Matrine-loaded bionic nano vesicle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano medicines, and particularly relates to matrine-loaded bionic nano vesicles as well as a preparation method and application thereof. The human skin fibroblast vesicles contain genetic materials and cell factors in the human fibroblast vesicles, can interact with various cells such as keratinocytes and mononuclear cells, and are combined with various mechanisms to promote the wound healing process; matrine is an effective component of radix sophorae flavescentis and has the effects of resisting inflammation, inhibiting excessive fibrosis and the like, the inflammation environment near a wound is adjusted through matrine, scar formation is adjusted while excessive damage of inflammation to tissue is avoided, then matrine is wrapped with the human skin fibroblast vesicles, and the matrine-loaded bionic nano vesicles are obtained. The strong anti-inflammatory effect of the sophocarpidine inhibits the oxidative stress level near a wound surface, the human skin fibroblast vesicles inhibit the oxidative stress level near the wound surface, and the sophocarpidine and the human skin fibroblast vesicles cooperate to effectively promote healing of the wound.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano medicine, and specifically relates to a matrine-loaded biomimetic nanovesicle, a preparation method thereof, and an application thereof. Background Art

[0002] The purpose of the inflammatory response in wound healing is to limit blood and fluid loss, remove dead or dying cells, and prevent infection. Although the inflammatory phase of wound healing is an inherent natural defense of the body against environmental attacks, an increase in inflammation continuously damages tissues in chronic wounds, thereby exacerbating the inflammatory environment of macrophage infiltration. The increase in inflammation will cause the destruction of wound tissue cells, thus promoting the production of extracellular matrix and exacerbating scar formation.

[0003] Matrine is an active ingredient of Sophora flavescens, belonging to tetracyclic quinolizidine alkaloids. It has been proven to have an obvious antagonistic effect on various acute exudative inflammations and has a certain stabilizing effect on the erythrocyte membrane. In addition, matrine also has an inhibitory effect on human hypertrophic scars, such as causing changes in the ultrastructure of fibroblasts and regulating functions such as protein synthesis and mitochondrial metabolism of scar fibroblasts. Therefore, matrine has the effects of anti-inflammation, inhibiting excessive fibrosis, and regulating scar formation, and has potential therapeutic effects on inhibiting the inflammatory response and hypertrophic scar formation in wound healing.

[0004] However, the direct application of matrine to the skin may lead to insufficient drug absorption. Although local treatment with exosomes can carry drugs to the epidermis and be effectively absorbed by human skin, the yield of exosomes is low and the controllability is poor under large-scale preparation conditions, and thus it cannot achieve the effect of effectively healing wounds. Summary of the Invention

[0005] The purpose of the present invention is to provide a matrine-loaded biomimetic nanovesicle, a preparation method thereof, and an application thereof. The matrine-loaded biomimetic nanovesicle contains human fibroblast genetic material and growth factors, and can effectively promote wound healing in synergy with the anti-inflammatory effect of matrine.

[0006] The present invention provides a matrine-loaded biomimetic nanovesicle, which includes a skin fibroblast vesicle and matrine encapsulated in the inner cavity of the skin fibroblast vesicle.

[0007] Preferably, the encapsulation efficiency of matrine in the matrine-loaded biomimetic nanovesicle is 77 ± 2.59%;

[0008] The particle size of the drug-loaded matrine nanovesicle is 245.6 ± 7.363 nm.

[0009] The present invention also provides a preparation method of the matrine-loaded biomimetic nanovesicle according to the above technical solution, including the following steps:

[0010] Co-culture skin fibroblasts with a cell culture medium solution containing matrine to obtain co-cultured skin fibroblasts;

[0011] Digest and centrifuge the co-cultured skin fibroblasts with trypsin, and resuspend the obtained precipitate in a PBS buffer solution containing matrine to obtain a cell suspension;

[0012] Perform extrusion treatment on the cell suspension by the gradient extrusion method to obtain the matrine-loaded biomimetic nanovesicles.

[0013] Preferably, the concentration of matrine in the cell culture medium solution containing matrine is 5-500 μg / mL; the concentration of matrine in the PBS buffer solution containing matrine is 5-500 μg / mL.

[0014] Preferably, the PBS buffer solution containing matrine further contains trehalose with a mass concentration of 5%.

[0015] Preferably, the extrusion treatment includes: repeatedly extruding the cell suspension through polycarbonate membranes with pore sizes of 1.2 μm, 0.6 μm, and 0.22 μm in sequence.

[0016] Preferably, the number of repeated extrusions is 1-5 times.

[0017] Preferably, the co-culture time is 4 h; after the extrusion treatment, it further includes centrifuging and washing the obtained vesicles, and the rotation speed of the centrifugation is (2-10)×10 4 g, and the time is 10-70 min.

[0018] The present invention also provides the use of the matrine-loaded biomimetic nanovesicles described in the above technical solution or the matrine-loaded biomimetic nanovesicles prepared by the preparation method described in the above technical solution in the preparation of anti-inflammatory and / or wound healing-promoting drugs.

[0019] The present invention also provides an anti-inflammatory and / or wound healing-promoting drug, and the active ingredient of the drug includes the matrine-loaded biomimetic nanovesicles described in the above technical solution or the matrine-loaded biomimetic nanovesicles prepared by the preparation method described in the above technical solution.

[0020] Beneficial effects:

[0021] The present invention provides a matrine-loaded biomimetic nanovesicle, which comprises a human skin fibroblast vesicle and matrine encapsulated in the lumen of the skin fibroblast vesicle. The skin fibroblast vesicle in the present invention contains genetic materials and cytokines in the fibroblast vesicle, and can interact with various cells such as keratinocytes and monocytes, and jointly promote the wound healing process through multiple mechanisms; matrine is an active ingredient of Sophora flavescens, which has anti-inflammatory and anti-fibrotic effects. The present invention uses matrine to regulate the inflammatory environment near the wound, avoid excessive damage to tissues caused by inflammation, and regulate scar formation at the same time. Then, the matrine-loaded biomimetic nanovesicle obtained by encapsulating matrine with the skin fibroblast vesicle, the strong anti-inflammatory effect of matrine inhibits the oxidative stress level near the wound surface, and the skin fibroblast vesicle inhibits the oxidative stress level near the wound surface. The two work synergistically to effectively promote wound healing. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0023] Figure 1 Schematic diagram of the preparation process of the matrine-loaded biomimetic nanovesicle in Example 1;

[0024] Figure 2 Transmission electron micrograph of the matrine-loaded biomimetic nanovesicle in Example 1;

[0025] Figure 3 High performance liquid analysis of matrine contained in the matrine-loaded biomimetic nanovesicle in Example 1;

[0026] Figure 4 Analysis of the uptake of drug-loaded vesicles by human skin fibroblasts in Example 1;

[0027] Figure 5 Cell viability graphs of fibroblasts and keratinocytes at different drug concentrations in Example 1;

[0028] Figure 6 Analysis of the promotion of fibroblast and keratinocyte migration by the matrine-loaded biomimetic nanovesicle in Example 1;

[0029] Figure 7 Fluorescence field view of the matrine-loaded biomimetic nanovesicle alleviating the oxidative stress level in Example 1;

[0030] Figure 8 Bright field view of the promotion of vascular endothelial cell tube formation by the matrine-loaded biomimetic nanovesicle in Example 1;

[0031] Figure 9Western blot analysis results of matrine-loaded biomimetic nanovesicles promoting type I collagen and transforming growth factor-β expression in fibroblasts in Example 1;

[0032] Figure 10 Wound healing promoted by matrine-loaded biomimetic nanovesicles in mice in Example 1;

[0033] Figure 11 Weight changes in mice caused by matrine-loaded biomimetic nanovesicles in Example 1. Detailed implementation manners

[0034] The present invention provides a matrine-loaded biomimetic nanovesicle, which includes a skin fibroblast vesicle and matrine encapsulated in the inner cavity of the skin fibroblast vesicle.

[0035] In the present invention, the encapsulation efficiency of matrine in the matrine-loaded biomimetic nanovesicle is preferably 77 ± 2.59%; the shape of the matrine-loaded biomimetic nanovesicle is preferably a vacuolar membrane shape, and the particle size is preferably 245.6 ± 7.363 nm. The skin fibroblast vesicle of the present invention preferably includes a human skin fibroblast vesicle.

[0036] The present invention also provides a preparation method of the matrine-loaded biomimetic nanovesicle described in the above technical solution, including the following steps:

[0037] Co-culture skin fibroblasts with a cell culture medium solution containing matrine to obtain co-cultured skin fibroblasts;

[0038] Digest and centrifuge the co-cultured skin fibroblasts with trypsin, and resuspend the obtained precipitate in a PBS buffer solution containing matrine to obtain a cell resuspension;

[0039] Perform extrusion treatment on the cell resuspension by gradient extrusion to obtain the matrine-loaded biomimetic nanovesicle.

[0040] In the present invention, skin fibroblasts are co-cultured with a cell culture medium solution containing matrine to obtain co-cultured skin fibroblasts. The skin fibroblast vesicles in the present invention preferably include human skin fibroblast vesicles. There are no special limitations on the sources of the skin fibroblasts and matrine in the present invention, and they can be purchased through conventional commercial channels in the art. The concentration of matrine in the cell culture medium solution containing matrine in the present invention is preferably 5 - 500 μg / mL, more preferably 100 - 400 μg / mL, and even more preferably 200 - 300 μg / mL; the cell culture medium preferably includes 89% high-glucose DMEM medium, 10% fetal bovine serum, and 1% double antibody; the double antibody preferably includes penicillin and streptomycin, and the mass ratio of penicillin to streptomycin is preferably 1:1. The co-culture time in the present invention is preferably 1 - 5 h, more preferably 4 h; there are no special limitations on other conditions for the co-culture, and it can be carried out according to the conventional culture conditions of human skin fibroblasts in the art.

[0041] After obtaining the co-cultured skin fibroblasts, in the present invention, the co-cultured skin fibroblasts are digested and centrifuged with trypsin. The digestion temperature in the present invention is preferably 37°C, and the skin fibroblasts are digested for 5 min with trypsin evenly covering them; there are no special limitations on the source of the trypsin, and conventional trypsin in the art can be used. There are no special limitations on the centrifugation conditions in the present invention, as long as the precipitated human skin fibroblasts after digestion are obtained.

[0042] After centrifugation, in the present invention, the precipitated digested skin fibroblasts are resuspended in a PBS buffer solution containing matrine to obtain a cell resuspension. The concentration of matrine in the PBS buffer solution containing matrine in the present invention is preferably 5 - 500 μg / mL, more preferably 100 - 400 μg / mL, and even more preferably 200 - 300 μg / mL. The PBS buffer solution containing matrine in the present invention preferably further includes trehalose with a mass concentration of 5%; the trehalose can protect the integrity of cell vesicles.

[0043] After obtaining the cell resuspension, in the present invention, the cell resuspension is subjected to extrusion treatment by the gradient extrusion method. The extrusion treatment in the present invention preferably includes: repeatedly extruding the cell resuspension through polycarbonate membranes with pore sizes of 1.2 μm, 0.6 μm, and 0.22 μm in sequence. The polycarbonate membranes in this order facilitate extruding cells into nano-vesicles in a gradient manner. The number of cycles of repeatedly extruding through the polycarbonate membranes with pore sizes of 1.2 μm, 0.6 μm, and 0.22 μm in sequence is preferably 1 - 5 times, more preferably 3 - 4 times, and even more preferably 3 times. The extrusion treatment in the present invention is preferably carried out using a pneumatic extruder or a high-pressure extruder.

[0044] After the extrusion treatment, the present invention preferably further includes centrifuging and washing the vesicles obtained by extrusion to obtain the matrine-loaded biomimetic nanovesicles. The rotation speed of the centrifugation in the present invention is preferably (2-10)×10 4 g, more preferably (4-10)×10 4 g; the centrifugation time is preferably 10-70 min, more preferably 50-70 min. The present invention preferably uses PBS solution for the washing, and the number of times of centrifugation and washing is preferably 2 times.

[0045] The present invention uses the gradient extrusion method to prepare the matrine-loaded biomimetic nanovesicles, which can load matrine into the vesicles while generating cell vesicles; at the same time, the gradient extrusion method can more efficiently generate fibroblast vesicles. Although its function is similar to that of exosomes, its yield is much higher than that of exosomes.

[0046] The mechanism of the present invention is as follows: The matrine-loaded nano-bionic vesicles prepared by the method of the present invention have the functions of regulating the synthesis and remodeling of the extracellular matrix scaffold (i.e., collagen) such as the genetic material and growth factors of fibroblasts, and the regeneration of the skin barrier by keratinocytes. This synergistically promotes wound healing with the anti-inflammatory effect of matrine. The present invention uses fibroblasts as the raw material for vesicles, provides exogenous fibroblast biofunctional molecules for the wound, improves the viability of fibroblasts while encapsulating matrine, and matrine is used for wound anti-inflammatory and regulating wound scar formation, ultimately rapidly and effectively accelerating wound healing.

[0047] Based on the above advantages, the present invention also provides the application of the matrine-loaded biomimetic nanovesicles described in the above technical solution or the matrine-loaded biomimetic nanovesicles prepared by the preparation method described in the above technical solution in the preparation of anti-inflammatory and / or wound healing drugs, preferably in the preparation of anti-inflammatory and wound healing drugs.

[0048] The present invention also provides an anti-inflammatory and / or wound healing drug, and the active ingredient of the drug includes the matrine-loaded biomimetic nanovesicles described in the above technical solution or the matrine-loaded biomimetic nanovesicles prepared by the preparation method described in the above technical solution. The present invention has no special limitation on the content of the matrine-loaded biomimetic nanovesicles in the drug, and various specifications of drugs containing the matrine-loaded biomimetic nanovesicles prepared as needed belong to the protection scope of the present invention.

[0049] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] Example 1

[0051] A preparation method of matrine-loaded biomimetic nanovesicles comprises the following steps:

[0052] 1) Preparation of matrine-loaded biomimetic nanovesicles: Human skin fibroblasts (HSF cells) are co-cultured with a culture medium solution containing 200 μg / mL matrine (the culture medium solution is 89% DMEM high-glucose medium, 10% fetal bovine serum, and 1% double antibody) for 4 h. Then, the HSF cells are digested with trypsin and resuspended in PBS buffer (containing 5 wt.% trehalose) containing 200 μg / mL matrine. The cell suspension is repeatedly extruded 1 - 5 times through a pneumatic or high-pressure extruder equipped with 1.2 μm, 0.6 μm, and 0.22 μm polycarbonate membranes. The obtained matrine-loaded fibroblast-derived vesicles (MHEM) are centrifuged at (2 - 10)×10 4 g for 10 - 70 min and washed to finally obtain matrine-loaded biomimetic nanovesicles. The schematic diagram of the preparation process of matrine-loaded biomimetic nanovesicles is as shown in Figure 1 Figure.

[0053] The transmission electron microscope image of MHEM prepared in Example 1 is as shown in Figure 2 Figure, Figure 2 and the scale bar in Figure 2 is 100 nm. It can be concluded from

[0054] that the morphology of the prepared vesicles is vacuolar membrane-like, and the particle size is about 245 nm. Qualitative analysis of matrine in the drug-loaded vesicles is carried out by high-performance liquid chromatography: The drug-loaded vesicles are resuspended in methanol and subjected to repeated freezing and thawing, and the obtained product is analyzed by high-performance liquid chromatography. The specific conditions are as follows: The mobile phase is acetonitrile - K2HPO4 solution (0.05 mol / L), the volume ratio of acetonitrile to potassium phosphate solution is 25:75, the flow rate is 1.0 mL / min, and the detection wavelength is 220 nm. The specification of the chromatographic column is 5 μm, 4.6×250 mm (from Agilent). A matrine standard product (from Solarbio) is used as a control.

[0055] The results are as shown in Figure 3 Figure. The peak time of matrine in the drug-loaded vesicles is the same as that of the standard product, confirming that matrine is successfully carried in the vesicles. Through detection, it is found that the encapsulation efficiency of matrine in the drug-loaded vesicles is 77 ± 2.59%.

[0056] 2) Seed the HSF cells in a Millicell EZ SLIDE 8-well culture plate (from Merck) overnight. The culture medium consists of 90% high-glucose DMEM medium + 10% fetal bovine serum. Then replace the medium with fresh medium containing MHEM stained with DiI (from Beyotime) (containing 10% fetal bovine serum and 1% double antibody, the same medium containing serum + double antibody is briefly described below). The protein concentration of the stained MHEM in the medium is 162 μg / mL, and incubate for 4 hours. Subsequently, wash the cells twice with PBS and incubate with Lyso-Tracker Green (from Beyotime) for 40 min. Finally, wash, fix, DAPI stain the cell samples, and image them with a confocal scanning microscope.

[0057] The results are as Figure 4 shown, Figure 4 The scale bar in the figure is 100 μm. MHEM aggregates on the cytoplasm and cell membrane, indicating that the MHEM aggregated in the cytoplasm has been taken up by the cells into the intracellular space, and the MHEM attached to the cell membrane will be released into the cell interior by binding to the cell membrane subsequently. This phenomenon indicates that the cells have successfully taken up and processed MHEM. In addition, considering that MHEM is composed of membranes, these nanovesicles have adhesion proteins homologous to fibroblasts, so they can target and bind homologously to fibroblasts. After MHEM fuses with the cells, its contents are released into the cells.

[0058] 3) Preparation of fibroblast vesicles (EM) without carrying matrine: Resuspend the human skin fibroblasts (HSF cells) digested with trypsin in PBS. Repeat extruding the cell suspension 1 - 5 times through a pneumatic or high-pressure extruder equipped with 1.2 μm, 0.6 μm, and 0.22 μm polycarbonate membranes. Centrifuge the obtained fibroblast-derived vesicles (EM) at (2 - 10)×10 4 g for 10 - 70 min and wash them to finally obtain fibroblast vesicles (EM) without carrying matrine.

[0059] 4) To verify the effects of MHEM and EM at different concentrations (using the BCA method to measure with the membrane protein concentration of MHEM and EM as the unified standard) on the viability of HSF and HaCat cells, the present invention adds the corresponding concentration of MHEM and EM solutions to 96-well plates containing HSF and HaCat (5×10 3 cells / well) respectively. After 24 h, add a medium containing 10% cytotoxicity test reagent (CCK8) (containing serum + double antibody) in the form of changing the medium for toxicity testing. The results are as Figure 5 shown.

[0060] When the protein concentration reaches 162 μg / mL, the cell proliferation ability is significantly enhanced; MHEM has a stronger effect on promoting cell proliferation; the decline in fibroblast viability is a factor in delayed wound healing. Therefore, increasing fibroblast viability can further promote wound healing.

[0061] 5) To detect the promoting effect of MHEM on the migration of fibroblasts and keratinocytes, the present invention uses a scratch assay, that is, HSF cells and HaCat cells are cultured in a 6-well plate at a density of 2×10 5 cells per well and cultured overnight. Then, the adherent cells in the well plate are scratched crosswise with a sterile 200 μL pipette tip. In an environment of 37 °C and 5% CO2, the cells are gently rinsed twice with PBS, and matrine, EM, and MHEM and blank PBS buffer are co-incubated with the cells, where the concentration of EM and MHEM is 162 μg / mL membrane protein concentration, and the concentration of matrine is 20 μg / mL. At 0 h, 12 h, and 24 h, the process of cell scratch healing is photographed.

[0062] The results are as Figure 6 shown, Figure 6 The scale bar length in the figure is 100 μm. After co-incubation for 12 h, matrine significantly promoted the migration of HSF cells, and the ability of MHEM to promote the migration of HSF was significantly stronger than that of EM. After co-incubation for 24 h, the scratches in the matrine group, EM group, and MHEM group were basically healed, indicating that matrine, EM, and MHEM have a significant promoting effect on the migration of HSF.

[0063] In addition, the migration ability of HaCat cells internalizing MHEM was significantly stronger than that of other groups. Although there was no significant difference between the matrine group and the PBS group in promoting the migration of HaCat cells, the effect of MHEM in promoting the migration of HaCat cells was more obvious than that of simple EM. This indicates that the synergistic mechanism of matrine and EM enhances the migration ability of HaCat cells, further revealing the potential of MHEM as a wound healing treatment option.

[0064] 6) To detect the alleviating effect of MHEM on the level of oxidative stress, the present invention uses M1 macrophages as an inflammation model for verification. RAW264.7 cells are treated with IFN-γ (final concentration: 2 ng / mL -1 ) and LPS (final concentration: 0.5 μg / mL -1 ) simultaneously for 24 h to induce macrophage polarization into the M1 type. Then, the M1 macrophages are incubated with DMEM containing matrine or MHEM for 4 h. To observe the presence of intracellular reactive oxygen species (ROS), the cells are stained with a ROS probe (from Beyotime), the cell samples are washed with PBS, and then fixed and stained with DAPI. The cells are observed under a fluorescence microscope.

[0065] The results are as Figure 7 shown, Figure 7 in which the scale bar is 100 μm. After M1 macrophages were co-incubated with matrine and ROS probe, the fluorescence intensity of the ROS probe decreased. Matrine effectively alleviated oxidative stress by inhibiting the pro-inflammatory function of M1 macrophages. In addition, when M1 macrophages were co-incubated with MHEM, the ROS levels in these macrophages were significantly reduced. Compared with matrine, MHEM had a stronger ROS inhibitory effect. Therefore, MHEM promoted the resolution of the inflammatory response and accelerated the transition of wound healing.

[0066] 7) To detect the effect of MHEM on promoting angiogenesis, the wells in a 96-well plate were pre-coated with Matrigel (from Corning). Human umbilical vein endothelial cells (from Yuan Chuang) were dispersed in a medium containing the corresponding drug (serum + double antibody), and seeded into the 96-well plate at a density of 2×10 5 cells per well and cultured for another 4 h. Then, the cells were observed using a microscope; the medium containing the corresponding drug (serum + double antibody) was PBS (accounting for 10% of the medium volume), matrine (20 μg / mL), vesicles (162 μg / mL), and matrine-containing cell vesicles (162 μg / mL), respectively.

[0067] The results are as Figure 8 shown, Figure 8 in which the scale bar is 100 μm. After human umbilical vein endothelial cells were co-incubated with vesicles or matrine for 4 h, tube formation was observed. When human umbilical vein endothelial cells were co-incubated with MHEM, the lumen structure and morphology were clearer, indicating that MHEM had a better effect on promoting angiogenesis than matrine or EM.

[0068] 8) Next, to verify whether MHEM can promote the expression of collagen and transforming growth factor in fibroblasts, the present invention adopted the Western blot method. The specific operation is as follows: After the HSF cells were co-cultured with the corresponding drug (the corresponding drug in step 6) for 24 h; the cells were digested, and proteins in the cells were extracted using cold RIPA buffer (from Biotech company) containing protease inhibitor mixture. Subsequently, the proteins were separated by 15% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (from Millipore company). After the membrane was blocked with 5% non-fat milk for 1 hour, the membrane was incubated overnight at 4 °C in the blocking solution with primary antibodies against GAPDH, type I collagen (COL-I), and transforming growth factor β (TGF-β) (from Abcam company). After thorough washing, the membrane was incubated with anti-rabbit or anti-mouse secondary antibody (from Abcam company) for 1 hour. Finally, a chemiluminescence kit (from Biotech company) was used to detect the proteins on the membrane, and a ChemiDoc MP imaging system (from BioRad company) was used to observe the intensity of the bands.

[0069] The results of Western blot detection are as Figure 9 shown. MHEM enhanced the expression of COL-I and TGF-β in HSF. COL-I and TGF-β are proteins and growth factors related to the wound healing process, and they play crucial roles in the physiology and pathology of wound healing. TGF-β is a transforming growth factor that regulates various cell functions during wound healing, including epithelialization, fibroblast migration, collagen synthesis and maturation, and extracellular matrix deposition. In addition, the collagen produced by fibroblasts, especially mature type I collagen (COL-I), helps to enhance tissue strength and helps to pull the wound tissue edges towards the center. The above results indicate that MHEM has the potential to promote granulation tissue proliferation, thereby accelerating wound healing.

[0070] To further verify the effect of MHEM on promoting wound healing in vivo and study the performance and mechanism of MHEM on promoting wound healing in vivo, the present invention made a full-thickness excision wound with a diameter of about 1.5 cm on the back of mice. MHEM, matrine, EM and other drugs were resuspended in PBS, and were injected subcutaneously around the mouse wounds at a dose corresponding to 5 mg / kg of membrane protein and 0.62 mg / kg of matrine. Thereafter, it was repeated every 1 day for a total of 3 times, and the wound size was recorded every other day.

[0071] The results are as Figure 10As shown in Table 1, compared with the PBS group, the wounds in the MHEM group and the EM group gradually healed starting from the 4th day. Starting from the 6th day, matrine promoted wound healing. On the 8th day, obvious wound margin contraction occurred in the mice treated with matrine and EM, and the wounds in the MHEM group completely healed on the 8th day, indicating that matrine and EM synergistically promoted wound healing.

[0072] Table 1 Wound healing rates of mice in each group during treatment

[0073]

[0074] During the wound healing process, we recorded the body weight changes of the mice every other day. The results are as Figure 11 shown. Subcutaneous injection of the vesicles did not cause any significant changes in the body weight of the mice, confirming the biosafety of MHEM.

[0075] It can be concluded from the above examples that the matrine-loaded biomimetic nanovesicles of the present invention contain human fibroblast genetic material and growth factors, and can effectively promote wound healing synergistically with the anti-inflammatory effect of matrine.

[0076] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A matrine-loaded biomimetic nanovesicle, characterized in that, The matrine-loaded biomimetic nanovesicles comprise dermal fibroblast vesicles and matrine encapsulated in the lumen of the dermal fibroblast vesicles.

2. The biomimetic nanovesicles loaded with matrine according to claim 1, wherein, The encapsulation efficiency of matrine in the matrine-loaded biomimetic nanovesicles is 77 ± 2.59%; The particle size of the matrine-loaded nanovesicles is 245.6 ± 7.363 nm.

3. The preparation method of the matrine-loaded biomimetic nanovesicles according to claim 1 or 2, characterized in that, It includes the following steps: Co-culturing dermal fibroblasts with a cell culture medium solution containing matrine to obtain co-cultured dermal fibroblasts; Digesting and centrifuging the co-cultured dermal fibroblasts with trypsin, and resuspending the obtained precipitate in a PBS buffer solution containing matrine to obtain a cell resuspension; Subjecting the cell resuspension to extrusion treatment by the gradient extrusion method to obtain the matrine-loaded biomimetic nanovesicles.

4. The preparation method according to claim 3, wherein, The concentration of matrine in the cell culture medium solution containing matrine is 5 - 500 μg / mL; the concentration of matrine in the PBS buffer solution containing matrine is 5 - 500 μg / mL.

5. According to the preparation method described in claim 3 or 4, the PBS buffer solution containing matrine further contains trehalose with a mass concentration of 5%.

6. The preparation method according to claim 3, characterized in that, The extrusion treatment includes: repeatedly extruding the cell resuspension through polycarbonate membranes with pore sizes of 1.2 μm, 0.6 μm, and 0.22 μm in sequence.

7. The preparation method according to claim 6, wherein The number of times of repeated extrusion is 1 - 5 times.

8. The preparation method according to claim 4, characterized in that, The time of the co-culture is 4 h; after the extrusion treatment, centrifugation and washing of the obtained vesicles are further included, and the rotation speed of the centrifugation is (2-10)×10 4 g, and the time is 10-70 min.

9. Use of the matrine-loaded biomimetic nanovesicles described in claim 1 or 2 or the matrine-loaded biomimetic nanovesicles prepared by the preparation method described in any one of claims 3 - 8 in the preparation of anti-inflammatory and / or wound healing-promoting drugs.

10. A drug for anti - inflammation and / or promoting wound healing, characterized in that, The active ingredient of the drug includes the matrine-loaded biomimetic nanovesicles described in claim 1 or 2 or the matrine-loaded biomimetic nanovesicles prepared by the preparation method described in any one of claims 3 - 8.

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