A traditional Chinese medicine exosome complex with anti-HPV and application thereof

Abstract

The application discloses a traditional Chinese medicine exosome compound with anti-HPV and application thereof, and relates to the technical field of biotechnology.The compound comprises functional components secreted by a human cell co-culture system and a traditional Chinese medicine source extract, and the traditional Chinese medicine source extract is derived from a combined traditional Chinese medicine extract of Astragalus membranaceus, Isatis indigotica Fort., Honeysuckle and Sophora flavescens Ait., and relates to the technical field of biotechnology.The traditional Chinese medicine exosome carries natural active ingredients, and a multi-target anti-virus network is formed by beta-defensin-2 and interferon-gamma generated by co-culture with human cells.Compared with single traditional Chinese medicine extract or chemically synthesized nanoparticles, the HPV inhibition rate is increased by more than 170%, and the direct cytotoxicity of traditional Chinese medicine is reduced through an exosome delivery system, and the IC 50 >200ug / mL, which is significantly lower than that of a water decoction, and mucous membrane irritation experiments show no damage.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a traditional Chinese medicine exosome complex with anti-HPV properties and its applications. Background Technology

[0002] Human papillomavirus (HPV) is a double-stranded circular DNA virus with over 200 subtypes identified to date. Persistent infection with high-risk HPV types (such as HPV16, 18, and 31) is a significant cause of malignant tumors such as cervical and anal cancers, while low-risk HPV types (such as HPV6 and 11) primarily cause benign lesions like genital warts. HPV infection is showing a trend towards affecting younger people and has a higher incidence rate, posing a serious threat to human health.

[0003] Currently, treatments for HPV infection mainly include chemotherapy, physical therapy, and immunomodulatory therapy. Chemotherapy, such as interferon and imiquimod, while possessing some antiviral activity, suffers from significant systemic adverse reactions, strong local irritation, and poor patient compliance. Physical therapy (such as laser, cryotherapy, and electrocautery) can only remove visible lesions and cannot eliminate the virus latent in the basal cell layer, resulting in a recurrence rate as high as 20%-50%. Immunomodulatory drugs, while enhancing the body's immune response to HPV, have a slow onset of action and significant individual variability. Furthermore, these traditional treatments lack specific targeting of the HPV virus and pose a potential risk of damage to normal tissues. Therefore, there is an urgent clinical need for safe, effective, and novel anti-HPV drugs with novel mechanisms of action.

[0004] Traditional Chinese medicine (TCM), a treasure of Chinese medicine, exhibits unique advantages in antiviral, immunomodulatory, and anti-inflammatory effects, characterized by multiple targets and low toxicity. In recent years, numerous studies have shown that active components of TCM (such as astragalus polysaccharides in Astragalus membranaceus, indirubin in Isatis indigotica, chlorogenic acid in Lonicera japonica, and oxymatrine in Sophora flavescens) can exert anti-HPV effects by inhibiting viral replication and regulating the body's immune function. For example, astragalus polysaccharides can enhance the phagocytic capacity of macrophages and the proliferative activity of T cells, while Isatis indigotica extract can interfere with HPV DNA polymerase activity. However, their use alone suffers from low bioavailability and insufficient targeting, and the direct cytotoxicity of decoctions or alcohol extracts is relatively high, limiting their clinical application. Exosomes, as important carriers of intercellular communication, are membrane vesicles with a diameter of 30-150 nm, widely present in body fluids. They can carry bioactive substances such as nucleic acids, proteins, and lipids, possessing good biocompatibility, low immunogenicity, and tissue targeting, making them a research hotspot in the biomedical field. In the field of antiviral research, exosomes have been proven to serve as drug delivery systems, targeting small molecule drugs, nucleic acids, or natural active ingredients to the site of infection to increase local drug concentration. Simultaneously, some cell-derived exosomes can carry antigen information, activating immune responses. However, current exosome research largely focuses on human or animal cells; the extraction, purification, activity verification, and antiviral mechanism studies of exosomes derived from traditional Chinese medicine (TCM) are still in-depth, and there is a lack of technical solutions for the synergistic application of TCM exosomes with functional immune components. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a traditional Chinese medicine exosome complex with anti-HPV activity and its application. This complex achieves high efficiency inhibition of HPV virus and low toxicity to normal tissues through the synergistic effect of traditional Chinese medicine exosomes and human cell co-culture system, providing a safe and effective new solution for the treatment of HPV infection-related diseases.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a traditional Chinese medicine exosome complex with anti-HPV properties and its preparation method, wherein the complex comprises functional components secreted by a co-culture system of traditional Chinese medicine extracts and human cells, and the traditional Chinese medicine extracts are derived from a combination of Astragalus membranaceus, Isatis indigotica, Lonicera japonica, and Sophora flavescens extracts. The preparation method steps are as follows:

[0007] (1) Mix the Chinese herbal raw materials in a mass ratio of (5-10):(3-6):(2-4):(1-3), dry them at 50-70℃ until the moisture content is ≤5%, pulverize them through an 80-120 mesh sieve, and extract them by reflux at 70-90℃ with 60%-80% ethanol (material-liquid ratio 1:8-1:12 g / mL) for 2-3 times, each time for 1.5-3 hours. Combine the extracts and concentrate them under reduced pressure to a crude drug concentration of 1-2 g / mL.

[0008] (2) Mix the concentrated solution with serum-free exosome culture medium at a volume ratio of 1:5-1:20, add H8 cells and T cell exosomes for co-culture, and the cell seeding density is (2-4)×10⁻⁶. 5 The cells / mL were cultured at 37°C, 5% CO2, and 95% humidity for 72 hours.

[0009] (3) The culture supernatant was collected and sterilized by centrifugation at 3000g for 15 minutes at 4℃ and filtering through a 0.22μm membrane. The precipitate was then collected by ultracentrifugation at 100000g for 70 minutes at 4℃. After resuspending in PBS, the ultracentrifugation was repeated once to obtain exosome particles with a diameter of 30-150nm. Nanoparticle tracking analysis showed the concentration to be (1.2-2.5)×10⁻⁶. 11 particles / mL, Western blot analysis showed positive results for expression of CD63, CD81 and HSP70 markers;

[0010] The functional components include β-defensin-2 and interferon-γ, whose concentrations in the complex, as detected by ELISA, were (10.2-15.8) ng / mL and (15.5-22.3) ng / mL, respectively. In in vitro experiments, the complex inhibited E6 / E7 protein expression in HPV-16-positive HeLa cells and HPV-18-positive SiHa cells by (69.2±2.6)% and (65.8±3.2)%, respectively. Its cytotoxicity (CCK-8 assay) to normal cervical cell line H8 cells was [not specified]. 50 >200μg / mL, all of which showed extremely significant differences compared with the blank control group.

[0011] The mass ratio of Astragalus membranaceus, Isatis indigotica, Lonicera japonica, and Sophora flavescens is 6:5:3:2.

[0012] In step (1), the ethanol extraction process involves an initial extraction time of 2 hours and a second extraction time of 1.5 hours, with 75% ethanol as the extraction solvent.

[0013] In step (2), the co-culture system contains 1% gentamicin by volume, and the pH of the culture medium is 7.2-7.6.

[0014] The application of a traditional Chinese medicine exosome complex in the preparation of an anti-HPV infection drug, wherein the drug is used to inhibit HPV-6, HPV-11, HPV-16 and HPV-18 infection and to prevent and treat diseases including cervical intraepithelial neoplasia (CIN I-III), condyloma acuminata and flat warts.

[0015] The pharmaceutical preparation is for external use, containing 0.5-5 mg / mL (based on CD63 protein) of a traditional Chinese medicine exosome complex, and the dosage forms include:

[0016] Lotion: Using purified water as solvent, add 1%-5% (v / v) propylene glycol and 0.1% (w / v) ethylparaben;

[0017] Gel agent: with 1%-3% by volume of carbomer 940 as the base, and 5%-10% by volume of glycerol to adjust the viscosity;

[0018] Suppositories: Based on semi-synthetic fatty acid glycerides, each suppository contains 5-20 mg of exosome complex after molding.

[0019] The drug formulation exhibits an adsorption rate of ≥85% for HPV virus particles in in vitro experiments and shows no irritation in vaginal mucosal stimulation experiments (rabbit model).

[0020] The herbal exosome complex comprises 1-10 wt%, a transdermal penetration enhancer comprises 0.5-5 wt% (selected from azone and laurocapram) and a pharmaceutical carrier comprises 85-98.5 wt%, wherein the pharmaceutical carrier is a sodium hyaluronate matrix.

[0021] In summary, this invention mainly has the following beneficial effects: Traditional Chinese medicine exosomes carry natural active ingredients, forming a multi-target antiviral network with β-defensin-2 and interferon-γ produced by co-culturing with human cells. Compared with single traditional Chinese medicine extracts or chemically synthesized nanoparticles, the HPV inhibition rate is increased by over 170%. Furthermore, the exosome delivery system reduces the direct cytotoxicity of traditional Chinese medicine, and the IC50... 50 >200μg / mL, significantly lower than the water decoction, and the mucosal irritation test showed no damage. Detailed Implementation

[0022] Example 1:

[0023] Astragalus membranaceus, Isatis indigotica, Lonicera japonica, and Sophora flavescens were mixed in a mass ratio of 6:5:3:2 and dried at 60℃ until the moisture content was 4%. The mixture was then pulverized and passed through a 100-mesh sieve. Extraction was performed using 75% ethanol (material-to-liquid ratio 1:10 g / mL) at 80℃ under reflux for 2 hours initially and 1.5 hours later. The extracts were combined and concentrated under reduced pressure to a crude drug concentration of 1.5 g / mL.

[0024] Mix the concentrated solution with serum-free exosome culture medium at a volume ratio of 1:10, add 1% (v / v) gentamicin, and adjust the pH to 7.4. Then, use 2 × 10⁻⁶... 5Add H8 cells and T cell exosomes at a cell seeding density of cells / mL and culture for 72 hours at 37°C, 5% CO2, and 95% humidity. Both H8 cells and T cell exosomes can be purchased directly and added to the co-culture system.

[0025] The culture supernatant was collected and centrifuged at 3000g for 15 minutes at 4°C to remove cell debris, followed by sterilization using a 0.22μm filter. Then, it was ultracentrifuged at 100,000g for 70 minutes at 4°C (speed error controlled within ±3%, temperature fluctuation ≤ ±1°C) to collect the precipitate. The precipitate was resuspended in PBS and ultracentrifuged once more to obtain exosome particles. Nanoparticle tracking analysis showed that the exosome diameter was 30-150 nm and the concentration was 1.8 × 10⁻⁶. 11 The particles / mL were detected by Western blot analysis, and the expression of CD63, CD81 and HSP70 markers was verified.

[0026] The concentrations of β-defensin-2 and interferon-γ were measured by ELISA and were 13.5 ng / mL and 18.2 ng / mL, respectively.

[0027] Example 2:

[0028] The mass ratio of Astragalus membranaceus, Isatis indigotica, Lonicera japonica, and Sophora flavescens was 10:3:2:1. The herbs were dried at 50℃ to a moisture content of 5% and then pulverized through an 80-mesh sieve. The extracts were extracted three times at 70℃ with 60% ethanol (material-to-liquid ratio 1:8 g / mL), for 3 hours each time. The extracts were combined and concentrated under reduced pressure to a crude drug concentration of 1 g / mL.

[0029] The volume ratio of concentrated solution to serum-free exosome culture medium was 1:5, and the cell seeding density was 3 × 10⁶ cells / year. 5 The cells / mL concentration was 1.2 × 10⁻⁶ cells / mL, and the culture time was 72 hours, with other conditions the same as in Example 1. 11 The concentration of particles / mL, β-defensin-2 was 10.2 ng / mL, and interferon-γ was 15.5 ng / mL.

[0030] Example 3:

[0031] The astragalus, isatis root, honeysuckle, and sophora flavescens were dried at 70℃ to a moisture content of 3% and then pulverized through a 120-mesh sieve. The extract was then refluxed twice at 90℃ for 1.5 hours each time with 80% ethanol (solid-liquid ratio 1:12 g / mL), and concentrated to a crude drug concentration of 2 g / mL.

[0032] The volume ratio of concentrated solution to serum-free exosome culture medium was 1:20, and the cell seeding density was 4 × 10⁶. 5The cells / mL concentration was 2.5 × 10⁻⁶ cells / mL, and the culture time was 96 hours, with other conditions the same as in Example 1. 11 The concentration of particles / mL, β-defensin-2 was 15.8 ng / mL, and interferon-γ was 22.3 ng / mL.

[0033] Example 4:

[0034] The exosome complex prepared in Example 1 was used as a matrix with 1.5% (w / v) carbomer 940 and 8% (w / v) glycerol to adjust the viscosity, resulting in a gel containing 2 mg / mL (based on CD63 protein) of the complex. The gel showed an 88% adsorption rate for HPV virus particles and no irritation in rabbit vaginal mucosa irritation tests.

[0035] Comparative Example 1:

[0036] The mass ratio of Astragalus membranaceus, Isatis indigotica, and Lonicera japonica was 6:5:3, and the extraction conditions were the same as in Example 1.

[0037] The conditions were the same as in Example 1, but the concentrations of the functional components β-defensin-2 were 6.8 ng / mL and interferon-γ were 9.5 ng / mL, resulting in an inhibition rate of 42.1 ± 3.5% against H8 cell proteins and an IC50 concentration of 9.5%. 50 At a concentration of 180 μg / mL, it showed increased toxicity to normal cells.

[0038] Comparative Example 2:

[0039] Same as Example 1, but exosomes were isolated directly from the Chinese herbal extract without H8 cell co-culture.

[0040] The absence of β-defensin-2 and interferon-γ expression and the 18.3±2.1% inhibition rate of H8 cell proteins demonstrate that the functional components originated from the co-culture system.

[0041] Comparative Example 3:

[0042] CD63-containing nanoparticles were prepared using chemical synthesis methods to mimic the structure of exosomes, but without the active ingredients of traditional Chinese medicine. The inhibitory rate on H8 cell proteins was 25.6±1.8%, and no β-defensin-2 or interferon-γ expression was observed, indicating that the extract derived from traditional Chinese medicine possesses unique activity.

[0043] Comparative Example 4:

[0044] Astragalus membranaceus, Isatis indigotica, Lonicera japonica, and Sophora flavescens were decocted twice with water in a ratio of 6:5:3:2. The decoctions were combined and concentrated to a crude drug concentration of 1.5 g / mL, and then directly applied to H8 cells. The H8 cell protein inhibition rate was 35.7 ± 2.9%, and the IC50 was [missing value]. 50It has a concentration of 150 μg / mL (higher toxicity) and no exosome structure (NTA detection showed no particles of 30-150 nm).

[0045] Table 1: Exosome characteristics and concentrations of functional components

[0046]

[0047] Table 2: Comparison of in vitro experimental results

[0048]

[0049]

[0050] As shown in Tables 1 and 2, Examples 1-3 all meet the requirements of the co-culture system of four traditional Chinese medicine combinations (Astragalus membranaceus, Isatis indigotica, Lonicera japonica, and Sophora flavescens) and human cells, with exosome diameters of 30-150 nm and concentrations of 1.2-2.5 × 10⁻⁶. 11 The particles / mL and markers CD63, CD81 and HSP70 were all positively expressed.

[0051] Comparative Example 1 lacked Sophora flavescens, resulting in a decrease of over 49% in the concentration of functional components and a reduction of over 39% in the HPV inhibition rate.

[0052] Comparative Example 2 was not co-cultured, lacked the corresponding functional components, and exosomes were not detected with an inhibition rate of less than 20%.

[0053] The gel in Example 4 shows a virus adsorption rate of ≥85% and is non-irritating to the mucosa. When combined with a transdermal penetration enhancer, its performance is superior to the traditional formulations in Comparative Examples 3-4.

[0054] Furthermore, gels can be replaced with lotions and suppositories;

[0055] The preparation steps of the lotion are as follows: Take the exosome complex prepared in Example 1, use purified water as solvent, add 3% volume fraction of propylene glycol and 0.1% mass fraction of ethylparaben, and stir evenly to make the complex concentration 1 mg / mL.

[0056] In vitro experiments showed that the wash had an adsorption rate of 86% for HPV virus particles, and rabbit vaginal mucosal irritation tests showed that it was non-irritating.

[0057] The preparation steps of the suppositories are as follows: Weigh 1000mg of semi-synthetic fatty acid glyceride matrix, heat and melt it, then cool it to about 40℃, add 10mg of exosome complex, stir evenly, inject the mixture into the suppository mold, cool and solidify it, and then take it out to obtain a suppository containing 10mg of complex per tablet.

[0058] In vitro experiments showed that the suppository had an adsorption rate of 89% for HPV virus particles, and rabbit vaginal mucosal irritation tests showed that it was non-irritating.

[0059] Compared with the chemically synthesized nanoparticles in Comparative Example 3, the Chinese herbal exosomes, carrying natural functional components, namely β-defensin-2 and interferon-γ, showed an inhibition rate of over 170% and more comprehensive expression of biomarkers.

[0060] The decoction in Comparative Example 4 lacked exosome structure, and its inhibition rate was only 51.6% of that in Example 1. Moreover, its cytotoxicity was significantly increased, demonstrating the key role of the exosome delivery system in reducing toxicity and improving targeting, effectively distinguishing it from traditional Chinese medicine dosage forms.

[0061] All examples showed IC50 values ​​for normal cervical cells H8. 50 All of them were greater than 200 μg / mL, while the comparative examples 1-4 were all ≤190 μg / mL, highlighting the low toxicity advantage of this scheme.

[0062] HPVCD63, CD81, and HSP70 infections primarily target cervical epithelial cells, such as CD63, CD81, HSP70HeLa and SiHa CD63, CD81, HSP70 cells. Direct application of these cells will indiscriminately affect normal cells, leading to low antiviral efficiency and damage to normal cells. However, exosomes produced by co-culturing with traditional Chinese medicine extracts can serve as natural protective carriers. Their lipid bilayer membrane structure can encapsulate functional components and isolate them from external enzymatic digestion. Compared to direct application of the components, exosome delivery can prolong the duration of action of functional components on the cervical mucosa.

[0063] Therefore, this invention significantly enhances anti-HPV activity by combining the synergistic effect of traditional Chinese medicine exosomes and co-cultured functional components, while ensuring safety, with an inhibition rate of 65%-71%. Moreover, key indicators such as virus adsorption rate and mucosal compatibility are superior to traditional methods.

[0064] Although embodiments of the present invention have been shown and described, these specific embodiments are merely explanations of the invention and are not intended to limit it. The specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. After reading this specification, those skilled in the art may make modifications, substitutions, and variations to the embodiments as needed without departing from the principles and spirit of the invention, but such modifications, substitutions, and variations are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A traditional Chinese medicine exosome complex with anti-HPV properties, characterized in that: The complex contains functional components secreted by a co-culture system of traditional Chinese medicine extracts and human cells. The traditional Chinese medicine extracts are derived from a combination of Astragalus membranaceus, Isatis indigotica, Lonicera japonica, and Sophora flavescens extracts. The preparation method is as follows: (1) Mix the Chinese herbal raw materials in a mass ratio of (5-10):(3-6):(2-4):(1-3), dry them at 50-70℃ until the moisture content is ≤5%, pulverize them through an 80-120 mesh sieve, and extract them by reflux at 70-90℃ with 60%-80% ethanol (material-liquid ratio 1:8-1:12 g / mL) for 2-3 times, each time for 1.5-3 hours. Combine the extracts and concentrate them under reduced pressure to a crude drug concentration of 1-2 g / mL. (2) Mix the concentrated solution with serum-free exosome culture medium at a volume ratio of 1:5-1:20, add H8 cells and T cell exosomes for co-culture, and the cell seeding density is (2-4)×10⁻⁶. 5 The cells / mL were cultured at 37°C, 5% CO2, and 95% humidity for 72-96 hours. (3) The culture supernatant was collected and sterilized by centrifugation at 3000g for 15 minutes at 4℃ and filtering through a 0.22μm membrane. The precipitate was then collected by ultracentrifugation at 100000g for 70 minutes at 4℃. After resuspending in PBS, the ultracentrifugation was repeated once to obtain exosome particles with a diameter of 30-150nm. Nanoparticle tracking analysis showed the concentration to be (1.2-2.5)×10⁻⁶. 11 particles / mL, Western blot analysis showed positive results for expression of CD63, CD81 and HSP70 markers; The functional components include β-defensin-2 and interferon-γ. ELISA analysis showed that the concentrations of β-defensin-2 and interferon-γ in the complex were 10.2-15.8 ng / mL and 15.5-22.3 ng / mL, respectively. In in vitro experiments, the complex inhibited E6 / E7 protein expression in HPV-16-positive HeLa cells and HPV-18-positive SiHa cells by (69.2±2.6)% and (65.8±3.2)%, respectively. The complex also showed an IC50 inhibitory effect on normal cervical cell line H8 cells, as detected by the CCK-8 assay. 50 >200 μg / mL, all of which showed highly significant differences compared to the blank control group.

2. The traditional Chinese medicine exosome complex with anti-HPV properties according to claim 1, characterized in that: The mass ratio of Astragalus membranaceus, Isatis indigotica, Lonicera japonica, and Sophora flavescens is 6:5:3:

2.

3. The traditional Chinese medicine exosome complex with anti-HPV properties according to claim 1, characterized in that: In step (1), the ethanol extraction process involves an initial extraction time of 2 hours and a second extraction time of 1.5 hours, with 75% ethanol as the extraction solvent.

4. The traditional Chinese medicine exosome complex with anti-HPV properties according to claim 1, characterized in that: Gentamicin with a final concentration of 1% (v / v) was added to the co-culture system described in step (2), and the pH of the culture medium was 7.2-7.

6.

5. A method for preparing the traditional Chinese medicine exosome complex according to any one of claims 1-4, characterized in that: Includes the following steps: Step 1: Pretreatment and extraction of Chinese herbal raw materials; Step 2: Human cell co-culture and supernatant collection; Step 3: Exosome isolation, purification, and quality control.

6. The application of the traditional Chinese medicine exosome complex according to any one of claims 1-4 in the preparation of anti-HPV infection drugs, characterized in that: The drug is used to inhibit HPV-6, HPV-11, HPV-16, and HPV-18 infections, and to prevent and treat diseases including cervical intraepithelial neoplasia, condyloma acuminata, and flat warts.

7. The application of the traditional Chinese medicine exosome complex according to claim 6 in the preparation of anti-HPV infection drugs, characterized in that: The drug is a topical preparation containing 0.5-5 mg / mL of a traditional Chinese medicine exosome complex, and the dosage forms include: Lotion: Using purified water as solvent, add 1%-5% by weight of propylene glycol and 0.1% by weight of ethylparaben; Gel agent: with 1%-3% by volume of carbomer 940 as the base, and 5%-10% by volume of glycerol to adjust the viscosity; Suppositories: Based on semi-synthetic fatty acid glycerides, each suppository contains 5-20 mg of exosome complex after molding.

8. The application of the traditional Chinese medicine exosome complex according to claim 7 in the preparation of anti-HPV infection drugs, characterized in that: The drug showed an adsorption rate of ≥85% for HPV virus particles in in vitro experiments and was non-irritating in vaginal mucosal stimulation tests.

9. A pharmaceutical composition for treating HPV infection, characterized in that: The pharmaceutical carrier comprises 1-10 wt% of the traditional Chinese medicine exosome complex as described in any one of claims 1-4, 0.5-5 wt% of the transdermal penetration enhancer, and 85-98.5 wt% of sodium hyaluronate matrix.

Images