the present invention (A, anti-inflammatory effects of RES, RES CDs, and RCD@PCy@EVs in the testes of cryptorchid mice; B, detection of blood-testis barrier integrity in seminiferous tubules after treatment with fluorescein isothiocyanate (FITC)-labeled dextran; C, ultrastructure of tight junctions observed using transmission electron microscopy, with red arrows indicating tight junction sites). DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] The drugs, reagents, etc. used in the examples of the present invention are described as follows: resveratrol was purchased from Boer; FOXO1 (14952S), pFOXO1 (9461S), AMPKα (2793S), pAMPKα (2535S), SIRT1 (8469S), β-actin (3700S), HRP-labeled anti-mouse IgG secondary antibody (7076S) and HRP-labeled anti-rabbit IgG secondary antibody (7074S) were purchased from CellSignaling Technology; β-Catenin monoclonal antibody (66379-1-Ig) and FITC-labeled goat anti-mouse IgG (H+L) (SA00003-1) were purchased from Proteintech Group; testosterone (ELK1332) and inhibin B (INHB) (ELK5434) ELISA kits were purchased from ELK Biotechnology; SYBR Green qPCR Mix (BL698A) and reverse transcription kit (containing gDNase) (BL699A) were purchased from Biosharp Life Science; Balb / c mice were provided by Guangzhou Ruige Biotechnology Co., Ltd.; FITC-dextran was purchased from MCE; Calcein / PI live-dead cell staining kit (C2015M) and CCK-8 cell viability detection kit (C0038) were purchased from Beyotime Biotechnology Co., Ltd.

[0024] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0025] Example 1, resveratrol carbon dots (RES CDs) and cell membrane biomimetic nanovesicles RCD@PCy@EVs loaded with resveratrol carbon dots, the preparation method thereof is as follows Figure 1 As shown, the following steps are included:

[0026] (1) 100 mg of pure trans-resveratrol was mixed with 20 mL of ultrapure water, and 10 M NaOH was added dropwise to adjust the pH until the solution was completely dissolved. The solution was reacted at 180°C for 8 hours. The resulting carbon dot solution was centrifuged at 4000 RPM and filtered through a 0.22 μm PES membrane to remove aggregates and impurities. The solution was then dialyzed against ultrapure water multiple times, and the residual organic solvent was removed by rotary evaporation. The solution was then freeze-dried, and the dried carbon dot powder was weighed to determine the concentration.

[0027] (2) 100 μL of 1 mg / mL Cy7 was added dropwise to 1 mL of 1 mg / mL DSPE-PEG solution. After stirring overnight, the free Cy7 was removed by dialysis to obtain Cy7@PEG. 500 μL of TM4-M (TM4 cell membrane, extracted using a membrane protein extraction kit (Biyuntian Company), and the membrane concentration was determined by the BCA method) was mixed with 250 μL of 1 mg / mL RES CDs and 250 μL of Cy7@PEG. The mixture was repeatedly extruded 20 times on 400 nm and 100 nm polycarbonate membranes using a micro-extruder. The final product RCD@PCy@EVs was stored in PBS at 4°C for later use.

[0028] Experimental analysis process:

[0029] Characterization: Dynamic light scattering (DLS) was used to analyze the particle size and zeta potential of RES CDs and RCD@PCy@EVs. Transmission electron microscopy (TEM) was used to observe the morphology. X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) were used to characterize the carbon dot components and surface groups. SDS-PAGE was used to verify the construction of RCD@PCy@EVs.

[0030] TM4 cell and macrophage culture: TM4 Sertoli cells were cultured at 37°C, 5% CO2, in DMEM / F12 medium supplemented with 5% horse serum, 2.5% fetal bovine serum, and 1% double-antibody. Macrophages were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum. The experimental groups were control, H2O2 group (150 μM), H2O2+RCD@PCy@EVs group (20 μg / mL), H2O2+RES CDs group (20 μg / mL), and H2O2+RES group (20 μg / mL).

[0031] Animal experimentation: The experiment was approved by the Animal Ethics Committee of Huanghuai University (202412200001) and followed the National Institutes of Health (NIH) Guide for the Use of Laboratory Animals. 25-day-old male BALB / c mice (Guangzhou Ruige Biotechnology Co., Ltd.) were housed at 20–26°C with a 12-h light–dark cycle.

[0032] The cryptorchidism model was constructed according to the method of Gao et al.: 18 mice were anesthetized and the testicles were fixed to the abdominal wall through abdominal incision. Six mice in the sham operation group had their testicles simply retracted. After surgery, the cryptorchidism mice were divided into three groups and injected intraperitoneally with RES, RES CDs, and RCD@PCy@EVs (200 mg / kg body weight) once every other day for a total of five times; the sham operation group was injected with an equal amount of PBS.

[0033] Hemolysis assay: Different concentrations of RES and RES CDs (5-50 μg / mL) were mixed with a 1:50 red blood cell suspension, incubated at 37°C for 2-6 hours, and then centrifuged. The supernatant was collected and the absorbance was measured at 450 nm. The hemolysis rate formula is:

[0034] Hemolysis rate (%) = (sample group OD - negative control OD) / (positive control OD - negative control OD) × 100;

[0035] CCK-8 cell viability assay: TM4 cells were cultured at 5×10 3 / well of a 96-well plate was inoculated, and 0-50 mg / L RES and RES CDs were added 24 hours later for treatment, and 10% CCK-8 solution was added for incubation for 2 hours, and the OD value was measured at 450 nm;

[0036] Transmission electron microscopy: Testicular tissue or TM4 cells were fixed with 2.5% glutaraldehyde, stained with osmium phosphate, uranyl acetate, and lead citrate, and then sectioned and observed using a Hitachi transmission electron microscope.

[0037] FITC-dextran permeability assessment: TM4 cells were seeded into Transwell chambers to form a barrier and then treated in groups. FITC-dextran was added to the apical side 24 hours later, and basolateral samples were collected 4 hours later to measure fluorescence intensity at 520 nm. FITC-dextran was injected into the tail vein to assess the integrity of the blood-testis barrier. 2 hours later, testicular cryosections were obtained, stained with Hoechst 33342, and imaged using a confocal microscope.

[0038] RT-qPCR: Tissue and cell RNA was extracted using the Trizol method and reverse transcribed, followed by qPCR using the SYBR Green kit. β-actin was used as an internal control. The primer sequences are shown in Table 1.

[0039] Table 1

[0040]

[0041]

[0042] Western blot: Tissue and cell lysates were transferred to the membrane via SDS-PAGE, blocked with 5% skim milk, incubated with primary antibody at 4°C overnight, and incubated with secondary antibody at room temperature for 2 hours. ECL development was performed. Primary antibody information is shown in Table 2:

[0043] Table 2

[0044]

[0045] ROS detection: TM4 cells were loaded with DCFH-DA probe for 30 minutes, washed with PBS, and then the green fluorescence intensity was observed under a fluorescence microscope;

[0046] Serum reproductive hormone detection: Mouse serum was centrifuged at 4000 rpm, and INHB and testosterone levels were measured using ELISA kits;

[0047] Sperm quality assessment: Epididymal sperm were released in PBS and tested for motility using a computer-assisted semen analysis system, with simultaneous verification using Calcein / PI live-dead staining.

[0048] Ultrasound imaging: Mice were anesthetized with 1.5% isoflurane and subjected to application of coupling agent. Ultrasound imaging of the reproductive system was performed using WirelessUSG Flash software.

[0049] Histological analysis: The testes were fixed with modified Bouin's solution for 48 hours, dehydrated with graded ethanol, embedded in paraffin, and then sectioned. The tissue structure was observed under a light microscope after H&E staining.

[0050] In vivo targeting verification of RCD@PCy@EVs: 50 μL of RCD@PCy@EVs were injected into the tail vein, and the fluorescence distribution of major organs was dynamically monitored by a small animal in vivo imaging system for 0-24 hours;

[0051] Mating experiment: Female mice in estrus were caged with male mice from the treatment group at a ratio of 1:1, and the mating success rate was determined by vaginal plugs or semen detection;

[0052] Statistical analysis: GraphPad Prism 8 was used for graphing, and the data were expressed as mean ± standard deviation. One-way analysis of variance (LSD or Games-Howell test) was performed using SPSS 17.0, and P < 0.05 was considered significant.

[0053] Experimental analysis results:

[0054] Characterization results:

[0055] X-ray photoelectron spectroscopy (XPS) is used to study its chemical composition, chemical bonding behavior and other chemical properties. The overall XPS spectrum is as follows Figure 2 As shown in A, resveratrol carbon dots are mainly composed of carbon (C) and oxygen (O); the high-resolution XPS spectrum of the C element is shown in Figure 2 As shown in B, characteristic peaks appear at 284.6eV, 285.9eV, and 289.3eV in the C1s spectrum, indicating the presence of CC, CO, and C=O vibrations in the carbon dots; Figure 2 As shown in Figure C, peaks corresponding to the O element were observed at 533.6eV and 531.5-532eV, indicating the presence of vibrations associated with CO and C=O functional groups, respectively. These results confirm the presence of abundant oxygen and carbon functional groups on the surface of carbon dots, strongly proving their significant hydrophilicity and excellent water solubility; Transmission electron microscopy (TEM) analysis shows Figure 2As shown in D, the average particle size of resveratrol carbon dots is about 15 nm, showing aggregation characteristics; the TEM image of RCD@PCy@EVs is shown in Figure 2 As shown in E, the surface of resveratrol carbon dots is wrapped by cell membrane, and RCD@PCy@EVs are spherical with a diameter of about 70 nm. Based on the dynamic light scattering (DLS) analysis of the particle size, the final particle size of the prepared RCD@PCy@EVs after extrusion from TM4 cell exosomes (EVs) is about 190 nm, which is consistent with the TEM observation results; the absorbance spectrum data are shown in Figure 2 As shown in F, the characteristic peak of resveratrol shifted in the resveratrol carbon dots and RCD@PCy@EVs samples; Figure 2 The Fourier transform infrared (FTIR) spectroscopy data of G showed that at 3384 cm -1 The peaks at about 1140 and 1320 cm-1 are attributed to the stretching vibration mode of OH in resveratrol carbon dots. -1 The peak at is attributed to the phenolic residues of resveratrol. The FTIR data provide evidence for the existence of residual resveratrol units on the surface of resveratrol carbon dots. Figure 2 As shown in Figure ​H, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that TM4 cell membrane shells, TM4 cell exosomes and RCD@PCy@EVs displayed highly consistent protein bands, indicating that almost all membrane proteins were retained during the preparation of RCD@PCy@EVs.

[0056] In vitro and in vivo biocompatibility evaluation of RCD@PCy@EVs:

[0057] Biocompatibility, cell viability, and oxidative stress response analyses were performed to compare resveratrol carbon dots with pure resveratrol, with the goal of evaluating whether carbon dots exhibited improved biological properties compared to single molecules, such as Figure 3 As shown in A, the hemolysis rates of resveratrol carbon dots and RCD@PCy@EVs (concentrations of 0-25 μg / mL) were less than 5% after 24 h, indicating that they have good blood compatibility;

[0058] The cytotoxicity of resveratrol carbon dots and RCD@PCy@EVs is crucial to evaluate their potential for drug delivery. Figure 3 As shown in Figure B, cell counting kit-8 (CCK-8) analysis showed that when the concentration was 20 μg / mL, the inhibition rate of resveratrol on cells reached about 80%, while resveratrol carbon dots and RCD@PCy@EVs had no toxicity to cells;

[0059] like Figure 3As shown in C, hematoxylin-eosin (H&E) staining further confirmed that all examined tissues (heart, liver, spleen, lung, and kidney) were intact;

[0060] Taken together, these results strongly demonstrate that RCD@PCy@EVs has strong biocompatibility and good clinical translation potential.

[0061] RCD@PCy@EVs alleviate blood-testis barrier dysfunction and improve spermatogenesis in cryptorchidism mice:

[0062] A wireless probe handheld color ultrasound device (Sonoxin Medical) was used to image the testicles in the abdominal cavity of normal mice, the testicles in the scrotum of normal mice, and the testicles in the abdominal cavity of cryptorchid mice. The image results are as follows: Figure 4 As shown, no testicles were found in the abdominal cavity of normal mice, while the size of the testicles in the abdominal cavity of cryptorchid mice was significantly smaller than the testicle volume in the scrotum of normal mice. The results showed that the cryptorchid mouse model was successfully established.

[0063] Figure 5 A shows the in vivo administration schemes of different treatments. The targeting and aggregation ability of RCD@PCy@EVs in the testis is the key to the function of cell membrane biomimetic nanovesicles. Fluorescence imaging of mice was performed at 0 hours, 12 hours and 24 hours after injection. Figure 5 As shown in BC, RCD@PCy@EVs can be delivered to the testicular tissue of the sham operation group, and the RCD@PCy@EVs group has a stronger targeting ability compared with the Cy7@PEG-injected group;

[0064] To evaluate any changes in sperm density and motility, after tail vein injection of resveratrol, resveratrol carbon dots, and RCD@PCy@EVs, the epididymis was cut into small pieces and placed in phosphate buffer in a 37 °C water bath for 10 min. Then, the volume and weight of the testes, the morphology, density, and motility of the sperm were measured, and the pathological changes, serum hormone levels, and the number of offspring mice were detected, e.g. Figure 5 As shown in D and 6D, the testicular volume and weight of cryptorchid mice were significantly reduced compared with the sham operation group, but there was no significant change in the RCD@PCy@EVs group; Figure 6 As shown in A, the metabolic process of RES CDs@PCy@EVs in mice. RCD@PCy@EVs can accelerate spermatogenesis. Figure 6 As shown in BC, the sperm density and survival rate were higher than those in the cryptorchidism group (CRY group); testosterone (T) is deeply involved in every step of male sexual response, inhibin B (INHB) is a glycoprotein hormone secreted by reproductive system cells and is one of the most significant serum markers of seminiferous tubule function and spermatogenesis in cryptorchidism mice, such as Figure 6As shown in EF, the testosterone levels of cryptorchid mice increased significantly after surgery, and the inhibin B levels of cryptorchid mice were lower than those of normal men, indicating that the function and number of supporting cells and germ cells decreased. After injection of RCD@PCy@EVs, the serum testosterone and inhibin B levels of cryptorchid mice increased significantly.

[0065] The live-death ratio of sperm was detected using the Calcein / PI dead cell viability assay kit. Figure 5 As shown in E, there were no viable sperm in the cryptorchidism group and the resveratrol-treated group; hematoxylin-eosin (H&E) staining of testicular tissue of cryptorchidism mice was shown in Figure 5 As shown in Figure F, the seminiferous tubules were atrophied and accompanied by extensive degeneration. The atrophied seminiferous tubules contained degenerated supporting cells and very few germ cells. Testicular tissue sections showed only supporting cells, and most seminiferous tubules showed cellular ballooning. As shown in Table 3, male mice treated with RCD@PCy@EVs regained their reproductive capacity. Cryptorchid mice and the resveratrol-treated group had no offspring, but male mice treated with RCD@PCy@EVs gave birth to offspring after mating with female mice.

[0066] Table 3 The number of offspring after mating between male and female mice in each group

[0067]

[0068] In summary, the drug-targeted delivery system prepared using TM4 cell membrane and resveratrol carbon dots has a good repair effect on spermatogenesis and reproductive disorders caused by cryptorchidism.

[0069] RCD@PCy@EVs restore blood-testis barrier integrity

[0070] In order to explore the molecular mechanism of drug effects on the reproduction of cryptorchid mice, different drugs were injected into the tail vein of cryptorchid mice, such as Figure 7 As shown in Figure A, the transcriptional levels of inflammatory factors in the testis are consistent with those in TM4 cells, indicating that RCD@PCy@EVs can significantly reduce the inflammatory response in the testis. Fluorescein isothiocyanate (FITC)-labeled dextran was injected through the tail vein to determine whether RCD@PCy@EVs can repair the damage of the blood-testis barrier. After the mice were sacrificed, the testicular tissue was immediately fixed and embedded in Sakura Tissue- OCT embedding medium, and then, use frozen sections to examine the integrity of the blood-testis barrier, such as Figure 7As shown in B, in the cryptorchidism group, FITC-dextran diffused freely into all areas of the seminiferous tubules, even the lumen. However, in the RCD@PCy@EVs group, as in the sham-operated testis, FITC-dextran was only present in the peripheral compartments of the seminiferous tubules and the interstitial tissue. Resveratrol (RES) was less effective in repairing the blood-testis barrier damage caused by cryptorchidism than in the RCD@PCy@EVs-treated group. Transmission electron microscopy (TEM) results were used to evaluate the effect of RCD@PCy@EVs on the restoration of blood-testis barrier function. Figure 7 As shown in Figure C, the typical "sandwich" structure of the blood-testis barrier was intact in the sham-operated and RCD@PCy@EVs groups. Sertoli cell necrosis was higher in the cryptorchidism and resveratrol-treated groups than in the other groups. RCD@PCy@EVs significantly promoted the repair of blood-testis barrier damage and protected the tight junctions between Sertoli cells.

[0071] In summary, the RCD@PCy@EVs prepared in the examples of the present invention have great potential as a new nanoformulation for the treatment of cryptorchidism. They can reduce oxidative stress, restore the function of the seminiferous tubules, and promote spermatogenesis. In addition, sperm function tests as well as in vitro mating and reproduction experiments confirmed the therapeutic effect of RCD@PCy@EVs on sperm dysfunction in cryptorchid mice, which will help develop the next generation of drugs for the treatment of spermatogenesis disorders.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cell membrane biomimetic nanovesicle loaded with resveratrol carbon dots, characterized in that: It is prepared by the following preparation process: Pure trans-resveratrol was mixed with ultrapure water, the pH was adjusted to completely dissolve, and the solution was reacted to obtain a carbon dot solution, which was then purified by centrifugation, membrane filtration, and dialysis, followed by rotary evaporation and freeze-drying to obtain RES CDs; Cy7 was added dropwise to the DSPE-PEG solution, stirred overnight, and dialyzed to remove free Cy7 to obtain Cy7@PEG. TM4-M was mixed with RES CDs and Cy7@PEG to obtain cell membrane biomimetic nanovesicles RCD@PCy@EVs loaded with resveratrol carbon dots.

2. The cell membrane biomimetic nanovesicles loaded with resveratrol carbon dots according to claim 1, characterized in that: NaOH is used for adjusting the pH.

3. The cell membrane biomimetic nanovesicles loaded with resveratrol carbon dots according to claim 1, characterized in that: In the step of reacting the solution to obtain the carbon dot solution, the reaction temperature is 180° C. and the reaction time is 8 hours.

4. The cell membrane biomimetic nanovesicles loaded with resveratrol carbon dots according to claim 1, characterized in that: The concentrations of Cy7, DSPE-PEG solution and RES CDs were 1 mg / mL, respectively, and the volume ratio of TM4-M to RES CDs and Cy7@PEG was 2:1:

1.

5. Use of the cell membrane biomimetic nanovesicles loaded with resveratrol carbon dots according to any one of claims 1 to 4 in preparing a drug for treating cryptorchidism.

6. The use according to claim 5, characterized in that The cell membrane biomimetic nanovesicles loaded with resveratrol carbon dots achieve the treatment of cryptorchidism by targetedly repairing the blood-testis barrier.

7. A drug for treating cryptorchidism, characterized in that: The drug comprises the cell membrane biomimetic nanovesicles loaded with resveratrol carbon dots as described in any one of claims 1 to 4.

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