Doxorubicin scavenger for protecting ovarian fertility and method of making same

Abstract

The application discloses a doxorubicin scavenger for protecting ovarian fertility and a preparation method thereof, comprising a DNA complex wrapped by a red blood cell membrane, wherein the red blood cell membrane is surface-modified by an anti-Mullerian duct antibody alphaAMH; the doxorubicin scavenger is specifically combined with AMH which is highly expressed in ovarian tissues through alphaAMH, is enriched in the ovarian part in a targeted manner, adsorbs doxorubicin through a DNA fragment in the DNA complex, reduces toxic damage of doxorubicin to ovarian cells, and further protects ovarian fertility. The doxorubicin scavenger can adsorb injected doxorubicin Dox, reduce side effects of doxorubicin Dox on ovarian granulosa cells, and further protect ovarian function and fertility.

Description

Technical Field

[0001] This invention relates to biomedicine, specifically to doxorubicin scavenger for protecting ovarian fertility and its preparation method. Background Technology

[0002] As women are increasingly diagnosed with cancer at younger ages, more and more women may receive cancer-related treatments during their peak reproductive years. As one of the main treatment methods for cancer, studies have shown that many chemotherapy drugs can damage ovarian and uterine function, leading to adverse consequences such as acute amenorrhea, premature ovarian failure, infertility, and chronic estrogen deficiency.

[0003] Doxorubicin (Dox), a widely used anthracycline antibiotic, is used to treat various cancers, including breast cancer, lung cancer, gastric cancer, lymphoma, ovarian cancer, and leukemia. It induces apoptosis by intercalating into DNA and preventing its replication and transcription. Studies have shown that Dox treatment can induce apoptosis in mouse ovarian granulosa cells and stromal cells. Granulosa cells are rapidly proliferating somatic cells surrounding the oocyte, providing nutrients and maturation factors for oocyte development and playing important roles such as estrogen secretion. Dox-induced apoptosis in granulosa cells leads to the loss of protection and nutrition for the oocyte, resulting in damage and even apoptosis. Dox has dose-dependent toxicity on growing follicles, disrupting them, upregulating the PI3K / PTEN / Akt signaling pathway, and losing the inhibition of primordial follicle growth activation. This leads to the activation and over-recruiting of primordial follicles, resulting in decreased ovarian reserve and infertility.

[0004] Regarding the preservation of ovarian function and fertility during chemotherapy, embryo cryopreservation and mature oocyte cryopreservation are the only methods recognized by the American Society for Reproductive Medicine for fertility preservation. However, both methods sacrifice ovarian endocrine function to some extent and are not suitable for pre-pubertal women. Furthermore, their invasiveness, high cost, and the need to postpone chemotherapy limit their clinical use. Therefore, exploring a method that can protect ovarian function during chemotherapy has become a key focus of research on protecting the fertility of women undergoing chemotherapy. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an adriamycin scavenger for protecting ovarian fertility and its preparation method, which can adsorb injected doxorubicin (Dox), reduce the toxic side effects of Dox on ovarian granulosa cells, and thus protect ovarian function and fertility.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an adriamycin scavenger for protecting ovarian fertility, comprising a DNA complex encapsulated by a red blood cell membrane, the surface of which is modified with an anti-Müllerian tube antibody αAMH; the adriamycin scavenger specifically binds to AMH highly expressed in ovarian tissue via αAMH, targeting and enriching itself in the ovarian region, and adsorbs Dox via DNA fragments in the DNA complex, thereby reducing the toxic damage of Dox to ovarian cells and thus protecting ovarian fertility.

[0007] As a further improvement of the present invention, the DNA complex adopts a DNA & protamine complex GCD / P.

[0008] As a further improvement of the present invention, the DNA fragment is a high-GC-content DNA fragment, with a GC content of 65%; and / or The DNA fragment is 657 bp in length.

[0009] As a further improvement of the present invention, the charge ratio of DNA to protamine is 0.4.

[0010] As a further improvement of the present invention, the particle size of the doxorubicin scavenger particles is 200 nm.

[0011] As a further improvement of the present invention, the erythrocyte membrane and αAMH are modified and connected by a biotin-streptavidin bridging method.

[0012] A method for preparing an adriamycin scavenger for protecting ovarian fertility includes the following steps: Extraction of red blood cell membranes (RM); Red blood cell membranes were modified to obtain αAMH-modified red blood cell membranes RM-αAMH; Prepare a DNA complex, wherein the DNA complex is a DNA-protamine complex GCD / P; The doxorubicin scavenger was obtained by mixing αAMH-modified erythrocyte membrane RM-αAMH with a DNA complex and extruding the mixture through a microextruder.

[0013] As a further improvement of the present invention, the DNA:RM-αAMH ratio is 1:50 w / w.

[0014] The beneficial effects of this invention are that the prepared biomimetic ovarian scavenger (BOS) can achieve ovarian targeting by binding to AMH highly expressed in ovarian granulosa cells through surface-modified αAMH, thereby neutralizing the subsequently injected chemotherapy drug Dox, reducing the chemotherapy-induced damage of Dox to the ovaries, and protecting ovarian fertility. Attached Figure Description

[0015] Figure 1 This invention provides a targeting map of RM-αAMH vesicles of different particle sizes on the ovary (a: RM-αAMH vesicles of different particle sizes prepared using DLS detection; b: quantitative fluorescence analysis of RM-αAMH vesicles of different particle sizes (DiD labeled) enriched in female mouse (E stage) ovarian tissue). Figure 2 The images show the preparation and characterization of the doxorubicin scavenger BOS of this invention (a: agarose gel electrophoresis analysis of GCD / P complexes prepared under different charge ratios; b: size distribution of BOS containing biotin-labeled GCD / P complexes or under different DNA to RM-αAMH mass ratios during incubation with streptavidin; c: hydrodynamic diameters and zeta potentials of GCD / P complexes, RM-αAMH vesicles, and BOS determined by dynamic light scattering; d: representative transmission electron microscopy images of BOS and RM-αAMH vesicles). Figure 3 The images show the targeting ability of BOS in vitro and in vivo (a: uptake of primary mouse ovarian granulosa cells by BOS pretreated with PBS, BOS (without αAMH), BOS or free αAMH for 30 minutes; b: quantitative fluorescence analysis of microscopic images; c: analysis of uptake of primary mouse ovarian granulosa cells by BOS pretreated with PBS, BOS (without αAMH), BOS or free αAMH for 30 minutes by flow cytometry; d: quantitative fluorescence analysis by flow cytometry; e: representative images of crystal violet staining of exfoliated vaginal cell smears from mice in different estrous cycles (top) and estrous cycle-related BOS accumulation in the ovary (bottom); f: fluorescence imaging of isolated mouse ovaries and uterus 24 hours after treatment with PBS, BOS (without αAMH) or BOS; g: quantitative fluorescence analysis of ovarian tissue). Figure 4The protective effects of BOS on GCs cells and ovaries in vitro and in vivo are shown in the figures: (a: Survival rate of mouse primary ovarian granulosa cells after 24 hours of treatment with different concentrations of BOS; b: Dose-dependent cytotoxicity of mouse primary ovarian granulosa cells after 24 hours of Dox treatment; c: Survival rate of mouse primary ovarian granulosa cells exposed to IC50 dose (0.2438 μM) Dox after pre-incubation with different concentrations of BOS; d: Survival rate of mouse primary ovarian granulosa cells exposed to IC50 dose (0.2438 μM) Dox after pre-incubation with different concentrations of BOS.) estradiol secretion from primary mouse ovarian granulosa cells (μM); e: time-dependent damage to mouse ovarian tissue weight caused by Dox treatment; f: time-dependent damage to mouse ovarian tissue volume caused by Dox treatment; g: representative histological images used for follicular phase determination criteria; h: ovarian weight, volume, and follicle count 1 day after treatment with PBS, Dox, or BOS+Dox; i: follicle count, ovarian weight, and volume 30 days after treatment with PBS, Dox, or BOS+Dox; j: TUNEL staining of ovarian sections showing apoptosis signals 24 hours after treatment with PBS, BOS, or BOS+Dox (green: TUNEL; blue: cell nucleus), scale bar = 100 µm. Figure 5 The following figures illustrate the protective effect of BOS on the fertility of female mice during Dox administration: (a: Schematic diagram of vaginal exfoliated cell smear preparation; b: Daily monitoring of estrous cycle changes over 30 days after treatment with PBS, Dox, or BOS+Dox; c: Timeline of in vivo reproductive experiment: female mice were pretreated with BOS on day 0, exposed to Dox on day 1, and mated with healthy male mice on day 30, followed by three consecutive rounds of reproductive testing; d: Conception rate of female mice after three consecutive rounds of reproductive treatment with PBS, Dox, or BOS+Dox; e: Representative images of pups born to female mice in the three rounds of reproductive treatment with PBS, Dox, or BOS+Dox; f: Statistical analysis of the number of pups per litter in the three consecutive rounds of reproductive treatment with PBS, Dox, or BOS+Dox.) Detailed Implementation

[0016] The present invention will now be described in further detail with reference to the embodiments shown in the accompanying drawings.

[0017] Many studies have utilized drugs to protect ovarian function during chemotherapy, preserving ovarian endocrine function while offering advantages such as being relatively non-invasive, convenient, and having high patient compliance. Currently, the ovarian protective drugs that have been proven to protect ovarian function during chemotherapy and approved for clinical use are mainly gonadotropin-releasing hormone agonists (GnRHa), but GnRHa still face issues of efficacy controversy and hypothalamic-pituitary-ovarian axis side effects.

[0018] Therefore, to meet the need for fertility protection in women undergoing chemotherapy, this invention utilizes the characteristic of Dox to insert into the DNA double-stranded structure to design a biomimetic ovarian scavenger (BOS) targeting ovarian tissue. BOS consists of a DNA complex encapsulated by a erythrocyte membrane, with an anti-Müllerian duct antibody (AMH) (αAMH) modified on the erythrocyte membrane surface. Before Dox injection, BOS is pre-distributed into the ovarian tissue via intravenous injection. The high-GC base-pair DNA fragments contained within its structure can adsorb subsequently injected Dox, reducing the toxic side effects of Dox on ovarian granulosa cells, thereby protecting ovarian function and fertility.

[0019] This invention utilizes repeated hypotonic treatment to separate and purify erythrocyte membranes and modify them with αAMH (RM-αAMH). Simultaneously, it uses ultrasonic titration to prepare a DNA & protamine complex (GCD / P). Finally, it employs a physical extrusion method to encapsulate GCD / P with RM-αAMH to construct a (GCD / P)-RM-αAMH doxorubicin scavenger (BOS). The BOS prepared by this invention can achieve ovarian targeting by binding to AMH highly expressed in ovarian granulosa cells through surface-modified αAMH. This neutralizes subsequent injections of the chemotherapy drug Dox, reducing chemotherapeutic damage to the ovaries and protecting ovarian fertility.

[0020] 1. Extraction of red blood cell membrane (RM).

[0021] Fresh red blood cells were obtained from adult C57BL / 6J mice. The procedure was as follows: Fresh blood was collected from the submandibular vein in the cheek. Red blood cells were separated by centrifugation at 3000-5000 rpm for 15 min, and white blood cells and platelets were removed from the serum. The separated red blood cells were aliquoted into 1.5 mL EP tubes (200 µL per tube), and 1 mL of ultrapure water was added to thoroughly resuspend the red blood cells. Then, 20×PBS was added to adjust the osmotic pressure to 1×PBS. The cells were centrifuged at 13000 rpm for 10 min at 4°C. The supernatant was discarded, and the above steps were repeated until the supernatant was clear and free of hemoglobin. The precipitate obtained after the final centrifugation was collected as the red blood cell membrane (RM). Protein concentration was measured using a BCA protein quantification kit and stored at -80°C for subsequent experiments.

[0022] 2. RM-αAMH was constructed by modifying RM with anti-Müllerian tube antibody (αAMH). Prepare red blood cell membranes modified with anti-AMH polyclonal antibody (αAMH) (RM-αAMH). RM and DSPE-PEG2000-Biotin (2 mg / mL) were incubated at a mass ratio of 20:1 in a 37°C incubator for 30 min. After the reaction was complete, 20×PBS was added to adjust the osmotic pressure to 1×PBS. The mixture was then centrifuged at 13000 rpm at 4°C for 10 min to completely remove free DSPE-PEG2000-Biotin. Prepare a 1 mg / mL streptavidin aqueous solution and αAMH diluted at a ratio of 1:200. Resuspend the precipitate obtained from the previous centrifugation step using the streptavidin solution and incubate again at 37°C for 30 min. After the reaction is complete, add 20×PBS to adjust the osmotic pressure to 1×PBS and centrifuge at 13000 rpm and 4°C for 10 min to remove free streptavidin. Finally, add the diluted αAMH and incubate at 37°C for 30 min to obtain αAMH-modified erythrocyte membranes (RM-αAMH).

[0023] 3. Targeting properties of RM-αAMH with different particle sizes to the ovary DiD-labeled RM was prepared according to a DiD to RM membrane protein mass ratio of 2:1000, and then RM-αAMH was prepared by modification with αAMH. Subsequently, RM-αAMH vesicles of different particle sizes were constructed by extrusion through 100 nm, 200 nm, and 400 nm core-porous polyester membranes in a micro-extruder, and the particle size distribution of each group of vesicles was determined using a Nano Zetasizer. The results showed that RM-αAMH vesicles with particle sizes of approximately 100 nm, 200 nm, and 400 nm could be obtained after extrusion through different core-porous polyester membranes. Figure 1 a). Subsequently, RM-αAMH vesicles of different particle sizes were injected via the tail vein into female Estrus (E) mice in estrus. Ovarian tissue was isolated 24 h later, homogenized, and the DiD fluorescence values ​​of the ovarian tissues from different groups were measured using a microplate reader. The results showed that 200 nm RM-αAMH vesicles could more effectively target and accumulate within the ovarian tissue. Figure 1 b). Therefore, RM-αAMH vesicles with a particle size of 200 nm were prepared as the optimal particle size for targeted enrichment into ovarian tissue.

[0024] 4. Preparation and characterization of doxorubicin scavenger BOS.

[0025] Amplification of *E. coli* carrying the green fluorescent protein plasmid pLVX-IRES-ZsGreen was performed. 1-5 mL of *E. coli* culture carrying pLVX-IRES-ZsGreen was collected and transferred to 2 mL centrifuge tubes. Plasmids were extracted using a plasmid extraction kit according to the manufacturer's instructions, and the extracted plasmid DNA was stored at -20°C. A high-GC content (65%) target DNA fragment (GCD) of approximately 657 bp from the pLVX-IRES-ZsGreen protein plasmid was amplified by PCR. The DNA-protamine binding ratio was then determined: under sonication in a water bath, a fixed volume (20 µL) of protamine (P) aqueous solutions of different concentrations (calculated according to different charge ratios) was dropwise added to 20 µL of DNA solution (0.1 µg / µL), and the mixture was incubated at room temperature for 5 min to prepare the DNA & protamine complex (GCD / P). The charge ratio is expressed as the ratio of lysine residues in protamine to phosphate groups in DNA. Agarose gel electrophoresis was performed to determine an optimal charge ratio of 0.4 (at which there is no free DNA) for subsequent particle preparation. Figure 2 a).

[0026] The optimal ratio of RM-αAMH to GCD / P was determined using biotin-avidin polymerization experiments. Biotin-labeled GCD / P (DNA mass: 5 µg) was mixed with different masses of RM-αAMH (membrane protein mass: 0, 250, 500 µg) and allowed to stand at room temperature for 10 min before extrusion through a 200 nm nuclear-porous polyester membrane in a microextruder. Excess streptavidin was added to the prepared nanoparticle solution, and the hydrodynamic diameter distribution of each group of particles was determined using a Nano Zetasizer. The ratio in which the addition of excess streptavidin did not significantly change the particle size was selected. Figure 2 b). The results showed that a DNA:RM-αAMH ratio of 1:50 (w / w) ensured that RM-αAMH completely encapsulated GCD / P to form (GCD / P)RM-αAMH, abbreviated as BOS. The prepared BOS underwent basic physical characterization, including observation of the surface morphology of the nanosystem using transmission electron microscopy (TEM) and characterization using a NanoZetasizer (Malvern) via dynamic light scattering (DLS). The results showed that (GCD / P)RM-αAMH (BOS) exhibited a membrane vesicle structure with a particle size of approximately 200 nm and a surface potential of approximately -12 mV. Figure 2 c,d).

[0027] 5. Detection of BOS targeting ability in vivo and in vitro DiD-labeled (GCD / P)RM (without αAMH targeting modification) and (GCD / P)RM-αAMH (BOS) were prepared at a DiD:PLGA mass ratio of 2:1000. The targeting affinity of BOS was studied in vitro using primary ovarian granulosa cells (GCs). Equal amounts of (GCD / P)RM and (GCD / P)RM-αAMH (BOS) were added to GCs cells and incubated for 30 min. Fluorescence microscopy results showed that the phagocytosis rate of BOS by GCs cells was significantly higher than that of the (GCD / P)RM group (without αAMH targeting modification). Furthermore, the phagocytosis rate of the BOS group decreased significantly after GCs cells were pretreated with αAMH, verifying that the binding of BOS to GCs is mainly mediated by αAMH-mediated endocytosis. Flow cytometry results also confirmed that the targeting effect of BOS to GCs is mediated by αAMH. Figure 3 ad).

[0028] To verify whether estrous cycle alternation affects the targeted enrichment effect of BOS on the ovary, this invention injected DiD-labeled BOS into female mice at different estrous cycle stages (Diestrus / Proestrus / Estrus / Metestrus, denoted as D / P / E / M stages, respectively) via the tail vein. In vivo imaging results showed that BOS injection in the E stage achieved the highest accumulation in the ovary and uterus. Figure 3 Therefore, subsequent in vivo experiments all used BOS injection during the E phase in female mice. Subsequently, this invention synchronized the estrous cycle of female mice to the E phase on the fourth day by replacing male mouse cages with female mice. Then, DiD-labeled (GCD / P)RM and (GCD / P)RM-αAMH (BOS) were injected respectively, with an equal volume of PBS injected as a control group. In vivo imaging results of the ovary and uterus showed that the fluorescence values ​​of BOS in the ovary and uterus were significantly higher than those in the control group and the (GCD / P)RM group. Figure 3 (f,g), confirming that the ovarian-targeting function of BOS in vivo is also mediated by modification of αAMH.

[0029] 6. In vitro and in vivo protective effects of BOS on GCs cells and ovaries This invention evaluated the ability of BOS to mitigate Dox-induced cytotoxicity in GCs in vitro. First, GCs were incubated with BOS for 24 h, and cell viability was assessed using CCK8 assays to study the cytotoxicity of BOS itself. The results showed that even at a concentration of 1 mg / ml, BOS did not produce any cytotoxicity in GCs, indicating that BOS has extremely high biocompatibility. Figure 4a). Meanwhile, different concentrations of Dox were incubated with GCs for 24 h, and the effect of Dox on GC cell survival was detected using CCK8 assay. The 50% inhibitory concentration (IC50) of Dox was calculated to be 0.2438 μM. Figure 4 b). At the IC50 level, BOS pre-incubation of GCs showed a dose-dependent inhibitory effect on Dox-induced GC cytotoxicity, and BOS at 1 mg / ml completely restored GC cell viability. Figure 4 c). Simultaneously, an estrogen assay kit was used to further verify that BOS can effectively protect against Dox-induced GC apoptosis and maintain the GCs' ability to secrete estrogen. Figure 4 d).

[0030] Furthermore, this invention investigated the ability of BOS to neutralize Dox and protect the ovaries in vivo. Firstly, this invention demonstrated that under the influence of 10 mg / kg Dox, ovarian tissue exhibited a time-dependent atrophy trend, with the most significant damage observed on day 30. Figure 4 Therefore, days 1 and 30 after Dox injection were selected as the detection time points for the subsequent experimental study. BOS was pre-injected into B6 female mice at a dose of 300 mg / kg, followed by an injection of 10 mg / kg Dox 24 hours later. Dox alone (10 mg / kg) and PBS alone served as experimental control groups. On day 1 after Dox treatment, this invention assessed the ovarian weight and volume, as well as the number of follicles at each stage, in each group of female mice. Figure 4 The level of follicular apoptosis in ovarian tissue was also detected using TUNEL reagent (gh). Figure 4 The results showed that the levels of the above indicators were similar between female mice pretreated with BOS and those in the PBS control group. While the Dox-only treatment group had no effect on ovarian tissue weight and volume, it significantly inhibited the number of primordial follicles. On day 30 after Dox treatment, the ovarian weight and volume, as well as the follicle counts at each stage, were assessed in each group of female mice. Figure 4 i). The results showed that the levels of the above indicators were similar between female mice pretreated with BOS and those in the PBS control group, while the Dox-only treatment group showed a significant decrease in ovarian tissue weight and volume, as well as a significant decrease in the number of follicles at each stage, on day 30. These results indicate that BOS can effectively protect against Dox-induced ovarian damage.

[0031] 7. The protective effect of BOS on fertility in female mice during Dox administration. BOS was pre-injected into female B6 mice at a dose of 300 mg / kg, followed by an injection of 10 mg / kg Dox 24 hours later. Dox alone (10 mg / kg) and PBS alone served as experimental controls. On day 30 after Dox treatment, healthy, age-appropriate male mice were selected and placed in cages with female mice from the different treatment groups. The estrous cycle changes in the female mice over 30 days were recorded. Figure 5 a, b), indicators such as the number of pups born each time the same female mouse conceived three times, pup survival rate, and gestation time ( Figure 5 c). The results showed that the pregnancy rate of female mice treated with Dox was ( Figure 5 d) The number of offspring per birth has decreased significantly. Figure 5 The results showed that BOS pretreatment resulted in similar levels of ovarian fertility as the PBS-treated group. These results indicate that BOS can effectively protect against Dox-induced ovarian fertility decline.

[0032] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A doxorubicin scavenger for protecting ovarian fertility, characterized in that, It includes a DNA complex encapsulated by a red blood cell membrane, the surface of which is modified with an anti-Müllerian tube antibody αAMH; the doxorubicin scavenger specifically binds to AMH, which is highly expressed in ovarian tissue, through αAMH, targeting and enriching itself in the ovarian region, and adsorbs doxorubicin through DNA fragments in the DNA complex, thereby reducing the toxic damage of doxorubicin to ovarian cells and protecting ovarian fertility.

2. The doxorubicin scavenger according to claim 1, characterized in that, The DNA complex uses a DNA & protamine complex GCD / P.

3. The doxorubicin scavenger according to claim 2, characterized in that, The DNA fragment is a high-GC-content DNA fragment, with a GC content of 65%; and / or The DNA fragment is 657 bp in length.

4. The doxorubicin scavenger according to claim 2, characterized in that, The charge ratio of DNA to protamine is 0.

4.

5. The doxorubicin scavenger according to claim 1, characterized in that, The particle size of the doxorubicin scavenger particles is 200 nm.

6. The doxorubicin scavenger according to claim 1, characterized in that, Red blood cell membranes are modified and linked to αAMH via a biotin-streptavidin bridging mechanism.

7. A method for preparing an adriamycin scavenger for protecting ovarian fertility, characterized in that, Includes the following steps Extraction of red blood cell membranes (RM); Red blood cell membranes were modified to obtain αAMH-modified red blood cell membranes RM-αAMH; Prepare a DNA complex, wherein the DNA complex is a DNA-protamine complex GCD / P; The doxorubicin scavenger was obtained by mixing αAMH-modified erythrocyte membrane RM-αAMH with a DNA complex and extruding the mixture through a microextruder.

8. The preparation method according to claim 7, characterized in that, When extruded using a micro extruder, the DNA:RM-αAMH ratio is 1:50 w / w.

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