Schisandrin B-loaded nano-vesicle and functional hydrogel as well as preparation method and application of schizandrin B-loaded nano-vesicle and functional hydrogel

Abstract

The invention provides a schisandrin B-loaded nano-vesicle and functional hydrogel as well as a preparation method and application thereof, and belongs to the technical field of medical dressings. The preparation method comprises the following steps: blending human umbilical cord mesenchymal stem cells with schisandrin B, and carrying out gradient extrusion through a microfiltration membrane to obtain the schisandrin B-loaded nano-vesicles. The engineered stem cell-derived nanovesicles prepared by the invention can accelerate angiogenesis and promote liver cell proliferation, thereby promoting injury repair after hepatic resection. As a novel visceral wound dressing, the functional hydrogel provided by the invention not only has better biocompatibility, but also can adsorb blood cells and activate a blood coagulation cascade system so as to achieve a hemostatic effect.

Description

Technical Field

[0001] The present invention relates to the technical field of medical dressings, and particularly relates to a nano-vesicle loaded with schisandrin B, a functional hydrogel, and a preparation method and application thereof. Background Art

[0002] Partial hepatectomy is a key surgical procedure widely used in the treatment of liver diseases, including liver cancer, liver transplantation donor acquisition, and other liver-related diseases. Although the liver has remarkable regenerative ability, enabling the remaining hepatocytes to proliferate and restore the mass and function of the liver, postoperative complications such as bleeding and wound infection are still major problems, leading to increased morbidity and mortality. After partial hepatectomy, the ability of the liver to regenerate rapidly is crucial for postoperative recovery, as insufficient liver regeneration may lead to liver failure and even death. In addition, small-for-size syndrome (SFSS), a complication related to liver transplantation, occurs when the graft is too small to meet the metabolic needs of the recipient, resulting in postoperative liver dysfunction and a significant reduction in survival rate.

[0003] The regeneration process of the liver is affected by many factors, such as immune response, cytokine release, oxidative stress, and the overall postoperative environment. Currently, in clinical practice, there is a lack of treatment options or drugs that can properly address such problems. Traditional liver wound dressings such as gelatin sponges play a physical barrier role in healing, providing certain hemostatic and protective functions, but they cannot provide bioactive substances to promote liver repair, have a potential risk of immune response, and cannot effectively prevent microbial infection. In addition, traditional dressings have poor plasticity and are difficult to fit complex liver wounds, so their efficacy in the treatment after hepatectomy is not good. Therefore, in clinical practice, to solve these problems, it mainly relies on precise electrocoagulation hemostasis and the widespread use of antibiotics, which inevitably causes hepatocyte death caused by burning and antibiotic-resistant infections. Therefore, there is an urgent need to develop a multifunctional injectable wound dressing to address the main problems such as hemostasis control, infection prevention, and promotion of liver regeneration.

[0004] Due to their ability to mediate cell - cell communication and regulate immune responses, stem - cell - derived exosomes have emerged as promising therapeutic tools in the field of liver disease treatment. They promote liver regeneration and repair by delivering bioactive molecules such as proteins and microRNAs to target cells, thereby reducing inflammation and fibrosis. However, exosome - based liver disease therapies are restricted in clinical translation by factors such as high production costs, low yields, and large inter - batch variations. In contrast, engineered stem - cell - derived nanovesicles (NVs) have advantages such as high yields, low costs, and good inter - batch quality consistency, making them a potentially promising therapeutic alternative. In addition, this novel nanovesicle - based therapy has shown remarkable progress in the treatment of neurological disorders and wound healing. With this technology, we aim to develop a novel postoperative strategy to effectively promote the regeneration and functional recovery of liver tissue after hepatectomy.

[0005] Engineered stem - cell - derived nanovesicles are natural lipid - bilayer vesicles with excellent biocompatibility and stability, making them ideal drug - delivery systems. Schisandrin B is a lignan compound derived from Schisandra chinensis and has been extensively studied for its therapeutic properties, especially in the application of liver diseases. As a potent bioactive molecule, Schisandrin B has shown significant hepatoprotective effects through its antioxidant, anti - inflammatory, and antifibrotic actions. In addition, studies have shown that Schisandrin B can promote hepatocyte regeneration and reduce hepatocyte apoptosis, thus promoting the recovery after liver injury.

[0006] Hydrogels have become ideal drug - delivery dressings due to their excellent biomimetic structure, biocompatibility, and drug - controlled release properties. Gelatin methacrylate (GelMA) is a photocrosslinkable material prepared by introducing methacryloyl groups into gelatin molecules. Under the action of a photoinitiator and ultraviolet light, it can rapidly crosslink to form a gel structure, showing excellent biocompatibility and degradability. CS - CA is chitosan (CS) modified by dihydrocaffeic acid (CA), which has strong tissue adhesiveness and endows it with antibacterial, antioxidant, and independent hemostatic functions, and is widely used in antibacterial hemostatic dressings. In addition, the positively charged amino groups on chitosan can interact with the negatively charged phospholipid membrane of cell vesicles through electrostatic adsorption, prolonging the drug - loading time and stability. The strontium ions in strontium chloride can bind to the phenolic hydroxyl groups on CS - CA to form a coordination network, enhancing the strength of the hydrogel. In addition, the addition of strontium also endows the hydrogel with additional antibacterial and tissue - repair functions. Summary of the Invention

[0007] The object of the present invention is to improve the bioavailability of schisandrin B, enhance its therapeutic effect and reduce side effects. For this purpose, the present invention has developed a functional hydrogel containing nanovesicles loaded with schisandrin B, its preparation method and application. The functional hydrogel has good biocompatibility and biological activity, can effectively stop bleeding, antibacterial and promote liver regeneration, and shows broad application potential in visceral wound repair. The use of a functional hydrogel dressing containing nanovesicles provides a more efficient and controllable solution for the repair of liver resection injury.

[0008] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing nanovesicles loaded with schisandrin B. After mixing human umbilical cord mesenchymal stem cells with schisandrin B, they are extruded through a microporous membrane gradient to obtain nanovesicles loaded with schisandrin B.

[0010] Preferably, the human umbilical cord mesenchymal stem cells are cells at passage 2-3. After being cultured in MSCM medium containing 10% FBS until the cell fusion rate reaches 85%-95%, they are digested, and the cells are collected and resuspended in phosphate buffer containing schisandrin B.

[0011] Preferably, the density of human umbilical cord mesenchymal stem cells in the mixed system is 1.0×10 6 ~6.0×10 7 cells / mL.

[0012] Preferably, the concentration of schisandrin in the mixed system is 200 μM.

[0013] Preferably, the pore sizes of the microporous membrane gradient are 3-5 μm, 1.0-1.2 μm and 0.2-0.4 μm in sequence; the number of extrusion repetitions is 3-5 times.

[0014] The present invention also provides nanovesicles loaded with schisandrin B obtained by the above preparation method.

[0015] The present invention also provides a method for preparing a functional hydrogel containing the nanovesicles loaded with schisandrin B. Methacrylated gelatin and chitosan modified with dihydrocaffeic acid are dissolved in phosphate buffer containing LAP photoinitiator, and then strontium chloride and the nanovesicles loaded with schisandrin B are added to obtain a prepolymer solution of the multifunctional hydrogel. The prepolymer solution is irradiated with ultraviolet light at 405 nm for 10-30 seconds for crosslinking to obtain the functional hydrogel.

[0016] Preferably, in the prepolymer solution, the mass-volume concentration of methacrylic anhydride-modified gelatin is 3-10%, the mass-volume concentration of dihydrocaffeic acid-modified chitosan is 1-5%, the mass-volume concentration of LAP photoinitiator is 2-3 mg / mL, the concentration of strontium chloride is 5-20 mM, and the concentration of nanovesicles loaded with schisandrin B is 100-500 μg / mL.

[0017] The present invention also provides a functional hydrogel obtained by the preparation method described above.

[0018] The present invention also provides the application of the nanovesicles loaded with schisandrin B or the functional hydrogel described above in the preparation of liver wound dressings.

[0019] The present invention relates to a method for preparing nanovesicles loaded with schisandrin B and a functional hydrogel and their applications. Research shows that the engineered stem cell-derived nanovesicles prepared in the present invention have a rich yield, an efficient preparation process, can achieve large-scale production, and have greater potential for transformation. The schisandrin B loaded in the vesicles can effectively prevent the degradation or inactivation of the drug during transportation, thereby improving the effectiveness of schisandrin B. Moreover, the nanoscale size of the vesicles helps them to be absorbed by cells, enhancing the effect of schisandrin B on target cells. In terms of application effects, the engineered stem cell-derived nanovesicles prepared in the present invention can accelerate angiogenesis and promote hepatocyte proliferation, thus promoting the repair of liver resection-induced injury. The experimental results show that the engineered stem cell-derived nanovesicles of the present invention can significantly promote the migration of vascular endothelial cells, simultaneously promote hepatocyte proliferation, inhibit oxidative stress injury and apoptosis of hepatocytes, effectively regulate the liver regeneration microenvironment, and demonstrate excellent tissue repair and anti-injury potential, providing a novel and highly potential clinical solution for the treatment of liver wounds.

[0020] In addition, the engineered stem cell-derived nanovesicles provide an ideal carrier for the delivery of schisandrin B, with an encapsulation efficiency of schisandrin B of over 90%, significantly improving its bioavailability and reducing the potential side effects of schisandrin B. The nanovesicles loaded with schisandrin B ensure that the controlled release process provides a continuous therapeutic concentration and effect at the liver injury site, playing a role in promoting hepatocyte proliferation and inhibiting oxidative damage of hepatocytes, thereby promoting the best liver regeneration efficacy.

[0021] The functional hydrogel provided by the present invention, as a novel visceral wound dressing, not only has good biocompatibility, but also can adsorb blood cells and activate the coagulation cascade system to achieve a hemostatic effect. The functional hydrogel provided by the present invention is a glycopeptide functional hydrogel that combines GelMA, CS-CA, strontium chloride, schisandrin B, and engineered stem cell-derived nanovesicles, and the loaded chitosan and strontium chloride also have a certain function of preventing microbial infection. The functional hydrogel, as a matrix carrier for controllably releasing nanovesicles loaded with schisandrin B, provides a powerful liver injury repair effect for the irregular and complex wounds after liver resection, and shows broad application potential in visceral wound repair. It can become an alternative choice for clinical liver resection dressings. Therefore, the present invention provides a novel and highly potential clinical solution.

[0022] The present invention innovatively applies a multifunctional hydrogel loaded with schisandrin nanovesicles to the wound treatment of partial hepatectomy. This multifunctional hydrogel has direct and efficient characteristics, can significantly promote the wound healing process of partial hepatectomy, effectively stop bleeding, promote hepatocyte regeneration, prevent hepatocyte apoptosis, and prevent postoperative microbial infection. Through this innovative application, the present invention provides a new and more potential solution for the field of wound treatment of partial hepatectomy.

[0023] The present invention also provides the application of the engineered nanovesicles loaded with schisandrin B described in the above technical solution or the functional hydrogel described in the above technical solution in the preparation of drugs for liver wound healing. In the present invention, the liver wound includes the liver wound surface generated after partial hepatectomy due to liver diseases of various etiologies or the acquisition of living donor livers, and is characterized by complex and irregular wound surfaces, continuous and hidden bleeding, potential possibility of peritoneal infection, hepatocyte regeneration disorder, and delayed liver function recovery, etc. Description of the Drawings

[0024] Figure 1 It is a high-performance liquid chromatography result diagram for detecting the encapsulation efficiency of schisandrin B in the nanovesicles loaded with schisandrin B obtained in Example 1;

[0025] Figure 2 It is a transmission electron microscope imaging diagram of the nanovesicles loaded with schisandrin B obtained in Example 1 (the scale bar in the figure is 100 nm);

[0026] Figure 3 It is a particle size distribution diagram of the nanovesicles loaded with schisandrin B obtained in Example 1;

[0027] Figure 4 It is a polyacrylamide gel electrophoresis detection result diagram obtained in Example 1;

[0028] Figure 5Result graph of nano-vesicles loaded with schisandrin B promoting the proliferation of AML12 mouse hepatocytes in Example 2;

[0029] Figure 6 Result graph of nano-vesicles loaded with schisandrin B promoting the migration of HUVEC human umbilical vein endothelial cells in Example 2;

[0030] Figure 7 Fluorescence result graph of nano-vesicles loaded with schisandrin B taken up by AML12 mouse hepatocytes;

[0031] Figure 8 Result graph of nano-vesicles loaded with schisandrin B inhibiting apoptosis of AML12 mouse hepatocytes caused by oxidative stress in Example 2;

[0032] Figure 9 UV spectrum characterization graph of CS-CA provided in Example 3 of the present invention;

[0033] Figure 10 Scanning electron microscope imaging graph of the functional hydrogel provided in Example 3 of the present invention;

[0034] Figure 11 Rate graph of the functional hydrogel provided in Example 3 of the present invention releasing nano-vesicles loaded with schisandrin B;

[0035] Figure 12 Bacterial spread plate result graph of the functional hydrogel provided in Example 3 of the present invention inhibiting bacteria;

[0036] Figure 13 Hemostasis result graph of the functional hydrogel provided in Example 3 of the present invention for the liver and tail of rats;

[0037] Figure 14 Result graph of the functional hydrogel provided in Example 3 of the present invention promoting liver regeneration after hepatectomy. Detailed implementation manners

[0038] The present invention provides a preparation method of nano-vesicles loaded with schisandrin B. After mixing human umbilical cord mesenchymal stem cells with schisandrin B, they are extruded through a microporous filter membrane gradient to obtain nano-vesicles loaded with schisandrin B.

[0039] In the present invention, the human umbilical cord mesenchymal stem cells are cells at passages 2-3. After being cultured in 10% FBS MSCM medium until the cell fusion rate reaches 85%-95%, they are digested, and the cells are collected and resuspended in phosphate buffer containing schisandrin B.

[0040] In the present invention, schisandrin B is dissolved with a small amount of DMSO and added to PBS to prepare a phosphate buffer containing schisandrin B.

[0041] In the present invention, after the cell fusion rate reaches 85% to 95%, the cells are digested in a gentle manner, centrifuged, the culture medium is discarded, and the prepared phosphate buffer containing schisandrin B is added to fully resuspend the cells.

[0042] In the present invention, in order to obtain nanovesicles of better quality, the MSCM culture medium is preferably a mesenchymal stem cell-specific culture medium with mesenchymal stem cell growth additives; the FBS, MSCM culture medium, and stem cell growth additives used in the present invention are of the Science Cell brand.

[0043] In the present invention, the stem cell growth additive is 5 ml of mesenchymal stem cell growth factor, which is mesenchymal stem cell growth factor (MSCGS, Cat. #7552) matched with the MSCM special culture medium of science cell on the market.

[0044] In the present invention, the density of human umbilical cord mesenchymal stem cells in the mixed system is 1.0×10 6 ~6.0×10 7 Pieces / mL.

[0045] In the present invention, the concentration of Schisandra chinensis in the mixed system is 200 μM.

[0046] In the present invention, the pore sizes of the microporous membrane gradient are 3-5 μm, 1.0-1.2 μm and 0.2-0.4 μm, preferably 5 μm, 1.2 μm and 0.2 μm; each gradient is preferably extruded 3-5 times, preferably 4 times.

[0047] The main advantages of vesicle-loaded Schisandrin B are targeted delivery, extended drug release time, protection of drug stability, improved biocompatibility, and promotion of cell absorption. By using vesicles as carriers, Schisandrin B can be accurately delivered to the site of liver damage, increase local drug concentration, enhance therapeutic effects, and reduce systemic side effects. In addition, the sustained-release properties of the vesicles help maintain stable drug concentrations and promote long-term efficacy. The good biocompatibility of the vesicle material reduces the risk of immune response, while the nanoscale size helps cells absorb the drug, thereby further enhancing the therapeutic effect of Schisandrin B. The present invention, by constructing engineered nanovesicles loaded with Schisandrin B, can not only achieve better therapeutic effects, but also realize a more stable and efficient preparation process.

[0048] The present invention also provides a method for preparing a functional hydrogel containing the nano-vesicles loaded with schisandrin B. Methacrylated gelatin (GelMA, brand: EFL) and dihydrocaffeic acid-modified chitosan (CS-CA) are dissolved in phosphate buffer solution (PBS, pH = 7.4) containing LAP photoinitiator (brand: EFL), and then strontium chloride (SrCl2, brand: Sigma) and the nano-vesicles loaded with schisandrin B are added to obtain a prepolymer solution of the multifunctional hydrogel. The prepolymer solution is placed under a 405 nm ultraviolet lamp for 10 - 30 seconds of light cross-linking to obtain the functional hydrogel.

[0049] In the present invention, the irradiation time of the ultraviolet lamp is preferably 15 - 20 s.

[0050] In the present invention, the preparation method of dihydrocaffeic acid-modified chitosan includes: chitosan (CS, 275.0 mg, 3.25 mmol) is dissolved in 5.0 mL of 1 mol / L HCl solution, and then 45.5 mL of double-distilled water is added; then the pH of the solution is adjusted to 5.0 with 5 mol / L NaOH, and an equal volume of ethanol is added; dihydrocaffeic acid (CA, 295.0 mg, 1.62 mmol) is dissolved in 3.0 mL of double-distilled water, and EDC (128.0 mg, 0.65 mmol) is dissolved in 50.0 mL of double-distilled water; the three solutions are mixed, and the mixture is continuously stirred at room temperature for 12 hours; after the reaction, the solution is purified with HCl acidified double-distilled water (pH 5.0) using a dialysis membrane (cut-off molecular weight 3500 Da) for 2 days, then purified with PBS (pH 7.4) for 4 hours, and finally purified with double-distilled water for 4 hours; the finally obtained CS-CA is freeze-dried and stored in a refrigerator at 4 °C for use.

[0051] In the present invention, the mass-volume concentration of methacrylated gelatin in the prepolymer solution is 3 - 10%, the mass-volume concentration of dihydrocaffeic acid-modified chitosan is 1 - 5%, the mass-volume concentration of LAP photoinitiator is 2 - 3 mg / mL, the concentration of strontium chloride is 5 - 20 mM, and the concentration of the nano-vesicles loaded with schisandrin B is 100 - 500 μg / mL.

[0052] In the present invention, first, a phosphate buffer solution containing LAP photoinitiator is prepared, and then methacrylated gelatin and dihydrocaffeic acid-modified chitosan are added. Among them, the concentration of LAP photoinitiator is 2 - 3 mg / mL, specifically it can be 2.5 mg / mL, and the preparation method is to mix 20 mL of phosphate buffer solution with 0.05 g of photoinitiator LAP evenly. To better dissolve the LAP standard solution of the photoinitiator, a method of heating the LAP standard solution of the photoinitiator in a water bath at 40 - 50 °C for 15 minutes with several oscillations during this period is adopted.

[0053] In the present invention, 1 g of methacrylated gelatin (GelMA) was added to 20 mL of the above-prepared phosphate buffer containing LAP photoinitiator, and dissolved by heating in a water bath at 60 - 70 °C in the dark for 20 - 30 minutes, with several oscillations during this period; subsequently, 400 mg of chitosan modified with dihydrocaffeic acid (CS-CA) was added to 20 mL of the above solution, and stirred and dissolved at 25 - 50 °C in the dark for 1 - 2 hours; then, 31.7 mg of strontium chloride (SrCl2) and 4 mg of engineered nanovesicles loaded with schisandrin B were added to 20 mL of the above solution, and stirred evenly. The setting of the above conditions can better obtain a prepolymer solution of a functional hydrogel with uniform dissolution.

[0054] The functional hydrogel of the present invention has good biocompatibility and does not cause immune rejection reactions. Moreover, the functional hydrogel has the characteristics of direct hemostasis and antibacterial properties, can quickly block the emergency bleeding of wounds, and prevent subsequent microbial infections. At the same time, the functional hydrogel can continuously release engineered nanovesicles loaded with schisandrin B, ensuring the stable efficacy and long-term slow release of schisandrin B, and significantly improving the treatment effect.

[0055] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0056] Example 1

[0057] An engineered nanovesicle loaded with schisandrin B was prepared as follows:

[0058] I. Preparation of phosphate buffer containing schisandrin B

[0059] First, schisandrin B was dissolved with a small amount of DMSO, and the specific ratio was 1 mg of schisandrin B dissolved in 0.02497 ml of DMSO. Then, an appropriate amount of PBS was added to make the concentration of schisandrin B 200 μM, and a phosphate buffer containing schisandrin B was prepared.

[0060] II. Preparation of engineered nanovesicles loaded with schisandrin B (S@NV)

[0061] (1) After resuscitating human umbilical cord mesenchymal stem cells, they were passaged 3 times and then continued to be cultured in MSCM medium containing 10% FBS (containing 5 ml of mesenchymal stem cell growth factor). When the fusion rate of human umbilical cord mesenchymal stem cells reached 85% - 95%, they were digested with trypsin at 37 °C for 5 min, and then centrifuged (1000 rpm, 5 min) to discard the culture medium, and resuspended with the above phosphate buffer (PBS) containing schisandrin B to form a single-cell suspension; at this time, the density of human umbilical cord mesenchymal stem cells was 8.0×10 6 cells / mL.

[0062] (2) Pass the single-cell suspension successively through microporous membranes with pore sizes of 5 μm, 1.2 μm, and 0.2 μm.

[0063] (3) Repeat the operation in step (2) 4 times to obtain nano-vesicles loaded with schisandrin B (S@NV).

[0064] III. Detection of the encapsulation efficiency of schisandrin B in the nano-vesicles loaded with schisandrin B

[0065] Use Agilent HPLC 1260 to measure the encapsulation efficiency of schisandrin B in the nano-vesicles loaded with schisandrin B by an indirect method. Centrifuge the solution containing the nano-vesicles loaded with schisandrin B obtained above at 4 °C and 100,000 g for 70 minutes. Collect the supernatant of the centrifuged solution. At this time, the supernatant contains unencapsulated free schisandrin B. Detect the concentration of schisandrin B in the supernatant on the above high-performance liquid chromatography instrument. The chromatographic conditions are as follows: methanol-water (72:28 / v:v) as the mobile phase, the flow rate is 1.0 mL / min, the detection wavelength is 254 nm, and the column temperature is 30 °C. Continuously analyze the supernatants after centrifugation of the nano-vesicle solutions loaded with schisandrin B prepared in three different batches, and take the average value as the proportion of unencapsulated schisandrin B.

[0066] Figure 1 It shows that, after calculation, the average encapsulation efficiency of schisandrin B is 95.8%.

[0067] IV. Identification of the nano-vesicles loaded with schisandrin B by transmission electron microscopy

[0068] (1) Take 10 μL of the freshly prepared nano-vesicle solution loaded with schisandrin B and drop it on the clean surface of the sealing film.

[0069] (2) Place the copper grid on top of the nano-vesicle droplet loaded with schisandrin B, with the film surface in contact with the droplet, and gently blot it dry with filter paper after suspending for 10 minutes.

[0070] (3) Transfer the copper grid onto the droplet of 3% glutaraldehyde fixative, fix it for 5 minutes, and then blot dry the fixative.

[0071] (4) Then transfer the copper grid to the surface of the water droplet and wash it repeatedly 10 times, suspending for 2 minutes each time and blotting dry the water with filter paper each time.

[0072] (5) Place the copper grid on the surface of the 4% uranyl acetate solution droplet and suspend it for 10 minutes, then blot dry the remaining solution.

[0073] (6) Finally, place the copper grid on the droplet of 1% methyl cellulose, let it stand for 5 minutes, and then blot dry the water.

[0074] (7) The copper mesh was left standing at room temperature for more than 30 min, air-dried, observed under a transmission electron microscope and photographed. The results are as Figure 2 shown.

[0075] Figure 2 The results showed that the isolated nanovesicles loaded with schisandrin B presented a typical round shape.

[0076] V. Detection of the particle size of the nanovesicles loaded with schisandrin B

[0077] The particle size of the nanovesicles loaded with schisandrin B was measured using ZetaVIEW PMX 110. 100 μL of the PBS solution containing the nanovesicles loaded with schisandrin B was diluted by an appropriate multiple to make its final concentration about 10 7 vesicles per milliliter. The set parameters were a particle size range of 50 - 200 nm, a molecular weight range of 1000 - 20107 Da, a temperature of 25 °C, a 4.0 mV He-Ne laser, and a wavelength of 633 nm. Each sample was analyzed continuously 3 times, the NTA values were recorded and analyzed at different positions. ZetaVIEW 8.04.02 software was used for data analysis.

[0078] Figure 3 The results showed that the particle size of the nanovesicles loaded with schisandrin B was about 195.9 nm.

[0079] VI. Preparation of nanovesicles (NV) derived from human umbilical cord mesenchymal stem cells

[0080] (1) After resuscitating human umbilical cord mesenchymal stem cells, they were passaged 3 times and then continued to be cultured in MSCM medium containing 10% FBS (containing 5 ml of mesenchymal stem cell growth factor). When the fusion rate of human umbilical cord mesenchymal stem cells reached 85% - 95%, they were digested with trypsin at 37 °C for 5 min, centrifuged (1000 rpm, 5 min), and resuspended with PBS to form a single-cell suspension; at this time, the density of human umbilical cord mesenchymal stem cells was 8.0×10 6 cells / mL.

[0081] (2) The single-cell suspension was successively passed through microporous membranes with pore sizes of 5 μm, 1.2 μm, and 0.2 μm;

[0082] (3) The operation in step (2) was repeated 4 times to obtain nanovesicles (NV) derived from human umbilical cord mesenchymal stem cells.

[0083] VII. Identification of the nanovesicles loaded with schisandrin B by polyacrylamide gel electrophoresis

[0084] (1) Add 100 μL of lysis buffer to 50 μL of human umbilical cord mesenchymal stem cell (abbreviated as HUMSC), nanovesicle (NV), and nanovesicle loaded with schisandrin B (S@NV) solutions respectively, and place them on ice for 30 min for lysis.

[0085] (2) Add 20 μL of loading buffer to an appropriate amount of the lysed mixed solution, boil it in a metal bath at 100 °C for 10 min, and it can be stored at -80 °C after cooling.

[0086] (3) After completing the preparation of the protein electrophoresis gel, take 5 μL of electrophoresis samples of human umbilical cord mesenchymal stem cells (abbreviated as HUMSC), nanovesicles (NV), and nanovesicles loaded with schisandrin B (S@NV) and add them to the gel lanes, and add 5 μL of protein Maker to the adjacent lane. Run the stacking gel at 80 V and the separating gel at 120 V.

[0087] (4) Remove the gel in the lane where the sample is located, add Coomassie Brilliant Blue, incubate it in the dark on a shaker for color development, wash off the excess dye with pure water, and observe and photograph for record.

[0088] The results are as Figure 4 shown. Human umbilical cord mesenchymal stem cells (abbreviated as HUMSC), nanovesicles (NV), and nanovesicles loaded with schisandrin B (S@NV) have the same protein expression.

[0089] Based on the comprehensive electron microscopy results, average particle size, and protein expression, the product finally prepared in this example conforms to the characteristics of engineered nanovesicles, indicating that what is finally obtained in this example is indeed nanovesicles loaded with schisandrin B.

[0090] Example 2

[0091] Cytological experiment: In the following experiments, the nanovesicles loaded with schisandrin B are the nanovesicles loaded with schisandrin B prepared in Example 1.

[0092] I. Cell proliferation detection experiment

[0093] After resuspending AML12 mouse hepatocytes with good growth status, seed them in a 96-well plate at a cell density of 100,000 cells / mL, 100 μL per well, and place them in an incubator for 12 h. Observe the cell adhesion situation, renew the medium, and add 100 μL of medium containing 75 μg / mL of S@NV or NV to each well as the experimental group, with 4 replicates in each group. At the same time, use AML12 cells treated with 100 μL of medium as the control group, and use the wells with 100 μL of medium without cells as the blank control group, with 4 replicates respectively.

[0094] After incubation in a constant temperature incubator for 24 h or 48 h, discard the old culture medium and add 100 μL of serum-free medium containing 10% CCK-8 working solution. Then transfer it to the incubator for incubation for 2 h. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of each well at 405 nm, detect cell viability, record and calculate the cell proliferation rate. The formula for calculating the cell proliferation rate is: (absorbance value of the experimental group - absorbance value of the blank control) / (absorbance value of the control group - absorbance value of the blank control) × 100%.

[0095] The experimental results are as Figure 5 shown. Adding an additional 75 μg / mL of S@NV or NV can significantly promote the proliferation of AML12 cells. In contrast, the proliferation-promoting effect of S@NV on AML12 is more significant, with the highest proliferation rate being 159.97% at 24 h and 390.55% at 48 h.

[0096] II. Cell scratch assay

[0097] Closely attach the Culture-Insert to a 24-well plate. Prepare HUVEC cell suspensions with a density of 1 million / mL respectively, and add 100 μL of the suspension to each well of the Culture-Insert according to the standard of 100 μL per well, taking care to avoid touching and causing the insert to move. Then, transfer the insert and the 24-well plate to a constant temperature incubator and incubate at 37 °C and 5% CO2 for 24 h. Check the cell density under a microscope. When the ideal confluence is reached, gently remove the Culture-Insert with sterile forceps. Then, wash with PBS solution to remove cell debris and non-attached cells. Finally, add serum-free medium containing 75 μg / mL S@NV or NV with a protein concentration of 1% fetal bovine serum, place it in a constant temperature incubator, and at the same time add the same volume of 1% fetal bovine serum medium for treatment as the control group. Each treatment group has 3 replicates. After 48 h, use an inverted microscope to observe and photograph the changes in the scratch, and calculate the scratch healing rate according to the following formula:

[0098] Scratch healing rate (%) = (initial scratch area - final scratch area) / initial scratch area × 100%. Among them, the initial scratch area is the scratch area set before the experiment, and the final scratch area is the scratch area after treatment in different treatment groups. The results are shown in Figure 6 .

[0099] The experimental results are as Figure 6 shown. Both 75 μg / mL of S@NV or NV can significantly promote the scratch healing of HUVEC cells, and the effect of S@NV is significantly stronger than that of NV, showing a significant migration-promoting effect of S@NV.

[0100] III. Cell uptake experiment

[0101] After resuspending the well-grown AML12 mouse hepatocytes, they were plated in confocal dishes at a cell density of 200,000 / mL, 1 mL per well, and placed in an incubator for 24 h. Prepare the DIO working solution. Take 1 μL each of DIO and the staining enhancer, add them to 500 μL of the S@NV solution (or NV solution, with a concentration of 75 μg / ml), vortex, and then add 500 μL of cell culture medium (using the non-vesicle-added group as the control group), and incubate in the dark for 15 min. Aspirate the culture medium in the confocal dish, and add the above-prepared incubated solution to the confocal dish, and co-incubate for 4 h. Prepare the Lyso-Tracker Red working solution: Take 1 μl of Lyso-Tracker Red and add it to 15 ml of warm cell culture medium, mix well to obtain the Lyso-Tracker Red working solution (the Lyso-Tracker Red working solution needs to be pre-incubated at 37 °C before use). Remove the culture medium in the confocal dish, and wash it twice with PBS. Add the prepared and pre-incubated Lyso-Tracker Red staining working solution, and co-incubate with the cells at 37 °C for 40 minutes. Then remove the Lyso-Tracker Red staining working solution, wash it twice with PBS, and add paraformaldehyde to fix for 30 min. Nuclear staining: Remove the fixative, wash it twice with PBS, then add a small amount (500 uL) of DAPI staining solution to cover the sample, and place it at room temperature for 3 - 5 minutes. Aspirate the DAPI staining solution, wash it three times with PBS, 4 minutes each time, and then use it for confocal microscopy.

[0102] The results are as Figure 7 shown, S@NV can be well taken up by AML12 mouse hepatocytes.

[0103] IV. Cell anti-apoptosis experiment

[0104] After resuspending the well-grown AML12 mouse hepatocytes, they were plated in 6-well plates at a cell density of 500,000 / mL, 2 mL per well, and placed in an incubator for 12 h. Observe the cell adhesion situation, renew the culture medium, and add 2 mL of the culture medium containing 75 μg / mL of S@NV or NV to each well as the experimental group, with 3 replicates in each group. At the same time, use the AML12 cells treated with 2 mL of the culture medium as the control group, with 6 replicates. After culturing for 48 h, renew the culture medium. Except for adding 2 mL of the culture medium to treat 3 replicates in the above control group as the negative control group, add 2 mL of the culture medium containing 500 μM hydrogen peroxide to the remaining culture wells to induce reactive oxygen species stress apoptosis. After culturing for 6 h, according to the instructions of the manual, use the AnnexinV-APC / 7-AAD apoptosis detection kit (brand: Biolegend) to stain the apoptotic cells, and then use the Beckman Coulter CytoFLEX flow cytometer to detect the proportion of apoptotic cells.

[0105] The experimental results are as follows Figure 8 shown. After pre-treating AML12 cells with 75 μg / mL of S@NV or NV, the antioxidant stress apoptosis ability of AML12 was significantly enhanced, and the effect of S@NV was significantly stronger than that of NV, as manifested by a significant decrease in the number of AnnexinV-APC / 7-AAD double-positive apoptotic cells in the S@NV group.

[0106] Example 3

[0107] This example provides a functional hydrogel, and the preparation method of this functional hydrogel is as follows:

[0108] I. Preparation of chitosan modified with dihydrocaffeic acid (CS-CA)

[0109] Chitosan (CS, 275.0 mg, 3.25 mmol) was dissolved in 5.0 mL of 1 mol / L HCl solution, and then 45.5 mL of double-distilled water was added; then the pH of the solution was adjusted to 5.0 using 5 mol / L NaOH, and an equal volume of ethanol was added; dihydrocaffeic acid (CA, 295.0 mg, 1.62 mmol) was dissolved in 3.0 mL of double-distilled water, and EDC (128.0 mg, 0.65 mmol) was dissolved in 50.0 mL of double-distilled water; the three solutions were mixed, and the mixture was continuously stirred at room temperature for 12 hours; after the reaction, the solution was purified with double-distilled water acidified with HCl (pH 5.0) using a dialysis membrane (cut-off molecular weight 3500 Da) for 2 days, followed by purification with PBS (pH 7.4) for 4 hours, and finally purification with double-distilled water for 4 hours; the obtained CS-CA was lyophilized and stored in a refrigerator at 4 °C for use.

[0110] The characteristic absorption (benzene ring structure) of dihydrocaffeic acid in the product was detected by ultraviolet-visible spectroscopy (UV-Vis), and unmodified chitosan was used as a blank control to observe the absorption peak at 280 nm. The experimental results are as follows Figure 9 shown. Chitosan modified with dihydrocaffeic acid had a significant absorption peak at 280 nm, while unmodified chitosan did not have this characteristic, indicating the successful synthesis of chitosan modified with dihydrocaffeic acid.

[0111] II. Preparation of the functional hydrogel prepolymer solution

[0112] Take 20 mL of PBS and add it to a brown bottle containing 50 mg of the photoinitiator LAP. Heat the solution in a water bath at 45 °C for 15 minutes, shaking it several times during this period to completely dissolve the photoinitiator LAP, and prepare a phosphate buffer solution containing 0.25% (w / v) of the photoinitiator LAP. Take 1 g of methacrylated gelatin (GelMA) and add it to 20 mL of the above solution. Heat and dissolve it in the dark in a water bath at 65 °C for 30 minutes, shaking it several times during this period to completely dissolve the methacrylated gelatin. Subsequently, add 400 mg of dihydrocaffeic acid-modified chitosan (CS-CA) to 20 mL of the above solution, stir and dissolve it in the dark at 40 °C for 2 hours, immediately sterilize it through a 0.22 μm sterile needle filter (to prevent low-temperature gelation), then add 31.7 mg of strontium chloride (SrCl2) and 4 mg of the engineered nanovesicles loaded with schisandrin B prepared in Example 1, and stir evenly to obtain a prepolymer solution of the functional hydrogel.

[0113] III. Preparation of the Functional Hydrogel

[0114] Irradiate the above prepolymer solution with a 405 nm light source for 30 s to cause gelation and obtain the functional hydrogel. Observe the structure of the functional hydrogel through a scanning electron microscope, and the results are as Figure 10 shown. Figure 10 The results show that the gel surface of the functional hydrogel presents a reticular pore structure.

[0115] IV. Release Efficiency of the Engineered Nanovesicles Loaded with Schisandrin B in the Functional Hydrogel

[0116] Place 1 mL of the functional hydrogel in 20 mL of PBS and incubate it in an incubator at 37 °C. Collect 1 mL of PBS at the predetermined time points and supplement it with an equal volume of fresh PBS. Detect the particle number using NTA (Nanoparticle Tracking Analysis, ZetaView, particle Metrix), and repeat it 3 times. Calculate the ratio of the released nanovesicles to the total number of loaded nanovesicles.

[0117] Figure 11 The results show that the functional hydrogel can continuously release the engineered nanovesicles loaded with schisandrin B, and the maximum release amount is basically reached on the sixth day.

[0118] Example 4

[0119] In vitro antibacterial experiment, in vivo hemostasis experiment, and in vivo liver wound repair treatment. In the following experiments, the functional hydrogel is the functional hydrogel prepared in Example 3.

[0120] I. In Vitro Antibacterial Experiment of the Functional Hydrogel

[0121] In vitro antibacterial activity tests were carried out using two common pathogenic bacteria (Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus). Prepare bacteria (in LB medium) at a concentration of 10 8 CFU / mL. Take 1 mL and add 1 mL of the functional hydrogel, and culture it in a shaker at 37 °C for 6 hours. The bacterial solution in the LB medium without the functional hydrogel was used as the control group. Dilute the co-cultured bacterial solution by 10 6 times, evenly coat it on the bacterial culture plate. After culturing for 24 hours, count the formed colony units and calculate the antibacterial rate.

[0122] The results are as Figure 12 shown. The antibacterial rates of the functional hydrogel against Escherichia coli and Staphylococcus aureus both reached over 95%, showing good antibacterial effects.

[0123] II. In vivo hemostasis experiment of the functional hydrogel

[0124] Select SD rats, 6 weeks old, weighing about 70 g, male. Continuously inhale anesthetic (2% isoflurane / O2) to anesthetize the SD rats. Weigh a piece of filter paper and place it under the bleeding site. In the tail bleeding model, quickly cut off the distal two-thirds of the tail using a surgical blade. In the liver bleeding model, make a vertical midline abdominal incision, with a range of continuously opening about 2 cm long along the lower part of the xiphoid process. Use surgical scissors to free the left lobe of the liver, and use a scalpel to quickly excise a 2-mm-wide piece of liver tissue from the left lobe of the liver. Subsequently, immediately inject the pre-polymer solution of the functional hydrogel at the bleeding site and continuously irradiate the area with 405-nm light. The negative control group received no treatment, and the commercial control group was given cyanoacrylate bioadhesive. Take pictures of the blood-infiltrated situation on the filter paper at the predetermined time points to reflect the hemostatic performance of the functional hydrogel.

[0125] The results are as Figure 13 shown. The functional hydrogel has strong hemostatic performance, which can significantly shorten the bleeding time and reduce the blood loss.

[0126] III. In vivo liver wound repair treatment experiment of the functional hydrogel

[0127] C57BL / 6J mice, 6 weeks old, weighing about 20 g, male, were selected. Modeling method: After continuously inhaling 2% isoflurane / O2 to anesthetize the mice, a transverse incision of about 1 cm was made below the xiphoid process to open the abdominal cavity. The left lateral lobe and middle lobe of the liver (including the gallbladder) were ligated and resected using 4-0 surgical silk thread. Subsequently, the peritoneum and skin were sequentially closed using 4-0 needle-bearing surgical silk thread, and the skin of the surgical area was disinfected. Drug administration grouping: Control group (without additional treatment), functional hydrogel group (functional hydrogel treatment was given before closing the surgical incision). Drug administration plan: After partial hepatectomy, 200 μL of the functional hydrogel prepolymer solution was injected into the hepatectomy site under 405 nm light irradiation. After the hydrogel was formed, the abdominal cavity was normally closed. Detection plan: The mice were euthanized at different time points after the operation, and the remaining liver was taken to calculate the liver / body weight ratio, and the peripheral blood was collected to detect liver function (AST and ALT).

[0128] The results are as Figure 14 shown. The liver / body weight ratio of the functional hydrogel group began to be significantly higher than that of the control group on the second day, indicating that the functional hydrogel promoted hepatocyte proliferation. The liver function indexes ALT and AST of the functional hydrogel group were significantly lower than those of the control group starting from the second day, indicating that the functional hydrogel promoted the repair of liver function.

[0129] Overall, it can be seen that the engineered nanovesicles loaded with schisandrin B used in the functional hydrogel of the present invention have high preparation efficiency. And the engineered nanovesicles loaded with schisandrin B can be well taken up by cells, and at the same time show excellent effects in promoting cell proliferation, migration and anti-apoptosis. Moreover, the functional hydrogel can achieve the sustained release of engineered nanoapoptotic vesicles derived from nanovesicles loaded with schisandrin B, and has good antibacterial, hemostatic and liver repair-promoting functions.

[0130] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of nano-vesicles loaded with schisandrin B, characterized in that, After mixing human umbilical cord mesenchymal stem cells with schisandrin B and extruding them through a microporous membrane in a gradient manner, nanovesicles loaded with schisandrin B are obtained.

2. The preparation method according to claim 1, characterized in that, The human umbilical cord mesenchymal stem cells are cells at passages 2 to 3. After being cultured in MSCM medium containing 10% FBS until the cell fusion rate reaches 85% to 95%, they are digested, and the cells are collected and resuspended in phosphate buffer containing schisandrin B.

3. The preparation method according to claim 2, wherein The density of human umbilical cord mesenchymal stem cells in the mixed system is 1.0×10 6 ~6.0×10 7 cells / mL.

4. The preparation method according to claim 3, characterized in that, The concentration of schisandrin in the mixing system is 200 μM.

5. The preparation method according to any one of claims 1 to 4, characterized in that The pore sizes of the microporous membrane gradient are successively 3 to 5 μm, 1.0 to 1.2 μm, and 0.2 to 0.4 μm; the number of extrusion repetitions is 3 to 5 times.

6. Nanovesicles loaded with schisandrin B obtained by the preparation method according to any one of claims 1 to 5.

7. A method for preparing a functional hydrogel containing the nanovesicles loaded with schisandrin B as described in claim 6, characterized in that, Methacrylated gelatin and dihydrocaffeic acid-modified chitosan are dissolved in phosphate buffer containing LAP photoinitiator, and then strontium chloride and the nanovesicles loaded with schisandrin B are added to obtain a prepolymer solution of the multifunctional hydrogel. The prepolymer solution is irradiated with ultraviolet light at 405 nm for 10 to 30 seconds for photocrosslinking to obtain the functional hydrogel.

8. The preparation method according to claim 7, characterized in that, In the prepolymer solution, the mass-volume concentration of methacrylated gelatin is 3 to 10%, the mass-volume concentration of dihydrocaffeic acid-modified chitosan is 1 to 5%, the mass-volume concentration of LAP photoinitiator is 2 to 3 mg / mL, the concentration of strontium chloride is 5 to 20 mM, and the concentration of nanovesicles loaded with schisandrin B is 100 to 500 μg / mL.

9. A functional hydrogel obtained by the preparation method according to claim 7 or 8.

10. Use of the nanovesicles loaded with schisandrin B according to claim 6 or the functional hydrogel according to claim 9 in the preparation of liver wound dressings.

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