Composition for improving vaginal dryness and relaxation and preparation method thereof
By using the synergistic network design of the RGD-Collagen-FGF2 fusion protein, the problem of the inability of existing technologies to simultaneously achieve localized retention and epithelial regeneration in the vagina was solved, thus achieving pathological reversal of vaginal dryness symptoms and enhancing the vagina's moisturizing ability and barrier function.
Patent Information
- Application Number
- CN202511474060.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-15
- Publication Date
- 2026-01-09
AI Technical Summary
Current treatment options cannot address the pathological mechanisms and simultaneously achieve localized retention in the vagina, ECM scaffold reconstruction, and epithelial cell regeneration. As a result, vaginal dryness symptoms cannot be fundamentally resolved, and there are issues such as hormone-dependent side effects or a lack of targeted retention capabilities.
The RGD-Collagen-FGF2 fusion protein was designed to achieve localization and retention through integrin adhesion. Collagen constructs the ECM scaffold and regulates metabolic balance, while FGF2 activates cell proliferation and mucin synthesis to achieve epithelial regeneration, forming a synergistic network of retention-repair-regeneration, reversing vaginal mucosal atrophy and loss of moisturizing ability.
It significantly increases vaginal epithelial thickness, promotes collagen fiber synthesis and mucin expression, increases ECM water content, and enhances vaginal barrier function, thus improving vaginal dryness symptoms from a pathological mechanism perspective, with better effects than traditional hormone drugs.
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Figure CN121287883A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and more specifically, to a composition for improving vaginal dryness and laxity and its preparation method. Background Technology
[0002] Vaginal dryness is a common symptom in gynecological clinics, frequently occurring in perimenopausal women, patients after bilateral oophorectomy, and individuals with low estrogen levels, with an incidence rate as high as 30%-50%. Its core pathological mechanism involves estrogen deficiency leading to atrophy and thinning of the vaginal mucosal epithelium, degradation of extracellular matrix (ECM) collagen fibers, reduced synthesis of mucins (MUC1 / MUC5AC), and decreased ECM water content, thereby causing symptoms such as dryness, burning pain, and difficulty in sexual intercourse, severely impacting patients' physiological health and quality of life. Current treatments primarily focus on symptomatic relief: hormonal medications such as estriol cream are commonly used clinically. While they can improve dryness in the short term, they have side effects such as hormone dependence, breast tenderness, and endometrial hyperplasia. Furthermore, they only relieve symptoms and cannot reverse the pathological processes of mucosal atrophy and ECM damage. Non-hormonal moisturizers lack targeted retention capabilities, making it difficult to achieve long-term effects locally in the vagina. They also cannot simultaneously achieve ECM repair and epithelial regeneration, resulting in short-lived and limited treatment effects. Currently, there is no treatment that can address the pathological mechanism and simultaneously achieve multi-stage synergistic treatment involving "local vaginal fixation - ECM scaffold reconstruction - epithelial cell regeneration." This approach cannot fundamentally solve the problems of vaginal dryness, such as mucosal atrophy, loss of moisturizing ability, and weakened barrier function. Therefore, there is an urgent need to develop a safe, efficient, and novel biological agent that can improve vaginal dryness from a pathological perspective. Summary of the Invention
[0003] This invention addresses the progressive pathological characteristics of vaginal dryness, namely "mucosal atrophy - ECM damage - barrier failure," and overcomes the limitations of existing technologies that "intervene in a single step and cannot achieve synergistic effects." It designs and constructs an RGD-Collagen-FGF2 fusion protein with a triple synergistic function of "positioning retention - scaffold repair - epithelial regeneration," reversing dryness symptoms from the root cause of the pathology.
[0004] To address the above problems, the present invention first provides a composition for improving vaginal dryness and laxity; In some embodiments, the composition comprises an RGD-Collagen-FGF2 fusion protein, the fusion protein comprising an RGD element, a Collagen element, an FGF2 element, and a linker.
[0005] In some embodiments, the RGD element is an RGD repeating structural unit with the amino acid sequence shown in SEQ ID NO:1; the Collagen element is an active fragment of human type III collagen with the amino acid sequence shown in SEQ ID NO:2; the FGF2 element is an active fragment of FGF2 with the amino acid sequence shown in SEQ ID NO:3; and the linker is... .
[0006] In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO:4.
[0007] The present invention also provides a preparation method, which includes the following steps: (1) designing and synthesizing a fusion gene sequence (5'-RGD-(Gly4Ser)3-Collagen-(Gly4Ser)3-FGF2-3') containing a (Gly4Ser)3 linker based on the codon preference of Escherichia coli; (2) digesting the synthesized gene with the pET-28a (+) vector using restriction endonucleases NdeI and XhoI, reacting at 37℃ for 2h, and then recovering the target fragment and the vector backbone; (3) ligating with T4 DNA ligase at 16℃ for 12h to construct the recombinant expression vector pET-28a-RGD-Collagen-FGF2, and verifying by sequencing; (4) transforming the recombinant vector into BL21 (DE3) competent cells, inducing at 37℃ for 4h with a final IPTG concentration of 0.5 mmol / L, sonicating and purifying by Ni-NTA affinity chromatography, and dialysis with a 10kDa dialysis bag to obtain the pure product.
[0008] Finally, the present invention provides an application of the above-described composition or fusion protein in the preparation of products for treating vaginal dryness and laxity.
[0009] Compared with the prior art, the present invention has at least the following beneficial effects: This invention is the first to propose that the design principle of the RGD-Collagen-FGF2 fusion protein lies in the synergistic effect of the three components, rather than their independent functions. They primarily form a synergistic network of "retention-repair-regeneration." Specifically, the RGD peptide achieves "localization and retention" through integrin adhesion, providing the basis for the action of Collagen and FGF2. Collagen constructs the ECM scaffold and regulates metabolic balance, providing the "soil" for FGF2-mediated cell proliferation. FGF2 activates cell proliferation and mucin synthesis, achieving "epithelial regeneration" on the Collagen scaffold, while further promoting ECM synthesis. Ultimately, through multi-stage synergy, it reverses vaginal mucosal atrophy (epithelial thickening, ECM reconstruction), restores moisturizing ability (increased mucin synthesis, increased ECM water content), and enhances barrier function, treating vaginal dryness from a pathological mechanism perspective, rather than merely providing symptomatic relief.
[0010] In summary, this invention achieves a breakthrough in the treatment of vaginal dryness from "symptomatic relief" to "pathological reversal" through the complete logic of "component co-design - preparation to ensure activity - application for precise efficacy," providing a novel biological agent that conforms to the pathological mechanism for the clinical treatment of this condition. Attached Figure Description
[0011] Figure 1 SDS-PAGE image of RGD-Collagen-FGF2 fusion protein.
[0012] Figure 2 The results of HE staining of mouse vaginal tissue to detect epithelial thickness are shown in the figure. The horizontal axis represents the experimental groups (blank group, model group, positive group, low / medium / high dose group), and the vertical axis represents the epithelial thickness (μm).
[0013] Figure 3 The graph shows the results of Masson staining of mouse vaginal tissue to detect the proportion of collagen fiber area. The horizontal axis represents the experimental group, and the vertical axis represents the percentage of collagen fiber area in the lamina propria area (%).
[0014] Figure 4 The results of immunohistochemical detection of mucin (MUC1 / MUC5AC) expression in mouse vaginal tissue are shown in the figure. The horizontal axis represents the experimental group, and the vertical axis represents the average optical density value (IOD / Area).
[0015] Figure 5 The graph shows the calculated ECM water content of mouse vaginal tissue. The horizontal axis represents the experimental group, and the vertical axis represents the ECM water content (%).
[0016] Figure 6 The graph shows the results of mouse vaginal epithelial permeability detection (fluorescein sodium method). The horizontal axis represents the experimental group, and the vertical axis represents the fluorescence intensity (FI). Detailed Implementation
[0017] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.
[0018] Example 1: Preparation of RGD-Collagen-FGF2 fusion protein The RGD element is selected from the repeating structural unit of RGD, and its amino acid sequence is as follows: RGDRGDRGDRGDRGD (SEQ ID NO:1); The Collagen element is selected from the active fragment of human type III collagen, and its amino acid sequence is as follows: GAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGAN (SEQ ID NO:2). The FGF element is selected from the active fragment of EGF2, and its amino acid sequence is as follows: PALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS (SEQ ID NO: 3); Full length of the RGD-Collagen-FGF2 fusion protein: RGDRGDRGDRGDRGDGGGGSGGGGGSGGGGSGDGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGGGGSGGGGSGGGGSPALPEDGG SGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS (SEQ ID NO:4) Using pET-28a (+) as the vector and Escherichia coli BL21 (DE3) as the host bacterium, a design containing pET-28a (+) was created based on codon preference. The fusion gene sequence of the linker: 5'-RGD- -Collagen- The full-length gene of -FGF2-3' was synthesized by Nanjing Genscript Biotech Co., Ltd. The synthesized gene was digested with restriction endonucleases NdeⅠ and XhoⅠ and incubated with the pET-28a (+) vector at 37℃ for 2 h. The target fragment and vector backbone were recovered by agarose gel electrophoresis. Ligation was performed using T4 DNA ligase at 16℃ for 12 h to construct the recombinant expression vector pET-28a-RGD-Collagen-FGF2. Sequencing was used to verify the sequence correctness.
[0019] Mix 10 μL of the recombinant vector with 100 μL of BL21 (DE3) competent cells, incubate at ice for 30 min, heat shock at 42 °C for 90 s, ice bath for 2 min, add 800 μL of LB medium, and culture with shaking at 37 °C for 1 h. Take 200 μL of the bacterial solution and spread it on an LB plate containing kanamycin (50 μg / mL), culture at 37 °C for 12 h, pick a single colony and inoculate it into 5 mL of LB medium, and culture with shaking at 37 °C until = 0.6 - 0.8. Add IPTG to a final concentration of 0.5 mmol / L, induce at 37 °C for 4 h, and collect the bacterial solution. Centrifuge the induced bacterial solution at 4 °C and 8000 r / min for 10 min, discard the supernatant, resuspend the cells with PBS, ultrasonically disrupt (power 300 W, working for 3 s, interval 5 s, for a total of 30 min), centrifuge at 4 °C and 12000 r / min for 20 min, collect the supernatant, load the supernatant onto a Ni-NTA affinity chromatography column, equilibrate the column with PBS containing 20 mmol / L imidazole, and then elute the target protein with PBS containing 200 mmol / L imidazole, collect the elution peak; Dialyze the eluate with a dialysis bag with a molecular weight cut-off of 10 kDa in PBS at 4 °C for 24 h, change the solution 3 times, obtain the purified fusion protein, and freeze-dry it for subsequent implementation. At the same time, detect by SDS-PAGE, see Figure 1 .
[0020] Figure 1 The results showed that a target band corresponding to the molecular weight of the fusion protein 27.75 kDa calculated theoretically appeared in the SDS-PAGE electrophoresis, and a highly pure RGD-Collagen-FGF2 fusion protein was obtained.
[0021] Example 2 Application of RGD-Collagen-FGF2 fusion protein in improving vaginal dryness Forty-eight SPF-grade female BALB / c mice, 6 - 8 weeks old, weighing 20 - 22 g, breeding environment: temperature 22 - 25 °C, humidity 50 - 60%, 12 h light-dark cycle, free access to food and water; RGD-Collagen-FGF2 fusion protein: prepared according to the method of Example 1, purity ≥ 95%, diluted with sterile PBS to three concentrations of 0.1 mg / mL, 0.5 mg / mL, and 1.0 mg / mL; Positive control drug: estriol cream (purchased from Jinlvjiayuan of Nanjing Tongrentang, batch number: (Yu) Weixiaozhengzi (2021) No. 0112); Construction of vaginal dryness model, randomly divide 48 mice into 6 groups (n = 8 / group): Blank control group (blank group): Only expose the ovaries by laparotomy, do not excise, suture layer by layer; Model control group (model group): Bilateral oophorectomy (oophorectomy) was performed to establish an estrogen deficiency-induced vaginal dryness model; Positive control group (positive group): After ovariectomy, patients were given Honglilai conjugated estrogen cream, specification 1g:0.625mg*4g*1 tube / box, purchased from Xinjiang Xinzhiyuan Biopharmaceutical Co., Ltd. Low-dose fusion protein group (low-dose group): 0.1 mg / mL RGD-Collagen-FGF2 fusion protein was administered after ovariectomy; Medium-dose fusion protein group: 0.5 mg / mL RGD-Collagen-FGF2 fusion protein was administered after ovariectomy; High-dose fusion protein group (high-dose group): 1.0 mg / mL RGD-Collagen-FGF2 fusion protein was administered after ovariectomy.
[0022] Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), and a midline incision (about 1 cm) was made in the lower abdomen. The bilateral ovaries and fallopian tubes were separated, the fallopian tubes were ligated, and the ovaries were removed. In the blank control group, only the ovaries were separated and returned to the peritoneum. After the operation, penicillin sodium (100,000 U / kg) was injected intramuscularly to prevent infection. The mice were fed in a routine manner for 1 week, and the serum estradiol (E2) level was detected by ELISA (the model group was considered to have successfully established the model if E2 < 10 pg / mL).
[0023] Once the model is successfully validated, drug administration will begin, with all medications administered vaginally. Positive control group (positive group) administration regimen: ① Take a sterile centrifuge tube and add 100 mg of Honglilai cream (accurately weighed using a sterile spatula); ② Add 1150 μL of sterile PBS (at a ratio of 1:11.5, i.e., 1 part cream + 11.5 parts diluent, total dilution factor 12.5 times); ③ Vortex for 30 seconds, let stand for 5 minutes, and then vortex again for 20 seconds to ensure complete dissolution of the cream and formation of a homogeneous suspension; ④ Concentration verification after dilution: The concentration was detected by high performance liquid chromatography (HPLC) and found to be 50 μg / g (±2 μg / g), which meets the target concentration requirement; Take 50 μL (mass ≈ 50 mg) of the diluted cream with a concentration of 50 μg / g each time to ensure that the dosage for each mouse is: conjugated estrogen dose = 50 mg × 50 μg / g = 2.5 μg, administered once daily for 21 consecutive days.
[0024] Model control group: An equal volume (50 μL) of sterile PBS (the same diluent as the positive control group to exclude solvent interference) was slowly injected into the vagina of mice once a day for 21 consecutive days. Fusion protein dosage groups: 50 μL of RGD-Collagen-FGF2 fusion protein solutions (corresponding to doses of 5 μg / mouse, 25 μg / mouse, and 50 μg / mouse) were prepared using sterile PBS solution and slowly injected into the vagina of mice once daily for 21 consecutive days.
[0025] Use a sterile pipette to draw 50 μL of drug / control solution and slowly inject it into the vagina of mice. During the administration period, observe the mice's activity, diet, weight, and whether there is redness, swelling, or abnormal discharge in the vagina.
[0026] Twenty-four hours after the last administration, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), blood was collected from the eyeballs, and the mice were euthanized. The intact vaginal tissue was quickly separated: the upper 1 / 3 of the vaginal tissue was taken, fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin, and used for HE staining, Masson staining, and immunohistochemical detection; the middle 1 / 3 of the vaginal tissue was taken and frozen at -80℃ for Western blot detection; the lower 1 / 3 of the vaginal tissue was taken, weighed immediately (recorded as wet weight W1), and then dried in a 60℃ oven to constant weight (recorded as dry weight W2) for ECM water content calculation.
[0027] SPSS 26.0 software was used for statistical analysis. Quantitative data are expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons among multiple groups, and LSD-t test was used for pairwise comparisons between groups. P < 0.05 was considered statistically significant.
[0028] The following is a histopathological examination of the vaginal tissue: HE staining: Paraffin sections were dewaxed to water, stained with hematoxylin for 5 min, differentiated with hydrochloric acid alcohol for 30 s, stained with eosin for 3 min, dehydrated and cleared, and then mounted. Under an inverted microscope (200× field of view), 5 sections were randomly selected from each group, and 3 fields of view were selected from each section. The vaginal epithelial thickness (μm) was measured using Image-Pro Plus 6.0 software, and the average value was calculated. See [link to image]. Figure 2 . Figure 2 The results showed that the epithelial thickness in the model control group was significantly lower than that in the blank control group (P < 0.05), indicating that the model was successfully constructed. Meanwhile, the epithelial thickness in the low, medium and high dose groups of the fusion protein was significantly higher than that in the model control group (P < 0.05), and there was no significant difference from the blank control group. The effect of the RGD-Collagen-FGF2 fusion protein on promoting epithelial thickness showed a dose-dependent effect.
[0029] Masson staining: Paraffin sections were dewaxed to water, stained with iron hematoxylin for 10 min, stained with Ponceau S and Acid Fuchsin for 5 min, treated with phosphomolybdic acid solution for 5 min, stained with aniline blue for 5 min, dehydrated and cleared, and then mounted. At 200× field of view, 5 sections were randomly selected from each group, and 3 fields of view were selected from each section. Image-Pro Plus 6.0 software was used to calculate the percentage of collagen fiber area to the lamina propria area (reflecting ECM reconstruction). See [link to image]. Figure 3 .
[0030] Figure 3 The results showed that the proportion of collagen fiber area in the model control group was significantly lower than that in the blank control group (P<0.05), indicating that the model was successfully constructed. The proportion of collagen fiber area in the low, medium and high dose fusion protein groups was significantly higher than that in the model control group (P<0.01), and the recovery effect of collagen fiber area in the low, medium and high dose fusion protein groups was higher than that in the positive control group.
[0031] Vaginal moisturizing ability test: Mucin expression detection (immunohistochemistry): Paraffin sections were dewaxed to water, 3% Endogenous peroxidase was blocked by incubation at room temperature for 10 min, followed by antigen retrieval (citrate buffer, heated at 95°C for 20 min), and blocking with 5% BSA for 30 min. MUC1 primary antibody (Thermo Fisher Scientific, MA5-14077) and MUC5AC primary antibody (Thermo Fisher Scientific, MA5-12178) were added, and incubation was performed overnight at 4°C. Secondary antibody Goat Anti-Mouse IgG-HRP (Sigma-Aldrich, A4416) was added and incubated at room temperature for 1 h. DAB staining was performed, followed by hematoxylin counterstaining and mounting. At 200× field of view, five slides were randomly selected from each group, and three fields of view were selected from each slide. The average optical density (IOD / Area, reflecting the amount of mucin synthesis) was calculated using Image-Pro Plus 6.0 software. See [link to image]. Figure 4 ; Figure 4 The results showed that the amount of mucin synthesis in the model control group was significantly lower than that in the blank control group (P<0.05), indicating that the model was successfully constructed. Meanwhile, the amount of mucin synthesis in the high-dose fusion protein group was significantly higher than that in the model control group (P<0.01), and there was no significant difference from the blank control group. The RGD-Collagen-FGF2 fusion protein had a better effect on promoting mucin synthesis than the positive control group.
[0032] ECM moisture content calculation: Calculated using the formula "Moisture content (%) = (W1 - W2) / W1 × 100%". For each group of 8 mice, the average value was taken. See below. Figure 5 .
[0033] Figure 5 The results showed that the ECM water content in the model control group was significantly lower than that in the blank control group (P<0.05), indicating that the model was successfully constructed. The ECM water content in the low, medium and high dose groups of the positive control group of fusion protein (P<0.05) was significantly increased, and the ECM water content in the medium and high dose groups of fusion protein was close to that in the blank control group.
[0034] Vaginal mucosal barrier function test: Vaginal epithelial permeability assay (fluorescein sodium method): One hour before the last administration, mice were injected intravenously with 0.1% fluorescein sodium solution (100 μL / mouse). One hour later, the mice were sacrificed, and vaginal tissue was collected. 1 mL of PBS was added to homogenate the tissue, and the mixture was centrifuged at 12000 r / min for 20 min at 4°C. The supernatant was collected, and the fluorescence intensity (FI) was measured using a fluorescence microplate reader (excitation wavelength 485 nm, emission wavelength 535 nm). A higher FI value indicates higher epithelial permeability and weaker barrier function. (See [link to relevant documentation]). Figure 6 .
[0035] Figure 6 The results showed that the fluorescence intensity of the model control group was significantly higher than that of the blank control group (P<0.05), indicating that the model was successfully constructed. The fluorescence intensity of the positive control group and the low, medium and high dose groups of fusion protein were significantly lower than that of the model control group, with the medium and high dose groups of fusion protein showing the most significant decrease (P<0.01). Moreover, the reduction effect was better than that of the positive control group.
[0036] This embodiment validates the results using an ovariectomized mouse model of vaginal dryness, demonstrating that the RGD-Collagen-FGF2 fusion protein can significantly increase vaginal epithelial thickness, promote collagen fiber synthesis (ECM reconstruction), enhance MUC1 / MUC5AC mucin expression and ECM water content (restoring moisturizing ability), and reduce epithelial permeability (enhancing barrier function), thereby improving vaginal dryness symptoms. Furthermore, the improvement effect of the high-dose fusion protein group is comparable to or better than that of the positive control drug (combined with estrogen ointment), indicating its promising application prospects in the treatment of vaginal dryness.
[0037] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A composition for improving vaginal dryness and laxity, characterized in that, The composition comprises an RGD-Collagen-FGF2 fusion protein, the fusion protein comprising an RGD element, a Collagen element, an FGF2 element, and a linker.
2. The composition according to claim 1, characterized in that, The RGD element is an RGD repeating structural unit, and its amino acid sequence is shown in SEQ ID NO:1; the Collagen element is an active fragment of human type III collagen, and its amino acid sequence is shown in SEQ ID NO:2; the FGF2 element is an active fragment of FGF2, and its amino acid sequence is shown in SEQ ID NO:3; the linker is... .
3. The composition according to claim 1 or 2, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:
4.
4. A method for preparing the RGD-Collagen-FGF2 fusion protein according to any one of claims 1-3, characterized in that, The method includes the following steps: (1) Design and synthesize a fusion gene sequence (5'-RGD-(Gly4Ser)3-Collagen-(Gly4Ser)3-FGF2-3') containing the (Gly4Ser)3 linker according to the codon preference of E. coli; (2) Digest the synthesized gene with the pET-28a (+) vector using restriction endonucleases NdeI and XhoI, react at 37℃ for 2h, and then recover the target fragment and the vector backbone; (3) Ligate with T4 DNA ligase at 16℃ for 12h to construct the recombinant expression vector pET-28a-RGD-Collagen-FGF2, and verify by sequencing; (4) Transform the recombinant vector into BL21 (DE3) competent cells, induce at 37℃ for 4h with IPTG final concentration of 0.5mmol / L, sonicate and purify by Ni-NTA affinity chromatography, and obtain the pure product by dialysis with a 10kDa dialysis bag.
5. The use of the composition or fusion protein according to any one of claims 1-3 in the preparation of a product for treating vaginal dryness and laxity.