A responsive hydrogel loaded with biomimetic nanovesicles and its preparation method and application

Abstract

The present invention relates to a responsive hydrogel loaded with biomimetic nanovesicles, a preparation method and an application, and belongs to the technical field of medical hydrogels. The method comprises: stimulating dental mesenchymal stem cells with a chemical vesicle stimulator and then mixing and extruding them with a melatonin mother solution to obtain biomimetic nanovesicles, wherein the chemical vesicle stimulator contains a buffer solution, a dithiothreitol solution and a paraformaldehyde solution, and the buffer solution contains 8.5-9 g / L sodium chloride, 0.2-0.24 g / L calcium dichloride and 2.3-2.4 g / L 4-hydroxyethylpiperazineethanesulfonic acid; 6-arm-PEG-pp-NH2 and six-arm polyethylene glycol succinimidyl glutarate coupled with a polypeptide cleavable by matrix metalloproteinases are respectively prepared with the biomimetic nanovesicles into the first and second precursor solutions, and the two are mixed and allowed to stand to form a gel to obtain the responsive hydrogel. The present invention obtains a responsive hydrogel that can significantly enhance the bone regeneration ability.

Description

Technical Field

[0001] The invention belongs to the technical field of medical hydrogels, and in particular relates to a responsive hydrogel loaded with bionic nanovesicles, a preparation method and an application thereof. Background Art

[0002] Bone is an important part of the human locomotor system and plays a supporting and protective role for the human body. However, inflammation, trauma, tumors, surgery, etc. can often lead to bone defects and related functional disorders. Bone defects in the oral and maxillofacial region will seriously affect the physiological functions of the oral and maxillofacial region, such as swallowing and pronunciation. In addition, they will seriously affect the appearance and have a negative impact on the patient's mental health. Although bone tissue has a certain self-repair ability, larger bone defects are often difficult to repair on their own. In response to this challenge, treatment strategies such as autologous bone transplantation and allogeneic bone transplantation have been developed clinically, but the trauma of secondary surgery and immune rejection reactions limit their clinical application. The concept of bone tissue engineering implants a biomaterial-seed cell-growth factor complex into the bone defect area, but the source and survival rate of seed cells still puzzle researchers. In comparison, acellular bone tissue scaffold materials show unique advantages, with lower immune risks and no ethical issues.

[0003] In bone tissue engineering, a good scaffold material should be able to regulate the local immune microenvironment and promote osteogenic differentiation while acting as a drug carrier. In recent years, the potential of hydrogel as a scaffold material has been widely studied due to its good mechanical properties, cell compatibility and other characteristics. However, the traditional hydrogel drug release mode is single, the cell utilization rate of drugs is relatively low, and the drug effect is not targeted. Inflammation-responsive hydrogels can achieve on-demand drug release. If stem cell vesicles encapsulating therapeutic drugs are loaded at the same time, the drug delivery of the system can be further made stem cell-targeted, so that the therapeutic factors can play the maximum role in regulating the bone defect environment. However, in previous studies, hydrogels with local delivery of stem cell vesicles are often simple in structure, with a single vesicle release mode and instability. At the same time, the delivered vesicles are often exosomes, whose extraction steps are cumbersome and the yield is limited. There are one or more problems such as low purity of extracellular vesicles and low efficiency of transport to target cells (for example, low efficiency of targeting bone marrow mesenchymal stem cells), which greatly limit their clinical application.

[0004] Therefore, it is very necessary to provide a responsive hydrogel loaded with biomimetic nanovesicles and a preparation method and application thereof. Summary of the invention

[0005] In order to solve one or more technical problems existing in the prior art, the present invention provides a responsive hydrogel loaded with biomimetic nanovesicles, a preparation method and an application thereof.

[0006] In a first aspect, the present invention provides a method for preparing a responsive hydrogel loaded with biomimetic nanovesicles, the preparation method comprising the following steps:

[0007] (1) Add chemical vesicle stimulators to dental mesenchymal stem cells and place them in CO 2 Stimulation is performed in a constant temperature incubator to obtain a giant plasma membrane vesicle solution; the chemical vesicle stimulator comprises a buffer solution, a dithiothreitol solution and a paraformaldehyde solution, and the buffer solution comprises 8.5-9 g / L sodium chloride, 0.2-0.24 g / L calcium chloride, 2.3-2.4 g / L 4-hydroxyethylpiperazineethanesulfonic acid and deionized water;

[0008] (2) mixing the melatonin mother solution with the giant plasma membrane vesicle solution and extruding the mixture to obtain biomimetic nanovesicles;

[0009] (3) 6-arm-PEG-NH 2 The compound 6-arm-PEG-pp-NH3 is reacted with a peptide cleavable by matrix metalloproteinase activated by EDCI / NHS to obtain a peptide cleavable by matrix metalloproteinase coupled to the compound 6-arm-PEG-pp-NH3 2 ;

[0010] (4) 6-arm-PEG-pp-NH 2 The first precursor solution is prepared with biomimetic nanovesicles, and the second precursor solution is prepared with six-arm polyethylene glycol succinimidyl glutarate and biomimetic nanovesicles. The first precursor solution and the second precursor solution are then mixed and allowed to stand to form a gel, thereby preparing a responsive hydrogel loaded with biomimetic nanovesicles.

[0011] Preferably, in step (1), the method for obtaining the dental mesenchymal stem cells is: digesting and centrifuging the periodontal ligament tissue or dental pulp tissue, discarding the supernatant, then adding α-MEM medium containing fetal bovine serum and penicillin-streptomycin and mixing evenly, and then subjecting the mixture to CO 2 After the primary stem cells were cultured in a constant temperature incubator until they reached 80% to 90% fusion, they were digested with trypsin-EDTA digestion solution and cultured and expanded to P4 to P6 generations at a ratio of 1:2 or 1:3 to obtain dental mesenchymal stem cells.

[0012] Preferably, the volume fraction of fetal bovine serum contained in the α-MEM culture medium is 10-20%, the volume fraction of penicillin-streptomycin contained in the α-MEM culture medium is 0.8-1.2%; and / or the trypsin-EDTA digestion solution is a digestion solution containing 0.25wt% trypsin and 0.1wt% EDTA.

[0013] Preferably, in step (1): the dithiothreitol solution is a dithiothreitol aqueous solution, the concentration of the dithiothreitol aqueous solution is 150-160 mg / mL; the paraformaldehyde solution is a 4% paraformaldehyde fixative; and / or the volume ratio of the buffer solution, the dithiothreitol solution and the paraformaldehyde solution is 1000:(1.5-2.5):(16-20).

[0014] Preferably, in step (1): the CO 2 The incubator contains CO 2 The volume fraction of CO is 5%. 2 The temperature of the constant temperature incubator is 37°C and the humidity is 100%; the stimulation time is 18-30 hours; the amount of the chemical vesicle stimulator added is 18-25 microliters of the chemical vesicle stimulator per square centimeter of dental mesenchymal stem cells; and / or the concentration of giant plasma membrane vesicles in the giant plasma membrane vesicle solution is 6.0×10 6 ~6.7×10 6 Pieces / mL.

[0015] Preferably, in step (2): the melatonin mother solution uses dimethyl sulfoxide and PBS buffer as solvents, and the concentration of melatonin in the melatonin mother solution is 0.0008-0.0012 mol / L; the volume ratio of the melatonin mother solution to the giant plasma membrane vesicle solution is 1:(8-10); the particle size of the biomimetic nanovesicle is not greater than 200 nm; and / or the extrusion is repeated extrusion through a polycarbonate membrane using a liposome extruder, the number of repeated extrusions is 8-12 times, and the polycarbonate membrane is a polycarbonate membrane with a pore size of 200 nm.

[0016] Preferably, in step (3): the 6-arm-PEG-NH 2 The mass ratio of the EDCI / NHS activated polypeptide cleavable by matrix metalloproteinase is (30-40):1; and / or the reaction temperature is 30-40°C, and the reaction time is 18-30h.

[0017] Preferably, in step (4), the first precursor solution is prepared by using PBS buffer to prepare a PBS buffer containing biomimetic nanovesicles, and then adding 6-arm-PEG-pp-NH 2 and mixed evenly to obtain a first precursor solution, wherein the protein concentration of the biomimetic nanovesicles in the PBS buffer containing the biomimetic nanovesicles is 1-1.5 mg / mL, and the PBS buffer containing the biomimetic nanovesicles and the 6-arm-PEG-pp-NH 2The mass ratio of the biomimetic nanovesicles to the biomimetic nanovesicles is 100: (10-18); the second precursor solution is prepared by using a PBS buffer to prepare a PBS buffer containing biomimetic nanovesicles, and then adding six-arm polyethylene glycol succinimidyl glutarate to the PBS buffer containing the biomimetic nanovesicles and mixing them evenly to obtain a second precursor solution, wherein the protein concentration of the biomimetic nanovesicles in the PBS buffer containing the biomimetic nanovesicles is 1-1.5 mg / mL, and the mass ratio of the PBS buffer containing the biomimetic nanovesicles to the six-arm polyethylene glycol succinimidyl glutarate is 100: (10-18); and / or the 6-arm-PEG-pp-NH 2 The mass ratio of the six-arm polyethylene glycol succinimidyl glutarate contained in the second precursor solution is (0.5~2): (0.5~2).

[0018] In a second aspect, the present invention provides a responsive hydrogel loaded with biomimetic nanovesicles prepared by the preparation method described in the first aspect of the present invention.

[0019] The present invention provides, in a third aspect, the use of a responsive hydrogel loaded with biomimetic nanovesicles prepared by the preparation method described in the first aspect of the present invention in bone tissue repair materials.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects:

[0021] (1) The present invention induces spontaneous foaming of dental mesenchymal stem cells and combines it with extrusion technology to successfully obtain nano-scale stem cell bionic nanovesicles and successfully load melatonin. At the same time, a hydrogel with inflammation-responsive release characteristics is produced through a relatively simple process. The bionic nanovesicles loaded with melatonin are loaded into the responsive hydrogel, thereby obtaining a responsive hydrogel drug delivery system loaded with bionic nanovesicles that responds to the local inflammatory microenvironment, can release melatonin on demand, and can efficiently deliver melatonin to target cells, thereby significantly enhancing bone regeneration ability.

[0022] (2) The present invention obtains a hydrogel responsive to matrix metalloproteinases, which can responsively release bionic nanovesicles loaded with melatonin, locally deliver bionic nanovesicles loaded with melatonin, and proposes a new method for efficient intracellular delivery of melatonin and for enhancing the antioxidant capacity of cells, thereby greatly promoting the repair capacity of local bone defects. The present invention solves one or more technical problems existing in the prior art, such as the single and unstable drug release pattern of the hydrogel drug delivery system, the poor drug targeting of the hydrogel drug delivery system, the single melatonin application method, and the low efficiency of intracellular delivery of melatonin.

[0023] (3) The present invention generates and separates giant plasma membrane vesicles from dental mesenchymal stem cells by using a chemical vesicle stimulator, and then obtains biomimetic nanovesicles with nanometer size by extrusion. Compared with the traditional method of extracting extracellular vesicles from cell culture supernatant, the method of the present invention can obtain vesicles with higher yield and higher purity. The polyformaldehyde solution contained in the chemical vesicle stimulator used in the present invention can induce non-specific cross-linking of proteins in the cell membrane, and the dithiothreitol contained in the chemical vesicle stimulator can act as a small molecule reducing agent to cut the thioester bond, promote the cell membrane to foam to form giant plasma membrane vesicles GPMV and fall into the supernatant; at the same time, the buffer solution contained in the chemical vesicle stimulator contains calcium ions Ca 2+ The present invention found that in Ca 2+ Under the condition of presence of Ca, cells were exposed to paraformaldehyde solution and dithiothreitol solution, which can make continuous Ca 2+ The inflow is beneficial to promote the blistering of the cell plasma membrane. The chemical vesicle stimulator used in the present invention makes the giant plasma membrane vesicle GPMV produced by the present invention contain no cytoskeleton or nuclear material. The cell membrane is directly extruded or the cell is lysed to extract the cell membrane, which will inevitably be contaminated by organelles or nuclear material. Therefore, compared with other cell membrane vesicle production methods, the method of the present invention produces purer cell membrane vesicles (bionic nanovesicles); and the obtained bionic nanovesicles retain many stem cell markers such as CD90, CD105, etc., as well as chemokine CXCR4, and have a targeting effect on stem cells; the present invention finds that loading the bionic nanovesicles with higher purity in the responsive hydrogel can utilize the stem cell targeting effect of the bionic nanovesicles to increase the uptake of drugs by stem cells, realize the targeted delivery and sustained release of drugs in bone tissue, and thus can significantly enhance the bone regeneration ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a scanning electron microscope image of the responsive hydrogel loaded with biomimetic nanovesicles obtained in Example 1 of the present invention;

[0025] Figure 2 The morphological diagram of the giant plasma membrane vesicle solution in Example 1 of the present invention after being labeled with the fluorescent dye DiI and thioflavin T is observed by confocal microscopy (scale bar, 1 μm); in the figure, Figure (a) corresponds to the cell membrane image labeled with the DiI dye, Figure (b) corresponds to the cytoplasm image labeled with thioflavin T, and Figure (c) corresponds to the image after being labeled with the fluorescent dye DiI and thioflavin T at the same time;

[0026] Figure 3 is a transmission electron microscope image of the bionic nanovesicle in Example 1 of the present invention;

[0027] Figure 4The Micro-CT detection results of the blank control group and the responsive hydrogel PEG-pp-nMSC@MT group loaded with biomimetic nanovesicles prepared in Example 1 of the present invention after repairing the mandibular bone defect in mice for 8 weeks; (a) is the CT scan of the blank control group, (b) is the CT scan of the PEG-pp-nMSC@MT group, and (c) is a bar graph of the new bone volume percentage (BV / TV). DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] In a first aspect, the present invention provides a method for preparing a responsive hydrogel loaded with biomimetic nanovesicles, the preparation method comprising the following steps:

[0030] (1) Add chemical vesicle stimulators to dental mesenchymal stem cells and place them in CO 2 Stimulation is performed in a constant temperature incubator to obtain a giant plasma membrane vesicle solution; the chemical vesicle stimulator comprises a buffer solution (also referred to as GPMV buffer), a dithiothreitol solution and a paraformaldehyde solution, wherein the buffer solution comprises sodium chloride (NaCl) 8.5-9 g / L (e.g., 8.5, 8.6, 8.7, 8.9 or 9 g / L), calcium chloride (CaCl 2 ) 0.2~0.24g / L (for example, 0.2, 0.21, 0.22, 0.23 or 0.24g / L), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) 2.3~2.4g / L (for example, 2.3, 2.2, 2.36, 2.38 or 2.4g / L) and the balance deionized water; in the present invention, preferably, the buffer solution is a buffer solution with a pH of 7.4, specifically, for example, by adding 0.5~1M NaOH aqueous solution to the buffer solution to adjust the pH of the buffer solution to 7.4; the present invention does not specifically limit the amount and concentration of the sodium hydroxide aqueous solution, as long as the pH of the buffer solution reaches the target value;

[0031] (2) Mixing the melatonin mother solution with the giant plasma membrane vesicle solution and extruding the mixture to obtain biomimetic nanovesicles (also referred to as biomimetic nanovesicles loaded with melatonin or nMSC@MT); in the present invention, for example, a liposome extruder can be used to repeatedly extrude through a polycarbonate membrane to obtain biomimetic nanovesicles with a particle size of no more than 200 nm; the present invention generates and separates giant plasma membrane vesicles from dental mesenchymal stem cells by using a chemical vesicle stimulator, and then extrude the mixture to obtain biomimetic nanovesicles with nanometer size. Compared with the traditional method of extracting extracellular vesicles from cell culture supernatant, the method of the present invention can obtain vesicles with higher yield and higher purity. The polyformaldehyde solution contained in the chemical vesicle stimulator used in the present invention can induce non-specific cross-linking of proteins in the cell membrane, and the dithiothreitol contained in the chemical vesicle stimulator can act as a small molecule reducing agent to cut the thioester bond, thereby promoting cell membrane foaming to form giant plasma membrane vesicles GPMV and shedding them into the supernatant; at the same time, the buffer solution contained in the chemical vesicle stimulator contains calcium ions Ca 2+ The present invention found that in Ca 2+ Under the condition of presence of Ca, cells were exposed to paraformaldehyde solution and dithiothreitol solution, which can make continuous Ca 2+ Inflow is conducive to promoting cell plasma membrane blistering. The chemical vesicle stimulator used in the present invention can make the giant plasma membrane vesicle GPMV produced by the present invention contain no cytoskeleton or nuclear material. Directly extruding the cell membrane or extracting the cell membrane after lysing the cell will inevitably be contaminated by organelles or nuclear material. Therefore, compared with other cell membrane vesicle production methods, the method of the present invention produces purer cell membrane vesicles (bionic nanovesicles); the present invention finds that loading the bionic nanovesicles with higher purity in the responsive hydrogel can make the loaded melatonin more efficiently targeted to bone marrow mesenchymal stem cells, realize the targeted delivery and sustained release of the drug in bone tissue, thereby significantly enhancing the bone regeneration ability;

[0032] (3) 6-arm-PEG-NH 2 The compound 6-arm-PEG-pp-NH3 is reacted with a peptide cleavable by matrix metalloproteinase activated by EDCI / NHS to obtain a peptide cleavable by matrix metalloproteinase coupled to the compound 6-arm-PEG-pp-NH3 2 In the present invention, step (3) is to make 6-arm-PEG-NH 2 A peptide cleavable by matrix metalloproteinases was coupled to obtain 6-arm-PEG-pp-NH 2In the present invention, the polypeptide cleavable by matrix metalloproteinase is abbreviated as pp; specifically, the polypeptide cleavable by matrix metalloproteinase is activated by EDCI / NHS in order to fully activate the carboxyl group of the polypeptide cleavable by matrix metalloproteinase, and the terminal carboxyl group of the polypeptide cleavable by matrix metalloproteinase activated by EDCI / NHS is reacted with 6-arm-PEG-NH 2 The amino groups of the 6-arm-PEG-NH 2 To obtain the compound 6-arm-PEG-pp-NH 2 ; The present invention does not make specific requirements for the EDCI / NHS activation of the polypeptide that can be cleaved by matrix metalloproteinases, which is a conventional technology in the field; the present invention does not make specific restrictions on the type and source of the polypeptide that can be cleaved by matrix metalloproteinases, and any polypeptide that can be specifically recognized and specifically cleaved by matrix metalloproteinases can be used, so that the present invention can obtain a responsive hydrogel loaded with biomimetic nanovesicles that is responsive to matrix metalloproteinase stimulation; in some specific embodiments, the polypeptide that can be cleaved by matrix metalloproteinases is, for example, the polypeptide GPLGIAGQ (also recorded as octapeptide GPLGIAGQ), which is a polypeptide known in the prior art and can be purchased from Qiangyao Biotechnology Co., Ltd.; for example, the site between glycine and isoleucine in the polypeptide GPLGIAGQ used in some specific embodiments of the present invention can be specifically recognized and specifically cleaved by matrix metalloproteinases (MMPs);

[0033] (4) 6-arm-PEG-pp-NH 2The first precursor solution is prepared with biomimetic nanovesicles, and six-arm polyethylene glycol succinimidyl glutarate (6-arm-PEG-SG) is prepared with biomimetic nanovesicles to form a first precursor solution, and six-arm polyethylene glycol succinimidyl glutarate (6-arm-PEG-SG) is prepared with biomimetic nanovesicles to form a second precursor solution, and then the first precursor solution and the second precursor solution are mixed and allowed to stand to form gel, so as to obtain a responsive hydrogel loaded with biomimetic nanovesicles; in the present invention, the first precursor solution and the second precursor solution are both made of PBS buffer as solvent; in the present invention, the mixing in step (4) can be performed, for example, by a circular oscillator; in the present invention, the standing time for gelation is, for example, 10 to 20 seconds (for example, 10 seconds, 15 seconds or 20 seconds). , the unit "s" represents the time "second"; in the present invention, the responsive hydrogel loaded with biomimetic nanovesicles is abbreviated as PEG-pp-nMSC@MT hydrogel or directly abbreviated as PEG-pp-nMSC@MT; it should be noted that the order of preparing the first precursor solution and the second precursor solution has no effect on the preparation method of the present invention. It should be understood that the preparation effect of preparing the second precursor solution first and then the first precursor solution, and preparing the first precursor solution and the second precursor solution at the same time has no effect on the preparation method as preparing the first precursor solution first and then the second precursor solution; the present invention has no effect on the preparation method of 6-arm-PEG-NH 2 There is no particular restriction on the sources of the polypeptides cleavable by matrix metalloproteinases, six-arm polyethylene glycol succinimidyl glutarate, EDCI (Chinese name: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (Chinese name: N-hydroxysuccinimide), etc., and they are all products that can be directly purchased on the market.

[0034] The present invention induces spontaneous foaming of dental mesenchymal stem cells and combines extrusion technology to successfully obtain nano-scale stem cell bionic nanovesicles and successfully load melatonin. At the same time, a hydrogel with inflammation-responsive release characteristics is produced through a relatively simple process. The bionic nanovesicles loaded with melatonin are loaded into the responsive hydrogel, and a responsive hydrogel drug delivery system loaded with bionic nanovesicles is obtained, which has the characteristics of responding to the local inflammatory microenvironment, releasing melatonin on demand, and efficiently delivering melatonin to target cells, thereby significantly enhancing bone regeneration ability; the responsive hydrogel loaded with bionic nanovesicles loaded with melatonin in the present invention has better promotion of bone defect repair than the responsive hydrogel loaded with phosphatidylserine in the prior art CN117159802A. The complex characteristics make the responsive hydrogel loaded with bionic nanovesicles described in the present invention have greater application prospects in bone tissue repair materials. This is because when CN117159802A simply loads free small molecule drugs, the drug's effect lacks targeting, and the efficiency of mobilizing local stem cells for proliferation or differentiation is relatively low. Therefore, the present invention uses bionic nanovesicles that can target stem cells to deliver melatonin, which can further promote bone defect repair; the present invention obtains a hydrogel with matrix metalloproteinase responsiveness, which can responsively release bionic nanovesicles loaded with melatonin, and locally deliver bionic nanovesicles loaded with melatonin, and proposes a new method for efficient intracellular delivery of melatonin and enhancing the antioxidant capacity of cells, which greatly promotes the repair ability of local bone defects.

[0035] According to some preferred embodiments, in step (1), the method for obtaining the dental mesenchymal stem cells is:

[0036] The periodontal ligament tissue or dental pulp tissue was digested and centrifuged, the supernatant was discarded, and then α-MEM medium containing fetal bovine serum and penicillin-streptomycin was added and mixed evenly, and then CO 2 After the primary stem cells are cultured in a constant temperature incubator until they reach 80% to 90% fusion, they are digested with trypsin-EDTA digestion solution and cultured and expanded to P4 to P6 generations at a ratio of 1:2 or 1:3 to obtain dental mesenchymal stem cells; in some specific embodiments, the CO 2 The incubator contains CO 2 The volume fraction of CO is 5%. 2 The temperature of the constant temperature incubator was 37°C and the humidity was 100%.

[0037] According to some preferred embodiments, the volume fraction of fetal bovine serum contained in the α-MEM culture medium is 10-20%, preferably 15%, and the concentration (volume fraction) of penicillin-streptomycin contained in the α-MEM culture medium is 0.8-1.2% (for example, 0.8%, 0.9%, 1%, 1.1% or 1.2%); in the present invention, the penicillin-streptomycin is, for example, commercially available penicillin-streptomycin dual antibody (10000U / mL); and / or the trypsin-EDTA digestion solution is a digestion solution containing 0.25wt% trypsin and 0.1wt% EDTA (ethylenediaminetetraacetic acid); in the present invention, the digestion solution containing 0.25wt% trypsin and 0.1wt% EDTA is recorded as 0.25% trypsin / 0.1% EDTA.

[0038] According to some preferred embodiments, the dental mesenchymal stem cells are periodontal ligament mesenchymal stem cells or dental pulp mesenchymal stem cells.

[0039] According to some specific embodiments, obtaining periodontal ligament mesenchymal stem cells (PDLSCs) from periodontal ligament tissue as raw material comprises the following steps:

[0040] S1. After the tooth is removed from the body, it is placed in a PBS buffer containing 2 vol% penicillin-streptomycin (i.e., penicillin-streptomycin dual antibody), transferred to an ultra-clean bench, and repeatedly rinsed with a PBS buffer containing 2 vol% penicillin-streptomycin in the ultra-clean bench. Then, 1 / 3 of the periodontal ligament tissue in the tooth root is scraped with a scalpel while moistening with a serum-free cell culture medium (i.e., serum-free cell culture medium); in the present invention, unless otherwise specified, the PBS buffer used is a 1×PBS buffer with a pH of 7.4;

[0041] S2. Cut the scraped periodontal ligament tissue into small pieces of 1 mm (length) × 1 mm (width) × 1 mm (height), and place them in a mixed enzyme solution containing collagenase I and neutral protease II for digestion at 37°C for 45 to 60 minutes; in the present invention, the mixed enzyme solution containing collagenase I and neutral protease II is formed by mixing a collagenase I solution and a neutral protease II solution, the collagenase I solution uses α-MEM culture medium (i.e., α-MEM liquid culture medium) as a solvent, the concentration of collagenase I in the collagenase I solution is 8 mg / mL, the neutral protease II solution uses PBS buffer as a solvent, the concentration of neutral protease II in the neutral protease II solution is 6 mg / mL, and the volume ratio of the collagenase I solution to the neutral protease II solution is 1:1;

[0042] S3, terminating the digestion in step S2 with a serum-containing α-MEM medium, and then centrifuging at 300g for 3 min; in the present invention, the serum-containing α-MEM medium can be, for example, an α-MEM medium containing 10-20 vol% fetal bovine serum; in some specific embodiments, the serum-containing α-MEM medium is an α-MEM medium containing 15 vol% fetal bovine serum;

[0043] S4. After centrifugation in step S3, discard the supernatant, add an appropriate amount (e.g., 3-4 mL) of α-MEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin, mix well by pipetting, and place in a culture dish with a diameter of 6 cm, and incubate at 4 °C for 1 h. 2 In a constant temperature incubator (containing CO 2 volume fraction of 5%, 37°C, 100% humidity); in some specific embodiments of the present invention, the primary cells with the supernatant discarded are placed in a cell culture dish with a diameter of 6 cm, and each culture dish finally contains 3 mL of α-MEM medium with a volume fraction of 15% fetal bovine serum and a volume fraction of 1% penicillin-streptomycin;

[0044] S5. Replace the medium every 2-3 days (i.e., replace the α-MEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin). When the cells reach 80-90% confluence, digest them with 0.25% trypsin / 0.1% EDTA at 37°C for 60-80s, then passage them at a ratio of 1:2 or 1:3, and culture and expand them in vitro to P4-P6 to obtain uniform periodontal ligament mesenchymal stem cells (PDLSCs).

[0045] In the present invention, when the dental mesenchymal stem cells are dental pulp mesenchymal stem cells, the steps of obtaining dental pulp mesenchymal stem cells DPSCs using dental pulp tissue as raw material are basically the same as the above steps S1 to S5, except that: S1, after the tooth is removed from the body, the dental tissue is split with a dental turbine handpiece, and the tooth is placed in a PBS buffer containing 2 vol% penicillin-streptomycin, and then transferred to an ultra-clean bench. After repeated rinsing with a PBS buffer containing 2 vol% penicillin-streptomycin in the ultra-clean bench, the dental pulp tissue is separated from the pulp cavity with a small fine forceps or a probe while being moistened with a serum-free cell culture medium (i.e., serum-free cell culture medium); and then the separated dental pulp tissue is used to perform subsequent steps S2 to S5.

[0046] According to some preferred embodiments, in step (1): the dithiothreitol solution is a dithiothreitol aqueous solution, and the concentration of the dithiothreitol aqueous solution is 150-160 mg / mL (for example, 150, 152, 154, 156, 158 or 160 mg / mL); the paraformaldehyde solution is 4% paraformaldehyde fixative (4% PFA); and / or the volume ratio of the buffer, the dithiothreitol solution and the paraformaldehyde solution is 1000:(1.5-2.5):(16-20) (for example, 1000:1.5:16, 1000:1.5:18, 1000:1.5:20, 1000:2:16, 1000:2:18, 1000:2:20, 1000:2.5:16, 1000:2.5:18 or 1000:2.5:20).

[0047] In the present invention, the rational selection of the components of the chemical vesicle stimulator and the optimization of their concentration and ratio are helpful to obtain high-concentration, high-purity, high-quality giant plasma membrane vesicles, thereby improving the efficiency of the bionic nanovesicles finally obtained in targeting stem cells, promoting the intracellular delivery of melatonin, more efficiently promoting stem cell proliferation and osteogenic differentiation, and enhancing bone regeneration ability; wherein the NaCl in the buffer solution with a concentration of 8.5-9 g / L is beneficial to maintaining the extracellular osmotic pressure and preventing cell stress caused by osmotic pressure changes, and the CaCl in the buffer solution with a concentration of 0.2-0.24 g / L is beneficial to maintaining the extracellular osmotic pressure and preventing cell stress caused by osmotic pressure changes. 2 By providing calcium ions, it helps promote the vesicle formation and release of giant plasma membrane vesicles. 2 If the concentration of CaCl is too low, the cell's ability to respond to chemical stimuli will be weakened, thereby reducing the formation of giant plasma membrane vesicles. 2If the concentration is too high, it will lead to excessive activation of intracellular calcium-dependent enzymes and may induce cell apoptosis. HEPES with a concentration of 2.3-2.4 g / L can avoid plasma membrane damage caused by pH fluctuations during vesicle formation. In addition, the chemical vesicle stimulator also contains a suitable concentration of dithiothreitol, which is conducive to cutting thioester bonds and promoting cell membrane blistering, which is sufficient to induce the formation of a large number of giant plasma membrane vesicles in a short time, but will not cause excessive cell lysis or non-specific reactions, thereby maintaining the purity and structural integrity of the vesicles. The chemical vesicle stimulator also contains a suitable concentration of paraformaldehyde, which can effectively fix the structure of the vesicles while inducing cell blistering relative to low-concentration aldehyde solutions, making the structure of the vesicles more stable. The high Bionic nanovesicles (mesenchymal stem cell membrane vesicles) with high purity and integrity can interact with targeted cells (such as bone marrow mesenchymal stem cells) more effectively. The natural mesenchymal stem cell membrane proteins and lipid molecules retained on the vesicle membrane help to bind to the specific receptors of the stem cells, thereby improving the targeting efficiency. In addition, the present invention can avoid the rapid ineffectiveness of the drug melatonin due to nonspecific degradation or rapid clearance by the immune system by using high-purity and structurally intact bionic nanovesicle drug carriers, and can be maintained in the body for a long time to avoid early release or degradation, thereby extending the in vivo half-life of the drug melatonin. The present invention can accurately deliver melatonin to bone marrow mesenchymal stem cells through the high targeting of bionic nanovesicles, which is beneficial to enhancing the bone regeneration effect.

[0048] According to some preferred embodiments, in step (1): the concentration of the dithiothreitol solution is 154 mg / mL; the volume ratio of the buffer, the dithiothreitol solution and the paraformaldehyde solution is 1000:2:18; and / or the buffer comprises 8.75 g / L sodium chloride, 0.22 g / L calcium chloride, 2.38 g / L 4-hydroxyethylpiperazineethanesulfonic acid and the remainder deionized water.

[0049] According to some preferred embodiments, in step (1): the CO 2 The incubator contains CO 2 The volume fraction of CO is 5%. 2 The temperature of the constant temperature incubator is 37° C. and the humidity is 100%; the stimulation time is 18 to 30 hours (e.g., 18, 20, 22, 24, 26, 28 or 30 hours); and / or the amount of the chemical vesicle stimulator added is 18 to 25 microliters of the chemical vesicle stimulator per square centimeter of dental mesenchymal stem cells.

[0050] According to some preferred embodiments, in step (1), the concentration of giant plasma membrane vesicles in the giant plasma membrane vesicle solution is 6.0×10 6 ~6.7×10 6The purity is not less than 95%, preferably not less than 99%.

[0051] According to some specific embodiments, in step (1), PDLSCs and / or DPSCs cultured in vitro and expanded to P4-P6 are selected. When the cells reach 90% fusion, the culture medium is discarded, and the cells are rinsed with PBS buffer for 3 times, and then the cells are washed with PBS buffer at a dosage of 20 uL / cm 2 Add chemical vesicle stimulants and CO 2 In a constant temperature incubator (CO 2 The cells were stimulated with 5% sodium chloride (volume fraction), 37°C, and 100% humidity) for 24 hours, the supernatant was collected, and the cell debris was removed by centrifugation at 100g for 3 minutes. The supernatant was taken to obtain a giant plasma membrane vesicle solution (GPMV solution); wherein the chemical vesicle stimulator is prepared by a buffer solution, a dithiothreitol solution, and a paraformaldehyde solution in a volume ratio of 1000:2:18, the buffer solution uses deionized water as a solvent, contains 8.75 g / L sodium chloride, 0.22 g / L calcium chloride, and 2.38 g / L 4-hydroxyethylpiperazineethanesulfonic acid, and the buffer solution has a pH value of 7.4 after adjustment with a NaOH aqueous solution; the dithiothreitol solution uses deionized water as a solvent, contains 154 mg / mL dithiothreitol, and the paraformaldehyde solution is 4% PFA.

[0052] According to some specific embodiments, step (1) obtains micrometer-sized giant plasma membrane vesicles GPMV with a particle size distribution range of 5 to 28 μm, an average particle size of 12.25 μm, and a vesicle concentration of 6.0×10 6 ~6.7×10 6 The protein concentration was 1-1.5 mg / mL as determined by A280 (nm) ultraviolet absorption method. The present invention obtains high-concentration GPMV, which is beneficial for obtaining high-yield bionic nanovesicles after nano-sizing.

[0053] According to some preferred embodiments, in step (2): the melatonin mother solution uses dimethyl sulfoxide and PBS buffer as solvents, and the concentration of melatonin in the melatonin mother solution is 0.0008-0.0012 mol / L (for example, 0.0008, 0.0009, 0.001, 0.0011 or 0.0012 mol / L), preferably 0.001 mol / L; in some specific embodiments, the preparation of the melatonin mother solution is, for example, as follows: 5.8 mg of melatonin powder is added to 120 μL-200 μL of dimethyl sulfoxide (DMSO) to dissolve, and then the solution is fixed to 25 mL with PBS buffer to obtain a melatonin mother solution with a concentration of 0.001 mol / L; the volume ratio of the melatonin mother solution to the giant plasma membrane vesicle solution is 1:(8-10) (for example, 1:8, 1:9 or 1:10); and / or the particle size of the biomimetic nanovesicles is not greater than 200 nm.

[0054] According to some preferred embodiments, in step (2), the extrusion is repeated extrusion through a polycarbonate membrane using a liposome extruder, and the number of repeated extrusions is 8 to 12 times (for example, 8, 9, 10, 11 or 12 times), preferably 10 times; the polycarbonate membrane is a polycarbonate membrane with a pore size of 200 nm.

[0055] According to some specific embodiments, step (2) is: mixing the melatonin mother solution with the giant plasma membrane vesicle solution, and then using a liposome extruder to pass through a 200 nm polycarbonate membrane and repeatedly co-extrude for 10 times to obtain biomimetic nanovesicles.

[0056] In the present invention, when the giant plasma membrane vesicle solution is directly extruded through a 200nm polycarbonate membrane using a liposome extruder for 10 times, nanovesicles nMSC without melatonin loading can be obtained. The present invention finds that compared with erythrocyte nanovesicles, bone marrow mesenchymal stem cells BMSC can take up more nanovesicles nMSC, and the nanovesicles nMSC have a better effect of targeting bone marrow mesenchymal stem cells; in a specific study, the erythrocyte membrane nanovesicles and the nanovesicles nMSC of the present invention are respectively labeled with the fluorescent dye DiI, and then respectively compared with mouse bone marrow. After co-culture with bone marrow mesenchymal stem cells (BMSC) for 24 hours, the cells were fixed with 4% PFA, permeabilized with 0.1% TritonX-100 for 15 minutes, blocked with 5% bovine serum albumin for 1 hour, and sealed with a mounting medium containing DAPI (4',6-diamidino-2-phenylindole). The cells were photographed using a laser confocal microscope, and the relative fluorescence intensity in the same field of view was measured. The results are shown in Table 1. As can be seen from Table 1, compared with red blood cell membrane nanovesicles, mouse bone marrow mesenchymal stem cells (BMSC) can take up more nanovesicles nMSC.

[0057] Table 1: Comparison of the results of BMSC uptake of erythrocyte membrane nanovesicles and nanovesicle nMSCs.

[0058]

[0059] According to some specific embodiments, the EDCI / NHS activation is as follows: a polypeptide cleavable by matrix metalloproteinases, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCI and N-hydroxysuccinimide NHS are mixed with water and stirred at 37° C. for 1 to 3 hours to fully activate the carboxyl group of the polypeptide cleavable by matrix metalloproteinases.

[0060] According to some preferred embodiments, the mass ratio of the polypeptide cleavable by matrix metalloproteinase, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCI and the N-hydroxysuccinimide NHS is (8-12): (110-120): (95-105).

[0061] According to some preferred embodiments, when performing the EDCI / NHS activation, the amount of water used is 80 to 100 times the sum of the mass amounts of the polypeptide cleavable by matrix metalloproteinase, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCI and the N-hydroxysuccinimide NHS.

[0062] According to some preferred embodiments, in step (3): the 6-arm-PEG-NH 2 The mass ratio of the EDCI / NHS activated polypeptide cleavable by matrix metalloproteinase is (30-40):1; and / or the reaction temperature is 30-40°C, and the reaction time is 18-30h.

[0063] According to some preferred embodiments, in step (4): the first precursor solution is prepared by using PBS buffer to prepare a PBS buffer containing biomimetic nanovesicles, and then adding 6-arm-PEG-pp-NH 2 and mixing them evenly to obtain a first precursor solution, wherein the protein concentration of the biomimetic nanovesicles in the PBS buffer containing biomimetic nanovesicles is 1-1.5 mg / mL (e.g., 1, 1.1, 1.2, 1.3, 1.4 or 1.5 mg / mL), and the PBS buffer containing biomimetic nanovesicles and the 6-arm-PEG-pp-NH 2The mass ratio of the biomimetic nanovesicles to the biomimetic nanovesicles is 100: (10-18) (for example, 100: 10, 100: 11, 100: 12, 100: 13, 100: 14, 100: 15, 100: 16, 100: 17 or 100: 18); the second precursor solution is prepared by using a PBS buffer to prepare a PBS buffer containing biomimetic nanovesicles, and then adding six-arm polyethylene glycol succinimidyl glutarate to the PBS buffer containing the biomimetic nanovesicles and mixing them uniformly to obtain a second precursor solution, wherein the biomimetic nanovesicles are contained in the PBS buffer containing the biomimetic nanovesicles. The protein concentration of the rice vesicles is 1-1.5 mg / mL (e.g., 1, 1.1, 1.2, 1.3, 1.4 or 1.5 mg / mL), the mass ratio of the PBS buffer containing the biomimetic nanovesicles to the six-arm polyethylene glycol succinimidyl glutarate is 100:(10-18) (e.g., 100:10, 100:11, 100:12, 100:13, 100:14, 100:15, 100:16, 100:17 or 100:18); and / or the 6-arm-PEG-pp-NH 2 The mass ratio of the six-arm polyethylene glycol succinimidyl glutarate contained in the second precursor solution is (0.5~2): (0.5~2); in the present invention, the protein concentration contained in the bionic nanovesicle is measured by A280 (nm) ultraviolet absorption method (i.e. 280 nanometer ultraviolet absorption method); in the present invention, before using the bionic nanovesicle to configure the first precursor solution and the second precursor solution, the bionic nanovesicle obtained in step (2) is dialyzed with PBS buffer. The amount of PBS buffer used during dialysis is: 1000 mL / time, replaced every 30~40 minutes, and dialyzed 4 times in total to remove residual dithiothreitol, polyformaldehyde and unloaded melatonin.

[0064] In a second aspect, the present invention provides a responsive hydrogel loaded with biomimetic nanovesicles prepared by the preparation method described in the first aspect of the present invention.

[0065] The present invention provides, in a third aspect, the use of a responsive hydrogel loaded with biomimetic nanovesicles prepared by the preparation method described in the first aspect of the present invention in bone tissue repair materials.

[0066] The present invention will be further described below by way of examples, but the protection scope of the present invention is not limited to these embodiments. The present invention may also have other various embodiments, and those skilled in the art may make various corresponding changes and deformations according to the present invention without departing from the spirit and essence of the present invention, but these corresponding changes and deformations shall all belong to the protection scope of the claims attached to the present invention.

[0067] Example 1

[0068] ① Preparation of periodontal ligament mesenchymal stem cells PDLSCs:

[0069] After the tooth was removed from the body, it was placed in a PBS buffer containing 2 vol% penicillin-streptomycin, and then transferred to a clean bench. After repeated rinsing with a PBS buffer containing 2 vol% penicillin-streptomycin in the clean bench, 1 / 3 of the periodontal ligament tissue in the tooth root was scraped with a scalpel under the condition of moistening with a serum-free cell culture medium (i.e., serum-free cell culture medium); the scraped periodontal ligament tissue was cut into small pieces of 1 mm (length) × 1 mm (width) × 1 mm (height), and placed in a mixed enzyme solution containing collagenase I and neutral protease II for digestion at 37°C for 60 min; the digestion was terminated with an α-MEM medium containing serum, and then centrifuged at 300g for 3 min. The α-MEM medium containing serum was an α-MEM medium containing 15 vol% fetal bovine serum; after centrifugation, the supernatant was discarded, and 3 mL of an α-MEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin by volume was added, and the mixture was mixed by pipetting and placed in a culture dish with a diameter of 6 cm, and incubated at CO 2 In a constant temperature incubator (containing CO 2 The cells were cultured at 37°C with a volume fraction of 5%, 37°C, and 100% humidity; the α-MEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin was replaced every 2 days. When the cells reached 80% confluence, they were digested with 0.25% trypsin / 0.1% EDTA at 37°C for 1 min, then passaged at a ratio of 1:3, and cultured in vitro to expand to the P6 generation to obtain uniform periodontal ligament mesenchymal stem cells (PDLSCs).

[0070] ② Preparation of giant plasma membrane vesicle solution (GPMV solution):

[0071] Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2 Add chemical vesicle stimulants and CO 2 In a constant temperature incubator (CO 2The cells were stimulated with a volume fraction of 5%, 37°C, and 100% humidity for 24 hours, and the supernatant was collected and centrifuged at 100g for 3 minutes to remove cell debris. The supernatant was taken to obtain a giant plasma membrane vesicle solution; wherein the chemical vesicle stimulator was prepared from a buffer solution, a dithiothreitol aqueous solution, and a paraformaldehyde solution in a volume ratio of 1000:2:18, and the buffer solution used deionized water as a solvent and contained 8.75 g / L sodium chloride, 0.22 g / L calcium chloride, and 4-hydroxyethylpiperazine 2.38 g / L of ethanesulfonic acid, and the buffer solution is a buffer solution with a pH value of 7.4 after adjustment with a NaOH aqueous solution; the dithiothreitol aqueous solution uses deionized water as a solvent, and contains a dithiothreitol concentration of 154 mg / mL, and the paraformaldehyde solution is 4% PFA; in this step, giant plasma membrane vesicles with a particle size distribution range of 5 to 28 μm and an average particle size of 12.25 μm are obtained, and the concentration of giant plasma membrane vesicles in the giant plasma membrane vesicle solution is 6.7×10 6 / mL, and the protein concentration was 1.5mg / mL determined by A280 (nm) UV absorption method.

[0072] ③ Preparation of biomimetic nanovesicles:

[0073] The melatonin mother solution was mixed with the giant plasma membrane vesicle solution obtained in step ② and then co-extruded 10 times through a 200 nm polycarbonate membrane using a liposome extruder to obtain biomimetic nanovesicles (nMSC@MT); wherein the melatonin mother solution used dimethyl sulfoxide (DMSO) and PBS as solvents, and the concentration of melatonin in the melatonin mother solution was 0.001 mol / L. The preparation of the melatonin mother solution of 0.001 mol / L is as follows: 5.8 mg of melatonin powder is added to 150 μL of dimethyl sulfoxide (DMSO) to dissolve, and then the solution is fixed to 25 mL with PBS buffer to obtain a melatonin mother solution with a concentration of 0.001 mol / L; the volume ratio of the melatonin mother solution to the giant plasma membrane vesicle solution is 1:9; after obtaining the bionic nanovesicles, the obtained bionic nanovesicles are dialyzed with PBS buffer to obtain dialyzed bionic nanovesicles for standby use; the amount of PBS buffer used during dialysis: 1000 mL / time, replaced every 35 minutes, and dialyzed 4 times in total to remove residual dithiothreitol, paraformaldehyde and unloaded melatonin.

[0074] ④ Preparation of solution of activated polypeptide cleavable by matrix metalloproteinase:

[0075] Weigh 10 mg of polypeptide GPLGIAGQ, 114 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 100 mg of N-hydroxysuccinimide (NHS), dissolve in 20 mL of distilled water, and stir at 37° C. for 2 h to obtain a solution of an activated polypeptide that can be cleaved by matrix metalloproteinases.

[0076] ⑤ Compound 6-arm-PEG-pp-NH coupled with a peptide cleavable by matrix metalloproteinase 2 Preparation:

[0077] Weigh 6-arm-PEG-NH 2 400 mg was added to the solution of the activated polypeptide cleavable by matrix metalloproteinases, and distilled water was added to make the total volume of the solution reach 150 mL, and the reaction was stirred at 37°C for 24 hours to obtain a reactant; the reactant was placed in a dialysis bag with a molecular weight cutoff of 20 kD for dialysis, and the distilled water was replaced every 12 hours, and the dialysis was continued for 72 hours; the dialyzed reactant was lyophilized to obtain a purified material, which is a compound 6-arm-PEG-pp-NH coupled with a polypeptide cleavable by matrix metalloproteinases 2 , store at -20℃ for future use.

[0078] ⑥ Preparation of PEG-pp-nMSC@MT hydrogel:

[0079] Prepare the first precursor solution: use PBS buffer to prepare the dialyzed biomimetic nanovesicles into PBS buffer containing biomimetic nanovesicles, and then add 100 mg of 6-arm-PEG-pp-NH 2 and mixed evenly to obtain a first precursor solution, wherein the protein concentration of the biomimetic nanovesicles in the PBS buffer containing the biomimetic nanovesicles is 1.5 mg / mL, and the PBS buffer containing the biomimetic nanovesicles and the 6-arm-PEG-pp-NH 2The mass ratio of the six-arm polyethylene glycol succinimidyl glutarate is 100:10; preparing a second precursor solution: using PBS buffer to prepare the dialyzed biomimetic nanovesicles into a PBS buffer containing biomimetic nanovesicles, and then adding 100 mg of six-arm polyethylene glycol succinimidyl glutarate to the PBS buffer containing the biomimetic nanovesicles and mixing them evenly to obtain a second precursor solution, wherein the protein concentration of the biomimetic nanovesicles in the PBS buffer containing the biomimetic nanovesicles is 1.5 mg / mL, and the mass ratio of the PBS buffer containing the biomimetic nanovesicles to the six-arm polyethylene glycol succinimidyl glutarate is 100:10; mixing the prepared first precursor solution and the prepared second precursor solution, quickly mixing them using a circular oscillator, and letting them stand for 15 seconds to form a gel, to obtain a responsive hydrogel loaded with biomimetic nanovesicles (PEG-pp-nMSC@MT hydrogel).

[0080] The scanning electron microscopy image of the responsive hydrogel loaded with biomimetic nanovesicles obtained in this example is as follows: Figure 1 The giant plasma membrane vesicle solution in this embodiment was observed by confocal microscopy after being labeled with fluorescent dye DiI and thioflavin T, as shown in FIG. Figure 2 As shown; the transmission electron microscopy image of the biomimetic nanovesicle in this embodiment, as shown Figure 3 shown.

[0081] Example 2

[0082] Embodiment 2 is substantially the same as Embodiment 1, except that:

[0083] ② Preparation of giant plasma membrane vesicle solution (GPMV solution):

[0084] Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2 Add chemical vesicle stimulants and CO 2 In a constant temperature incubator (CO 2 The cells were stimulated with 5% sodium chloride (volume fraction), 37°C, and 100% humidity) for 24 hours, the supernatant was collected, and the cell debris was removed by centrifugation at 100g for 3 minutes. The supernatant was taken to obtain a giant plasma membrane vesicle solution; wherein the chemical vesicle stimulator is prepared by a buffer solution, a dithiothreitol aqueous solution, and a paraformaldehyde solution in a volume ratio of 1000:4:30, the buffer solution uses deionized water as a solvent, contains 8.75 g / L sodium chloride, 0.22 g / L calcium chloride, and 2.38 g / L 4-hydroxyethylpiperazineethanesulfonic acid, and the buffer solution has a pH value of 7.4 after adjustment with a NaOH aqueous solution; the dithiothreitol aqueous solution uses deionized water as a solvent, contains 180 mg / mL dithiothreitol, and the paraformaldehyde solution is 4% PFA.

[0085] Example 3

[0086] Embodiment 3 is substantially the same as Embodiment 1, except that:

[0087] ② Preparation of giant plasma membrane vesicle solution (GPMV solution):

[0088] Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2 Add chemical vesicle stimulants and CO 2 In a constant temperature incubator (CO 2 The cells were stimulated with 5% sodium chloride (volume fraction), 37°C, and 100% humidity) for 24 hours, the supernatant was collected, and the cell debris was removed by centrifugation at 100g for 3 minutes. The supernatant was taken to obtain a giant plasma membrane vesicle solution; wherein the chemical vesicle stimulator is prepared by a buffer solution, a dithiothreitol aqueous solution, and a paraformaldehyde solution in a volume ratio of 1000:2:10, the buffer solution uses deionized water as a solvent, contains 8.75 g / L sodium chloride, 0.22 g / L calcium chloride, and 2.38 g / L 4-hydroxyethylpiperazineethanesulfonic acid, and the buffer solution has a pH value of 7.4 after adjustment with a NaOH aqueous solution; the dithiothreitol aqueous solution uses deionized water as a solvent, contains 120 mg / mL dithiothreitol, and the paraformaldehyde solution is 4% PFA.

[0089] Comparative Example 1

[0090] Comparative Example 1 is substantially the same as Example 1, except that:

[0091] ② Preparation of giant plasma membrane vesicle solution (GPMV solution):

[0092] Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2 Add chemical vesicle stimulants and CO 2 In a constant temperature incubator (CO 2The cells were stimulated with 5% water (volume fraction, 5%, 37°C, 100% humidity) for 24 hours, the supernatant was collected, and the cell debris was removed by centrifugation at 100g for 3 minutes. The supernatant was taken to obtain a giant plasma membrane vesicle solution; wherein the chemical vesicle stimulator was prepared from a buffer solution and a paraformaldehyde solution in a volume ratio of 1000:18, the buffer solution used deionized water as solvent, contained 8.75 g / L sodium chloride, 0.22 g / L calcium chloride, 2.38 g / L 4-hydroxyethylpiperazineethanesulfonic acid, and the buffer solution was a buffer solution with a pH value of 7.4 after adjustment with a NaOH aqueous solution; the paraformaldehyde solution was 4% PFA.

[0093] Comparative Example 2

[0094] Comparative Example 2 is substantially the same as Example 1, except that:

[0095] ② Preparation of giant plasma membrane vesicle solution (GPMV solution):

[0096] Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2 Add chemical vesicle stimulants and CO 2 In a constant temperature incubator (CO 2 The cells were stimulated with 5% sodium chloride (volume fraction), 37°C, and 100% humidity) for 24 hours, the supernatant was collected, and the cell debris was removed by centrifugation at 100g for 3 minutes. The supernatant was taken to obtain a giant plasma membrane vesicle solution; wherein the chemical vesicle stimulator is prepared by a buffer solution, a dithiothreitol aqueous solution, and a paraformaldehyde solution in a volume ratio of 1000:2:18, the buffer solution uses deionized water as a solvent, contains 8.75 g / L sodium chloride, 2.38 g / L 4-hydroxyethylpiperazineethanesulfonic acid, and the buffer solution has a pH value of 7.4 after adjustment with a NaOH aqueous solution; the dithiothreitol aqueous solution uses deionized water as a solvent, contains 154 mg / mL dithiothreitol, and the paraformaldehyde solution is 4% PFA.

[0097] Comparative Example 3

[0098] Comparative Example 3 is substantially the same as Example 1, except that:

[0099] ② Preparation of giant plasma membrane vesicle solution (GPMV solution):

[0100] Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2 Add chemical vesicle stimulants and CO 2In a constant temperature incubator (CO 2 The cells were stimulated with 5% volume fraction, 37°C, and 100% humidity) for 24 hours, the supernatant was collected, and the cell debris was removed by centrifugation at 100g for 3 minutes. The supernatant was taken to obtain a giant plasma membrane vesicle solution; wherein the chemical vesicle stimulator was prepared from deionized water and paraformaldehyde solution in a volume ratio of 1000:18, and the paraformaldehyde solution was 4% PFA.

[0101] Comparative Example 4

[0102] Comparative Example 4 is substantially the same as Example 1, except that:

[0103] ② Preparation of giant plasma membrane vesicle solution (GPMV solution):

[0104] Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2 Add chemical vesicle stimulants and CO 2 In a constant temperature incubator (CO 2 The cells were stimulated with 5% sodium chloride (volume fraction), 37°C, and 100% humidity) for 24 hours, the supernatant was collected, and the cell debris was removed by centrifugation at 100g for 3 minutes. The supernatant was taken to obtain a giant plasma membrane vesicle solution; wherein the chemical vesicle stimulator is prepared by a buffer solution, a dithiothreitol aqueous solution, and a paraformaldehyde solution in a volume ratio of 1000:2:18, the buffer solution uses deionized water as a solvent, contains 8.75 g / L sodium chloride, 13.5 g / L calcium chloride, and 2.6 g / L 4-hydroxyethylpiperazineethanesulfonic acid, and the buffer solution has a pH value of 7.4 after adjustment with a NaOH aqueous solution; the dithiothreitol aqueous solution uses deionized water as a solvent, contains 154 mg / mL dithiothreitol, and the paraformaldehyde solution is 4% PFA.

[0105] Comparative Example 5

[0106] ① Preparation of a solution of activated polypeptide cleavable by matrix metalloproteinases:

[0107] Weigh 10 mg of polypeptide GPLGIAGQ, 114 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 100 mg of N-hydroxysuccinimide (NHS), dissolve in 20 mL of distilled water, and stir at 37° C. for 2 h to obtain a solution of an activated polypeptide that can be cleaved by matrix metalloproteinases.

[0108] ② Tetra-PEG-pp-NH 2 Preparation:

[0109] Weigh Tetra-PEG-NH 2 400 mg of the activated peptide cleavable by matrix metalloproteinases was added to the solution, and distilled water was added to make the total volume of the solution reach 150 mL. The reaction was stirred at 37° C. for 24 hours to obtain a reactant. The reactant was placed in a dialysis bag with a molecular weight cutoff of 20 kD for dialysis, and the distilled water was replaced every 12 hours for 72 hours. The dialyzed reactant was lyophilized to obtain a purified material, which is a compound Tetra-PEG-pp-NH coupled with a peptide cleavable by matrix metalloproteinases. 2 , store at -20℃ for future use.

[0110] ③ Preparation of PEG-pp-PS hydrogel:

[0111] Weigh the compound Tetra-PEG-pp-NH obtained in step ②, which is coupled with a peptide cleavable by matrix metalloproteinases. 2 100 mg of PS was dissolved in 1 mL of distilled water to prepare the first precursor solution; 0.5 mg of phosphatidylserine (PS) was weighed and evenly mixed in 1 mL of distilled water, and then 100 mg of tetra-PEG-SG was added and fully dissolved to prepare the second precursor solution; the first precursor solution and the second precursor solution were mixed and quickly mixed using an orbital oscillator, and allowed to stand for 15 seconds to form a gel, to obtain a responsive hydrogel (PEG-pp-PS hydrogel).

[0112] Comparative Example 6

[0113] Comparative Example 6 is substantially the same as Comparative Example 5, except that:

[0114] ② Compound 6-arm-PEG-pp-NH coupled with a peptide cleavable by matrix metalloproteinase 2 Preparation:

[0115] Weigh 6-arm-PEG-NH 2 400 mg was added to the solution of the activated polypeptide cleavable by matrix metalloproteinases, and distilled water was added to make the total volume of the solution reach 150 mL, and the reaction was stirred at 37°C for 24 hours to obtain a reactant; the reactant was placed in a dialysis bag with a molecular weight cutoff of 20 kD for dialysis, and the distilled water was replaced every 12 hours, and the dialysis was continued for 72 hours; the dialyzed reactant was lyophilized to obtain a purified material, which is a compound 6-arm-PEG-pp-NH coupled with a polypeptide cleavable by matrix metalloproteinases 2 , store at -20℃ for future use.

[0116] ③ Preparation of PEG-pp-PS hydrogel:

[0117] Weigh the compound 6-arm-PEG-pp-NH obtained in step ② and coupled with a peptide cleavable by matrix metalloproteinases 2 100 mg of PS was dissolved in 1 mL of distilled water to prepare the first precursor solution; 0.5 mg of phosphatidylserine (PS) was weighed and evenly mixed in 1 mL of distilled water, and then 100 mg of six-arm polyethylene glycol succinimidyl glutarate 6-arm-PEG-SG was added and fully dissolved to prepare the second precursor solution; the first precursor solution and the second precursor solution were mixed and quickly mixed using an orbital oscillator, and allowed to stand for 15 seconds to form a gel to obtain a responsive hydrogel (PEG-pp-PS hydrogel).

[0118] Comparative Example 7

[0119] Weigh 6-arm-PEG-NH 2 100 mg was dissolved in 1 mL PBS buffer to prepare the first precursor solution; 100 mg of six-arm polyethylene glycol succinimidyl glutarate 6-arm-PEG-SG was weighed and dissolved in 1 mL PBS buffer to prepare the second precursor solution; the first precursor solution and the second precursor solution were mixed and quickly mixed using an orbital oscillator, and allowed to stand for 15 seconds to gel, to obtain PEG hydrogel.

[0120] Comparative Example 8

[0121] ①Same as step ① of Example 1.

[0122] ②Same as step ② of Example 1.

[0123] ③ is the same as step ③ of Example 1.

[0124] ④ Preparation of PEG-nMSC@MT hydrogel:

[0125] Prepare the first precursor solution: use PBS buffer to prepare the dialyzed biomimetic nanovesicles into PBS buffer containing biomimetic nanovesicles, and then add 100 mg of 6-arm-PEG-NH 2 and mixed evenly to obtain a first precursor solution, wherein the protein concentration of the biomimetic nanovesicles in the PBS buffer containing the biomimetic nanovesicles is 1.5 mg / mL, and the PBS buffer containing the biomimetic nanovesicles and the 6-arm-PEG-NH 2The mass ratio of the six-arm polyethylene glycol succinimidyl glutarate is 100:10; preparing a second precursor solution: using PBS buffer to prepare the dialyzed biomimetic nanovesicles into a PBS buffer containing biomimetic nanovesicles, and then adding 100 mg of six-arm polyethylene glycol succinimidyl glutarate 6-arm-PEG-SG to the PBS buffer containing the biomimetic nanovesicles and mixing them evenly to obtain a second precursor solution, wherein the protein concentration of the biomimetic nanovesicles in the PBS buffer containing the biomimetic nanovesicles is 1.5 mg / mL, and the mass ratio of the PBS buffer containing the biomimetic nanovesicles to the six-arm polyethylene glycol succinimidyl glutarate is 100:10; mixing the prepared first precursor solution and the prepared second precursor solution, quickly mixing them using a circular oscillator, and letting them stand for 15 seconds to gel, to obtain PEG-nMSC@MT hydrogel.

[0126] The present invention tests the concentration and purity of giant plasma membrane vesicles in the giant plasma membrane vesicle solutions obtained in Examples 1 to 3 and Comparative Examples 1 to 4, and the results are shown in Table 2.

[0127] Table 2: The concentration and purity results of giant plasma membrane vesicles in the giant plasma membrane vesicle solutions obtained in Examples 1 to 3 of the present invention and Comparative Examples 1 to 4.

[0128]

[0129] The hydrogels finally prepared in Examples 1 to 3 and Comparative Examples 1 to 8 were subjected to an animal experiment on the repair of mandibular defects in mice. The experimental method was as follows: an 8-week-old C57BL / 6 male mouse was used to prepare a mandibular bone defect model. After general anesthesia, the left mandible was shaved and disinfected, a 5 mm long incision was made, the masseter muscle was separated, and the mandible was exposed. A high-speed mobile phone was used to make a critical size bone defect with a diameter of 2.5 mm and a thickness of about 1 mm. The punching was stopped when there was a sense of falling. During the punching process, attention was paid to timely cooling and removing the remaining bone powder. The experimental groups were: A: bone defect blank control group (no hydrogel was implanted), B: bone defect PEG-pp-nMSC@MT hydrogel group (implanted with the hydrogel sample in Example 1), C: bone defect PEG-pp-nMSC@MT hydrogel group (implanted with the hydrogel sample in Example 2), D: bone defect PEG-pp-nMSC@MT hydrogel group (implanted with the hydrogel sample in Example 3), E: bone defect hydrogel group Gel group (implanted with the hydrogel sample in Comparative Example 1), F: bone defect hydrogel group (implanted with the hydrogel sample in Comparative Example 2), G: bone defect hydrogel group (implanted with the hydrogel sample in Comparative Example 3), H: bone defect hydrogel group (implanted with the hydrogel sample in Comparative Example 4), I: bone defect hydrogel group (implanted with the hydrogel sample in Comparative Example 5), J: bone defect hydrogel group (implanted with the hydrogel sample in Comparative Example 6), K: bone defect PEG hydrogel group (implanted with the hydrogel sample in Comparative Example 7), L: bone defect PEG-nMSC@MT hydrogel group (implanted with the hydrogel sample in Comparative Example 8), the mice were killed 8 weeks after the operation, and the new bone volume after 8 weeks of repair was tested by imaging detection; in the animal experiment of mandibular bone defect repair in mice, hydrogel samples with a shape and size matching the bone defect were implanted; the imaging detection process was: the tissue was placed in a fixative to be fully fixed, and the mandibular sample was scanned using Skyscan1174. The parameters of Skyscan 1174 were set as 8 μm pixel size, 50 kVp kilovolt peak, and 800 μA current. The scanned data were reconstructed using NRecon and CTvox software and imported into CTAn software for image acquisition (Micro-CT scanning) and new bone volume percentage analysis. The new bone volume percentage results of each experimental group after 8 weeks of bone defect repair (n=3) are shown in Table 3. The present invention also provides a blank control group and a Micro-CT detection result diagram of the PEG-pp-nMSC@MT hydrogel prepared in Example 1 of the present invention repairing the mandibular defect of mice for 8 weeks, as shown in FIG. Figure 4 shown.

[0130] Table 3: Comparison of the ability to repair mandibular defects in mice.

[0131]

[0132] It can be seen from the data in Table 3 that the responsive hydrogel loaded with biomimetic nanovesicles prepared in the present invention has an excellent repair effect on bone defects. The responsive hydrogel loaded with biomimetic nanovesicles prepared in some preferred embodiments can make the new bone volume percentage BV / TV reach more than 40% after 8 weeks of bone defect repair, which is of great significance for promoting local bone defect repair and has great application prospects in bone tissue repair materials.

[0133] Parts of the present invention that are not described in detail are well known to those skilled in the art.

[0134] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a responsive hydrogel loaded with biomimetic nanovesicles, characterized in that: The preparation method comprises the following steps: (1) adding a chemical vesicle stimulator to dental mesenchymal stem cells and placing the cells in a CO2 constant temperature incubator for stimulation to obtain a giant plasma membrane vesicle solution; the chemical vesicle stimulator comprises a buffer solution, a dithiothreitol solution and a paraformaldehyde solution; the buffer solution comprises 8.5-9 g / L sodium chloride, 0.2-0.24 g / L calcium chloride, 2.3-2.4 g / L 4-hydroxyethylpiperazineethanesulfonic acid and deionized water; (2) mixing the melatonin mother solution with the giant plasma membrane vesicle solution and extruding the mixture to obtain biomimetic nanovesicles; (3) reacting 6-arm-PEG-NH2 with a peptide cleavable by matrix metalloproteinases activated by EDCI / NHS to obtain a compound 6-arm-PEG-pp-NH2 coupled with a peptide cleavable by matrix metalloproteinases; (4) 6-arm-PEG-pp-NH2 and biomimetic nanovesicles are formulated into a first precursor solution, and six-arm polyethylene glycol succinimidyl glutarate and biomimetic nanovesicles are formulated into a second precursor solution. The first precursor solution and the second precursor solution are then mixed and allowed to stand to form a gel, thereby obtaining a responsive hydrogel loaded with biomimetic nanovesicles.

2. The preparation method according to claim 1, characterized in that: In step (1), the method for obtaining the dental mesenchymal stem cells is: The periodontal ligament tissue or dental pulp tissue was digested and centrifuged, the supernatant was discarded, and then α-MEM culture medium containing fetal bovine serum and penicillin-streptomycin was added and mixed evenly. Then, it was cultured in a CO2 constant temperature incubator until the primary stem cells reached 80%~90% fusion, and then digested with trypsin-EDTA digestion solution and cultured and expanded to P4~P6 generations at a ratio of 1:2 or 1:3 to obtain dental mesenchymal stem cells.

3. The preparation method according to claim 2, characterized in that: The volume fraction of fetal bovine serum contained in the α-MEM medium is 10-20%, and the volume fraction of penicillin-streptomycin contained in the α-MEM medium is 0.8-1.2%; and / or The trypsin-EDTA digestion solution is a digestion solution containing 0.25 wt % of trypsin and 0.1 wt % of EDTA.

4. The preparation method according to claim 1, characterized in that: In step (1): The dithiothreitol solution is a dithiothreitol aqueous solution, and the concentration of the dithiothreitol aqueous solution is 150-160 mg / mL; The paraformaldehyde solution is a 4% paraformaldehyde fixative; and / or The volume ratio of the buffer solution, the dithiothreitol solution and the paraformaldehyde solution is 1000:(1.5-2.5):(16-20).

5. The preparation method according to claim 1, characterized in that: In step (1): The volume fraction of CO2 contained in the CO2 constant temperature incubator is 5%, the temperature of the CO2 constant temperature incubator is 37°C, and the humidity is 100%; The stimulation time is 18 to 30 hours; The amount of the chemical vesicle stimulator added is 18-25 microliters of the chemical vesicle stimulator per square centimeter of dental mesenchymal stem cells; and / or The giant plasma membrane vesicle solution contains giant plasma membrane vesicles at a concentration of 6.0×10 6 ~6.7×10 6 Pieces / mL.

6. The preparation method according to claim 1, characterized in that: In step (2): The melatonin mother solution uses dimethyl sulfoxide and PBS buffer as solvents, and the concentration of melatonin in the melatonin mother solution is 0.0008-0.0012 mol / L; The volume ratio of the melatonin mother solution to the giant plasma membrane vesicle solution is 1:(8-10); and / or The particle size of the bionic nanovesicle is not greater than 200 nm; and / or The extrusion is repeated extrusion through a polycarbonate membrane using a liposome extruder, the number of repeated extrusions is 8 to 12 times, and the polycarbonate membrane is a polycarbonate membrane with a pore size of 200 nm.

7. The preparation method according to claim 1, characterized in that: In step (3): The mass ratio of the 6-arm-PEG-NH2 to the EDCI / NHS activated polypeptide cleavable by matrix metalloproteinase is (30-40):1; and / or The reaction temperature is 30-40° C., and the reaction time is 18-30 h.

8. The preparation method according to claim 1, characterized in that: In step (4): The first precursor solution is prepared by using a PBS buffer to prepare a PBS buffer containing biomimetic nanovesicles, and then adding 6-arm-PEG-pp-NH2 to the PBS buffer containing biomimetic nanovesicles and mixing them evenly to obtain a first precursor solution, wherein the protein concentration of the biomimetic nanovesicles in the PBS buffer containing biomimetic nanovesicles is 1-1.5 mg / mL, and the mass ratio of the PBS buffer containing biomimetic nanovesicles to the 6-arm-PEG-pp-NH2 is 100:(10-18); The second precursor solution is prepared by: preparing the biomimetic nanovesicles with PBS buffer to prepare a PBS buffer containing biomimetic nanovesicles, and then adding six-arm polyethylene glycol succinimidyl glutarate to the PBS buffer containing the biomimetic nanovesicles and mixing them evenly to obtain the second precursor solution, wherein the protein concentration of the biomimetic nanovesicles in the PBS buffer containing the biomimetic nanovesicles is 1-1.5 mg / mL, and the mass ratio of the PBS buffer containing the biomimetic nanovesicles to the six-arm polyethylene glycol succinimidyl glutarate is 100:(10-18); and / or The mass ratio of 6-arm-PEG-pp-NH2 contained in the first precursor solution to six-arm polyethylene glycol succinimidyl glutarate contained in the second precursor solution is (0.5~2): (0.5~2).

9. A responsive hydrogel loaded with biomimetic nanovesicles prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the responsive hydrogel loaded with biomimetic nanovesicles prepared by the preparation method according to any one of claims 1 to 8 in bone tissue repair materials.

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