FSH33 peptide modified DNA tetrahedron nano delivery system as well as preparation method and application thereof

Abstract

The invention provides an FSH33 peptide modified DNA tetrahedron nano delivery system as well as a preparation method and application thereof, and aims to provide a safe and efficient antioxidant nano drug and optimize the enrichment efficiency to promote ovarian aging and ovarian tissue damage repair. The system is a nano-scale nucleic acid compound composed of a tetrahedral DNA frame structure and an FSH targeting peptide. The tetrahedral DNA frame structure is a microscopic 3D stereoscopic tetrahedral structure formed by self-assembly of DNA single chains of a specific nucleic acid sequence, wherein each side extends out of a viscous end used for being connected with an FSH peptide. The research finds that the DNA tetrahedron can safely and effectively improve the ovarian endocrine function, ovarian reserve, fertility and the like of mice in a drug protection group, has no obvious toxic or side effect on all organs and tissues of the whole body, and can promote the repair of ovarian oxidative damage.

Description

Technical Field

[0001] The present invention relates to the technical fields of biology and pharmacy, and particularly relates to an FSH33 peptide-modified DNA tetrahedron nano-delivery system, a preparation method thereof, and an application thereof. Background Art

[0002] Ovarian aging is a process in which the ovarian reserve function of women related to age gradually declines until it fails, and it ages abnormally early and rapidly compared to any other organ of the female body system. In addition, ovarian aging is the pacemaker of female body aging and the initiating factor for the aging of multiple organs. The available treatment options for ovarian aging include hormone replacement therapy, stem cell therapy, antioxidants, melatonin, growth hormone, and mitochondrial therapy, etc. However, the curative effect of current drug intervention treatment is still very limited. Therefore, it is of great scientific significance to develop corresponding preventive and treatment measures for the pathogenesis of ovarian aging.

[0003] Oxidation caused by an increase in the intracellular reactive oxygen species (ROS) level is a key factor leading to cell senescence and aging. Research shows that excessive ROS can damage mitochondrial DNA, trigger the "ROS cycle", damage follicular granulosa cells, affect follicle reserve, and reduce estrogen production, thereby destroying the growth potential of oocytes. In addition, excessive ROS leads to apoptosis, telomere damage, inflammation, etc. Clearing ROS has become an important direction for preventing and treating senescent cells.

[0004] DNA nanomaterials are nanomaterials formed by folding a long single-stranded DNA scaffold and hundreds of short strands of DNA complementary to it into a specific spatial structure. Due to its good biocompatibility (low toxicity, low immunogenicity), precise addressability, and stability advantages, it stands out among other traditional nanostructures. In addition, DNA tetrahedron nucleic acid (DTN) also has the characteristics of simple synthesis, low cost, and high yield, and can also realize the functional design of materials through various technical means such as designing the DNA sequence, assembly method, modification method, and coupling with other materials. DNA nanomaterials also have redox properties. Some studies have shown that DNA nanomaterials exhibit ROS and NO scavenging abilities. However, the traditional DNA tetrahedron nucleic acid has limited therapeutic effects on ovarian aging and damage, and it is difficult to efficiently target the oxidative stress ovarian tissue. Currently, there are no reports on drug research based on DNA tetrahedron nucleic acid targeting ovarian granulosa cells to reduce the intracellular ROS level, thereby achieving an anti-aging effect.

[0005] In summary, there is an urgent need to develop a drug with high biocompatibility, strong targeting ability, and excellent therapeutic effects on ovarian damage and ovarian aging. Summary of the Invention

[0006] The object of the present invention is to provide an FSH33 peptide-modified DNA tetrahedron nanodelivery system, its preparation method and application. This nanodelivery system can reduce oxidative stress in ovarian granulosa cells, thereby delaying ovarian injury and aging.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, there is provided an FSH33 peptide-modified DNA tetrahedron nanodelivery system, which is composed of a tetrahedral DNA framework nucleic acid (DTN) and an FSH33-53 peptide covalently linked by click chemistry; the sequence of the FSH33-53 peptide is shown as SEQ ID NO.5.

[0009] Furthermore, the molar ratio of the tetrahedral DNA framework nucleic acid (abbreviated as DTN) to the FSH33-53 peptide (abbreviated as FSH) is 1:4 to 200. Preferably, the molar ratio is 1:4 to 40.

[0010] Furthermore, the tetrahedral DNA framework structure serving as the backbone in the delivery system is composed of four DNA single strands with sequences as shown in SEQ ID NO.1 to 4; 20 T bases are designed at the 5'-end nucleic acid sequence of the DNA single strand as the sticky end for connecting the FSH33 peptide with 20 A base sticky ends; according to the base complementary pairing principle, the four DNA single strands are folded by a one-step annealing method to form a microscopic 3D tetrahedral framework structure, and the unbound nucleic acid sequences in the DNA single strand extend from the four sides of the tetrahedron as sticky ends for connecting the 20 A bases at the end of the FSH peptide.

[0011] In the second aspect of the present invention, there is provided a preparation method of an FSH33 peptide-modified DNA tetrahedron nanodelivery system, and the method includes:

[0012] The first amino acid tyrosine at the N-terminus of the FSH33 peptide with 20 A base sticky ends is modified with azidoacetic acid, and it is mixed and incubated with a dibenzocyclooctyne-modified 20 A base chain and reacted by click chemistry method to obtain the reacted intermediate;

[0013] The tetrahedral DNA framework nucleic acid is mixed with the intermediate, vortexed, and ultrafiltered to obtain the FSH33 peptide-modified DNA tetrahedron nanodelivery system.

[0014] Furthermore, the synthesis method of the tetrahedral DNA framework nucleic acid includes the following steps: adding four DNA single strands into TM buffer solution, maintaining at 95°C for 10 min, and maintaining at 4°C for more than 20 min to obtain it.

[0015] Furthermore, the four DNA single strands are four DNA single strands with an equimolar ratio.

[0016] Furthermore, the reaction conditions of the click chemistry method include: the temperature is 36°C - 38°C; the time is 3 - 6 h.

[0017] Furthermore, the ultrafiltration uses a 100 kDa molecular weight membrane.

[0018] Furthermore, the synthesis concentration of the DNA tetrahedron modified with the targeting peptide FSH33 is 1 - 100 μM. The subsequent application concentration is diluted before use. Since the inhibitor has a significant impact on cell activity as the concentration increases, 10 nM FSH-DTN is finally selected for subsequent experiments.

[0019] In the third aspect of the present invention, there is provided the use of the DNA tetrahedron nanodelivery system modified with the FSH33 peptide in the preparation of a drug for protecting ovarian function.

[0020] The protection of ovarian function includes delaying ovarian aging and / or repairing ovarian damage and / or improving ovarian reserve function.

[0021] As a specific embodiment, the protection of ovarian function is to repair ovarian damage.

[0022] As a specific embodiment, the ovarian aging is ovarian aging caused by oxidative stress.

[0023] As a specific embodiment, the ovarian damage is manifested as the accumulation of oxidative stress and inflammation.

[0024] As a specific embodiment, the application is: inhibiting the decline in the number of follicles during ovarian aging, and / or, inhibiting the decrease in the secretion of estrogen and progesterone, and / or, inhibiting the decline in fertility function.

[0025] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0026] 1. In the present invention, the improvement effect of FSH-DTN on the D-gal-induced chronic ovarian aging model is detected. Using an in vitro ovarian aging cell model, by detecting the levels of oxidative stress, inflammation, and cell aging in ovarian granulosa cells, it is found that FSH-DTN has a significant promoting effect on the proliferation of ovarian granulosa cells, can significantly reduce the ROS level in ovarian granulosa cells, and increase the expression of antioxidant-related proteins Nrf2, CAT, SOD2, and GPX1.

[0027] 2. The present invention uses a D-gal-induced ovarian senescence mouse model to explore the safety of FSH-DTN and its protective effect on ovarian senescence, including the detection of mouse health status, ovarian endocrine function, ovarian reserve, fertility, etc. FSH-DTN exhibits excellent in vivo antioxidant capacity; the estrous cycle is improved; the ovarian reserve function index E2 increases and FSH decreases; the number of primordial follicles increases and the number of atretic follicles decreases; the pregnancy rate increases and the litter size increases. FSH-DTN can significantly improve the reduction of ovarian reserve, endocrine, and fertility functions during ovarian senescence, and plays an important role in delaying ovarian senescence.

[0028] 3. The FSH33 peptide-modified DNA tetrahedron nanodelivery system of the present invention has good biosafety, strong targeting, and high bioavailability.

[0029] 4. The preparation method of the FSH33 peptide-modified DNA tetrahedron nanodelivery system of the present invention is simple, effective, economical, and applicable.

[0030] 5. The FSH33 peptide-modified DNA tetrahedron nanodelivery system of the present invention has broad application prospects in the preparation of drugs for protecting ovarian function, including delaying ovarian senescence and / or repairing ovarian damage and / or improving ovarian reserve function. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 It is a schematic diagram of the synthesis and characterization results of FSH-DTN; Figure 1 A is the schematic diagram of the synthesis; Figure 1 B is the result of high-performance liquid chromatography; Figure 1 C is the result of agarose gel electrophoresis detection. 1 represents the S1 DNA single strand, 2 represents the conjugate of the S1 DNA single strand and the S2 DNA single strand, 3 represents the conjugate of the S1 DNA single strand, the S2 DNA single strand, and the S3 DNA single strand, and DTN represents the tetrahedral framework nucleic acid; Figure 1 D is the agarose gel electrophoresis characterization diagram of DTN and FSH-DTN; Figure 1 E is the ultraviolet absorption spectrum of DTN and FSH-DTN. In the figure, "out" represents the liquid in the outer tube of the ultrafiltration tube after centrifugation when preparing TT. Figure 1 F is the transmission electron microscope picture; Figure 1 G is the atomic force microscope picture; Figure 1H is the test results of particle size and Zeta potential; Figure 1 I is the structural stability in different environments.

[0033] Figure 2 For the results of cell uptake and fluorescence distribution of FSH-DTN in the whole body: Among them, Figure 2 A is the image of the laser confocal microscope for cell uptake; Figure 2 B - C show that after intravenous injection of DTN and FSH-DTN, more FSH-DTN enters the ovary; Figure 2 D is the statistical comparison chart. Data are expressed as mean ± SD (n = 3). Student's t-test was used for statistical analysis. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001.

[0034] Figure 3 For the results of ROS labeled by DCFH-DA probe in cells and the CCK-8 detection results. Among them, Figure 3 A is the image of the fluorescence microscope; Figure 3 B is the fluorescence statistical comparison chart, Figure 3 C is the result of ROS flow cytometry detection, Figure 3 D is the CCK8 detection result. Data are expressed as mean ± SD (n = 3). Student's t-test was used for statistical analysis. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001.

[0035] Figure 4 For the improvement effect of FSH-DTN on the D-gal-induced mouse ovarian senescence model. Detection of mouse endocrine function; Among them, Figure 4 A is the proportion of regular and irregular estrous cycles in each group, Figure 4 B is the serum E2 level of mice in each group, Figure 4 C is the serum FSH level of mice in each group, Figure 4 D is the change of ovarian index in each group, Figure 4 E is the follicle counting results in each group. PMF: primordial follicle; PF: primary follicle; SF: secondary follicle; AF: antral follicle; ATF: atretic follicle.

[0036] Figure 5 For the results of antioxidant experiment in vivo of ovarian senescent mice. Among them, Figure 5 A is the detection of reactive oxygen species by DHE staining; Figure 5 B is the statistical result comparison chart of DHE staining experiment, Figure 5 C is the detection of protein expression levels of Nrf2, CAT, GPX1, and SOD2 by immunohistochemistry, Figure 5D is a comparative graph of the statistical results of immunohistochemistry experiments. The data are expressed as mean ± SD (n = 3). Student's t-test was used for statistical analysis. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001. Detailed implementation manners

[0037] The following will specifically elaborate on the present invention in combination with the detailed implementation manners and embodiments, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and embodiments are used to illustrate the present invention rather than limit the present invention.

[0038] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. In case of any contradiction, this specification shall prevail.

[0039] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or by existing methods.

[0040] Definition and use of terms

[0041] Ovarian senescence: In the present invention, it mainly refers to the process in which the ovarian function of women gradually declines with age, affected by multiple factors such as genetics, environment, and lifestyle. Based on the decrease in the number of follicles and the quality of eggs, it ultimately manifests as menopause and affects multiple organs throughout the body, leading to the occurrence of related diseases.

[0042] Ovarian function impairment: In the present invention, it mainly refers to the process in which, affected by multiple factors such as genetics, environment, and lifestyle, based on the decrease in the number of follicles and the quality of eggs, the endocrine function, ovarian reserve, and fertility of women's ovaries decline. Ultimately, it manifests as menopause and affects multiple organs throughout the body, leading to the occurrence of related diseases.

[0043] Improving or enhancing ovarian reserve function: The ovarian reserve function in the present invention refers to the ability of primordial follicles in the ovarian cortex to develop into fertilizable oocytes. Currently, the main indicators for clinically evaluating ovarian reserve function include age, basal endocrine hormones, cytokines, antral follicle count (AFC), etc.

[0044] D-gal: D-galactose is a hexosamine with the molecular formula C 6 H 12 O 6, with a relative molecular mass of 180.16, is widely used in biological experiments to create oxidative stress function damage models in various organs, such as liver injury, neurodegeneration, ovarian aging, etc.

[0045] The general idea of the present invention is as follows:

[0046] According to a typical embodiment of the present invention, a DNA tetrahedron nanodelivery system modified with FSH33 peptide is provided. The nanodelivery system is composed of a tetrahedral DNA framework nucleic acid (DTN) and an FSH33-53 peptide covalently linked by click chemistry. The DNA tetrahedron nanodelivery system modified with FSH33 peptide is abbreviated as FSH-DTN; the amino acid sequence of the FSH33-53 peptide is shown in SEQ ID NO.5.

[0047] Furthermore, the tetrahedral DNA framework structure serving as the backbone in the delivery system is composed of four DNA single strands with sequences shown in SEQ ID NO.1-4; 20 T bases are designed at the 5'-end nucleic acid sequence of the DNA single strand as the sticky end for connecting the FSH33 peptide with 20 A base sticky ends; the four DNA single strands are folded into a microscopic 3D tetrahedral framework structure by one-step annealing according to the base complementary pairing principle. The unbound nucleic acid sequences in the DNA single strand extend from the four sides of the tetrahedron as sticky ends for connecting the 20 A bases at the end of the FSH peptide.

[0048]

[0049] According to another typical embodiment of the present invention, a preparation method of a DNA tetrahedron nanodelivery system modified with FSH33 peptide is provided. The method includes:

[0050] Step S1: Modify the first amino acid tyrosine at the N-terminus of the FSH33 peptide with 20 A base sticky ends by azidoacetic acid, and mix and incubate it with a 20 A base chain modified with dibenzocyclooctyne, and react by click chemistry method to obtain the reacted intermediate;

[0051] In the step S1,

[0052] The molar ratio of the 20 A base chain modified with dibenzocyclooctyne to the FSH peptide is 1:2.

[0053] The reaction conditions of the click chemistry method include: the temperature is 36°C - 38°C; the time is 3 - 6 h.

[0054] Step S2: Mix the tetrahedral DNA framework nucleic acid with the intermediate, vortex, and ultrafilter to obtain the DNA tetrahedron nanodelivery system modified with FSH33 peptide;

[0055] In the step S2,

[0056] The preparation method of the tetrahedral DNA framework nucleic acid includes:

[0057] Adding four DNA single strands into TM buffer, maintaining at 95 °C for 10 min, and maintaining at 4 °C for more than 20 min to obtain the product.

[0058] Furthermore, the final concentration of each of the four DNA single strands is 1 μM.

[0059] Furthermore, the molar ratio of the tetrahedral DNA framework nucleic acid (DTN) to the FSH33-53 peptide is 1:4 to 200.

[0060] Preferably, through experiments in the embodiments of the present invention, it is found that when the molar ratio of DTN to the FSH33-53 peptide is 1:4, the ligation efficiency between the two is the highest.

[0061] Through the above steps, an FSH33 peptide-modified DNA tetrahedron nanodelivery system can be successfully constructed. The FSH33 peptide-modified DNA tetrahedron nanodelivery system has the application prospect of preparing drugs for protecting ovarian function.

[0062] Next, the present application will be described in detail in combination with examples and experimental data.

[0063] Example 1, FSH33 peptide-modified DNA tetrahedron nanodelivery system and its preparation method

[0064] I. Preparation method of FSH33 peptide-modified DNA tetrahedron nanodelivery system

[0065] 1. Dissolve four DNA single strands (S1, S2, S3, S4) in TM buffer (10 mM Tris-HCl, 50 mM MgCl, pH = 8.0), control the final concentration of the four DNA single strands to be 1 μM, mix well, and then quickly heat to 95 °C and keep for 10 minutes, and then quickly cool to 4 °C and maintain for 20 minutes to self-assemble into a tetrahedral framework nucleic acid DTN with a final concentration of 1 μM (confirmed by AGE electrophoresis and HPLC, Figure 1 B and Figure 1 C).

[0066] 2. A DNA strand modified with DBCO (dibenzocyclooctyne) and 20 consecutive A bases can react with an FSH33-53 peptide modified with azide at the N-terminus through a strain-promoted 1,3-dipolar cycloaddition reaction in aqueous solution (the molar ratio of DBCO-A20 to the FSH peptide is 1:2). After reaction and oscillation, react at 37 °C for 3 h in a thermal cycler through this copper-free click reaction to generate an intermediate FSH-A20.

[0067] 3. The complex obtained by co-incubating 1 μM DTN solution with FSH-A20 was shaken at room temperature for 5 min and ultrafiltered to obtain the tetrahedral framework nucleic acid complex FSH-DTN modified with FSH peptide. The molar ratio of the tetrahedral DNA framework nucleic acid (abbreviated as DTN) to the FSH33-53 peptide (abbreviated as FSH) was 1:4.

[0068] II. Characterization of the DNA tetrahedron nanodelivery system modified with FSH33 peptide

[0069] For the synthesized FSH-DTN, atomic force microscopy, transmission electron microscopy and particle size ( Figure 1 D, E, F) showed that its size was about 32.2 nm. The hydrated particle size of FSH-DTN increased slightly compared with that of FSH. It can be considered that FSH-DTN was successfully synthesized, and the Zeta potential was about -15.2 mV. From the results of the Zeta potential of DTN and FSH-DTN, it can be seen that both DTN and FSH-DTN had good dispersibility and stability in aqueous solution.

[0070] The structural stability of FSH-DTN in the biomimetic fluid was detected by AGE ( Figure 1 G). The structural stability within 24 hours was tested at 37 °C in PBS and 10% fetal bovine serum (FBS) respectively. FSH-DTN could maintain its structural stability within 24 hours in PBS at 37 °C and within 12 hours in 10% fetal bovine serum (FBS) at 37 °C.

[0071] Example 2

[0072] In this example, the molar ratio of the tetrahedral DNA framework nucleic acid to the FSH33-53 peptide was 1:40, and the other steps were the same as in Example 1.

[0073] Example 3

[0074] In this example, the molar ratio of the tetrahedral DNA framework nucleic acid to the FSH33-53 peptide was 1:200, and the other steps were the same as in Example 1.

[0075] Experimental Example 1. Detection of the ligation rate of FSH33 peptide at different DTN:FSH ratios

[0076] The ligation efficiency of FSH33 peptide and DTN at different ratios in Example 1, Example 2 and Example 3 was examined by AGE electrophoresis ( Figure 1 C) and ultraviolet spectrophotometer ( Figure 1 E). The ultraviolet spectra of DTN, FSH33 peptide, FSH-DTN, and the liquid outside the ultrafiltration tube after centrifugation during the preparation of FSH-DTN (FSH-DTN out) were measured by ultraviolet spectrophotometer to determine whether TPP was successfully ligated.

[0077] From Figure 1 the results of E ultraviolet spectrophotometry, it can be seen that under the condition of equal amounts of tFNAs, the peak of FSH-DTN shifts to the left, and the absorption peak at 260 nm is higher than that of DTN.

[0078] In Example 1, when the molar ratio is 1:4 (DTN:FSH), the absorption peak at 260 nm increases significantly and the peak shifts to the left, indicating that the FSH33 peptide is successfully linked to DTN, and the ligation efficiency is the highest at this time.

[0079] In Example 2, when the molar ratio is 1:40 (DTN:FSH), the absorption peak at 260 nm is still higher than that of pure DTN but lower than that at the 1:4 ratio, and the peak shifts to the left, suggesting that partial ligation is successful, but the efficiency decreases with the excess of FSH (possibly due to steric hindrance or reaction competition).

[0080] In Example 3, when the molar ratio is 1:200 (DTN:FSH), the absorption peak at 260 nm is lower than that of pure DTN and shifts to the left, indicating that the ligation may not be successful (excess FSH interferes with the reaction to form a precipitate).

[0081] Experimental Example 2: Improvement effect of FSH-DTN on Dgal-induced in vitro ovarian senescence model

[0082] Research shows that D-galactose (D-gal) can create an accelerated aging model in vitro and in vivo. The main mechanism is to increase the level of intracellular reactive oxygen species, which further causes oxidative stress, inflammatory response, mitochondrial dysfunction and apoptosis. Based on the literature, in the following, a 200 mM concentration of D-gal was used to intervene for 48 hours to construct an ovarian senescence granulosa cell model.

[0083] 1. Cell uptake experiment

[0084] Using Cy3-labeled S4, Cy3-DTN and Cy3-FSH-DTN were synthesized according to Example 1. The COV cell suspension was inoculated in a confocal dish, pre-cultured in an incubator for 24 h, and then Cy3-labeled DTN and FSH-DTN (concentration 100 nM) were added. After culturing in the incubator for 1.5, 6, 12, and 24 h, the culture medium was aspirated, and the cells were washed three times with PBS for 5 min each time; then fixed with 4% paraformaldehyde for 10 min, the paraformaldehyde was aspirated, and the cells were washed three times with PBS for 5 min each time; then treated with 33258 for 10 min in the dark, the 33258 was aspirated, and the cells were washed three times with PBS for 5 min each time; then mounted with 10% glycerol, protected from light, and stored at 4 °C. The distribution of Cy5 fluorescence signal in the cells was observed using a confocal microscope (FV3000, Olympus, Japan).

[0085] From the images of the laser confocal microscope ( Figure 2 A), it can be observed that Cy5-labeled FSH-DTN is widely distributed in the cytoplasm of cells, and FSH-DTN has stronger targeting ability.

[0086] 2. ROS and CCK-8 experiments

[0087] The results are as Figure 3 shown in A. D-gal-induced oxidative stress in the COV cells of the model group generated a large amount of ROS, and the ROS levels in the DTN and FSH-DTN treatment groups decreased.

[0088] Further, according to Figure 3 the fluorescence images in A, the relative ratios of the fluorescence intensities of the ROS levels in each treatment group were statistically analyzed. Each treatment group was statistically analyzed 3 times, and the average value was taken. The results are as Figure 3 shown in B. And the average fluorescence intensity was statistically analyzed by flow cytometry. The results are as Figure 3 shown in C.

[0089] It can be seen from Figure 3 B and Figure 3 C that compared with the D-gal group, the ROS levels in the DTN and FSH-DTN treatment groups decreased, and the ROS level in the FSH-DTN treatment group decreased more significantly, indicating that FSH-DTN has a better antioxidant effect.

[0090] Figure 3 It can be seen from the CCK-8 results in D that high concentrations of DTN and FSH-DTN significantly inhibited cell viability. Since the inhibitor had a significant effect on cell viability as the concentration increased, the application concentration of FSH-DTN in cells was finally selected as 10 nM for subsequent experiments.

[0091] Experimental Example 3: Improvement effect of FSH-DTN on D-gal-induced in vivo ovarian aging model

[0092] 1. In vivo distribution effect test

[0093] An oxidative stress-mediated ovarian aging model was established by subcutaneous injection of D-gal for 42 consecutive days. During the administration of D-gal, DTN (5 nmol / kg) and FSH-DTN (5 nmol / kg) were injected into the tail vein every other day until the end of D-gal administration. On the day of administration, D-gal was subcutaneously injected 1 hour after the tail vein injection of DTN and FSH-DTN.

[0094] As Figure 2As shown in Figure B, the targeting of FSH-DTN in a mouse model was verified by an in vivo fluorescence imaging system (IVIS). Cy5-labeled FSH-DTN (Cy5-FSH-DTN) and Cy5-labeled DTN (Cy5-DTN) synthesized according to Example 1 were administered to normal mice via the tail vein. At 0.5, 1, 3, 6, and 12 h respectively, mouse organs (heart, liver, spleen, lung, kidney) and uterus and ovaries were isolated, and their fluorescence distribution was observed. In mice, the fluorescence of Cy5-FSH-DTN and Cy5-DTN was mainly distributed in the liver and kidneys, and the fluorescence distribution of Cy5-FSH-DTN in the ovaries was significantly higher than that of Cy5-DTN, which fully demonstrated its targeting to the ovaries.

[0095] As Figure 2 As shown in C-D, after intravenous injection of DTN and FSH-DTN, compared with DTN, more FSH-DTN of the present invention entered the ovaries.

[0096] 2. Detection of endocrine function in mice

[0097] Through a 14-day monitoring of the estrous cycle, it was found that compared with the control group, the proportion of regular estrous cycles in the D-gal group was significantly reduced, and the proportion of regular estrous cycles in the DTN and FSH-DTN groups was significantly increased compared with the D-gal group ( Figure 4 A).

[0098] Hormone level detection found that: compared with the control group, the FSH level in the D-gal group was significantly increased, and the E2 level had no significant change; while compared with the D-gal group, the FSH level in the intervention group was reduced and the E2 level was increased; the FSH level in the FSH-DTN group was significantly reduced compared with the D-gal group, and the E2 levels were all significantly increased ( Figure 4 B, 4C).

[0099] The ovarian indices of mice in the DTN and FSH-DTN groups were significantly higher than those in the D-gal group ( Figure 4 D).

[0100] The follicle counting results showed that compared with the control group, the proportion of atretic follicles in the D-gal group was significantly increased. The proportion of atretic follicles in the DTN and FSH-DTN groups was reduced compared with the D-gal group, but there was no statistical trend ( Figure 4 E). It indicates that FSH-DTN can improve the ovarian function decline caused by D-gal.

[0101] 3. Evaluation of ROS and antioxidant-related proteins in mouse ovaries

[0102] Ovarian tissues were collected, and DHE staining, immunohistochemical staining (IHC), and qPCR were used to detect the changes in antioxidant-related proteins to evaluate the antioxidant ability of the drug. DHE staining ( Figure 5A) shows that the oxidative stress level of D-gal is significantly enhanced, while the oxidative stress levels in the DTN and FSH-DTN treatment groups are reduced.

[0103] The IHC results ( Figure 5 C) show that the basic expression levels of CAT, SOD2, GPX1, and Nrf2 are significantly decreased in the D-gal group and significantly increased in the DTN and FSH-DTN treatment groups.

[0104] Further, Figure 5 A and Figure 5 C are statistically analyzed. Each treatment group is statistically analyzed 3 times, and the average value is taken. The results are as Figure 5 B and Figure 5 shown in D, indicating that FSH-DTN has a better effect on the recovery of antioxidant function.

[0105] In summary, the FSH-DTN constructed based on tetrahedral framework nucleic acid in the present invention can target ovarian granulosa cells, reduce intracellular oxidative stress, improve the mitochondrial membrane potential of cells, alleviate cell senescence, deliver drugs to the ovary, improve the uptake efficiency of the ovary for tetrahedral framework nucleic acid, relieve the decline of ovarian endocrine function caused by D-gal, improve ovarian reserve in mice, enhance the therapeutic effect on ovarian injury and ovarian senescence, and is superior to tetrahedral framework nucleic acid in the treatment of ovarian injury and senescence, having practical popularization and application value.

[0106] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.

[0107] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0108] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A FSH33 peptide-modified DNA tetrahedral nano-delivery system, characterized in that: The nano-delivery system is composed of a tetrahedral DNA framework nucleic acid (DTN) and a FSH33-53 peptide covalently linked by click chemistry; the amino acid sequence of the FSH33-53 peptide is shown in SEQ ID NO.

5.

2. A FSH33 peptide-modified DNA tetrahedral nano-delivery system according to claim 1, characterized in that: The molar ratio of the tetrahedral DNA framework nucleic acid to the FSH33-53 peptide is 1:4-200.

3. A FSH33 peptide-modified DNA tetrahedral nano-delivery system according to claim 1, characterized in that: In the structure of FSH-DTN, the tetrahedral DNA framework nucleic acid is composed of four DNA single strands with sequences as shown in SEQ ID NO.1-4; the 5' end nucleic acid sequence of each DNA single strand is designed with 20 T bases as 20 A base sticky ends for connecting FSH33 peptide.

4. A method for preparing a FSH33 peptide-modified DNA tetrahedral nano-delivery system, characterized in that: The method comprises: The first amino acid tyrosine at the N-terminus of the FSH33 peptide with 20 A base sticky ends was modified with azidoacetic acid, and then mixed and incubated with a 20 A base chain modified with dibenzocyclooctyne and reacted by click chemistry to obtain a reaction intermediate. The tetrahedral DNA framework nucleic acid and the intermediate are mixed and vortexed, and ultrafiltered to obtain the FSH33 peptide-modified DNA tetrahedral nano delivery system.

5. The preparation method according to claim 4, characterized in that: The reaction conditions of the click chemistry method include: temperature of 36° C.-38° C.; reaction time of 3 to 6 hours.

6. The preparation method according to claim 4, characterized in that: The method for synthesizing the tetrahedral DNA framework nucleic acid comprises the following steps: adding four DNA single strands into TM buffer, maintaining at 95° C. for 10 minutes, and maintaining at 4° C. for more than 20 minutes, to obtain the tetrahedral DNA framework nucleic acid.

7. Use of the FSH33 peptide-modified DNA tetrahedron nano-delivery system according to any one of claims 1-2 in the preparation of a drug for protecting ovarian function.

8. The use according to claim 7, characterized in that: The ovarian function protection includes delaying ovarian aging and / or repairing ovarian damage and / or improving ovarian reserve function.

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