Hydrogel drug delivery system loaded with motherwort exosome as well as preparation method and application of hydrogel drug delivery system
Through the hydrogel delivery system loading motherwort exosomes, the double-layer loaded sustained-release cefotaxime of exosomes and temperature-sensitive hydrogels is used to solve the problems of poor efficacy, many side effects and short half-life of traditional drug dosage forms in the treatment of endometriitis, achieving long-term, anti-inflammatory and antibacterial synergies of the drug, and providing a new treatment plan.
Patent Information
- Application Number
- CN202510453053.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-08-19
AI Technical Summary
Traditional drug dosage forms have problems such as poor efficacy, many side effects, antibiotic resistance and secondary infection when treating endometriitis, and the drug has a short half-life, which cannot effectively solve the inflammatory response and insufficient tissue repair.
The hydrogel delivery system loading motherwort exosomes is adopted to achieve local enrichment and long-term release of drugs through double-layer encapsulation of exosomes and temperature-sensitive hydrogels.
It significantly extends the retention time of the drug in the body, enhances the antibacterial effect, reduces systemic side effects, realizes the comprehensive treatment of endometriitis, and breaks through the limitations of traditional single antibiotics.
Smart Images
Figure CN120501692A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of medical materials, and in particular to a hydrogel drug delivery system loaded with motherwort exosomes, and a preparation method and application thereof. Background Art
[0002] In modern clinical practice, the incidence of gynecological diseases remains high, significantly impacting women's physical and mental health. Infectious diseases are common among gynecological diseases, such as endometritis, cervicitis, and pelvic inflammatory disease. Abnormal inflammatory responses produce a large number of pro-inflammatory factors, which stimulate cell proliferation and inhibit apoptosis, thereby increasing the risk of gynecological tumors.
[0003] Motherwort, also known as Cnidium monnieri and Kuncao, is the fresh or dried aerial part of the Lamiaceae plant (Leonurus japonicus Houtt.). It has benefits such as promoting blood circulation and regulating menstruation, promoting diuresis and reducing swelling, and clearing away heat and detoxifying. It is used for menstrual disorders, amenorrhea, dysmenorrhea, and lochia retention. Motherwort contains a variety of chemical components, including alkaloids, diterpenes, and ferulic acid. Motherwort is primarily composed of leonurine and stachydrine hydrochloride. Motherwort and its preparations play an important role in the treatment of women's health problems and are the most commonly used Traditional Chinese Medicine in modern obstetrics and gynecology. Modern pharmacological studies have shown that motherwort has numerous benefits, including uterine protection, anti-inflammatory, antioxidant, and vasoprotective properties.
[0004] Exosomes, small nanoscale vesicles secreted by cells, have a phospholipid bilayer structure and contain proteins, lipids, RNA, and other molecules. They play a vital role in intercellular material exchange and information transmission, serving both as transport vehicles and possessing biological functions. The lipid composition of exosomes extracted from fresh motherwort plants suggests that motherwort exosomes have therapeutic effects on intrauterine inflammation. Furthermore, motherwort exosomes are easy to mass-produce, safe and reliable, highly stable, and have low immunogenicity, making them an ideal choice for future drug delivery vehicles.
[0005] Traditional drug formulations often have poor efficacy and many side effects in the treatment of endometritis. In addition, excessive use of antibiotics can lead to drug resistance, secondary infections, and problems related to drug metabolism. Therefore, there is an urgent need to provide patients with gynecological infections with new, safer and more effective treatment options. Summary of the Invention
[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a hydrogel drug delivery system loaded with motherwort exosomes, as well as its preparation method and application. The present invention prolongs the retention time of cefotaxime in the body through double-layer encapsulation and sustained release of exosomes and thermosensitive hydrogel, improves the problem of short half-life of cefotaxime, and achieves synergistic enhancement with the anti-inflammatory effect of motherwort exosomes, enhances the antibacterial effect, and has great application value in the treatment of endometritis.
[0007] The technical solution of the present invention to solve the above technical problems is as follows: providing a hydrogel drug delivery system loaded with motherwort exosomes, comprising a hydrogel and motherwort exosomes; the motherwort exosomes contain cefotaxime; the hydrogel is made by cross-linking chitosan and sodium β-glycerophosphate.
[0008] The present invention also provides a method for preparing the hydrogel drug delivery system loaded with motherwort exosomes, comprising the following steps:
[0009] (1) Mixing the Leonurus herb exosome solution with the cefotaxime solution, incubating overnight in the dark, and collecting the upper liquid after centrifugation to obtain drug-loaded exosomes;
[0010] (2) adding chitosan into acetic acid solution and stirring to dissolve to obtain chitosan solution;
[0011] (3) Under stirring in an ice-water bath, the sodium β-glycerophosphate solution is added to the chitosan solution obtained in step (2), and then the drug-loaded exosomes obtained in step (1) are added to obtain a hydrogel drug delivery system loaded with motherwort exosomes.
[0012] Furthermore, in step (1), the motherwort exosome solution is prepared by the following steps: extracting motherwort juice, removing impurities by gradient centrifugation, centrifuging the supernatant at 110,000-130,000 g for 9-12 hours, then resuspending the precipitate with PBS solution, and then layering it on the upper layer of the gradient sucrose solution, centrifuging at 110,000-130,000 g for 12-15 hours, taking the 30%-40% layer solution and centrifuging it at 150,000 g for 12-15 hours to wash away the sucrose, and finally resuspending the precipitate with PBS solution to obtain the motherwort exosome solution.
[0013] Furthermore, gradient centrifugation was performed at 6000 g for 30 min, 9000 g for 45 min, and 12000 g for 90 min.
[0014] Furthermore, the concentrations of the gradient sucrose solution include 8 wt %, 30 wt %, 45 wt % and 60 wt % from top to bottom.
[0015] The beneficial effect of adopting the above further technical solution is that the present invention effectively improves the content and purity of the obtained motherwort exosomes by ultracentrifugation purification of gradient concentration sucrose solution.
[0016] Furthermore, in step (1), the concentration of the Leonurus japonicus exosome solution is 1.6-3.3×10 12 / mL, the concentration of cefotaxime solution is 0.8-1.2mg / mL; the volume ratio of motherwort exosome solution to cefotaxime solution is 1:2-4.
[0017] Furthermore, in step (1), the solution is centrifuged at 3000-6000 g for 15-45 min at 0-4°C using an ultrafiltration tube (Mw=100 kD).
[0018] Furthermore, in step (2), the concentration of the acetic acid solution is 0.05-0.15 mol / L; the concentration of the chitosan solution is 1-3 wt%.
[0019] Furthermore, in step (3), the concentration of the β-sodium glycerophosphate solution is 50-60 wt%.
[0020] Furthermore, in step (3), the volume ratio of the sodium β-glycerophosphate solution, the chitosan solution, and the drug-loaded exosomes is 6-8:2-4:0.4-0.6.
[0021] The present invention also provides the use of the hydrogel drug delivery system loaded with motherwort exosomes in the preparation of a drug for treating endometritis.
[0022] The present invention has the following beneficial effects:
[0023] 1. This invention encapsulates the antibacterial drug cefotaxime within exosomes. Encapsulation in Leonurus exosomes enables sustained release of cefotaxime in vivo, reducing side effects caused by systemic drug exposure. The lipid bilayer structure of the exosomes not only provides a relatively stable environment for the encapsulated antibacterial drug cefotaxime but also possesses a certain ability to cross biological barriers. Furthermore, using a thermosensitive hydrogel as a drug delivery system and intrauterine perfusion as a drug delivery method, this method achieves dual sustained release, significantly prolonging the drug's duration of action and improving therapeutic efficacy. This provides a new approach for the treatment of endometritis and other intrauterine infectious diseases.
[0024] 2. The thermosensitive hydrogel synthesized by the present invention can change from liquid to solid in response to changes in body temperature, thereby achieving a sustained drug release effect in the uterine cavity. This changes the traditional antibiotic administration method that requires frequent injections and increases patient compliance. Local administration can also enrich the drug concentration and enhance the therapeutic effect. At the same time, through the double-layer encapsulation and sustained release of exosomes and thermosensitive hydrogels, the retention time of cefotaxime in the body is prolonged, the problem of the short half-life of cefotaxime is improved, and synergistic synergy is achieved with the anti-inflammatory effect of motherwort exosomes, thereby enhancing the antibacterial effect.
[0025] 3. The present invention fully combines the medicinal properties of motherwort and for the first time constructs its exosomes with ceftriaxone into a synergistic treatment system. Motherwort exosomes have multiple biological activities and can exert anti-inflammatory, antioxidant and anti-fibrotic effects, forming a complementary advantage with the potent antibacterial properties of ceftriaxone. This synergistic mechanism breaks through the limitations of traditional single antibiotic treatment, and realizes the comprehensive treatment of endometritis from multiple levels such as antibacterial, anti-inflammatory and tissue repair, significantly improving the treatment effect. This combined medication regimen not only retains the antibacterial advantages of ceftriaxone, but also effectively solves the problems of inflammatory response and insufficient tissue repair in single antibiotic treatment through the multiple pharmacological effects of motherwort exosomes, providing a new solution for the treatment of endometritis. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is a transmission electron microscopy image of Leonurus herb exosomes;
[0027] Figure 2 This is an analysis chart of the contents of leonurine and stachydrine hydrochloride in Leonurus herb exosomes;
[0028] Figure 3 is the drug release profile of drug-loaded exosomes;
[0029] Figure 4 This is a scanning electron microscope image of the drug delivery system of Example 1;
[0030] Figure 5 This is the lipid composition analysis diagram of motherwort exosomes;
[0031] Figure 6 Comparison of the retention time of the drug delivery system in rat uterus in vitro imaging
[0032] Figure 7 This is a quantitative graph showing the retention of the drug delivery system in the rat uterus. DETAILED DESCRIPTION
[0033] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.
[0034] Example 1
[0035] A hydrogel drug delivery system loaded with motherwort exosomes, comprising a hydrogel and motherwort exosomes; the motherwort exosomes contain cefotaxime; the hydrogel is prepared by cross-linking chitosan and sodium β-glycerophosphate;
[0036] The preparation method comprises the following steps:
[0037] (1) 3.3×10 12 The 100 mg / mL Leonurus herb exosome solution was mixed with 1 mg / mL cefotaxime solution at a volume ratio of 1:3, incubated in the dark at 4°C overnight, and centrifuged at 5000 g for 30 min at 4°C using an ultrafiltration tube (Mw = 100 kD) to collect the supernatant to obtain the drug-loaded exosomes.
[0038] (2) Add chitosan to 0.1 mol / L acetic acid solution and stir to dissolve to obtain chitosan solution; the concentration of chitosan solution is 2 wt%
[0039] (3) Under stirring in an ice-water bath, 56 wt% sodium β-glycerophosphate solution was added to the chitosan solution obtained in step (2), and then the drug-loaded exosomes obtained in step (1) were added to obtain a hydrogel drug delivery system loaded with motherwort exosomes; the volume ratio of sodium β-glycerophosphate solution, chitosan solution and drug-loaded exosomes was 7:3:0.5.
[0040] In step (1), the motherwort exosome solution is prepared by the following steps: extracting motherwort juice, centrifuging at 6000g for 30 minutes, 9000g for 45 minutes and 12000g for 90 minutes to remove impurities, centrifuging the supernatant at 120000g for 10 hours, then resuspending the precipitate with PBS solution, and then layering it on the upper layer of a gradient sucrose solution including 8wt%, 30wt%, 45wt% and 60wt% from top to bottom, centrifuging at 120000g for 13 hours, taking the 30%-40% layer solution and centrifuging at 150000g for 13 hours to wash away the sucrose, and finally resuspending the precipitate with PBS solution to obtain the motherwort exosome solution.
[0041] Example 2
[0042] A hydrogel drug delivery system loaded with motherwort exosomes, the preparation method of which comprises the following steps:
[0043] (1) 1.6×10 12 The 0.8 mg / mL Leonurus herb exosome solution was mixed with 0.8 mg / mL cefotaxime solution at a volume ratio of 1:2, incubated in the dark overnight, and centrifuged at 3000 g for 15 min at 0°C to collect the supernatant liquid to obtain the drug-loaded exosomes.
[0044] (2) Add chitosan to 0.05 mol / L acetic acid solution and stir to dissolve to obtain chitosan solution; the concentration of chitosan solution is 1 wt%
[0045] (3) Under stirring in an ice-water bath, 50 wt% sodium β-glycerophosphate solution was added to the chitosan solution obtained in step (2), and then the drug-loaded exosomes obtained in step (1) were added to obtain a hydrogel drug delivery system loaded with motherwort exosomes; the volume ratio of sodium β-glycerophosphate solution, chitosan solution and drug-loaded exosomes was 6:2:0.4.
[0046] Example 3
[0047] A hydrogel drug delivery system loaded with motherwort exosomes, the preparation method of which comprises the following steps:
[0048] (1) 2.8×10 12 The 1.5 mg / mL Leonurus herb exosome solution was mixed with 1.2 mg / mL cefotaxime solution at a volume ratio of 1:4, incubated overnight in the dark, and centrifuged at 6000 g for 45 min at 2°C to collect the supernatant liquid to obtain the drug-loaded exosomes.
[0049] (2) Add chitosan to 0.15 mol / L acetic acid solution and stir to dissolve to obtain chitosan solution; the concentration of chitosan solution is 3 wt%
[0050] (3) Under stirring in an ice-water bath, 60 wt% sodium β-glycerophosphate solution was added to the chitosan solution obtained in step (2), and then the drug-loaded exosomes obtained in step (1) were added to obtain a hydrogel drug delivery system loaded with motherwort exosomes; the volume ratio of sodium β-glycerophosphate solution, chitosan solution and drug-loaded exosomes was 8:4:0.6.
[0051] Experimental Example 1 Morphological Characterization of Motherwort Exosomes
[0052] The morphology of motherwort exosomes was observed using a transmission electron microscope: 15 μL of motherwort exosome sample was dropped onto the surface of the copper mesh. After the surface was dry, 1% phosphotungstic acid solution was added for counterstaining for 2 minutes. The excess dye was gently absorbed with filter paper. After the sample was completely dried at room temperature, it was placed under an electron transmission microscope to observe the morphology of the sample. The results are as follows: Figure 1 As shown, the prepared Leonurus herb exosomes have a complete and uniform morphology and a particle size of about 123 nm.
[0053] Experimental Example 2 Analysis of the Content of Leonurine and Stachydrine Hydrochloride in Leonurus Exosomes by High Performance Liquid Chromatography
[0054] (1) Leonurine: A standard solution with a concentration of 0.8 mg / mL was prepared with 24% chromatographic grade methanol and gradient dilution was performed to make the standard concentrations of 0.8 mg / mL, 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL and 0.05 mg / mL, respectively. 100 μL of Leonurine exosomes was placed in a 1.5 mL volumetric flask with a cannula. The sample was injected and analyzed using a Waters C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of acetonitrile (A) and 0.1% phosphate solution (B), with a gradient setting of 0-10 min: 100% A; 10-20 min: 100%-15% A; 20-40 min: 15% A; the injection volume was 10 μL, the flow rate was 1.0 mL / min, the column temperature was 30°C, and the detection wavelength was 277 nm.
[0055] (2) Stachydrine hydrochloride: A standard solution with a concentration of 0.1 mg / mL was prepared with 3% chromatographic grade methanol and gradient dilution was performed to make the standard concentrations of 1, 0.5, 0.25, 0.125, and 0.0625 mg / mL, respectively. 100 μL of Leonurus herb exosomes was placed in a 1.5 mL volumetric flask with a cannula. The sample was injected and analyzed using a Waters C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of acetonitrile (A) and 0.1% phosphate solution (B), with a gradient setting of 0-10 min: 100% A; 10-20 min: 100%-3% A; 20-40 min: 3% A; the injection volume was 10 μL, the flow rate was 1.0 mL / min, the column temperature was 30°C, and the detection wavelength was 200 nm.
[0056] Test results such as Figure 2 As shown, motherwort exosomes contain leonurine and stachydrine.
[0057] Experimental Example 3: Detection of in vitro drug release curve of drug-loaded exosomes by UV-visible spectrophotometry
[0058] The cefotaxime solution after gradient dilution was measured using an ultraviolet-visible spectrophotometer to prepare a standard curve; the drug-loaded exosomes obtained in step (2) of Example 1 were placed in a dialysis bag for testing, and the dialysis bag was immersed in 100 mL of dissolution medium and shaken in a shaking water bath at 37°C and 100 rpm. 1 mL of liquid was taken out at regular intervals and aliquoted, and the same volume of fresh release medium was added to maintain equilibrium. The absorbance of the solution was measured at 235 nm, and the cefotaxime release amount was calculated from the standard curve. The results are shown in FIG. Figure 3 As shown in the figure, drug-loaded exosomes can effectively release the drug under different pH conditions.
[0059] Experimental Example 4 Morphological Characterization of Thermosensitive Hydrogel Before and After Drug Loading
[0060] The morphological characteristics of the thermosensitive hydrogel before and after drug loading were observed using a scanning electron microscope: 3 mL of blank hydrogel solution and the hydrogel drug delivery system of Example 1 were respectively taken into a 5 mL EP tube, and the EP tube was placed in a 37°C oven for about 5 minutes until the gel became solid. The EP tube was then placed in liquid nitrogen for quick freezing for 5 minutes and freeze-dried at low temperature for 48 hours. The resulting hydrogel was freeze-dried and broken with tweezers. The surface morphology of the hydrogel cross section was observed using a scanning electron microscope at a voltage of 3kV after sputtering and gold plating. The results are shown in Figure 2. Figure 4 As shown, particle size statistics in the drug-loaded hydrogel SEM image were performed using Image J software. The particle size ranged from 73 to 180 nm, indicating that they were Leonurus exosomes. The results showed that Leonurus exosomes were evenly loaded in the hydrogel.
[0061] Experimental Example 5: Analysis of lipid components in exosomes of Leonurus japonicus
[0062] The lipid components of Leonurus herb exosomes were analyzed and the results were as follows Figure 5 As shown in the results, its lipid components such as monogalactosyldiacylglycerol, phosphatidylethanolamine, and sterol esters have anti-inflammatory, antioxidant, and immune regulation effects.
[0063] Experimental Example 6 Comparison of the retention time of thermosensitive hydrogel before and after drug loading in rats
[0064] (1) 15 μL of the drug-loaded exosomes in Example 1 labeled with a far-infrared cell membrane fluorescent probe (DiD) was diluted with 85 μL of PBS and then perfused into the rat uterus as a control group (PBS / motherwort exosomes@DiD dye);
[0065] (2) 15 μL of the hydrogel delivery system of Example 1 labeled with a far-infrared cell membrane fluorescent probe (DiD) was added dropwise to 85 μL of the thermosensitive hydrogel under magnetic stirring in an ice-water bath and intrauterine perfusion into the rat uterus as the experimental group (hydrogel / motherwort exosomes@DiD dye);
[0066] (3) Three rats were taken at 6h, 12h, 24h and 48h after intrauterine perfusion, and the rats were anesthetized with 3% pentobarbital intraperitoneal injection, and the uterine tissue was dissected. The retention effect of motherwort exosomes in the rats was recorded using a small animal in vivo imaging system. The results are as follows: Figure 6 and 7 shown.
[0067] Depend on Figure 6 and 7 It can be seen that the hydrogel drug delivery system of the present invention effectively increases the retention time of the drug.
[0068] In summary, the preparation route of the present invention is simple, has no toxic side effects, has low immunogenicity, and has a significant effect of prolonging the drug action time. Intrauterine localized treatment can not only avoid the potential side effects caused by systemic medication, but also locally enrich the effective concentration of the drug and enhance the efficacy. The encapsulation of the motherwort exosome delivery system not only enhances the antibacterial and anti-inflammatory activity of the thermosensitive hydrogel, but also alleviates the problem of reduced drug utilization caused by drug dispersion.
[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A hydrogel drug delivery system loaded with motherwort exosomes, characterized in that: The invention comprises a hydrogel and motherwort exosomes; the motherwort exosomes contain cefotaxime; and the hydrogel is prepared by cross-linking chitosan and sodium β-glycerophosphate.
2. The method for preparing the hydrogel drug delivery system loaded with Leonurus herb exosomes according to claim 1, characterized in that: The following steps are involved: (1) Mixing the Leonurus herb exosome solution with the cefotaxime solution, incubating overnight in the dark, and collecting the upper liquid after centrifugation to obtain drug-loaded exosomes; (2) adding chitosan into acetic acid solution and stirring to dissolve to obtain chitosan solution; (3) Under stirring in an ice-water bath, the sodium β-glycerophosphate solution is added to the chitosan solution obtained in step (2), and then the drug-loaded exosomes obtained in step (1) are added to obtain a hydrogel drug delivery system loaded with motherwort exosomes.
3. The method for preparing the hydrogel drug delivery system loaded with Leonurus herb exosomes according to claim 2, wherein: In step (1), the motherwort exosome solution is prepared by the following steps: extracting motherwort juice, removing impurities by gradient centrifugation, centrifuging the supernatant at 110,000-130,000g for 9-12h, then resuspending the precipitate with PBS solution, and then layering it on the upper layer of the gradient sucrose solution, centrifuging at 110,000-130,000g for 12-15h, taking the 30%-40% layer solution and centrifuging it at 150,000g for 12-15h to wash away the sucrose, and finally resuspending the precipitate with PBS solution to obtain the motherwort exosome solution.
4. The method for preparing the hydrogel drug delivery system loaded with Leonurus herb exosomes according to claim 2, wherein: In step (1), the concentration of the motherwort exosome solution is 1.6-3.3×10 12 / mL, the concentration of the cefotaxime solution is 0.8-1.2 mg / mL; the volume ratio of the motherwort exosome solution to the cefotaxime solution is 1:2-4.
5. The method for preparing the hydrogel drug delivery system loaded with Leonurus herb exosomes according to claim 2, wherein: In step (1), centrifuge at 3000-6000 g for 15-45 min at 0-4°C.
6. The method for preparing the hydrogel drug delivery system loaded with Leonurus herb exosomes according to claim 2, wherein: In step (2), the concentration of the acetic acid solution is 0.05-0.15 mol / L; the concentration of the chitosan solution is 1-3 wt%.
7. The method for preparing the hydrogel drug delivery system loaded with Leonurus herb exosomes according to claim 2, wherein: In step (3), the concentration of the β-sodium glycerophosphate solution is 50-60wt%.
8. The method for preparing the hydrogel drug delivery system loaded with Leonurus herb exosomes according to claim 2, wherein: In step (3), the volume ratio of the sodium β-glycerophosphate solution, chitosan solution and drug-loaded exosomes is 6-8:2-4:0.4-0.
6.
9. Use of the hydrogel drug delivery system loaded with Leonurus japonicus exosomes as claimed in claim 1 in the preparation of a drug for treating endometritis.