A mitochondrion-optimized targeted ovarian mitochondrion liposome and a preparation method and application thereof

Abstract

This invention discloses a liposome targeting ovarian mitochondria with mitochondrial optimization function, comprising a liposome body, PQQ, ovarian-specific targeting peptides YAGKLLISLSEERISAHHVP and TPP. PQQ is encapsulated within the liposome body, and the ovarian-specific targeting peptides YAGKLLISLSEERISAHHVP and TPP are respectively modified on the surface of the liposome body via amphiphilic polymer anchoring sites. This invention also discloses its preparation method and its application in the preparation of drugs for treating ovarian reserve insufficiency. The liposome of this invention significantly improves drug bioavailability, successfully overcomes the bottlenecks of existing delivery technologies, achieves highly efficient optimization of ovarian mitochondrial function, and provides a breakthrough solution for the precision treatment of diseases such as ovarian reserve insufficiency.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a targeted ovarian mitochondrial liposome with mitochondrial optimization function, its preparation method, and its application. Background Technology

[0002] Depleted ovarian reserve (DOR) refers to a condition in which a woman's ovaries have a reduced number and / or decreased quality of oocytes, leading to decreased fertility. With the increasing trend of later marriage and later childbearing, the adverse effects of DOR on female fertility are becoming increasingly serious.

[0003] Dysfunction of the ovary (DOR) is a degenerative disease of the ovary, and mitochondrial function is one of the most critical contributing factors in this process. As the most abundant organelle in granulosa cells and oocytes, mitochondria not only regulate granulosa cell metabolism, cell cycle, and cell signal transduction, but also provide all the energy for oocyte fertilization and embryonic development. Therefore, decreased mitochondrial function is often accompanied by a decline in the number and quality of primordial follicles, ultimately leading to decreased ovarian reserve.

[0004] Pyrroloquinoline quinone (PQQ) is one of the most potent antioxidants known. PQQ has been found to stimulate mitochondrial biogenesis, improving the quantity and quality of mitochondria in aging cells, thus potentially delaying cellular aging and treating degenerative diseases. PQQ can be taken orally as a nutritional supplement; however, oral nutrients are subject to the first-pass effect in the liver, leading to the metabolism or breakdown of some nutrients in the liver, resulting in a reduced amount entering systemic circulation and potentially decreased bioavailability.

[0005] With the development of nanotechnology in recent years, encapsulating drugs in nanocarriers such as liposomes to prepare nanodrug delivery systems has gradually become an emerging therapy. This method can not only improve drug solubility and stability, but also achieve drug aggregation in target tissues through passive and active targeting, greatly improving drug absorption and utilization.

[0006] Ovarian granulosa cells specifically express the AMH receptor AMHRII on their surface; therefore, targeted drug delivery to ovarian granulosa cells can be achieved by developing strategies that target AMHRII.

[0007] Triphenylphosphine (TPP) is one of the most commonly used mitochondrial targeting ligands. Due to its cationic properties, in addition to using the negative potential inside the mitochondrial membrane to introduce nanomedicine delivery systems into the mitochondria, it can also make the surface of liposomes carry a positive charge to facilitate adsorption and penetration of the basement membrane. Summary of the Invention

[0008] The purpose of this invention is to provide a targeted ovarian mitochondrial liposome with mitochondrial optimization function, its preparation method and application, in order to overcome the shortcomings of the prior art.

[0009] The present invention adopts the following technical solution:

[0010] The first aspect of the present invention provides a mitochondrial-targeting ovarian mitochondrial liposome with mitochondrial optimization function, comprising a liposome body, pyrrolquinolinone, an ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP, and triphenylphosphine. The pyrrolquinolinone is encapsulated in the liposome body, and the ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP and triphenylphosphine are respectively modified on the surface of the liposome body through amphiphilic polymer anchoring sites.

[0011] Furthermore, the amphiphilic polymer includes distearate phosphatidylethanolamine-polyethylene glycol 2000.

[0012] Furthermore, the liposome body is constructed from lecithin and cholesterol.

[0013] A second aspect of this invention provides a method for preparing the above-mentioned mitochondrial-targeted ovarian mitochondrial liposomes with mitochondrial optimization function, comprising the following steps:

[0014] (1) Synthesis of DSPE-PEG2000-YAGKLLISLSEERISAHHVP

[0015] Distearate phosphatidylethanolamine-polyethylene glycol 2000-succinimide ester, i.e., DSPE-PEG2000-NHS, was dissolved in a solvent, and ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP and triethylamine were added to carry out the reaction. After the reaction was completed, the reaction solution was post-processed to obtain the lyophilized product DSPE-PEG2000-YAGKLLISLSEERISAHHVP.

[0016] (2) Synthesis of DSPE-PEG2000-TPP

[0017] Distearate phosphatidylethanolamine-polyethylene glycol 2000-succinimide ester, i.e., DSPE-PEG2000-NHS, was dissolved in a solvent, and amino-modified triphenylphosphine, i.e., TPP-NH2, and triethylamine were added to carry out the reaction. After the reaction was completed, the reaction solution was post-treated to obtain the lyophilized product DSPE-PEG2000-TPP.

[0018] (3) Preparation of mitochondrial liposomes with mitochondrial optimization function targeting ovarian mitochondrial liposomes

[0019] Lecithin, cholesterol, DSPE-PEG2000-YAGKLLISLSEERISAHHVP lyophilized products, and DSPE-PEG2000-TPP lyophilized products were dissolved in a solvent, evaporated under reduced pressure to form a film, and then hydrated by adding an aqueous solution containing PQQ. After post-processing, targeted ovarian mitochondrial liposomes with mitochondrial optimization function were obtained.

[0020] Further, the solvent in step (1) includes N,N-dimethylformamide; the post-treatment includes dialyzing the reaction solution, collecting the dialysate, freeze-drying, and sealing under an inert gas atmosphere.

[0021] Further, the solvent in step (2) includes chloroform; the post-treatment includes concentrating the reaction solution by vacuum distillation, precipitating with ice-cold diethyl ether, filtering, collecting the product precipitate, and vacuum drying.

[0022] Furthermore, the solvent in step (3) includes chloroform; the post-processing includes sonication, extrusion, purification, and freeze drying.

[0023] The third aspect of this invention provides the application of the above-mentioned targeted ovarian mitochondrial liposomes with mitochondrial optimization function in the preparation of a medicine for treating ovarian reserve insufficiency.

[0024] Furthermore, the treatment for ovarian reserve insufficiency involves optimizing mitochondrial function in ovarian granulosa cells.

[0025] The beneficial effects of this invention are:

[0026] This invention provides a liposome with mitochondrial optimization function targeting ovarian mitochondria. It not only has ovarian mitochondrial targeting but also exhibits good biocompatibility and safety. The liposome comprises a liposome body, PQQ, an ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP, and TPP. PQQ is encapsulated within the liposome body, while the ovarian-specific targeting peptides YAGKLLISLSEERISAHHVP and TPP are modified onto the surface of the liposome body via amphiphilic polymer anchoring sites. The liposome body is constructed from lecithin and cholesterol, and PQQ, as the target drug, is uniformly encapsulated within it. The ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP is a self-validated ovarian-specific targeting peptide sequence. Modifying this peptide sequence onto the surface of the liposome body enables active targeting of ovarian granulosa cells. TPP, as a mitochondrial targeting agent, is modified onto the surface of the liposome body to achieve mitochondrial targeting within ovarian granulosa cells. This invention utilizes cell membrane compatibility and the targeted binding of the ovarian-specific targeting polypeptide sequence YAGKLLISLSEERISAHHVP to increase drug uptake by ovarian granulosa cells. Simultaneously, it leverages the mitochondrial targeting agent TPP to increase drug accumulation in intracellular mitochondria, further enhancing the bioavailability of PQQ. This invention achieves precise organ / cell-level localization using the independently validated ovarian-specific targeting polypeptide YAGKLLISLSEERISAHHVP, while integrating the mitochondrial targeting agent TPP to efficiently enrich the drug (PQQ) at its mitochondrial site of action. This synergistic targeting strategy significantly increases the uptake of PQQ by ovarian granulosa cells, substantially improving drug bioavailability. It successfully overcomes the bottlenecks of existing delivery technologies, achieving highly efficient optimization of ovarian mitochondrial function and providing a breakthrough solution for the precision treatment of diseases such as ovarian reserve insufficiency. Attached Figure Description

[0027] Figure 1 To observe the targeted binding of the ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP-FITC to the KGN cell line using laser confocal microscopy.

[0028] Figure 2 To observe the morphology of targeted ovarian mitochondrial liposomes with mitochondrial optimization function using transmission electron microscopy (TEM). The three images from left to right are three randomly selected fields of view.

[0029] Figure 3The results show the in vivo and in vitro safety of mitochondrial liposomes with mitochondrial optimization function. A shows the cytotoxicity results of different concentrations of mitochondrial liposomes with mitochondrial optimization function; B shows the weight change of mice after tail vein injection of mitochondrial liposomes with mitochondrial optimization function; C shows the serum biochemical results (including alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine) of mice after tail vein injection of mitochondrial liposomes with mitochondrial optimization function; D shows the HE staining results (magnification 20×) of paraffin-embedded sections of various organs (heart, liver, spleen, lung, kidney, and ovary) of mice after tail vein injection of mitochondrial liposomes with mitochondrial optimization function.

[0030] Figure 4 To observe the targeted binding of ovarian mitochondrial liposomes with mitochondrial optimization function to the KGN cell line and mitochondria using laser confocal microscopy. The top is the control group, and the bottom is the liposome group. From left to right, they represent DAPI fluorescence, FITC fluorescence, Mito-tracker fluorescence, and superimposed fluorescence.

[0031] Figure 5 To observe the targeting of mitochondrial liposomes with mitochondrial optimization function in mice using in vivo imaging. The top image shows the in vivo imaging results, and the bottom image shows the ex vivo imaging results.

[0032] Figure 6 This study aimed to detect the mitochondrial optimization capabilities of targeted ovarian mitochondrial liposomes with mitochondrial optimization function in the KGN cell line. Figure A shows the reactive oxygen species (ROS) levels in each treatment group, with DAPI fluorescence, DCF fluorescence, and superimposed fluorescence displayed from top to bottom. Figure B shows the JC-1 mitochondrial membrane potential levels in each treatment group, with JC-1 monomer fluorescence, JC-1 polymer fluorescence, and superimposed fluorescence displayed from top to bottom. Detailed Implementation

[0033] The present invention will be further explained below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0034] Example 1: Synthesis and Cell Targeting Identification of Ovarian-Specific Targeting Peptide YAGKLLISLSEERISAHHVP-FITC

[0035] (1) Synthesis of ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP-FITC

[0036] It was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. using a solid-phase synthesis method based on the polypeptide's amino acid sequence. FITC serves as a fluorescent label; however, it is not required for marketed drugs.

[0037] (2) Identification of the ovarian granulosa cell targeting ability of ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP-FITC

[0038] First, a 20mm diameter cell slide (Biosharp) was prepared. ® (Catalog No.: BS-20-RC) was placed at the bottom of the wells of a 12-well cell culture plate. KGN (human ovarian granulosa cell) cell lines were seeded at 50,000 cells / well in the 12-well cell culture plate slides. The culture medium was 1 ml of complete medium (complete medium is DMEM / F12 medium containing 10 v / v% fetal bovine serum, the same below). The plates were then incubated at 37°C and 5% CO2 for 24 hours until the cell density reached 60%. All cells were divided into 4 groups (i.e., 3 experimental groups and 1 control group), and each group had 3 replicate samples in 3 wells. The ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP-FITC prepared in Example 1 (1) was diluted to 20 μM, 40 μM, and 80 μM with complete culture medium, respectively, and added to the corresponding wells of each experimental group, 1 mL per well, and labeled as YAGKLLISLSEERISAHHVP-FITC 20 μM, YAGKLLISLSEERISAHHVP-FITC 40 μM, and YAGKLLISLSEERISAHHVP-FITC 80 μM, respectively. An equal volume of complete culture medium was added as a control group, labeled CK. The cells were cultured for 3 hours in a 37°C, 5% CO2 incubator. The supernatant from each well was then discarded, and the cells were cultured with PBS (Biosharp). ® The cells were washed twice with PBS (product number BL302A; hereinafter the same). 1 ml of 4 mV / v paraformaldehyde was added to each well for fixation for 15 minutes, then the 4 mV / v paraformaldehyde was discarded. The cells were then washed three times with PBS for 5 minutes each time. Finally, the cell slides were removed and placed on a glass slide. Approximately 5 μL of anti-fluorescence quenching mounting medium containing DAPI (Shanghai Beyotime Biotechnology Co., Ltd., product number: P0131) was added to the slide, and laser confocal microscopy was performed immediately. For the detection of the ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP-FITC, an excitation wavelength of 488 nm and an emission wavelength of 525 nm were used. For the detection of DAPI, an excitation wavelength of 405 nm and an emission wavelength of 480 nm were used.

[0039] Experimental results are as follows Figure 1As shown (DAPI appears blue, representing nuclear staining; FITC appears green, representing ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP-FITC staining). From Figure 1 It can be seen that, compared with the control group, the ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP-FITC can bind to and internalize the surface of KGN cell mesomy 1, indicating that this specific targeting peptide has a ovarian granulosa cell targeting effect.

[0040] Example 2: Preparation of Targeted Ovarian Mitochondrial Liposomes with Mitochondrial Optimization Function

[0041] (1) Synthesis of DSPE-PEG2000-YAGKLLISLSEERISAHHVP-FITC

[0042] Accurately weigh 60 mg of distearate phosphatidylethanolamine-polyethylene glycol 2000-succinimide ester, i.e., DSPE-PEG2000-NHS or DSPE-PEG2K-NHS (MW: 2900), dissolve it in 2 ml of N,N-dimethylformamide (DMF), add the ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP-FITC (1.1 eq.) and triethylamine (3.0 eq.) synthesized in Example 1, dissolve completely, and react at room temperature (300 rpm) for 12 h. The synthetic route is as follows:

[0043]

[0044] After the reaction is complete, the reaction solution is transferred to a dialysis bag (molecular weight cutoff 3000 Da) and dialyzed in deionized water at room temperature for 24 hours, replacing the deionized water every 2 hours. After dialysis, the dialysate is collected and freeze-dried (pre-freezing temperature: liquid nitrogen; primary drying temperature: -40°C, 24 hours; secondary drying temperature: -20°C, 6 hours; vacuum degree: 0.1 mbar). The product is sealed in an inert gas environment (such as nitrogen, in this example, nitrogen) to obtain the DSPE-PEG2000-YAGKLLISLSEERISAHHVP-FITC freeze-dried product, which is then stored under dry conditions at -20°C for later use.

[0045] (2) Synthesis of DSPE-PEG2000-TPP

[0046] Accurately weigh 500 mg of DSPE-PEG2000-NHS (i.e., DSPE-PEG2K-NHS) and dissolve it in 5 mL of chloroform. Add amino-modified triphenylphosphine (i.e., TPP-NH2) (1.0 eq.) and triethylamine (2.0 eq.) until completely dissolved. Stir at room temperature (300 rpm) for 1 h. The synthetic route is as follows:

[0047] After the reaction was completed, the reaction solution was concentrated by vacuum distillation (water bath temperature 37°C, vacuum pressure 0.1 bar, rotation speed 80 rpm, time 60 minutes). The solution was then precipitated at 4°C with 10 times the volume of ice-cold diethyl ether at room temperature for 2 hours (stirring speed 400 rpm). The product was filtered through a 0.22 μm microporous membrane, and the precipitate was retained on the membrane. The product was then dried under vacuum (pressure 0.05 mbar, temperature room temperature, time 48 hours) to obtain DSPE-PEG2000-TPP (i.e. DSPE-PEG2K-TPP) lyophilized product, which was then stored at -20°C for later use.

[0048] (3) Preparation of mitochondrial liposomes with mitochondrial optimization function targeting ovarian mitochondrial liposomes

[0049] Dissolve 130.5 mg of lecithin, 7.5 mg of cholesterol, 3 mg of CY5.5 (CY5.5 serves as a fluorescent marker, but is not required for marketed drugs), 4.5 mg of DSPE-PEG2000-YAGKLLISLSEERISAHHVP-FITC lyophilized product, and 4.5 mg of DSPE-PEG2000-TPP lyophilized product in 5 mL of chloroform and vortex at room temperature until completely dissolved. Place the chloroform solution in a round-bottom flask and evaporate under reduced pressure on a rotary evaporator (conditions: 40°C, vacuum pressure 0.1 bar, rotation speed 100 rpm, evaporation time 60 minutes) until a film forms. Add 5 mL of an aqueous solution containing PQQ (2 mg / mL) and hydrate at 50°C for 2 hours, stirring moderately at 300 rpm during hydration to promote hydration and ensure the lipid film is fully hydrated to form liposomes. After ultrasonic treatment (180W) for 10 min, the liposomes were extruded through a liposome extruder (polycarbonate membrane, 100nm pore size) (repeated 10 times) and then purified. Purification was performed using a nanodialysis device (polycarbonate membrane, 30nm pore size, dialysis at 4°C for 48 hours, with the dialysate (deionized water) replaced every 8 hours). The purified liposome suspension was collected. Deionized water was added to the purified liposome suspension to bring the volume to 15 mL, and 15 mg of 10wt% mannose was added as a freeze-drying protectant. The mixture was gently mixed and freeze-dried (pre-freezing temperature: liquid nitrogen; primary drying temperature: -40°C, 24 hours; secondary drying temperature: -20°C, 6 hours; vacuum: 0.1 mbar) to obtain ovarian mitochondrial liposomes with mitochondrial optimization function.

[0050] The particle size and surface potential of the prepared mitochondrial-optimized ovarian mitochondrial liposomes were determined using a nanoparticle size potentiometry meter (Brookhaven, USA, model: NanoBrook 90plus PALS). The drug encapsulation efficiency and drug loading of the prepared mitochondrial-optimized ovarian mitochondrial liposomes were calculated using the following formula:

[0051]

[0052] The morphology and distribution of the ovarian mitochondrial liposomes with mitochondrial optimization function were observed using transmission electron microscopy.

[0053] Table 1 shows the particle size, surface potential, drug encapsulation efficiency, and drug loading of mitochondrial liposomes with mitochondrial optimization function targeting ovarian mitochondrial liposomes. Figure 2 The image shows the morphology of ovarian mitochondrial liposomes with mitochondrial optimization function. Figure 2 As shown, the targeted ovarian mitochondrial liposomes with mitochondrial optimization function exhibit a uniform bilayer membrane structure under transmission electron microscopy.

[0054]

[0055] Example 3: In vivo and in vitro safety experiments of mitochondrial-targeting ovarian mitochondrial liposomes with mitochondrial optimization function.

[0056] KGN cell line was used at 1×10 4 Cells were seeded per well in 96-well plates with 100 μL of complete culture medium and incubated overnight at 37°C in a 5% CO2 cell culture incubator until the cell density reached 60%. All cells were divided into six groups (5 experimental groups and 1 negative control group), with 5 replicates per well for each group. The mitochondrial-targeting ovarian mitochondrial liposomes with mitochondrial optimization function prepared in Example 2 were diluted with complete culture medium to 50 μg / ml, 100 μg / ml, 150 μg / ml, 200 μg / ml, and 250 μg / ml, respectively, and added to the corresponding wells of each experimental group, 100 μL per well. An equal volume of complete culture medium was added as a negative control group. Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. After 24 hours, the supernatant of each well was discarded, and the cells were washed twice with PBS. The CCK-8 reagent (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 40203ES76) was diluted with basal medium (DMEM / F12 medium without fetal bovine serum, the same below) to make the CCK-8 reagent content 10v / v%. Then, 100μL of basal medium containing 10v / v% CCK-8 reagent was added to each well. After culturing for another 3 hours, the absorbance at a wavelength of 450nm was detected by microplate reader. The cell viability of each experimental group was obtained by comparing the OD value with that of the negative control group.

[0057] The results of the cytotoxicity assay are shown below. Figure 3 A, from Figure 3 As can be seen from A, the mitochondrial-targeted ovarian mitochondrial liposomes with mitochondrial optimization function prepared in Example 2 showed no significant cytotoxicity at any concentration and had a certain beneficial effect of promoting cell proliferation.

[0058] Twelve ICR mice (8 weeks old, purchased from Shanghai Silex Laboratory Animal Co., Ltd.) were randomly divided into two groups: a liposome group and a control group. The liposome group received a tail vein injection of mitochondrial-optimized targeted ovarian mitochondrial liposomes (dissolved in PBS at a concentration of 1.5 mg / mL), while the control group received a tail vein injection of PBS. The dosage was 5 mg of mitochondrial-optimized targeted ovarian mitochondrial liposomes / kg mouse for the liposome group and an equal volume of PBS for the control group. Administration was every other day for a total of 7 days (4 administrations). Mice were weighed daily at 09:00 until day 8. Administration was performed after weighing. Twenty-four hours after the end of the treatment course (day 8), all mice were anesthetized with isoflurane inhalation. 0.5 ml of whole blood was collected via the orbital vein, and the mice were euthanized via CO2 inhalation. Heart, liver, spleen, lungs, kidneys, and ovaries were collected and fixed with 4 mM / v% paraformaldehyde. Whole blood was collected and incubated overnight at 4°C, then centrifuged at 3000 rpm for 10 min at 4°C. The resulting supernatant was mouse serum, used for blood biochemical tests (including alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine). All tissues were fixed at room temperature for 48 h, then embedded in paraffin and sectioned for HE staining to assess tissue structure.

[0059] Results of in vivo toxicity tests are shown in Figure 3 BD, from Figure 3 As shown in Figure B, within one week of tail vein injection of mitochondrial-targeted ovarian mitochondrial liposomes with mitochondrial optimization function into mice, there was no significant increase or decrease in mouse body weight. Figure 3 CD showed that one week after tail vein injection of mitochondrial-targeted ovarian mitochondrial liposomes with mitochondrial optimization function into mice, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine in mice were not significantly increased compared with the control group. HE staining showed that after tail vein injection of mitochondrial-targeted ovarian mitochondrial liposomes with mitochondrial optimization function into mice, there were no obvious necrosis, lysis, or acute inflammation in the heart, liver, spleen, lungs, kidneys, and ovarian tissues, indicating that the liposomes had no obvious acute toxicity in mice.

[0060] Example 4: Cellular uptake and mitochondrial targeting experiment of mitochondrial-targeted ovarian mitochondrial liposomes with mitochondrial optimization function.

[0061] Cellular uptake and mitochondrial targeting experiments were conducted using the mitochondrial-optimized ovarian mitochondrial liposomes prepared in Example 2. First, 20 mm diameter biosharp cell slices were used... ® (Catalog No.: BS-20-RC) was placed at the bottom of a 12-well cell culture plate. KGN cell line was seeded at 50,000 cells / well in the 12-well cell culture plate slide, with 1 ml of complete culture medium. The plate was then incubated at 37°C and 5% CO2 for 24 hours until the cell density reached 60%. The mitochondrial-targeting ovarian mitochondrial liposomes with mitochondrial optimization function prepared in Example 2 were diluted to 200 μg / ml with complete culture medium and added as the experimental group, 1 ml per well. An equal volume of complete culture medium was added as the negative control group. Each group had 3 replicate samples. Each group was incubated at 37℃ in a 5% CO2 cell culture incubator for 3 hours. The supernatant was then discarded, and the cells were washed twice with PBS. Following the instructions of the Mito-Tracker Red CMXRos kit (Shanghai Beyotime Biotechnology Co., Ltd., product number: C1049B-50μg), the mitochondrial red fluorescent probe was diluted 1:1000 in basal culture medium and added to the wells of each cell culture plate (1 ml per well). The plates were then incubated at 37℃ in a 5% CO2 cell culture incubator for 20 minutes. After incubation, the culture medium was discarded, and the cells were washed twice with PBS. 1 ml of 4m / v% paraformaldehyde was added to each well for fixation for 15 minutes. The 4m / v% paraformaldehyde was then discarded, and the cells were washed three times with PBS for 5 minutes each time. Finally, the cell slides were removed and placed on a glass slide. At the same time, about 5 μL of anti-fluorescence quenching mounting solution containing DAPI (Shanghai Beyotime Biotechnology Co., Ltd., product number: P0131) was added to the glass slide. Laser confocal microscopy was then performed immediately. When detecting DAPI, the excitation light was set to 405 nm and the emission light to 461 nm; when detecting FITC, the excitation light was set to 488 nm and the emission light to 520 nm; and when detecting Mito-tracker, the excitation light was set to 561 nm and the emission light to 599 nm.

[0062] Experimental results are as follows Figure 4 As shown (DAPI appears blue, representing nuclear staining; FITC appears green, representing ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP-FITC staining; Mito-tracker appears red, representing mitochondrial staining in KGN cell lines; Merge is the result of all staining superposition). From Figure 4It can be seen that, compared with the control group, the ovarian mitochondrial liposome with mitochondrial optimization function can co-localize with mitochondria in the KGN cell line, indicating that the liposome has cell-targeting and mitochondrial-targeting effects in the KGN cell line.

[0063] Example 5: In vivo targeting of ovarian mitochondrial liposomes with mitochondrial optimization function

[0064] In vivo targeting experiments were conducted using the mitochondrial-optimized targeted ovarian mitochondrial liposomes prepared in Example 2. Twenty-five ICR mice (8 weeks old, purchased from Shanghai Silex Laboratory Animal Co., Ltd.) were randomly divided into five groups, designated as Group 1, Group 2, Group 3, Group 4, and Group 5. Group 1 served as the control group, with mice receiving a tail vein injection of PBS at the same volume as the other groups. Groups 2, 3, 4, and 5 served as the experimental groups (liposome groups), with mice receiving a tail vein injection of the mitochondrial-optimized targeted ovarian mitochondrial liposomes (dissolved in PBS at a concentration of 1.5 mg / mL). The dosage for each experimental group was 5 mg of the mitochondrial-optimized targeted ovarian mitochondrial liposomes / kg mouse. In vivo imaging was performed at 15 minutes (Group 2), 30 minutes (Group 3), 1 hour (Group 4), and 2 hours (Group 5) after administration. Before imaging, mice were placed in an induction chamber containing 5v / v% isoflurane (oxygen flow rate 1L / min) for 1-2 minutes until the righting reflex disappeared. After successful anesthesia, they were placed in an in vivo imaging system. Anesthesia was maintained via a nasal cone with 1.5-2v / v% isoflurane (oxygen flow rate 0.5L / min). The in vivo imaging system was purchased from PerkinElmer (model IVIS Lumina III). Fluorescence imaging mode was selected, with an excitation wavelength of 675nm, an emission filter of 695nm, and an exposure time of 3 seconds for in vivo imaging. After in vivo imaging, the mice were immediately euthanized by cervical dislocation under anesthesia. The uterus, bilateral fallopian tubes, and ovaries were dissected and removed. After cleaning with PBS to remove residual blood, the mice were reinserted into the imaging system, and in vitro imaging was performed again using the same parameters.

[0065] Experimental results are as follows Figure 5 As shown. From Figure 5 It can be seen that, compared with the control group, the CY5.5 fluorescence signal of the liposome group showed the most significant enrichment effect in the ovary of mice at 30 minutes, indicating that the liposome has an ovarian-targeting effect in mice.

[0066] Example 6: Identification of mitochondrial optimization capability of targeted ovarian mitochondrial liposomes with mitochondrial optimization function

[0067] The mitochondrial optimization capability of the ovarian mitochondrial-targeting liposomes prepared in Example 2 was tested. First, 20 mm diameter biosharp cell slices were used... ® (Catalog No.: BS-20-RC) was placed at the bottom of the wells of a 12-well cell culture plate. KGN cell lines were seeded at 50,000 cells / well in the 12-well cell culture plate slides, with 1 ml of complete culture medium. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours until the cell density reached 60%. All cells were divided into three groups: group 1 was the control group, group 2 was the senescence group (H2O2 group), and group 3 was the senescence + liposome group (H2O2 + liposome group), with three replicates in each group. First, the senescence model was constructed for groups 2 and 3. Specifically, the culture medium was discarded, and the cells were washed once with PBS. Then, 3 wt% hydrogen peroxide solution was diluted to 300 μM with complete culture medium and added to groups 2 and 3, 1 ml per well. Simultaneously, an equal volume of hydrogen peroxide-free complete culture medium was added to group 1. The plates were incubated at 37°C in a 5% CO2 incubator for 3 hours. At this point, the cell senescence models for the second and third groups were completed. Next, the cells in the third group were treated with ovarian mitochondrial liposomes with mitochondrial optimization function. Specifically, the culture medium was discarded, the cells were washed twice with PBS, and the ovarian mitochondrial liposomes with mitochondrial optimization function prepared in Example 2 were diluted to 200 μg / ml with complete culture medium and added to the third group, 1 ml per well. Simultaneously, an equal volume of complete culture medium was added to the first and second groups. The cells were incubated at 37°C in a 5% CO2 cell incubator for 24 hours to complete the treatment. Finally, the mitochondrial function of all KGN cell lines was assessed using two methods: intracellular reactive oxygen species (ROS) assay and mitochondrial membrane potential assay.

[0068] Intracellular reactive oxygen species (ROS) were measured using an ROS detection kit (Shanghai Beyotime Biotechnology Co., Ltd., product number: S0033S). DCFH-DA was diluted with PBS at a volume ratio of 1:1000. The culture medium was discarded, and the cells were washed once with PBS. 1 ml of diluted DCFH-DA was added to each well, and the cells were incubated at 37°C in a 5% CO2 cell culture incubator for 20 minutes. The diluted DCFH-DA was discarded, and the cells were washed three times with PBS to thoroughly remove any uninfiltrated DCFH-DA. Cell slides were then removed and placed on a glass slide. Approximately 5 μL of anti-fluorescence quenching mounting medium containing DAPI (Shanghai Beyotime Biotechnology Co., Ltd., product number: P0131) was added to the slide. Laser confocal microscopy was performed immediately, with excitation wavelength set to 488 nm and emission wavelength to 525 nm.

[0069] Mitochondrial membrane potential was detected using a mitochondrial membrane potential detection kit (Shanghai Beyotime Biotechnology Co., Ltd., product number: C2006). 50 μL of JC-1 (200X) was diluted to 1× with 8 ml of ultrapure water. The JC-1 was thoroughly dissolved and mixed by vigorous vortexing. Then, 2 ml of JC-1 staining buffer (5X) was added, and the mixture was thoroughly mixed to obtain the JC-1 staining working solution, which was then kept on ice. The culture medium was discarded, and the cells were washed once with PBS. 1 ml of JC-1 staining working solution was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator for 20 minutes. During incubation, an appropriate amount of JC-1 staining buffer (1X) was prepared by adding 4 ml of distilled water to every 1 ml of JC-1 staining buffer (5X), and this buffer was kept on ice. After incubation, the JC-1 staining working solution was discarded, and the cells were washed twice with JC-1 staining buffer (1X). Remove the cell smear and place it on a glass slide. Simultaneously, add approximately 5 μL of anti-fluorescence quenching mounting solution containing DAPI (Shanghai Beyotime Biotechnology Co., Ltd., product number: P0131) to the glass slide. Immediately perform laser confocal microscopy imaging. According to the instructions of the mitochondrial membrane potential detection kit (Shanghai Beyotime Biotechnology Co., Ltd., product number: C2006), the fluorescence intensity of JC-1 monomer and JC-1 polymer was detected, with the following parameter settings: for detecting JC-1 monomer, the excitation light was set to 490 nm and the emission light was set to 530 nm; for detecting JC-1 polymer, the excitation light was set to 525 nm and the emission light was set to 590 nm.

[0070] Experimental results are as follows Figure 6 As shown ( Figure 6 In A, DAPI appears blue, representing nuclear staining; DCF appears green, representing reactive oxygen species staining; and Merge is the result of superimposing all staining. Figure 6 In section B, JC-1 monomer appears green, indicating staining of the JC-1 monomer; JC-1 polymer appears red, indicating staining of the JC-1 polymer. Merge is the result of the superposition of all staining. Figure 6 As shown in Figure A, compared with the control group, the green fluorescence signal in the cells of the aging group was enhanced, indicating a significant increase in intracellular reactive oxygen species (ROS) content, while the ROS content in the cells of the aging + liposome group was significantly lower than that of the second group. Figure 6 As shown in Figure B, compared to the control group, the aging group showed enhanced green fluorescence and weakened red fluorescence, indicating a decrease in the number of healthy mitochondria and an increase in the number of abnormal mitochondria. Conversely, compared to the aging group, the aging + liposome group showed weakened green fluorescence and enhanced red fluorescence, indicating that after treatment with liposome-targeted ovarian mitochondria with mitochondrial optimization function, the number of healthy mitochondria increased and the number of abnormal mitochondria decreased. These two sets of results suggest that the liposomes optimize mitochondrial function in the KGN cell line.

Claims

1. A mitochondrial-targeting ovarian mitochondrial liposome with mitochondrial optimization function, characterized in that, It includes the liposome body, pyrroloquinoline quinone, ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP, and triphenylphosphine. Pyrroloquinoline quinone is encapsulated in the liposome body, and ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP and triphenylphosphine are modified on the surface of the liposome body through amphiphilic polymer anchoring sites. The amphiphilic polymer includes distearate phosphatidylethanolamine-polyethylene glycol 2000; The liposome body is constructed from lecithin and cholesterol; The targeted ovarian mitochondrial liposomes with mitochondrial optimization function are prepared by the following steps: (1) Synthesis of DSPE-PEG2000-YAGKLLISLSEERISAHHVP Distearate phosphatidylethanolamine-polyethylene glycol 2000-succinimide ester, i.e., DSPE-PEG2000-NHS, was dissolved in a solvent, and ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP and triethylamine were added to carry out the reaction. After the reaction was completed, the reaction solution was post-processed to obtain the lyophilized product DSPE-PEG2000-YAGKLLISLSEERISAHHVP. (2) Synthesis of DSPE-PEG2000-TPP Distearate phosphatidylethanolamine-polyethylene glycol 2000-succinimide ester, i.e., DSPE-PEG2000-NHS, was dissolved in a solvent, and amino-modified triphenylphosphine, i.e., TPP-NH2, and triethylamine were added to carry out the reaction. After the reaction was completed, the reaction solution was post-treated to obtain the lyophilized product DSPE-PEG2000-TPP. (3) Preparation of mitochondrial liposomes with mitochondrial optimization function targeting ovarian mitochondrial liposomes Lecithin, cholesterol, DSPE-PEG2000-YAGKLLISLSEERISAHHVP lyophilized products, and DSPE-PEG2000-TPP lyophilized products were dissolved in a solvent, evaporated under reduced pressure to form a film, and then hydrated by adding an aqueous solution containing pyrroloquinoline quinone (PQQ). After post-processing, targeted ovarian mitochondrial liposomes with mitochondrial optimization function were obtained.

2. The ovarian mitochondrial liposome with mitochondrial optimization function according to claim 1, characterized in that, The solvent in step (1) includes N,N-dimethylformamide; the post-treatment includes dialyzing the reaction solution, collecting the dialysate, freeze-drying, and sealing under an inert gas atmosphere.

3. The ovarian mitochondrial liposome with mitochondrial optimization function according to claim 1, characterized in that, The solvent in step (2) includes chloroform; the post-treatment includes concentrating the reaction solution by vacuum distillation, precipitating with ice-cold ether, filtering, collecting the product precipitate, and vacuum drying.

4. A targeted ovarian mitochondrial liposome with mitochondrial optimization function according to claim 1, characterized in that, The solvent in step (3) includes chloroform; the post-processing includes sonication, extrusion, purification, and freeze drying.

5. A method for preparing targeted ovarian mitochondrial liposomes with mitochondrial optimization function, characterized in that, Includes the following steps: (1) Synthesis of DSPE-PEG2000-YAGKLLISLSEERISAHHVP Distearate phosphatidylethanolamine-polyethylene glycol 2000-succinimide ester, i.e., DSPE-PEG2000-NHS, was dissolved in a solvent, and ovarian-specific targeting peptide YAGKLLISLSEERISAHHVP and triethylamine were added to carry out the reaction. After the reaction was completed, the reaction solution was post-processed to obtain the lyophilized product DSPE-PEG2000-YAGKLLISLSEERISAHHVP. (2) Synthesis of DSPE-PEG2000-TPP Distearate phosphatidylethanolamine-polyethylene glycol 2000-succinimide ester, i.e., DSPE-PEG2000-NHS, was dissolved in a solvent, and amino-modified triphenylphosphine, i.e., TPP-NH2, and triethylamine were added to carry out the reaction. After the reaction was completed, the reaction solution was post-treated to obtain the lyophilized product DSPE-PEG2000-TPP. (3) Preparation of mitochondrial liposomes with mitochondrial optimization function targeting ovarian mitochondrial liposomes Lecithin, cholesterol, DSPE-PEG2000-YAGKLLISLSEERISAHHVP lyophilized products, and DSPE-PEG2000-TPP lyophilized products were dissolved in a solvent, evaporated under reduced pressure to form a film, and then hydrated by adding an aqueous solution containing pyrroloquinoline quinone (PQQ). After post-processing, targeted ovarian mitochondrial liposomes with mitochondrial optimization function were obtained.

6. The method for preparing a targeted ovarian mitochondrial liposome with mitochondrial optimization function according to claim 5, characterized in that, The solvent in step (1) includes N,N-dimethylformamide; the post-treatment includes dialyzing the reaction solution, collecting the dialysate, freeze-drying, and sealing under an inert gas atmosphere.

7. The method for preparing a targeted ovarian mitochondrial liposome with mitochondrial optimization function according to claim 5, characterized in that, The solvent in step (2) includes chloroform; the post-treatment includes concentrating the reaction solution by vacuum distillation, precipitating with ice-cold ether, filtering, collecting the product precipitate, and vacuum drying.

8. The method for preparing a targeted ovarian mitochondrial liposome with mitochondrial optimization function according to claim 5, characterized in that, The solvent in step (3) includes chloroform; the post-processing includes sonication, extrusion, purification, and freeze drying.

9. The use of the mitochondrial-optimized ovarian mitochondrial liposomes according to any one of claims 1-4 in the preparation of a medicament for treating ovarian reserve insufficiency, characterized in that, The treatment for ovarian reserve insufficiency involves optimizing mitochondrial function in ovarian granulosa cells.

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