Cartilage-affinity cross-linked liposome nano-particles as well as preparation method and application of cartilage-affinity cross-linked liposome nano-particles

Abstract

The invention relates to cartilage affinity cross-linked liposome nanoparticles and a preparation method and application thereof.The cross-linked liposome nanoparticles are composed of multiple layers of lipid vesicles and ligands, the ligands are covalently conjugated to the multiple layers of lipid vesicles to form the cross-linked liposome nanoparticles, the ligands are sulfydryl modified polypeptides, and the sulfydryl modified polypeptides are covalently conjugated to the multiple layers of lipid vesicles to form the cross-linked liposome nanoparticles. Protein molecules and / or hydrophobic small molecule drugs are encapsulated by the cross-linked liposome nanoparticles to obtain the multi-layer cross-linked liposome, and the cross-linked liposome has cartilage affinity, can be combined with cartilage for a long time to prolong the residence time by delivering the drugs to cartilage where arthritis occurs in articular cavities, and can be used for preparing the cartilage drug. The staying time of the multi-layer cross-linked liposome in the articular cavity is prolonged to 40 days or above, and the cross-linked liposome nano-particles are non-toxic by increasing the local drug concentration and have a better drug delivery effect and excellent targeted binding performance.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to a cartilage-affinitive cross-linked liposome nanoparticle, a preparation method thereof, and uses thereof. Background Art

[0002] Osteoarthritis is a disease caused by multiple pathogenic factors. A series of mechanical and biological reactions related to various factors such as autoinflammation, aging, obesity, immunity, metabolism, genetics, and chronic strain will imbalance the balance between bone degradation and generation in joint tissues, thus accelerating the development of osteoarthritis in the body. At the same time, osteoarthritis may also experience stages or show progressive evolution over time, leading to further severity and deterioration of the degree and symptoms.

[0003] More than 500 million people worldwide suffer from osteoarthritis and related diseases, and women are particularly severely affected by the disease. The pain and limited mobility caused by osteoarthritis affect the physical and mental health of patients. Although osteoarthritis is not a fatal disease, with the increasing aging of the population, it has brought a huge burden all over the world, such as social burden, economic burden, etc., especially in some regions with a serious aging population. And currently, there is a trend of younger onset of osteoarthritis patients, especially among high-risk and susceptible occupations such as athletes. In addition, when osteoarthritis develops to the middle and late stages, it will become one of the main causes of disability. According to the United Nations' estimate, by 2050, 20% of the global population will be composed of people over 60 years old, which means that by 2050, at least 130 million people worldwide will be affected by osteoarthritis, and among them, it is estimated that 40 million people will suffer from severe disability due to the development of osteoarthritis to the middle and late stages.

[0004] Currently, the strategies for preventing and treating early-stage osteoarthritis mainly aim to reduce joint pain and restore joint function at the same time, so as to delay surgical treatment as much as possible. If there are no obvious lesions or abnormal conditions requiring surgical treatment, the widely used conservative treatment methods mainly include dietary supplements, drug therapies, and minimally invasive surgeries involving injecting various substances, aiming to restore joint homeostasis with the least trauma and provide clinical improvement and possible disease remission effects. Almost all osteoarthritis patients will experience continuous regular pain, which undoubtedly restricts their daily activities. Therefore, drug analgesic treatment is one of the most commonly used methods to improve the quality of life of patients. The medications for osteoarthritis are mainly divided into two categories: analgesic drugs such as paracetamol (acetaminophen) and ibuprofen (ibuprofen), and drugs for delaying cartilage degeneration such as glucosamine and chondroitin sulfate, but they can only temporarily relieve pain and are not long-term curative treatment strategies.

[0005] For the treatment of early cartilage damage and mid-late stage osteoarthritis, there is currently no optimal treatment plan clinically. It is mainly based on the personal preferences of clinicians, and most aim at cartilage repair and reconstruction of a healthy cartilage joint surface.

[0006] Inject chondrogenic drugs into the joint cavity through arthrocentesis to repair damaged cartilage and delay or avoid invasive treatment in the late stage of the disease. For example, Ma L, Zheng X, Lin R, et al. Knee osteoarthritis therapy: recent advances in intra-articular drug delivery systems[J]. Drug design, development and therapy, 2022: 1311-1347. However, during the injection process, the self-structure of articular cartilage and the clearance effect of surrounding capillaries and lymphatic vessels can hinder the penetration of drugs into the cartilage tissue within the joint cavity. Due to the lack of an effective drug delivery system to break through these obstacles, the drug efficacy is difficult to meet clinical needs.

[0007] Based on the above situation of the existing technology, there are technical problems in the existing technology that need to be solved urgently, such as the unsatisfactory treatment effect of traditional osteoarthritis and the inability of chondrogenic drugs to effectively deliver drugs to the lesion against the patient's own clearance effect. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides a cartilage-affinitive cross-linked liposome nanoparticle (CBP-stabilized ICMVs), which is composed of multilayer lipid vesicles and a ligand. The mass ratio of the multilayer lipid vesicles to the ligand is 2.2:(0.56 - 0.8). The ligand is covalently conjugated to the multilayer lipid vesicles to form the cross-linked liposome nanoparticle, and the ligand is a polypeptide modified with a thiol group.

[0009] The carboxyl terminus (-COOH, C-terminus) of the polypeptide is connected to cysteine, and the cysteine contains a thiol group. The thiol group undergoes a Michael addition reaction with the maleic anhydride bond on the multilayer lipid vesicles to form a covalent bond, realizing the covalent conjugation between the ligand and the multilayer lipid vesicles. The cross-linking ratio of the ligand to the multilayer lipid vesicles is 0.7 - 1, the grafting rate of the ligand in the covalent conjugation is 20 - 40%, the potential of the cross-linked liposome nanoparticle is greater than or equal to 1.21 mV, and the particle size is 200 - 240 nm.

[0010] Furthermore, the ligand is purchased from Wuhan Jipeptide Biotechnology Co., Ltd.

[0011] Further, the polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized from an amino acid sequence, which is SEQ ID NO:1.

[0012] Further, the multi-layered lipid vesicles include functionalized lipids (MPB), phosphatidylcholine, and phosphatidylglycerol.

[0013] Further, the functionalized lipid is a maleimide-functionalized lipid, the phosphatidylcholine is dioleoylphosphatidylcholine, and the phosphatidylglycerol is sodium 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol).

[0014] The present invention also provides a method for preparing the above-mentioned cartilage-affinitive cross-linked liposome nanoparticles, which comprises the following steps:

[0015] Step 1, preparing a solution: the solution includes one or more of bis-tris propane buffer solution (Bis-tris buffer), dithiothreitol solution (DTT solution), magnesium chloride solution, and calcium chloride solution;

[0016] Step 2, preparing a liposome solution: dissolving phosphatidylcholine, phosphatidylglycerol, and functionalized lipids in chloroform to obtain a liposome solution;

[0017] Step 3, preparing a lipid film: air-drying the liposome solution prepared in Step 2 at room temperature to obtain a lipid film;

[0018] Step 4, preparing multi-layered liposomes: hydrating the lipid film prepared in Step 3 with a buffer solution, and dissolving the lipid film in the buffer solution by oscillation to form a mixed solution A1, which contains multi-layered liposomes at the micron level;

[0019] Step 5, preparing single-layer liposomes: using a liposome extruder to repeatedly extrude the mixed solution A1 prepared in Step 4 to obtain a mixed solution B1, which contains single-layer liposomes;

[0020] Step 6, preparing multi-layered lipid vesicles: adding a divalent ion solution and a dithiothreitol solution to the mixed solution B1, incubating with stirring, centrifuging after incubation, discarding the supernatant to obtain a solid C1, washing the solid C1 with bis-tris propane buffer solution and then centrifuging again to obtain a solid D1, which is the multi-layered lipid vesicles;

[0021] Step 7, preparing cross-linked liposome nanoparticles: mixing the obtained multi-layered lipid vesicles with a ligand and incubating, covalently conjugating the ligand to the multi-layered lipid vesicles to obtain cross-linked liposome nanoparticles.

[0022] Further, the preparation method of the Bis-tris buffer solution in step 1 is as follows:

[0023] Weigh Bis-tris and prepare a 10 mM Bis-tris buffer solution with deionized water.

[0024] Further, the preparation method of the dithiothreitol solution (DTT solution) in step 1 is as follows:

[0025] Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL using the Bis-tris buffer solution.

[0026] Further, the preparation method of the magnesium chloride solution in step 1 is as follows:

[0027] Weigh magnesium chloride hexahydrate and prepare a magnesium chloride solution with a concentration of 0.085 mg / mL using the Bis-tris buffer solution.

[0028] Further, the preparation method of the calcium chloride solution in step 1 is as follows:

[0029] Weigh anhydrous calcium chloride and prepare a calcium chloride solution with a concentration of 0.116 mg / mL using the Bis-tris buffer solution.

[0030] Further, the molar ratio of choline phosphate, glycerol phosphate, and functionalized lipid in step 2 is (4 - 5):(1 - 2):(4 - 10).

[0031] Further, the total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution in step 2 is 0.0125 - 0.02125 mol / L.

[0032] Further, the functionalized lipid is maleimide-functionalized lipid (MPB).

[0033] Further, the preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0034] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, obtain organic solution a; the organic solution a is successively passed through a 5% mass concentration of Na 2 CO 3The organic solution b was obtained by washing with a solution, a 1% HCl solution, and a 1% NaCl solution, and the organic solution b was dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid.

[0035] wherein the molar ratio of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine is 1:1:2, and the total concentration of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L, and the used Na 2 CO 3 The volumes of the solution, the HCl solution, and the NaCl solution are the same as the volume of chloroform, the temperature of the drying and evaporation is 24 °C, and the time of the drying and evaporation is 24 h.

[0036] Furthermore, the air-drying time in step 3 is 12 - 24 h.

[0037] Furthermore, the concentration of the lipid membrane in the buffer solution in step 4 is 1 - 2 μmol / ml.

[0038] Furthermore, the buffer solution in step 4 is a bis-tris (hydroxymethyl) aminomethane buffer solution.

[0039] Furthermore, the oscillation conditions in step 4 are: oscillating once every 10 minutes during the hydration process, the time of oscillating once is 30 seconds, and the number of oscillations is 6 times.

[0040] Furthermore, the liposome extruder in step 5 contains a 100 μm filter membrane.

[0041] Furthermore, the number of repeated extrusions in step 5 is 20 - 30 times.

[0042] Furthermore, the divalent ion solution in step 6 is one or two of magnesium chloride solution and calcium chloride solution.

[0043] Furthermore, the volume ratio of the mixed solution B1, the divalent ion solution, and the dithiothreitol solution in step 6 is 1000:(5 - 20):(14.7 - 21).

[0044] Furthermore, the stirring speed in step 6 is 120 - 200 rpm, the incubation temperature is 37 °C, and the incubation time is 1 - 2 h; the conditions for centrifugation and re-centrifugation are both 14000 rpf and 4 minutes.

[0045] Furthermore, the incubation temperature in step 7 is 37 °C, and the incubation time is 0.5 - 1 h.

[0046] Further, the grafting rate of covalent conjugation in step 7 is 20-40%.

[0047] Further, the preparation method of the ligand in step 7 is as follows: Cysteine is connected to the C-terminus of the polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using existing techniques to obtain the ligand. The polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is SEQ ID NO:1.

[0048] Further, when preparing the liposome solution in step 2, rhodamine-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (Rhodamine-DSPE) is added for fluorescence labeling to facilitate observation under a confocal microscope.

[0049] Further, rhodamine-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine with 1 / 100 of the molar mass of choline phosphate is used to replace the functionalized lipid in an equimolar amount.

[0050] Further, the rhodamine-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine is purchased from Xi'an Ruixi Biotechnology Co., Ltd., and the model is DSPE-Rhodamine B R-DZP006.

[0051] The present invention also provides a multi-layer cross-linked liposome, which includes the above-mentioned cartilage-affinitive cross-linked liposome nanoparticles and proteinaceous molecules and / or hydrophobic small molecule drugs encapsulated in the cross-linked liposome nanoparticles, and the encapsulation rate of the multi-layer cross-linked liposome is ≥40%.

[0052] Further, the proteinaceous molecules and / or hydrophobic small molecule drugs are encapsulated in the multi-layer lipid vesicles of the cross-linked liposome nanoparticles.

[0053] Further, the proteinaceous molecules include one or more of the model proteinaceous drugs bovine serum albumin (BSA) and transforming growth factor (TGF-β3).

[0054] Further, the hydrophobic small molecule drug is 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid (kartogenin, KGN).

[0055] Further, the preparation method of the multi-layer cross-linked liposome includes the following steps:

[0056] Step 1, Prepare the solution: The solution includes one or more of bis-tris buffer solution, dithiothreitol solution (DTT solution), magnesium chloride solution, and calcium chloride solution;

[0057] Step 2, Prepare the liposome solution: Dissolve phosphatidylcholine, phosphatidylglycerol, and functionalized lipid in chloroform to obtain the liposome solution;

[0058] Step 3, Prepare the liposome mixture: Dissolve protein molecules and / or hydrophobic small molecule drugs in bis-tris buffer solution or chloroform to obtain a dissolved solution, and add the dissolved solution to the liposome solution prepared in Step 2 to obtain the liposome mixture;

[0059] Step 4, Prepare the lipid film containing the drug: Air-dry the liposome mixture prepared in Step 3 at room temperature to obtain the lipid film containing the drug;

[0060] Step 5, Prepare the multi-layer liposome containing the drug: Hydrate the lipid film containing the drug prepared in Step 4 with a buffer solution, and through oscillation, fully dissolve the lipid film containing the drug in the buffer solution to form a mixed solution A2, and the mixed solution A2 includes micron-sized multi-layer liposomes containing the drug;

[0061] Step 6, Prepare the single-layer liposome containing the drug: Use a liposome extruder to repeatedly extrude the mixed solution A2 prepared in Step 5 to obtain a mixed solution B2, and the mixed solution B2 contains single-layer liposomes containing the drug;

[0062] Step 7, Prepare the multi-layer lipid vesicle containing the drug: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B2, incubate with stirring, after the incubation ends, centrifuge, discard the supernatant to obtain a solid C2, wash the solid C2 with bis-tris buffer solution and then centrifuge again to obtain a solid D2, which is the multi-layer lipid vesicle containing the drug;

[0063] Step 8, Prepare the multi-layer cross-linked liposome: Mix the obtained multi-layer lipid vesicle containing the drug with a ligand and then incubate, covalently conjugate the ligand to the multi-layer lipid vesicle containing the drug to obtain the multi-layer cross-linked liposome.

[0064] Furthermore, the preparation method of the bis-tris buffer solution in Step 1 is as follows:

[0065] Weigh bis-tris and prepare a 10 mM bis-tris buffer solution with deionized water.

[0066] Further, the preparation method of the dithiothreitol solution (DTT solution) in Step 1 is as follows:

[0067] Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL of dithiothreitol using bis-tris propane buffer solution.

[0068] Further, the preparation method of the magnesium chloride solution in Step 1 is as follows:

[0069] Weigh magnesium chloride hexahydrate and prepare a magnesium chloride solution with a concentration of 0.085 mg / mL of magnesium chloride hexahydrate using bis-tris propane buffer solution.

[0070] Further, the preparation method of the calcium chloride solution in Step 1 is as follows:

[0071] Weigh anhydrous calcium chloride and prepare a calcium chloride solution with a concentration of 0.116 mg / mL of anhydrous calcium chloride using bis-tris propane buffer solution.

[0072] Further, the molar ratio of choline phosphate, glycerol phosphate, and functionalized lipid in Step 2 is (4 - 5):(1 - 2):(4 - 10). Further, the total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution in Step 2 is 0.0125 - 0.02125 mol / L.

[0073] Further, the functionalized lipid is maleimide-functionalized lipid (MPB).

[0074] Further, the preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0075] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, obtain organic solution a; the organic solution a is successively washed with a 5% (mass concentration) Na 2 CO 3 solution, a 1% (mass fraction) HCl solution, and a 1% (mass fraction) NaCl solution to obtain organic solution b. The organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid.

[0076] wherein the molar ratio of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine is 1:1:2, and the total concentration of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L, and the used Na2 CO 3 The volumes of the CO solution, HCl solution, and NaCl solution are the same as that of chloroform respectively. The temperature of drying and evaporation is 24 °C, and the time of drying and evaporation is 24 h.

[0077] Furthermore, in step 3, the proteinaceous molecules include one or more of the model protein drugs bovine serum albumin (BSA) and transforming growth factor (TGF-β3), and the hydrophobic small molecule drug is 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid (kartogenin, KGN).

[0078] Furthermore, when proteinaceous molecules are selected, they are mixed with the bis-tris buffer solution to obtain a proteinaceous molecule buffer solution, i.e., a dissolution solution. The concentration of the proteinaceous molecules in the proteinaceous molecule buffer solution is 0.25 - 1 mg / mL.

[0079] Among them, when the selected proteinaceous molecules are the model protein drugs bovine serum albumin (BSA) and transforming growth factor (TGF-β3), the ratio between them can be arbitrary, as long as the concentration value of the proteinaceous molecules in the proteinaceous molecule buffer solution is satisfied.

[0080] Furthermore, when the hydrophobic small molecule drug is selected, it is mixed with the chloroform to obtain a dissolution solution. The concentration of the hydrophobic small molecule drug in the dissolution solution is 0.5 - 1 mg / mL.

[0081] Furthermore, the ratio of the total mass of the proteinaceous molecules and / or hydrophobic small molecule drugs in the dissolution solution to the total moles of phosphocholine, phosphoglycerol, and functionalized lipid in the liposome solution is (0.5 - 1.7):(2.5 - 4.25), where the unit of the total mass is g and the unit of the total moles is mol.

[0082] Furthermore, the air-drying time in step 4 is 12 - 24 h.

[0083] Furthermore, the concentration of the lipid membrane containing drugs in the buffer solution in step 5 is 1 - 2 μmol / ml.

[0084] Furthermore, the buffer solution in step 5 is one of the bis-tris buffer solution or the proteinaceous molecule buffer solution.

[0085] Furthermore, the oscillation conditions in step 5 are: oscillating once every 10 minutes during the hydration process, the time for each oscillation is 30 seconds, and the number of oscillations is 6 times.

[0086] Furthermore, the liposome extruder in step 6 contains a 100 μm filter membrane.

[0087] Further, the number of repeated squeezes in step 6 is 20 - 30 times.

[0088] Further, the divalent ion solution in step 7 is one or both of a magnesium chloride solution and a calcium chloride solution.

[0089] Further, the volume ratio of the mixed solution B2, the divalent ion solution, and the dithiothreitol solution in step 7 is 1000:(5 - 20):(14.7 - 21).

[0090] Further, the stirring speed in step 7 is 120 - 200 rpm, the incubation temperature is 37 °C, and the incubation time is 1 - 2 h; the conditions for both centrifugation and re - centrifugation are 14000 rpf for 4 minutes.

[0091] Further, the incubation temperature in step 8 is 37 °C, and the incubation time is 0.5 - 1 h.

[0092] Further, the grafting rate of the covalent conjugation in step 8 is 20 - 40%.

[0093] Further, the preparation method of the ligand in step 8 is as follows: Cysteine is connected to the C - terminus of the polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using existing techniques to obtain the ligand. The polypeptide is a collagen - binding peptide (CBP), and the collagen - binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is SEQ ID NO:1.

[0094] Further, when preparing the liposome solution in step 2, rhodamine - modified 1,2 - distearoyl - sn - glycero - 3 - phosphoethanolamine (Rhodamine - DSPE) is added for fluorescence labeling to facilitate observation under a confocal microscope.

[0095] Further, rhodamine - modified 1,2 - distearoyl - sn - glycero - 3 - phosphoethanolamine with a molar mass of 1 / 125 of the functionalized lipid is used to replace 1 / 125 of the functionalized lipid in an equimolar amount.

[0096] Further, the rhodamine - modified 1,2 - distearoyl - sn - glycero - 3 - phosphoethanolamine is purchased from Xi'an Ruixi Biotechnology Co., Ltd., and the model is DSPE - Rhodamine B R - DZP006.

[0097] The present invention also provides a drug, and the drug includes the above - mentioned multi - layer cross - linked liposomes.

[0098] Further, the delivery system includes an oral drug or an injectable vaccine.

[0099] Furthermore, the multilamellar crosslinked liposomes are delivered into the joint cavity, and the multilamellar crosslinked liposomes target the arthritic cartilage in the joint cavity through the ligands on the crosslinked liposome nanoparticles, delivering the drug directly to the cartilage and continuously releasing the encapsulated protein molecules and / or hydrophobic small molecule drugs. The ligands in the multilamellar crosslinked liposomes achieve the cartilage affinity of the multilamellar crosslinked liposomes, such that the residence time of the multilamellar crosslinked liposomes in the joint cavity is ≥ 40 days.

[0100] The beneficial effects of the present invention are as follows:

[0101] 1. The cartilage-affinity crosslinked liposome nanoparticles prepared by the present invention can encapsulate protein molecules and / or hydrophobic small molecule drugs to obtain the multilamellar crosslinked liposomes. The ligands in the multilamellar crosslinked liposomes target the arthritic cartilage in the joint cavity, delivering the drug directly to the cartilage, being able to bind to the cartilage for a long time to extend the residence time, achieving the cartilage affinity of the multilamellar crosslinked liposomes of the present invention, increasing the residence time of the multilamellar crosslinked liposomes in the joint cavity to more than 40 days. By increasing the local drug concentration, and the crosslinked liposome nanoparticles prepared by the present invention are non-toxic, have excellent drug delivery effects and excellent target binding performance;

[0102] 2. The cartilage-affinity crosslinked liposome nanoparticles prepared by the present invention are composed of multilamellar lipid vesicles and ligands, and are multilamellar crosslinked liposomes with high matrix affinity. The ligands are covalently conjugated to the multilamellar lipid vesicles to form the crosslinked liposome nanoparticles. The ligands are thiol-modified polypeptides, and the polypeptides selected by the present invention are collagen-binding peptides, which can bind to the cartilage for a long time to extend the residence time;

[0103] 3. The drug prepared by the present invention using the multilamellar crosslinked liposomes can be widely applied in aspects such as cartilage regeneration. For example, in the treatment of cartilage defects caused by osteoarthritis, using the multilamellar crosslinked liposomes included in the drug as a carrier, the cartilage defects can be treated by minimally invasive means - intra-articular injection, improving the treatment effect, avoiding the impacts and problems brought by surgical operations such as joint replacement. Moreover, due to the cartilage affinity of the multilamellar crosslinked liposomes prepared by the present invention, the drug can be accurately enriched to a large extent, reducing the drug loss rate and improving the efficiency of cartilage regeneration. Description of the Drawings

[0104] Figure 1 It is a comparative diagram of the average particle size test results in Test Example 3 of the present invention;

[0105] Figure 2It is the comparison chart of potential test results in Test Example 4 of the present invention;

[0106] Figure 3 It is the comparison chart of encapsulation efficiency test results of Comparative Examples 4-12 in Test Example 5 of the present invention;

[0107] Figure 4 It is the comparison chart of release cycle test results in Test Example 6 of the present invention;

[0108] Figure 5 It is the comparison chart of the results observed by confocal microscopy in Test Example 7 of the present invention;

[0109] Figure 6 It is the comparison chart of the results observed by confocal microscopy in Test Example 8 of the present invention;

[0110] Figure 7 It is the schematic diagram of subcutaneous injection of liposomes into the left and right knee joints of mice in Test Example 9 of the present invention;

[0111] Figure 8 It is the in vivo imaging diagram of mice in Test Example 9 of the present invention;

[0112] Figure 9 It is the half-life curve graph in Test Example 9 of the present invention. Detailed implementation manners

[0113] Example 1

[0114] This example provides a preparation method of cartilage-affinity cross-linked liposome nanoparticles (CBP-stabilized ICMVs), including the following steps:

[0115] Step 1, prepare the solution: The solution includes bis-tris buffer solution, dithiothreitol solution (DTT solution), and magnesium chloride solution;

[0116] The preparation method of the bis-tris buffer solution: Weigh bis-tris and prepare a 10 mM bis-tris buffer solution with deionized water.

[0117] The preparation method of the dithiothreitol solution (DTT solution): Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL of dithiothreitol with bis-tris buffer solution.

[0118] The preparation method of the magnesium chloride solution: Weigh magnesium chloride hexahydrate and prepare a magnesium chloride solution with a concentration of 0.085 mg / mL of magnesium chloride hexahydrate with bis-tris buffer solution.

[0119] Step 2: Prepare liposome solution: Dissolve choline phosphate, glycerol phosphate, and functionalized lipid in chloroform at a molar ratio of 4:1:5, i.e., 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain a liposome solution. The total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution is 0.0125 mol / L;

[0120] In this example, the functionalized lipid is maleimide-functionalized lipid, the choline phosphate is dioleoylphosphatidylcholine, and the glycerol phosphate is 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt.

[0121] The preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0122] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, obtain organic solution a; the organic solution a is successively washed with a 5% (mass concentration) Na 2 CO 3 solution, a 1% (mass fraction) HCl solution, and a 1% (mass fraction) NaCl solution, to obtain organic solution b. The organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid.

[0123] Among them, the molar ratio of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine is 1:1:2. The total concentration of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L. The volumes of the used Na 2 CO 3 solution, HCl solution, and NaCl solution are the same as the volume of chloroform. The temperature of the drying and evaporation is 24 °C, and the time of the drying and evaporation is 24 h.

[0124] Step 3: Prepare lipid film: Air-dry the liposome solution prepared in Step 2 at room temperature for 12 h to obtain a lipid film;

[0125] Step 4: Prepare multilamellar liposomes: Hydrate the lipid film prepared in Step 3 with a buffer solution. The concentration of the lipid film in the buffer solution is 1 μmol / ml. The buffer solution is bis-tris propane buffer solution. By shaking, the lipid film is fully dissolved in the buffer solution to form a mixed solution A1. The mixed solution A1 contains micron-sized multilamellar liposomes;

[0126] The conditions for oscillation are as follows: during the hydration process, oscillation is performed once every 10 minutes, the oscillation time each time is 30 seconds, and the number of oscillations is 6 times.

[0127] Step 5: Prepare monolayer liposomes: Use a liposome extruder containing a 100 μm filter membrane to repeatedly extrude the mixed solution A1 prepared in Step 4 20 times to obtain a mixed solution B1, and the mixed solution B1 contains monolayer liposomes;

[0128] Step 6: Prepare multilamellar lipid vesicles: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B1. The volume ratio of the mixed solution B1 to the divalent ion solution is 1000:5:21. Incubate at 37 °C with a stirring speed of 120 rpm for 1 h. After incubation, centrifuge at 14000 rpf for 4 minutes, discard the supernatant to obtain a solid C1. Wash the solid C1 with a bis-tris buffer solution and then centrifuge again at 14000 rpf for 4 minutes to obtain a solid D1, which is the multilamellar lipid vesicles; in this example, the divalent ion solution is a magnesium chloride solution;

[0129] Step 7: Prepare cross-linked liposome nanoparticles: Prepare a ligand. The preparation method of the ligand is as follows: Cysteine is connected to the C-terminus of a polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using existing technology to obtain the ligand. The polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is SEQ ID NO:1; After mixing the obtained multilamellar lipid vesicles with the ligand and incubating at 37 °C for 0.5 h, covalently conjugate the ligand to the multilamellar lipid vesicles to obtain cross-linked liposome nanoparticles.

[0130] In this example, the mass of the polypeptide, i.e., the collagen-binding peptide (CBP), is 0.8 mg.

[0131] Example 2

[0132] This example provides a method for preparing cartilage-affinitive cross-linked liposome nanoparticles (CBP-stabilized ICMVs), including the following steps:

[0133] Step 1: Prepare solutions: The solutions include a bis-tris buffer solution, a dithiothreitol solution (DTT solution), and a calcium chloride solution;

[0134] The preparation method of the bis-tris buffer solution: Weigh bis-tris and prepare a 10 mM bis-tris buffer solution with deionized water.

[0135] The preparation method of the dithiothreitol solution (DTT solution) is as follows: Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL of dithiothreitol using bis-tris propane buffer solution.

[0136] The preparation method of the calcium chloride solution is as follows: Weigh anhydrous calcium chloride and prepare a calcium chloride solution with a concentration of 0.116 mg / mL of anhydrous calcium chloride using bis-tris propane buffer solution.

[0137] Step 2: Prepare the liposome solution: Dissolve choline phosphate, glycerol phosphate, and functionalized lipid in chloroform according to a molar ratio of 4:1:5, that is, 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain the liposome solution. The total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution is 0.0125 mol / L.

[0138] In this example, the functionalized lipid is maleimide-functionalized lipid, the choline phosphate is dioleoylphosphatidylcholine, and the glycerol phosphate is 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt.

[0139] The preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0140] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, obtain organic solution a; Organic solution a is washed successively with a 5% (mass concentration) Na 2 CO 3 solution, a 1% (mass fraction) HCl solution, and a 1% (mass fraction) NaCl solution to obtain organic solution b. Organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid.

[0141] where the molar ratio of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine is 1:1:2. The total concentration of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L. The volumes of the used Na 2 CO 3 solution, HCl solution, and NaCl solution are the same as the volume of chloroform. The temperature of the drying and evaporation is 24 °C, and the time of the drying and evaporation is 24 h.

[0142] Step 3: Preparation of lipid film: The liposome solution prepared in Step 2 is air-dried at room temperature for 12 h to obtain a lipid film;

[0143] Step 4: Preparation of multilamellar liposomes: Hydrate the lipid film prepared in Step 3 with a buffer solution. The concentration of the lipid film in the buffer solution is 1 μmol / ml. The buffer solution is bis-tris propane buffer solution. By shaking, the lipid film is fully dissolved in the buffer solution to form a mixed solution A1, and the mixed solution A1 contains micron-sized multilamellar liposomes;

[0144] The conditions for the shaking are: shake once every 10 minutes during the hydration process, the shaking time for one time is 30 seconds, and the number of shaking times is 6 times.

[0145] Step 5: Preparation of unilamellar liposomes: Use a liposome extruder containing a 100 μm filter membrane to repeatedly extrude the mixed solution A1 prepared in Step 4 20 times to obtain a mixed solution B1, and the mixed solution B1 contains unilamellar liposomes;

[0146] Step 6: Preparation of multilamellar lipid vesicles: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B1. The volume ratio of the mixed solution B1 to the divalent ion solution is 1000:5:21. In this example, the usage amount of the calcium chloride solution is 10 μL, and the usage amount of the dithiothreitol solution (DTT) is 16.8 μL. Incubate at 37 °C with a stirring speed of 120 rpm for 1 h. After the incubation, centrifuge at 14000 rpf for 4 minutes, discard the supernatant to obtain a solid C1, wash the solid C1 with bis-tris propane buffer solution and then centrifuge at 14000 rpf for 4 minutes again to obtain a solid D1, which is the multilamellar lipid vesicles; in this example, the divalent ion solution is a magnesium chloride solution;

[0147] Step 7: Preparation of cross-linked liposome nanoparticles: Prepare a ligand. The preparation method of the ligand is: Cysteine is connected to the C-terminus of a polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using existing technology to obtain a ligand. The polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is LRELHLNNN; After mixing the obtained multilamellar lipid vesicles with the ligand and incubating at 37 °C for 0.5 h, covalently conjugate the ligand to the multilamellar lipid vesicles to obtain cross-linked liposome nanoparticles.

[0148] In this example, the mass of the polypeptide, collagen-binding peptide (CBP), is 0.8 mg.

[0149] Example 3

[0150] This embodiment provides a method for preparing multi-layer cross-linked liposomes, wherein the multi-layer cross-linked liposomes encapsulate the model protein drug bovine serum albumin (BSA), and the method comprises the following steps:

[0151] Step 1, preparing solutions: The solutions include bis-tris buffer solution, dithiothreitol solution (DTT solution), and magnesium chloride solution;

[0152] The preparation method of the bis-tris buffer solution is as follows: Weigh bis-tris (hydroxymethyl) aminomethane and prepare a 10 mM bis-tris buffer solution with deionized water.

[0153] The preparation method of the dithiothreitol solution (DTT solution) is as follows: Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL of dithiothreitol using the bis-tris buffer solution.

[0154] The preparation method of the magnesium chloride solution is as follows: Weigh magnesium chloride hexahydrate and prepare a magnesium chloride solution with a concentration of 0.085 mg / mL of magnesium chloride hexahydrate using the bis-tris buffer solution.

[0155] Step 2, preparing a liposome solution: Dissolve phosphatidylcholine, phosphatidylglycerol, and functionalized lipid in chloroform according to a molar ratio of 4:1:5, that is, 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain a liposome solution. The total concentration of phosphatidylcholine, phosphatidylglycerol, and functionalized lipid in the liposome solution is 0.0125 mol / L;

[0156] In this embodiment, the functionalized lipid is maleimide-functionalized lipid, the phosphatidylcholine is dioleoylphosphatidylcholine, and the phosphatidylglycerol is 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt.

[0157] The preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0158] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, an organic solution a is obtained; the organic solution a is successively washed with a 5% Na 2 CO 3 solution, a 1% HCl solution, and a 1% NaCl solution to obtain an organic solution b. The organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid.

[0159] The molar ratio of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine is 1:1:2. The total concentration of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L. The Na 2 CO 3 solution, HCl solution, and NaCl solution have the same volume as that of chloroform respectively. The temperature of drying and evaporation is 24 °C, and the time of drying and evaporation is 24 h.

[0160] Step 3: Prepare the liposome mixture: Dissolve the model protein drug bovine serum albumin (BSA) in the bis-tris buffer solution to obtain a dissolved solution, and add the dissolved solution to the liposome solution prepared in Step 2 to obtain the liposome mixture;

[0161] The mass of the protein molecule in the protein molecule buffer solution is 0.5 mg;

[0162] Step 4: Prepare the lipid membrane containing BSA: Air-dry the liposome solution prepared in Step 3 at room temperature for 12 h to obtain the lipid membrane containing the drug;

[0163] Step 5: Prepare the multilamellar liposomes containing BSA: Hydrate the lipid membrane containing BSA prepared in Step 4 with the buffer solution. The concentration of the lipid membrane containing BSA in the buffer solution is 1 μmol / ml. The buffer solution is the bis-tris buffer solution. By shaking, the lipid membrane containing BSA is fully dissolved in the buffer solution to form the mixed solution A2. The mixed solution A2 includes micron-sized multilamellar liposomes containing BSA;

[0164] The conditions of the shaking are: shake once every 10 minutes during the hydration process, the shaking time for one time is 30 seconds, and the number of shaking times is 6 times.

[0165] Step 6: Prepare the unilamellar liposomes containing BSA: Use a liposome extruder containing a 100 μm filter membrane to extrude the mixed solution A2 prepared in Step 5 20 times repeatedly to obtain the mixed solution B2. The mixed solution B2 contains unilamellar liposomes containing BSA;

[0166] Step 7. Preparation of multi-layered lipid vesicles containing BSA: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B2. The volume ratio of the mixed solution B2 to the divalent ion solution is 1000:5. In this example, the amount of the magnesium chloride solution used is 5 μL, and the amount of the dithiothreitol solution (DTT) used is 16.8 μL. Incubate at 37 °C with a stirring speed of 120 rpm for 1 h. After incubation, centrifuge at 14000 rpf for 4 minutes, discard the supernatant to obtain solid C2. Wash solid C2 with a bis-tris buffer solution and then centrifuge again at 14000 rpf for 4 minutes to obtain solid D2, which is the multi-layered lipid vesicles containing BSA. In this example, the divalent ion solution is the magnesium chloride solution.

[0167] Step 8. Preparation of multi-layered cross-linked liposomes: Prepare a ligand. The preparation method of the ligand is as follows: Cysteine is connected to the C-terminus of a polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using an existing technique to obtain the ligand. The polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is SEQ ID NO:1. Mix the obtained multi-layered lipid vesicles containing BSA with the ligand and incubate at 37 °C for 0.5 h to covalently conjugate the ligand to the multi-layered lipid vesicles to obtain multi-layered cross-linked liposomes.

[0168] Example 4

[0169] This example provides a method for preparing multi-layered cross-linked liposomes. The multi-layered cross-linked liposomes encapsulate a model protein drug bovine serum albumin (BSA) and include the following steps:

[0170] Step 1. Prepare solutions: The solutions include a bis-tris buffer solution, a dithiothreitol solution (DTT solution), and a magnesium chloride solution.

[0171] The preparation method of the bis-tris buffer solution: Weigh bis-tris and prepare a 10 mM bis-tris buffer solution with deionized water.

[0172] The preparation method of the dithiothreitol solution (DTT solution): Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL of dithiothreitol using the bis-tris buffer solution.

[0173] The preparation method of the magnesium chloride solution: Weigh magnesium chloride hexahydrate and prepare a magnesium chloride solution with a concentration of 0.085 mg / mL of magnesium chloride hexahydrate using the bis-tris buffer solution.

[0174] Step 2: Prepare liposome solution: Dissolve choline phosphate, glycerol phosphate, and functionalized lipid in chloroform at a molar ratio of 4:1:5, i.e., 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain a liposome solution. The total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution is 0.0125 mol / L;

[0175] In this example, the functionalized lipid is maleimide-functionalized lipid, the choline phosphate is dioleoylphosphatidylcholine, and the glycerol phosphate is 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt.

[0176] The preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0177] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, obtain organic solution a; The organic solution a is successively washed with a 5% Na 2 CO 3 solution, a 1% HCl solution, and a 1% NaCl solution to obtain organic solution b. The organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid.

[0178] where the molar ratio of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine is 1:1:2, and the total concentration of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L. The volumes of the used Na 2 CO 3 solution, HCl solution, and NaCl solution are the same as the volume of chloroform. The temperature of the drying and evaporation is 24°C, and the time of the drying and evaporation is 24 h.

[0179] Step 3: Prepare liposome mixture: Dissolve the model protein drug bovine serum albumin (BSA) in bis-tris propane buffer solution to obtain a dissolution solution, and add the dissolution solution to the liposome solution prepared in Step 2 to obtain a liposome mixture;

[0180] The mass of the protein molecule in the protein molecule buffer solution is 0.5 mg;

[0181] Step 4. Preparation of lipid membrane containing BSA: The liposome solution prepared in Step 3 is air-dried at room temperature for 12 h to obtain a lipid membrane containing the drug.

[0182] Step 5. Preparation of multilamellar liposomes containing BSA: Hydrate the lipid membrane containing BSA prepared in Step 4 with a buffer solution. The concentration of the lipid membrane containing BSA in the buffer solution is 1 μmol / ml. The buffer solution is bis-tris propane buffer solution. By shaking, the lipid membrane containing BSA is fully dissolved in the buffer solution to form a mixed solution A2, and the mixed solution A2 contains micron-sized multilamellar liposomes containing BSA.

[0183] The conditions for the shaking are: shaking once every 10 minutes during the hydration process, the shaking time for one time is 30 seconds, and the number of shaking times is 6 times.

[0184] Step 6. Preparation of unilamellar liposomes containing BSA: Use a liposome extruder containing a 100 μm filter membrane to repeatedly extrude the mixed solution A2 prepared in Step 5 20 times to obtain a mixed solution B2, and the mixed solution B2 contains unilamellar liposomes containing BSA.

[0185] Step 7. Preparation of multilamellar lipid vesicles containing BSA: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B2. The volume ratio of the mixed solution B2 to the divalent ion solution is 1000:5. In this example, the usage amount of the magnesium chloride solution is 10 μL, and the usage amount of the dithiothreitol solution (DTT) is 16.8 μL. Incubate at 37 °C with a stirring speed of 120 rpm for 1 h. After the incubation, centrifuge at 14000 rpf for 4 minutes, discard the supernatant to obtain a solid C2, wash the solid C2 with bis-tris propane buffer solution and then centrifuge at 14000 rpf again for 4 minutes to obtain a solid D2, which is the multilamellar lipid vesicles containing BSA. In this example, the divalent ion solution is the magnesium chloride solution.

[0186] Step 8. Preparation of multilamellar cross-linked liposomes: Prepare a ligand. The preparation method of the ligand is: Cysteine is connected to the C-terminus of the polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using the prior art to obtain the ligand. The polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is SEQ ID NO:1; After mixing the obtained multilamellar lipid vesicles containing BSA with the ligand and incubating at 37 °C for 0.5 h, covalently conjugate the ligand to the multilamellar lipid vesicles to obtain multilamellar cross-linked liposomes.

[0187] Example 5

[0188] This example provides a method for preparing multilamellar cross-linked liposomes. The multilamellar cross-linked liposomes encapsulate the model protein drug bovine serum albumin (BSA), and the method includes the following steps:

[0189] Step 1, prepare the solutions: The solutions include bis-tris buffer solution, dithiothreitol solution (DTT solution), and magnesium chloride solution;

[0190] The preparation method of the bis-tris buffer solution: Weigh bis-tris and use deionized water to prepare a 10 mM bis-tris buffer solution.

[0191] The preparation method of the dithiothreitol solution (DTT solution): Weigh dithiothreitol (DTT) and use the bis-tris buffer solution to prepare a dithiothreitol solution with a concentration of 2.31 mg / mL.

[0192] The preparation method of the magnesium chloride solution: Weigh magnesium chloride hexahydrate and use the bis-tris buffer solution to prepare a magnesium chloride solution with a concentration of 0.085 mg / mL.

[0193] Step 2, prepare the liposome solution: Dissolve choline phosphate, glycerol phosphate, and functionalized lipid in chloroform according to a molar ratio of 4:1:5, that is, 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain a liposome solution. The total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution is 0.0125 mol / L;

[0194] In this example, the functionalized lipid is maleimide-functionalized lipid, the choline phosphate is dioleoylphosphatidylcholine, and the glycerol phosphate is 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt.

[0195] The preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0196] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, obtain organic solution a; The organic solution a is successively washed with a 5% Na 2 CO 3 solution, a 1% HCl solution, and a 1% NaCl solution, to obtain organic solution b. The organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid.

[0197] wherein the molar ratio of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine is 1:1:2, and the total concentration of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L. The Na 2 CO 3 solution, HCl solution, and NaCl solution have the same volume as the chloroform volume. The temperature for drying and evaporation is 24 °C, and the time for drying and evaporation is 24 h.

[0198] Step 3: Prepare the liposome mixture: Dissolve the model protein drug bovine serum albumin (BSA) in bis-tris propane buffer solution to obtain a dissolved solution, and add the dissolved solution to the liposome solution prepared in Step 2 to obtain a liposome mixture;

[0199] The mass of the protein molecule in the protein molecule buffer solution is 0.5 mg;

[0200] Step 4: Prepare the lipid film containing BSA: Air-dry the liposome solution prepared in Step 3 at room temperature for 12 h to obtain a lipid film containing the drug;

[0201] Step 5: Prepare the multilamellar liposomes containing BSA: Hydrate the lipid film containing BSA prepared in Step 4 with buffer solution. The concentration of the lipid film containing BSA in the buffer solution is 1 μmol / ml, and the buffer solution is bis-tris propane buffer solution. By shaking, the lipid film containing BSA is fully dissolved in the buffer solution to form a mixed solution A2, and the mixed solution A2 includes micron-sized multilamellar liposomes containing BSA;

[0202] The conditions for shaking are: shake once every 10 minutes during the hydration process, the shaking time for one time is 30 seconds, and the number of shaking times is 6 times.

[0203] Step 6: Prepare the unilamellar liposomes containing BSA: Use a liposome extruder containing a 100 μm filter membrane to repeatedly extrude the mixed solution A2 prepared in Step 5 20 times to obtain a mixed solution B2, and the mixed solution B2 contains unilamellar liposomes containing BSA;

[0204] Step 7. Preparation of multilamellar lipid vesicles containing BSA: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B2. The volume ratio of the mixed solution B2 to the divalent ion solution is 1000:5. In this example, the amount of the magnesium chloride solution used is 15 μL, and the amount of the dithiothreitol solution (DTT) used is 16.8 μL. Incubate at 37 °C with a stirring speed of 120 rpm for 1 h. After the incubation, centrifuge at 14000 rpf for 4 minutes, discard the supernatant to obtain solid C2. Wash solid C2 with a bis-tris propane buffer solution and then centrifuge again at 14000 rpf for 4 minutes to obtain solid D2, which is the multilamellar lipid vesicles containing BSA. In this example, the divalent ion solution is the magnesium chloride solution;

[0205] Step 8. Preparation of multilamellar crosslinked liposomes: Prepare a ligand. The preparation method of the ligand is as follows: Cysteine is connected to the C-terminus of a polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using existing techniques to obtain the ligand. The polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is SEQ ID NO:1. Mix the obtained multilamellar lipid vesicles containing BSA with the ligand and incubate at 37 °C for 0.5 h to covalently conjugate the ligand to the multilamellar lipid vesicles to obtain multilamellar crosslinked liposomes.

[0206] Example 6

[0207] This example provides a method for preparing multilamellar crosslinked liposomes. The multilamellar crosslinked liposomes encapsulate the model protein drug bovine serum albumin (BSA), and the method includes the following steps:

[0208] Step 1. Prepare solutions: The solutions include a bis-tris propane buffer solution (Bis-tris buffer), a dithiothreitol solution (DTT solution), and a magnesium chloride solution;

[0209] The preparation method of the bis-tris propane buffer solution (Bis-tris buffer) is as follows: Weigh bis-tris propane and prepare a 10 mM bis-tris propane buffer solution with deionized water.

[0210] The preparation method of the dithiothreitol solution (DTT solution) is as follows: Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL of dithiothreitol using the bis-tris propane buffer solution.

[0211] The preparation method of the magnesium chloride solution is as follows: Weigh magnesium chloride hexahydrate and prepare a magnesium chloride solution with a concentration of 0.085 mg / mL of magnesium chloride hexahydrate using the bis-tris propane buffer solution.

[0212] Step 2: Prepare liposome solution: Dissolve choline phosphate, glycerol phosphate, and functionalized lipid in chloroform at a molar ratio of 4:1:5, i.e., 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain liposome solution. The total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution is 0.0125 mol / L;

[0213] In this example, the functionalized lipid is maleimide-functionalized lipid, the choline phosphate is dioleoylphosphatidylcholine, and the glycerol phosphate is 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt.

[0214] The preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0215] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, obtain organic solution a; the organic solution a is washed successively with a 5% Na 2 CO 3 solution, a 1% HCl solution, and a 1% NaCl solution to obtain organic solution b. The organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid.

[0216] wherein the molar ratio of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine is 1:1:2, the total concentration of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L, the volumes of the used Na 2 CO 3 solution, HCl solution, and NaCl solution are the same as the volume of chloroform, the temperature of the drying and evaporation is 24 °C, and the time of the drying and evaporation is 24 h.

[0217] Step 3: Prepare liposome mixture: Dissolve the model protein drug bovine serum albumin (BSA) in bis-tris propane buffer solution to obtain a dissolved solution, and add the dissolved solution to the liposome solution prepared in Step 2 to obtain liposome mixture;

[0218] The mass of the protein molecule in the protein molecule buffer solution is 0.5 mg;

[0219] Step 4: Preparation of lipid membrane containing BSA: The liposome solution prepared in Step 3 is air-dried at room temperature for 12 h to obtain a lipid membrane containing the drug;

[0220] Step 5: Preparation of multilamellar liposomes containing BSA: Hydrate the lipid membrane containing BSA prepared in Step 4 with a buffer solution. The concentration of the lipid membrane containing BSA in the buffer solution is 1 μmol / ml. The buffer solution is bis-tris propane buffer solution. By oscillation, the lipid membrane containing BSA is fully dissolved in the buffer solution to form a mixed solution A2, and the mixed solution A2 includes micron-sized multilamellar liposomes containing BSA;

[0221] The conditions for the oscillation are: Oscillate once every 10 minutes during the hydration process. The time for each oscillation is 30 seconds, and the number of oscillations is 6 times.

[0222] Step 6: Preparation of unilamellar liposomes containing BSA: Use a liposome extruder containing a 100 μm filter membrane to repeatedly extrude the mixed solution A2 prepared in Step 5 20 times to obtain a mixed solution B2, and the mixed solution B2 contains unilamellar liposomes containing BSA;

[0223] Step 7: Preparation of multilamellar lipid vesicles containing BSA: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B2. The volume ratio of the mixed solution B2 to the divalent ion solution is 1000:5. In this example, the usage amount of the magnesium chloride solution is 20 μL, and the usage amount of the dithiothreitol solution (DTT) is 16.8 μL. Incubate at 37 °C with a stirring speed of 120 rpm for 1 h. After the incubation, centrifuge at 14000 rpf for 4 minutes, discard the supernatant to obtain a solid C2, wash the solid C2 with bis-tris propane buffer solution and then centrifuge at 14000 rpf again for 4 minutes to obtain a solid D2, which is the multilamellar lipid vesicles containing BSA; In this example, the divalent ion solution is the magnesium chloride solution;

[0224] Step 8: Preparation of multilamellar cross-linked liposomes: Prepare a ligand. The preparation method of the ligand is: Cysteine is connected to the C-terminus of the polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using the existing technology to obtain the ligand. The polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is SEQ ID NO:1; After mixing the obtained multilamellar lipid vesicles containing BSA with the ligand and incubating at 37 °C for 0.5 h, covalently conjugate the ligand to the multilamellar lipid vesicles to obtain multilamellar cross-linked liposomes.

[0225] Example 7

[0226] This embodiment provides a method for preparing multi-layer cross-linked liposomes, and the multi-layer cross-linked liposomes encapsulate the model protein drug bovine serum albumin (BSA). The method includes the following steps:

[0227] Step 1, Prepare solutions: The solutions include Bis-tris buffer solution, dithiothreitol solution (DTT solution), and calcium chloride solution.

[0228] The preparation method of the Bis-tris buffer solution: Weigh Bis-tris and prepare a 10 mM Bis-tris buffer solution with deionized water.

[0229] The preparation method of the dithiothreitol solution (DTT solution): Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL of dithiothreitol using the Bis-tris buffer solution.

[0230] The preparation method of the calcium chloride solution: Weigh anhydrous calcium chloride and prepare a calcium chloride solution with a concentration of 0.116 mg / mL of anhydrous calcium chloride using the Bis-tris buffer solution.

[0231] Step 2, Prepare liposome solution: Dissolve choline phosphate, glycerol phosphate, and functionalized lipid in chloroform at a molar ratio of 4:1:5, that is, 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain a liposome solution. The total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution is 0.0125 mol / L.

[0232] In this embodiment, the functionalized lipid is maleimide-functionalized lipid, the choline phosphate is dioleoylphosphatidylcholine, and the glycerol phosphate is 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt.

[0233] The preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0234] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, obtain organic solution a. The organic solution a is washed successively with a 5% Na 2 CO 3 solution, a 1% HCl solution, and a 1% NaCl solution to obtain organic solution b. The organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid.

[0235] The molar ratio of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine is 1:1:2. The total concentration of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L. The Na 2 CO 3 solution, HCl solution, and NaCl solution have the same volume as that of chloroform. The temperature of drying and evaporation is 24 °C, and the time of drying and evaporation is 24 h.

[0236] Step 3: Prepare a liposome mixture: Dissolve the model protein drug bovine serum albumin (BSA) in a bis-tris buffer solution to obtain a solution, and add the solution to the liposome solution prepared in Step 2 to obtain a liposome mixture;

[0237] The mass of the protein molecule in the protein molecule buffer solution is 0.5 mg.

[0238] Step 4: Prepare a lipid membrane containing BSA: Air-dry the liposome solution prepared in Step 3 at room temperature for 12 h to obtain a lipid membrane containing the drug;

[0239] Step 5: Prepare multilamellar liposomes containing BSA: Hydrate the lipid membrane containing BSA prepared in Step 4 with a buffer solution. The concentration of the lipid membrane containing BSA in the buffer solution is 1 μmol / ml. The buffer solution is a bis-tris buffer solution. By shaking, the lipid membrane containing BSA is fully dissolved in the buffer solution to form a mixed solution A2. The mixed solution A2 includes micron-sized multilamellar liposomes containing BSA;

[0240] The conditions of the shaking are: shake once every 10 minutes during the hydration process, the time of shaking once is 30 seconds, and the number of shaking times is 6 times.

[0241] Step 6: Prepare unilamellar liposomes containing BSA: Use a liposome extruder containing a 100-μm filter membrane to repeatedly extrude the mixed solution A2 prepared in Step 5 20 times to obtain a mixed solution B2. The mixed solution B2 contains unilamellar liposomes containing BSA;

[0242] Step 7. Preparation of multi-layered lipid vesicles containing BSA: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B2. The volume ratio of the mixed solution B2 to the divalent ion solution is 1000:5. In this example, the usage amount of the calcium chloride solution is 5 μL, and the usage amount of the dithiothreitol solution (DTT) is 16.8 μL. Incubate at 37 °C with a stirring speed of 120 rpm for 1 h. After the incubation, centrifuge at 14000 rpf for 4 minutes, discard the supernatant to obtain solid C2. Wash solid C2 with bis-tris buffer solution and then centrifuge at 14000 rpf again for 4 minutes to obtain solid D2, which is the multi-layered lipid vesicles containing BSA. In this example, the divalent ion solution is the calcium chloride solution;

[0243] Step 8. Preparation of multi-layered cross-linked liposomes: Prepare a ligand. The preparation method of the ligand is as follows: Cysteine is connected to the C-terminus of the polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using the prior art to obtain the ligand. The polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is SEQ ID NO:1. Mix the obtained multi-layered lipid vesicles containing BSA with the ligand and incubate at 37 °C for 0.5 h to covalently conjugate the ligand to the multi-layered lipid vesicles to obtain multi-layered cross-linked liposomes.

[0244] Example 8

[0245] This example provides a method for preparing multi-layered cross-linked liposomes. The multi-layered cross-linked liposomes encapsulate the model protein drug bovine serum albumin (BSA) and include the following steps:

[0246] Step 1. Prepare solutions: The solutions include bis-tris buffer solution, dithiothreitol solution (DTT solution), and calcium chloride solution;

[0247] The preparation method of the bis-tris buffer solution: Weigh bis-tris and prepare a 10 mM bis-tris buffer solution with deionized water.

[0248] The preparation method of the dithiothreitol solution (DTT solution): Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL of dithiothreitol using the bis-tris buffer solution.

[0249] The preparation method of the calcium chloride solution: Weigh anhydrous calcium chloride and prepare a calcium chloride solution with a concentration of 0.116 mg / mL of anhydrous calcium chloride using the bis-tris buffer solution.

[0250] Step 2: Prepare liposome solution: Dissolve choline phosphate, glycerol phosphate, and functionalized lipid in chloroform at a molar ratio of 4:1:5, i.e., 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain a liposome solution. The total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution is 0.0125 mol / L;

[0251] In this example, the functionalized lipid is maleimide-functionalized lipid, the choline phosphate is dioleoylphosphatidylcholine, and the glycerol phosphate is 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt.

[0252] The preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0253] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, obtain organic solution a; Organic solution a is washed successively with a 5% (mass concentration) Na 2 CO 3 solution, a 1% (mass fraction) HCl solution, and a 1% (mass fraction) NaCl solution to obtain organic solution b. Organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid.

[0254] wherein the molar ratio of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine is 1:1:2, and the total concentration of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L. The volumes of the used Na 2 CO 3 solution, HCl solution, and NaCl solution are the same as the volume of chloroform. The temperature of the drying and evaporation is 24 °C, and the time of the drying and evaporation is 24 h.

[0255] Step 3: Prepare liposome mixture: Dissolve the model protein drug bovine serum albumin (BSA) in bis-tris propane buffer solution to obtain a solution, and add the solution to the liposome solution prepared in Step 2 to obtain a liposome mixture;

[0256] The mass of the protein molecule in the protein molecule buffer solution is 0.5 mg.

[0257] Step 4: Preparation of lipid membrane containing BSA: The liposome solution prepared in Step 3 was air-dried at room temperature for 12 h to obtain a lipid membrane containing the drug.

[0258] Step 5: Preparation of multilamellar liposomes containing BSA: The lipid membrane containing BSA prepared in Step 4 was hydrated with a buffer solution. The concentration of the lipid membrane containing BSA in the buffer solution was 1 μmol / ml. The buffer solution was bis-Tris propane buffer solution. By oscillation, the lipid membrane containing BSA was fully dissolved in the buffer solution to form a mixed solution A2, and the mixed solution A2 included micron-sized multilamellar liposomes containing BSA.

[0259] The conditions of the oscillation were: oscillating once every 10 minutes during the hydration process, the oscillation time for one time was 30 seconds, and the number of oscillations was 6 times.

[0260] Step 6: Preparation of unilamellar liposomes containing BSA: Using a liposome extruder containing a 100 μm filter membrane, the mixed solution A2 prepared in Step 5 was extruded repeatedly 20 times to obtain a mixed solution B2, and the mixed solution B2 contained unilamellar liposomes containing BSA.

[0261] Step 7: Preparation of multilamellar lipid vesicles containing BSA: A divalent ion solution and a dithiothreitol solution were added to the mixed solution B2. The volume ratio of the mixed solution B2 to the divalent ion solution was 1000:5. In this example, the usage amount of the calcium chloride solution was 10 μL, and the usage amount of the dithiothreitol solution (DTT) was 16.8 μL. Incubation was carried out at 37 °C with a stirring speed of 120 rpm for 1 h. After the incubation ended, centrifugation was carried out at 14000 rpf for 4 minutes, and the supernatant was discarded to obtain a solid C2. The solid C2 was washed with bis-Tris propane buffer solution and then centrifuged again at 14000 rpf for 4 minutes to obtain a solid D2, which was the multilamellar lipid vesicles containing BSA. In this example, the divalent ion solution was a calcium chloride solution.

[0262] Step 8: Preparation of multilamellar crosslinked liposomes: Preparation of a ligand. The preparation method of the ligand was: Cysteine was connected to the C-terminus of the polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using the existing technology to obtain the ligand. The polypeptide was a collagen-binding peptide (CBP), and the collagen-binding peptide was synthesized through an amino acid sequence, and the amino acid sequence was SEQ ID NO:1. After mixing the obtained multilamellar lipid vesicles containing BSA with the ligand and incubating at 37 °C for 0.5 h, the ligand was covalently conjugated to the multilamellar lipid vesicles to obtain multilamellar crosslinked liposomes.

[0263] Example 9

[0264] This embodiment provides a method for preparing multilamellar crosslinked liposomes, and the multilamellar crosslinked liposomes encapsulate the model protein drug bovine serum albumin (BSA), comprising the following steps:

[0265] Step 1, preparing solutions: The solutions include bis-tris buffer solution, dithiothreitol solution (DTT solution), and calcium chloride solution;

[0266] The preparation method of the bis-tris buffer solution: Weigh bis-tris (hydroxymethyl) aminomethane and prepare a 10 mM bis-tris buffer solution with deionized water.

[0267] The preparation method of the dithiothreitol solution (DTT solution): Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL of dithiothreitol using the bis-tris buffer solution.

[0268] The preparation method of the calcium chloride solution: Weigh anhydrous calcium chloride and prepare a calcium chloride solution with a concentration of 0.116 mg / mL of anhydrous calcium chloride using the bis-tris buffer solution.

[0269] Step 2, preparing the liposome solution: Dissolve choline phosphate, glycerol phosphate, and functionalized lipid in chloroform according to a molar ratio of 4:1:5, that is, 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain the liposome solution. The total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution is 0.0125 mol / L;

[0270] In this embodiment, the functionalized lipid is maleimide-functionalized lipid, the choline phosphate is dioleoylphosphatidylcholine, and the glycerol phosphate is 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt.

[0271] The preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0272] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, obtain organic solution a; The organic solution a is washed successively with a 5% Na 2 CO 3 solution, a 1% HCl solution, and a 1% NaCl solution, to obtain organic solution b. The organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid.

[0273] wherein the molar ratio of sodium 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol), N-hydroxysuccinimide ester, and triethylamine is 1:1:2, and the total concentration of sodium 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol), N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L. The Na 2 CO 3 solution, HCl solution, and NaCl solution have the same volume as the chloroform. The temperature of drying and evaporation is 24 °C, and the time of drying and evaporation is 24 h.

[0274] Step 3: Prepare the liposome mixture: Dissolve the model protein drug bovine serum albumin (BSA) in a bis-tris propane buffer solution to obtain a dissolved solution, and add the dissolved solution to the liposome solution prepared in Step 2 to obtain a liposome mixture;

[0275] The mass of the protein molecule in the protein molecule buffer solution is 0.5 mg.

[0276] Step 4: Prepare the lipid membrane containing BSA: Air-dry the liposome solution prepared in Step 3 at room temperature for 12 h to obtain a lipid membrane containing the drug;

[0277] Step 5: Prepare the multilamellar liposomes containing BSA: Hydrate the lipid membrane containing BSA prepared in Step 4 with a buffer solution. The concentration of the lipid membrane containing BSA in the buffer solution is 1 μmol / ml, and the buffer solution is a bis-tris propane buffer solution. By shaking, the lipid membrane containing BSA is fully dissolved in the buffer solution to form a mixed solution A2, and the mixed solution A2 includes micron-sized multilamellar liposomes containing BSA;

[0278] The conditions of the shaking are: shake once every 10 minutes during the hydration process, the shaking time for one time is 30 seconds, and the number of shaking times is 6 times.

[0279] Step 6: Prepare the unilamellar liposomes containing BSA: Use a liposome extruder containing a 100 μm filter membrane to repeatedly extrude the mixed solution A2 prepared in Step 5 20 times to obtain a mixed solution B2, and the mixed solution B2 contains unilamellar liposomes containing BSA;

[0280] Step 7: Preparation of multilamellar lipid vesicles containing BSA: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B2. The volume ratio of the mixed solution B2 to the divalent ion solution is 1000:5. In this example, the usage amount of the calcium chloride solution is 15 μL, and the usage amount of the dithiothreitol solution (DTT) is 16.8 μL. Incubate at 37 °C with a stirring speed of 120 rpm for 1 h. After incubation, centrifuge at 14000 rpf for 4 minutes, discard the supernatant to obtain solid C2. Wash solid C2 with bis-tris propane buffer solution and then centrifuge again at 14000 rpf for 4 minutes to obtain solid D2, which is the multilamellar lipid vesicles containing BSA. In this example, the divalent ion solution is the calcium chloride solution;

[0281] Step 8: Preparation of multilamellar cross-linked liposomes: Prepare a ligand. The preparation method of the ligand is as follows: Cysteine is connected to the C-terminus of the polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using existing techniques to obtain the ligand. The polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is SEQ ID NO:1; After mixing the obtained multilamellar lipid vesicles containing BSA with the ligand, incubate at 37 °C for 0.5 h to covalently conjugate the ligand to the multilamellar lipid vesicles to obtain multilamellar cross-linked liposomes.

[0282] Example 10

[0283] This example provides a method for preparing multilamellar cross-linked liposomes. The multilamellar cross-linked liposomes encapsulate the model protein drug bovine serum albumin (BSA), and the method includes the following steps:

[0284] Step 1: Prepare solutions: The solutions include bis-tris propane buffer solution (Bis-tris buffer), dithiothreitol solution (DTT solution), and calcium chloride solution;

[0285] The preparation method of the bis-tris propane buffer solution (Bis-tris buffer) is as follows: Weigh bis-tris propane and prepare a 10 mM bis-tris propane buffer solution with deionized water.

[0286] The preparation method of the dithiothreitol solution (DTT solution) is as follows: Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL of dithiothreitol using bis-tris propane buffer solution.

[0287] The preparation method of the calcium chloride solution is as follows: Weigh anhydrous calcium chloride and prepare a calcium chloride solution with a concentration of 0.116 mg / mL of anhydrous calcium chloride using bis-tris propane buffer solution.

[0288] Step 2: Prepare liposome solution: Dissolve choline phosphate, glycerol phosphate, and functionalized lipid in chloroform at a molar ratio of 4:1:5, i.e., 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain a liposome solution. The total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution is 0.0125 mol / L;

[0289] In this example, the functionalized lipid is maleimide-functionalized lipid, the choline phosphate is dioleoylphosphatidylcholine, and the glycerol phosphate is 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt.

[0290] The preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0291] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, obtain organic solution a; the organic solution a is successively washed with a 5% Na 2 CO 3 solution, a 1% HCl solution, and a 1% NaCl solution to obtain organic solution b. The organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid.

[0292] wherein the molar ratio of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine is 1:1:2. The total concentration of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L. The volumes of the used Na 2 CO 3 solution, HCl solution, and NaCl solution are the same as the volume of chloroform. The temperature of the drying and evaporation is 24 °C, and the time of the drying and evaporation is 24 h.

[0293] Step 3: Prepare liposome mixture: Dissolve the model protein drug bovine serum albumin (BSA) in bis-tris propane buffer solution to obtain a dissolved solution, and add the dissolved solution to the liposome solution prepared in Step 2 to obtain a liposome mixture;

[0294] The mass of the protein molecule in the protein molecule buffer solution is 0.5 mg.

[0295] Step 4. Preparation of lipid membrane containing BSA: The liposome solution prepared in Step 3 is air-dried at room temperature for 12 h to obtain a lipid membrane containing the drug.

[0296] Step 5. Preparation of multilamellar liposomes containing BSA: Hydrate the lipid membrane containing BSA prepared in Step 4 with a buffer solution. The concentration of the lipid membrane containing BSA in the buffer solution is 1 μmol / ml, and the buffer solution is bis-tris propane buffer solution. By shaking, the lipid membrane containing BSA is fully dissolved in the buffer solution to form a mixed solution A2, and the mixed solution A2 contains micron-sized multilamellar liposomes containing BSA.

[0297] The conditions for the shaking are: shake once every 10 minutes during the hydration process, the shaking time for one time is 30 seconds, and the number of shaking times is 6 times.

[0298] Step 6. Preparation of unilamellar liposomes containing BSA: Use a liposome extruder containing a 100 μm filter membrane to repeatedly extrude the mixed solution A2 prepared in Step 5 20 times to obtain a mixed solution B2, and the mixed solution B2 contains unilamellar liposomes containing BSA.

[0299] Step 7. Preparation of multilamellar lipid vesicles containing BSA: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B2. The volume ratio of the mixed solution B2 to the divalent ion solution is 1000:5. In this example, the usage amount of the calcium chloride solution is 20 μL, and the usage amount of the dithiothreitol solution (DTT) is 16.8 μL. Incubate at 37 °C with a stirring speed of 120 rpm for 1 h. After the incubation, centrifuge at 14000 rpf for 4 minutes, discard the supernatant to obtain a solid C2, wash the solid C2 with bis-tris propane buffer solution and then centrifuge at 14000 rpf again for 4 minutes to obtain a solid D2, which is the multilamellar lipid vesicles containing BSA. In this example, the divalent ion solution is a calcium chloride solution.

[0300] Step 8. Preparation of multilamellar cross-linked liposomes: Prepare a ligand. The preparation method of the ligand is: Cysteine is connected to the C-terminus of a polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using the prior art to obtain the ligand. The polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is SEQ ID NO:1; After mixing the obtained multilamellar lipid vesicles containing BSA with the ligand and incubating at 37 °C for 0.5 h, covalently conjugate the ligand to the multilamellar lipid vesicles to obtain multilamellar cross-linked liposomes.

[0301] Example 11

[0302] This example provides a method for preparing cartilage-affinitive cross-linked liposome nanoparticles (CBP-stabilized ICMVs), which includes the following steps:

[0303] Step 1, preparing solutions: The solutions include Bis-tris buffer solution, dithiothreitol solution (DTT solution), and calcium chloride solution;

[0304] The preparation method of the Bis-tris buffer solution: Weigh Bis-tris and prepare a 10 mM Bis-tris buffer solution with deionized water.

[0305] The preparation method of the dithiothreitol solution (DTT solution): Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL of dithiothreitol using the Bis-tris buffer solution.

[0306] The preparation method of the calcium chloride solution: Weigh anhydrous calcium chloride and prepare a calcium chloride solution with a concentration of 0.116 mg / mL of anhydrous calcium chloride using the Bis-tris buffer solution.

[0307] Step 2, preparing liposome solution: Dissolve choline phosphate, glycerol phosphate, and functionalized lipid in chloroform according to a molar ratio of 4:1:5, that is, 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain a liposome solution. The total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution is 0.0125 mol / L;

[0308] In this example, the functionalized lipid is maleimide-functionalized lipid, the choline phosphate is dioleoylphosphatidylcholine, and the glycerol phosphate is 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt.

[0309] The preparation method of the maleimide-functionalized lipid (MPB) is:

[0310] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, an organic solution a is obtained; the organic solution a is successively washed with a 5% Na 2 CO 3 solution, a 1% HCl solution, and a 1% NaCl solution, to obtain an organic solution b. The organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid,

[0311] The molar ratio of sodium 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol), N-hydroxysuccinimide ester, and triethylamine is 1:1:2. The total concentration of sodium 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol), N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L. The Na 2 CO 3 solution, HCl solution, and NaCl solution have the same volume as the chloroform. The temperature for drying and evaporation is 24 °C, and the time for drying and evaporation is 24 h.

[0312] Step 3: Prepare a lipid film: Air-dry the liposome solution prepared in Step 2 at room temperature for 12 h to obtain a lipid film;

[0313] Step 4: Prepare multilamellar liposomes: Hydrate the lipid film prepared in Step 3 with a buffer solution. The concentration of the lipid film in the buffer solution is 1 μmol / ml. The buffer solution is a bis-tris propane buffer solution. By shaking, the lipid film is fully dissolved in the buffer solution to form a mixed solution A1, which includes micron-sized multilamellar liposomes;

[0314] The conditions for shaking are: shake once every 10 minutes during the hydration process, the shaking time for each time is 30 seconds, and the number of shaking times is 6 times.

[0315] Step 5: Prepare unilamellar liposomes: Use a liposome extruder containing a 100 μm filter membrane to repeatedly extrude the mixed solution A1 prepared in Step 4 20 times to obtain a mixed solution B1, which contains unilamellar liposomes;

[0316] Step 6: Prepare multilamellar lipid vesicles: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B1. The volume ratio of the mixed solution B1 to the divalent ion solution is 1000:5:21. In this example, the usage amount of the calcium chloride solution is 10 μL, and the usage amount of the dithiothreitol solution (DTT) is 21 μL. Incubate at 37 °C with a stirring speed of 120 rpm for 1 h. After incubation, centrifuge at 14000 rpf for 4 minutes, discard the supernatant to obtain a solid C1. Wash the solid C1 with a bis-tris propane buffer solution and then centrifuge again at 14000 rpf for 4 minutes to obtain a solid D1, which is the multilamellar lipid vesicles; In this example, the divalent ion solution is a magnesium chloride solution;

[0317] Step 7: Preparation of cross-linked liposome nanoparticles: Prepare a ligand, and the preparation method of the ligand is as follows: Cysteine is connected to the C-terminus of a polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using existing techniques to obtain the ligand. The polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is LRELHLNNN; After mixing the obtained multilamellar lipid vesicles with the ligand and incubating at 37 °C for 0.5 h, the ligand is covalently conjugated to the multilamellar lipid vesicles to obtain cross-linked liposome nanoparticles.

[0318] In this example, the mass of the polypeptide, which is a collagen-binding peptide (CBP), is 0.8 mg.

[0319] Example 12

[0320] This example provides a method for preparing cartilage-affinitive cross-linked liposome nanoparticles (CBP-stabilized ICMVs), which includes the following steps:

[0321] Step 1: Prepare a solution: The solution includes a bis-tris buffer solution, a dithiothreitol solution (DTT solution), and a calcium chloride solution;

[0322] The preparation method of the bis-tris buffer solution: Weigh bis-tris and prepare a 10 mM bis-tris buffer solution with deionized water.

[0323] The preparation method of the dithiothreitol solution (DTT solution): Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL of dithiothreitol using the bis-tris buffer solution.

[0324] The preparation method of the calcium chloride solution: Weigh anhydrous calcium chloride and prepare a calcium chloride solution with a concentration of 0.116 mg / mL of anhydrous calcium chloride using the bis-tris buffer solution.

[0325] Step 2: Prepare a liposome solution: Dissolve choline phosphate, glycerol phosphate, and functionalized lipid in chloroform according to a molar ratio of 4:1:5, that is, 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain a liposome solution. The total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution is 0.0125 mol / L;

[0326] In this embodiment, the functionalized lipid is a maleimide-functionalized lipid, the phosphatidylcholine is dioleoylphosphatidylcholine, and the phosphatidylglycerol is 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt.

[0327] The preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0328] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform, and react at room temperature for 3 hours. After the reaction is completed, an organic solution a is obtained; the organic solution a is successively washed with a 5% (mass concentration) Na 2 CO 3 solution, a 1% (mass fraction) HCl solution, and a 1% (mass fraction) NaCl solution to obtain an organic solution b. The organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain the maleimide-functionalized lipid.

[0329] wherein the molar ratio of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine is 1:1:2, and the total concentration of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L. The volumes of the used Na 2 CO 3 solution, HCl solution, and NaCl solution are the same as the volume of chloroform. The temperature of the drying and evaporation is 24 °C, and the time of the drying and evaporation is 24 h.

[0330] Step 3: Prepare a lipid film: Air-dry the liposome solution prepared in Step 2 at room temperature for 12 h to obtain a lipid film;

[0331] Step 4: Prepare multilamellar liposomes: Hydrate the lipid film prepared in Step 3 with a buffer solution. The concentration of the lipid film in the buffer solution is 1 μmol / ml. The buffer solution is a bis-tris propane buffer solution. By shaking, the lipid film is fully dissolved in the buffer solution to form a mixed solution A1, and the mixed solution A1 contains micron-sized multilamellar liposomes;

[0332] The conditions of the shaking are: shake once every 10 minutes during the hydration process, the shaking time for one time is 30 seconds, and the number of shaking times is 6 times.

[0333] Step 5: Prepare unilamellar liposomes: Use a liposome extruder containing a 100 μm filter membrane to repeatedly extrude the mixed solution A1 prepared in Step 4 20 times to obtain a mixed solution B1, and the mixed solution B1 contains unilamellar liposomes.

[0334] Step 6: Preparation of multilamellar lipid vesicles: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B1. The volume ratio of the mixed solution B1 to the divalent ion solution is 1000:5:21. In this example, the usage amount of the calcium chloride solution is 10 μL, and the usage amount of the dithiothreitol solution (DTT) is 19.95 μL. Incubate at 37 °C with a stirring speed of 120 rpm for 1 h. After the incubation, centrifuge at 14000 rpf for 4 minutes, discard the supernatant to obtain solid C1, wash solid C1 with a bis-tris buffer solution, and then centrifuge again at 14000 rpf for 4 minutes to obtain solid D1, which is the multilamellar lipid vesicles; in this example, the divalent ion solution is a magnesium chloride solution;

[0335] Step 7: Preparation of cross-linked liposome nanoparticles: Prepare a ligand. The preparation method of the ligand is as follows: Cysteine is connected to the C-terminus of a polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using existing techniques to obtain the ligand. The polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is LRELHLNNN; After mixing the obtained multilamellar lipid vesicles with the ligand and incubating at 37 °C for 0.5 h, covalently conjugate the ligand to the multilamellar lipid vesicles to obtain cross-linked liposome nanoparticles.

[0336] In this example, the mass of the polypeptide, i.e., the collagen-binding peptide (CBP), is 0.8 mg.

[0337] Example 13

[0338] This example provides a method for preparing cartilage-affinitive cross-linked liposome nanoparticles (CBP-stabilized ICMVs), including the following steps:

[0339] Step 1: Prepare solutions: The solutions include a bis-tris buffer solution, a dithiothreitol solution (DTT solution), and a calcium chloride solution;

[0340] The preparation method of the bis-tris buffer solution: Weigh bis-tris and prepare a 10 mM bis-tris buffer solution with deionized water.

[0341] The preparation method of the dithiothreitol solution (DTT solution): Weigh dithiothreitol (DTT) and prepare a dithiothreitol solution with a concentration of 2.31 mg / mL using the bis-tris buffer solution.

[0342] The preparation method of the calcium chloride solution is as follows: Weigh anhydrous calcium chloride and prepare a calcium chloride solution with a concentration of 0.116 mg / mL of anhydrous calcium chloride using a bis(tris(hydroxymethyl)aminomethane) buffer solution.

[0343] Step 2: Prepare a liposome solution: Dissolve choline phosphate, glycerol phosphate, and functionalized lipid in chloroform according to a molar ratio of 4:1:5, that is, 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain a liposome solution. The total concentration of choline phosphate, glycerol phosphate, and functionalized lipid in the liposome solution is 0.0125 mol / L.

[0344] In this example, the functionalized lipid is maleimide-functionalized lipid, the choline phosphate is dioleoylphosphatidylcholine, and the glycerol phosphate is sodium 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol).

[0345] The preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0346] Dissolve sodium 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol), N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, obtain organic solution a; Organic solution a is washed successively with a 5% (mass concentration) Na 2 CO 3 solution, a 1% (mass fraction) HCl solution, and a 1% (mass fraction) NaCl solution to obtain organic solution b. Organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain maleimide-functionalized lipid.

[0347] Among them, the molar ratio of sodium 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol), N-hydroxysuccinimide ester, and triethylamine is 1:1:2. The total concentration of sodium 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol), N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L. The volumes of the used Na 2 CO 3 solution, HCl solution, and NaCl solution are the same as the volume of chloroform. The temperature of the drying and evaporation is 24 °C, and the time of the drying and evaporation is 24 h.

[0348] Step 3: Prepare a lipid membrane: Air-dry the liposome solution prepared in Step 2 at room temperature for 12 h to obtain a lipid membrane.

[0349] Step 4. Preparation of multilamellar liposomes: Hydrate the lipid film prepared in Step 3 with a buffer solution. The concentration of the lipid film in the buffer solution is 1 μmol / ml. The buffer solution is bis-tris propane buffer solution. By shaking, the lipid film is fully dissolved in the buffer solution to form a mixed solution A1, which contains micron-sized multilamellar liposomes.

[0350] The conditions for shaking are as follows: Shake once every 10 minutes during the hydration process. The shaking time for each time is 30 seconds, and the number of shaking times is 6 times.

[0351] Step 5. Preparation of unilamellar liposomes: Use a liposome extruder containing a 100 μm filter membrane to repeatedly extrude the mixed solution A1 prepared in Step 4 for 20 times to obtain a mixed solution B1, which contains unilamellar liposomes.

[0352] Step 6. Preparation of multilamellar lipid vesicles: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B1. The volume ratio of the mixed solution B1 to the divalent ion solution is 1000:5:21. In this example, the usage amount of the calcium chloride solution is 10 μL, and the usage amount of the dithiothreitol solution (DTT) is 18.9 μL. Incubate at 37 °C with a stirring speed of 120 rpm for 1 h. After the incubation, centrifuge at 14000 rpf for 4 minutes, discard the supernatant to obtain a solid C1, wash the solid C1 with bis-tris propane buffer solution, and then centrifuge at 14000 rpf for 4 minutes again to obtain a solid D1, which is the multilamellar lipid vesicles. In this example, the divalent ion solution is a magnesium chloride solution.

[0353] Step 7. Preparation of cross-linked liposome nanoparticles: Prepare a ligand. The preparation method of the ligand is as follows: Cysteine is connected to the C-terminus of a polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using the existing technology to obtain the ligand. The polypeptide is a collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is LRELHLNNN; After mixing the obtained multilamellar lipid vesicles with the ligand and incubating at 37 °C for 0.5 h, covalently conjugate the ligand to the multilamellar lipid vesicles to obtain cross-linked liposome nanoparticles.

[0354] In this example, the mass of the polypeptide, i.e., the collagen-binding peptide (CBP), is 0.8 mg.

[0355] Example 14

[0356] This example provides a preparation method of cartilage-affinitive cross-linked liposome nanoparticles (CBP-stabilized ICMVs), which includes the following steps:

[0357] Step 1, Prepare the solution: The solution includes Bis-tris buffer solution, Dithiothreitol (DTT) solution, and Calcium chloride solution;

[0358] The preparation method of the Bis-tris buffer solution: Weigh Bis-tris and prepare a 10 mM Bis-tris buffer solution with deionized water.

[0359] The preparation method of the DTT solution: Weigh Dithiothreitol (DTT) and prepare a DTT solution with a concentration of 2.31 mg / mL of DTT using the Bis-tris buffer solution.

[0360] The preparation method of the Calcium chloride solution: Weigh anhydrous calcium chloride and prepare a calcium chloride solution with a concentration of 0.116 mg / mL of anhydrous calcium chloride using the Bis-tris buffer solution.

[0361] Step 2, Prepare the liposome solution: Dissolve phosphatidylcholine, phosphatidylglycerol, and functionalized lipid in chloroform according to a molar ratio of 4:1:5, that is, 1 μmol of DOPC, 0.25 μmol of DOPG, and 1.25 μmol of MPB, to obtain the liposome solution. The total concentration of phosphatidylcholine, phosphatidylglycerol, and functionalized lipid in the liposome solution is 0.0125 mol / L;

[0362] In this example, the functionalized lipid is maleimide-functionalized lipid, the phosphatidylcholine is dioleoylphosphatidylcholine, and the phosphatidylglycerol is 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt.

[0363] The preparation method of the maleimide-functionalized lipid (MPB) is as follows:

[0364] Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform and react at room temperature for 3 hours. After the reaction is completed, obtain the organic solution a; The organic solution a is washed successively with a 5% Na 2 CO 3 solution, a 1% HCl solution, and a 1% NaCl solution to obtain the organic solution b. The organic solution b is dried and evaporated with sodium sulfate or magnesium sulfate to obtain the maleimide-functionalized lipid.

[0365] The molar ratio of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine is 1:1:2. The total concentration of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, N-hydroxysuccinimide ester, and triethylamine in chloroform is 0.8 mol / L. The Na 2 CO 3 solution, HCl solution, and NaCl solution have the same volume as that of chloroform. The temperature of drying and evaporation is 24 °C, and the time of drying and evaporation is 24 h.

[0366] Step 3: Prepare a lipid membrane: Air-dry the liposome solution prepared in Step 2 at room temperature for 12 h to obtain a lipid membrane;

[0367] Step 4: Prepare multilamellar liposomes: Hydrate the lipid membrane prepared in Step 3 with a buffer solution. The concentration of the lipid membrane in the buffer solution is 1 μmol / ml. The buffer solution is a bis-tris propane buffer solution. By shaking, the lipid membrane is fully dissolved in the buffer solution to form a mixed solution A1. The mixed solution A1 contains micron-sized multilamellar liposomes;

[0368] The conditions for shaking are: Shake once every 10 minutes during the hydration process. The time for shaking once is 30 seconds, and the number of shaking times is 6 times.

[0369] Step 5: Prepare unilamellar liposomes: Use a liposome extruder containing a 100 μm filter membrane to repeatedly extrude the mixed solution A1 prepared in Step 4 20 times to obtain a mixed solution B1. The mixed solution B1 contains unilamellar liposomes;

[0370] Step 6: Prepare multilamellar lipid vesicles: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B1. The volume ratio of the mixed solution B1 to the divalent ion solution is 1000:5:21. In this example, the usage amount of calcium chloride solution is 10 μL, and the usage amount of dithiothreitol solution (DTT) is 14.7 μL. Incubate at 37 °C with a stirring speed of 120 rpm for 1 h. After incubation, centrifuge at 14000 rpf for 4 minutes, discard the supernatant to obtain a solid C1. Wash the solid C1 with a bis-tris propane buffer solution and then centrifuge again at 14000 rpf for 4 minutes to obtain a solid D1, which is the multilamellar lipid vesicles. In this example, the divalent ion solution is a magnesium chloride solution;

[0371] Step 7: Preparation of cross-linked liposome nanoparticles: Prepare a ligand, and the preparation method of the ligand is as follows: Cysteine is connected to the C-terminus of a polypeptide by Wuhan Jipeptide Biotechnology Co., Ltd. using existing techniques to obtain the ligand. The polypeptide is collagen-binding peptide (CBP), and the collagen-binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is LRELHLNNN; After mixing the obtained multilamellar vesicles with the ligand and incubating at 37 °C for 0.5 h, covalently conjugate the ligand to the multilamellar vesicles to obtain cross-linked liposome nanoparticles.

[0372] In this example, the mass of the polypeptide, which is collagen-binding peptide (CBP), is 0.8 mg.

[0373] Example 15

[0374] When preparing the liposome solution in Step 2 of Example 2, add rhodamine-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (Rhodamine-DSPE) to fluorescently label it for easy observation under a confocal microscope.

[0375] Use rhodamine-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine with 1 / 100 of the molar mass of phosphocholine to equimolarly replace the functionalized lipid. The rhodamine-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine is purchased from Xi'an Ruixi Biotechnology Co., Ltd., and the model is DSPE-Rhodamine B R-DZP006.

[0376] Comparative Example 1

[0377] This comparative example is for preparing traditional bilayer liposomes (SUV). The difference from Example 2 is that CBP is not grafted, divalent ion solution is not added, and DTT solution is not added, and the remaining preparation methods and steps are the same as those in Example 2.

[0378] Comparative Example 2

[0379] This comparative example is for preparing multilamellar vesicles (MLV). The difference from Example 2 is that CBP is not grafted and DTT solution is not added, and the remaining preparation methods and steps are the same as those in Example 2.

[0380] Comparative Example 3

[0381] This comparative example is for preparing ICMVs without grafted CBP. The difference from Example 2 is that CBP is not grafted, and the remaining preparation methods and steps are the same as those in Example 2.

[0382] Comparative Example 4

[0383] This comparative example is based on Comparative Example 1, encapsulating 0.5 mg of the model protein drug bovine serum albumin (BSA). The preparation method is the same as the above-mentioned preparation method. In this comparative example, a calcium ion solution is selected.

[0384] Comparative Example 5

[0385] This comparative example is based on Comparative Example 1, encapsulating 0.5 mg of transforming growth factor (TGF-β3). The preparation method is the same as the above-mentioned preparation method. In this comparative example, a calcium ion solution is selected.

[0386] Comparative Example 6

[0387] This comparative example is based on Comparative Example 1, encapsulating 1 mg of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid (kartogenin, KGN). The preparation method is the same as the above-mentioned preparation method. In this comparative example, a calcium ion solution is selected.

[0388] Comparative Example 7

[0389] This comparative example is based on Comparative Example 2, encapsulating 0.5 mg of the model protein drug bovine serum albumin (BSA). The preparation method is the same as the above-mentioned preparation method. In this comparative example, a calcium ion solution is selected.

[0390] Comparative Example 8

[0391] This comparative example is based on Comparative Example 2, encapsulating 0.5 mg of transforming growth factor (TGF-β3). The preparation method is the same as the above-mentioned preparation method. In this comparative example, a calcium ion solution is selected. Comparative Example 9

[0392] This comparative example is based on Comparative Example 2, encapsulating 1 mg of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid (kartogenin, KGN). The preparation method is the same as the above-mentioned preparation method. In this comparative example, a calcium ion solution is selected.

[0393] Comparative Example 10

[0394] This comparative example is based on Comparative Example 3, encapsulating 0.5 mg of the model protein drug bovine serum albumin (BSA). The preparation method is the same as the above-mentioned preparation method. In this comparative example, a calcium ion solution is selected.

[0395] Comparative Example 11

[0396] This comparative example is based on Comparative Example 3, encapsulating 0.5 mg of transforming growth factor (TGF-β3). The preparation method is the same as the above-mentioned preparation method. In this comparative example, a calcium ion solution is selected. Comparative Example 12

[0397] This comparative example is based on Comparative Example 3, encapsulating 1 mg of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid (kartogenin, KGN). The preparation method is the same as the above-mentioned preparation method. In this comparative example, a calcium ion solution is selected.

[0398] Comparative Example 13

[0399] This comparative example is for comparison with Example 15. 0.8 mg of collagen-binding peptide (CBP) in Example 15 is replaced with 1.3 mg of polyethylene glycol, and the remaining steps remain unchanged.

[0400] Comparative Example 14

[0401] This comparative example grafts CBP on the basis of Comparative Example 2 to obtain CBP-conjugated MLV.

[0402] Test Example 1

[0403] The encapsulation efficiency of Examples 3-10 was tested, and the test results are shown in Tables 1 and 2:

[0404] Table 1

[0405] Serial number Example 3 Example 4 Example 5 Example 6 Entrapment efficiency (%) 5-10 25-30 65-75 100

[0406] Table 2

[0407] Serial number Example 7 Example 8 Example 9 Example 10 Entrapment efficiency (%) 10-15 40-45 85-90 100

[0408] It can be seen from the comparison of the above table that the encapsulation efficiency of the multi-layer cross-linked liposomes increases with the increase of the added volume of Ca 2+ , Mg 2+ and when the added volume reaches 20 μL, the yield is close to 100%. However, if the dosage of divalent ions is too large, it is easy to cause too many layers and too large particle size of the multi-layer cross-linked liposomes, making the multi-layer cross-linked liposomes prone to precipitation in the suspension. While if the dosage of divalent ions is too small, it will lead to too few layers of liposome vesicles formed, resulting in too low encapsulation efficiency, poor sustained-release effect, and difficulty in precipitation after high-speed centrifugation, which makes it difficult to graft ligands in the subsequent operation. Therefore, the optimal dosage of divalent ions is selected as adding 15 μL of Mg 2+ or 10 μL of Ca 2+ .

[0409] Test Example 2

[0410] The yields of Examples 2 and Examples 11-14 were tested. The test results are shown in Table 3:

[0411] Table 3

[0412] Serial number Dosage (μL) Yield (%) Crosslinking ratio Grafting rate (%) Example 11 21 45-50 1 20 Example 12 19.95 70 0.95 25 Example 13 18.9 95 0.9 30 Example 2 16.8 100 0.8 35 Example 14 14.7 90 0.7 40

[0413] From the comparison in the above table, it can be seen that: as the amount of DTT used decreases, the encapsulation efficiency of the multi-layer cross-linked liposomes increases. And according to the cross-linking ratio of DTT for subsequent ligand grafting rate calculation, as the amount of DTT used decreases, the subsequent grafting rate increases. However, if the addition amount of DTT is 14.7 μL, the cross-linking ratio is about 0.7, which will sacrifice the cross-linking effect of the multi-layer cross-linked liposomes, resulting in a part of the non-cross-linked multi-layer cross-linked liposomes, accelerating the drug release rate and making it unstable. Therefore, the optimal addition amount of DTT is 16.8 μL.

[0414] Test Example 3

[0415] The average particle size of the products prepared in Comparative Examples 1-3 and Example 1 was tested, and the test results are shown in Figure 1 and Table 4:

[0416] Table 4

[0417] Serial number Average particle size (y) / nm Error (yEr±) Comparative example 1 102.43 3.493 Comparative example 2 167.296 1.098 Comparative example 3 187.48 2.432 Example 1 232.72 2.875

[0418] Test Example 4

[0419] The potential of the products prepared in Comparative Examples 1-3 and Example 1 was tested, and the test results are shown in Figure 2 and Table 5:

[0420] Table 5

[0421] Serial number Zeta potential / mV Comparative example 1 -0.2624 Comparative example 2 -0.274 Comparative example 3 -0.5656 Example 1 1.21

[0422] Among them, the potential test can characterize the stability and dispersibility of liposomes; stably dispersed liposomes usually have a relatively high Zeta potential.

[0423] Test Example 5

[0424] The encapsulation efficiency of Comparative Examples 4-12 for the model protein drugs bovine serum albumin BSA, transforming growth factor TGF-β3, and the hydrophobic small molecule drug kartogenin KGN was tested, and the test results are as Figure 3 shown, and the encapsulation efficiency of ICMVs is significantly higher than that of SUV and MLV.

[0425] Test Example 6

[0426] The release cycle of the model drug bovine serum albumin BSA encapsulated in Comparative Example 4, Comparative Example 14, and Example 3 was tested, and the test results are as Figure 4 shown: Comparative Example 4: 3 days; Comparative Example 14: 14 days; Example 3: 40 days.

[0427] Test Example 7

[0428] Coat the culture dish with a 0.012 mg / mL solution of rat tail collagen type I at a coating concentration of 2 μg / cm². Ensure that the collagen solution covers the surface of the culture dish. After leaving it at room temperature for 1 hour, wash it 3 - 4 times with phosphate buffer PBS. Then block the control group with a 2% BSA solution for 1 hour. Drop the comparative example 13 and example 15 onto the culture dish coated with the rat tail collagen type I solution, incubate at 37°C and 100 rpm, take samples at 1 h, 30 d, and 60 d respectively. After taking the samples, wash them 3 - 4 times with PBS, and then observe under a confocal microscope. The observation results are as Figure 5 shown.

[0429] Test example 8

[0430] Determine the binding effect of the rhodamine B-labeled multi-layer cross-linked liposomes grafted with CBP or PEG prepared in example 15 and comparative example 13 on rabbit cartilage sections. Drop ICMV(Rho)-PEG and ICMV(Rho)-CBP onto the rabbit cartilage sections, incubate at 37°C and 100 rpm, take samples at 2 h and 30 d respectively. After taking the samples, wash them 3 - 4 times with PBS, and then observe under a confocal microscope. The observation results are as Figure 6 shown.

[0431] Test example 9

[0432] Determine the in vivo cartilage-targeting binding effect of the rhodamine B-labeled multi-layer cross-linked liposomes grafted with CBP or PEG prepared in example 15 and comparative example 13.

[0433] Experimental animals: 6 male Balb / c mice, weighing about 30 grams, all purchased from the Experimental Animal Center of Peking University.

[0434] Experimental method: Before the experiment, depilate the mice. Depilate the experimental-related parts of the mice, such as the abdomen, back, and legs.

[0435] Conduct the experiment. Anesthetize the mice by intraperitoneal injection of 0.4 mL of 4% chloral hydrate solution, as Figure 7 shown. Subcutaneously inject 0.2 mL of the products prepared in example 15 and comparative example 13 into the left and right knee joints of the mice respectively. After the injection, gas-anesthetize the mice with isoflurane 2 hours later. Adjust the posture of the anesthetized mice and place them in the imaging chamber of a small animal in vivo imager, ensuring that the mice lie on their backs with their abdomens facing up and their limbs stretched out. Through the imaging software of the in vivo imager, take pictures under the condition of emitting an acceptance spectrum of EX / EM 543 / 598 nm. Then place the mice in the in vivo imager for taking pictures according to the above operations at 24, 48, 72, 96 h, and 7 d respectively, as Figure 8Observe the enrichment of liposomes grafted with different ligands at the knee joint, and draw a half-life curve as Figure 9 shown. It can be seen that the half-life of Comparative Example 13 is 0.67 days, and the half-life of Example 15 is 3.01 days. It is proved that the polypeptide selected in the present invention can bind to cartilage for a long time to extend the residence time, achieve the precise enrichment of the drug at the knee joint in mice, realize the cartilage affinity of the multi-layer cross-linked liposomes prepared in the present invention, reduce drug loss, and improve the efficiency of cartilage regeneration.

[0436] It should be understood that the present invention is not limited to what has been described above and shown in the drawings, and various modifications and changes can be made without departing from its scope. The scope of the present invention is only limited by the appended claims.

Claims

1. A cartilage - affinity cross - linked liposome nanoparticle, characterized in that, the cross - linked liposome nanoparticle is composed of multilamellar lipid vesicles and a ligand, the mass ratio of the multilamellar lipid vesicles to the ligand is 2.2:(0.56 - 0.8), the ligand is covalently conjugated to the multilamellar lipid vesicles to form the cross - linked liposome nanoparticle, and the ligand is a thiol - modified polypeptide; the carboxyl terminus of the polypeptide is connected to cysteine, the cysteine contains a thiol group, and the thiol group undergoes a Michael addition reaction with the maleic anhydride bond on the multilamellar lipid vesicles to form a covalent bond, realizing the covalent conjugation between the ligand and the multilamellar lipid vesicles, and the grafting rate of the ligand in the covalent conjugation is 20 - 40%.

2. The cartilage - affinity cross - linked liposome nanoparticle according to claim 1, characterized in that, the polypeptide is a collagen - binding peptide, and the collagen - binding peptide is synthesized through an amino acid sequence, and the amino acid sequence is SEQ ID NO:

1.

3. The cartilage - affinity cross - linked liposome nanoparticle according to claim 1, characterized in that, the multilamellar lipid vesicles include functionalized lipids, phosphatidylcholine, and phosphatidylglycerol.

4. The cartilage - affinity cross - linked liposome nanoparticle according to claim 3, characterized in that, the functionalized lipid is a maleimide - functionalized lipid, the phosphatidylcholine is dioleoylphosphatidylcholine, and the phosphatidylglycerol is 1,2 - dioleoyl - sn - glycero - 3 - phospho - rac - (1 - glycerol) sodium salt.

5. A preparation method of the cartilage - affinity cross - linked liposome nanoparticle according to any one of claims 1 - 4, characterized in that, it includes the following steps: Step 1, preparing a solution: the solution includes one or more of bis - tris buffer solution, dithiothreitol solution (DTT solution), magnesium chloride solution, and calcium chloride solution; Step 2, preparing a liposome solution: dissolving phosphatidylcholine, phosphatidylglycerol, and functionalized lipid in chloroform to obtain a liposome solution; Step 3, preparing a lipid film: air - drying the liposome solution prepared in Step 2 at room temperature to obtain a lipid film; Step 4, preparing multilamellar liposomes: hydrating the lipid film prepared in Step 3 with a buffer solution, and through oscillation, making the lipid film fully dissolve in the buffer solution to form a mixed solution A1, and the mixed solution A1 includes micron - sized multilamellar liposomes; Step 5, preparing unilamellar liposomes: using a liposome extruder to repeatedly extrude the mixed solution A1 prepared in Step 4 to obtain a mixed solution B1, and the mixed solution B1 contains unilamellar liposomes; Step 6, preparing multilamellar lipid vesicles: adding a divalent ion solution and a dithiothreitol solution to the mixed solution B1, incubating with stirring, centrifuging after the incubation ends, discarding the supernatant to obtain a solid C1, washing the solid C1 with a bis - tris buffer solution and then centrifuging again to obtain a solid D1, which is the multilamellar lipid vesicles; Step 7. Preparation of cross-linked liposome nanoparticles: The obtained multilamellar vesicles are mixed with a ligand and incubated, and the ligand is covalently conjugated to the multilamellar vesicles to obtain cross-linked liposome nanoparticles.

6. A multilamellar cross-linked liposome, characterized in that the multilamellar cross-linked liposome comprises the cartilage-affinity cross-linked liposome nanoparticles according to any one of claims 1-4, and protein molecules and / or hydrophobic small molecule drugs encapsulated in the cross-linked liposome nanoparticles, and the encapsulation efficiency of the multilamellar cross-linked liposome is ≥40%.

7. The multilamellar cross-linked liposome according to claim 6, characterized in that the protein molecules and / or hydrophobic small molecule drugs are encapsulated in the multilamellar vesicles of the cross-linked liposome nanoparticles.

8. The multilamellar cross-linked liposome according to claim 6, characterized in that the protein molecules include one or more of the model protein drugs bovine serum albumin and transforming growth factor; the hydrophobic small molecule drug is 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid.

9. The multilamellar cross-linked liposome according to claim 6, characterized in that the preparation method of the multilamellar cross-linked liposome comprises the following steps: Step 1. Prepare a solution: The solution includes one or more of bis-tris buffer solution, dithiothreitol solution (DTT solution), magnesium chloride solution, and calcium chloride solution; Step 2. Prepare a liposome solution: Phosphatidylcholine, phosphatidylglycerol, and functionalized lipids are dissolved in chloroform to obtain a liposome solution; Step 3. Prepare a liposome mixture: Protein molecules and / or hydrophobic small molecule drugs are dissolved in bis-tris buffer solution or chloroform to obtain a dissolved solution, and the dissolved solution is added to the liposome solution prepared in Step 2 to obtain a liposome mixture; Step 4. Prepare a lipid film containing drugs: The liposome mixture prepared in Step 3 is air-dried at room temperature to obtain a lipid film containing drugs; Step 5. Prepare multilamellar liposomes containing drugs: Hydrate the lipid film containing drugs prepared in Step 4 with a buffer solution, and by shaking, the lipid film containing drugs is fully dissolved in the buffer solution to form a mixed solution A2, and the mixed solution A2 includes micron-sized multilamellar liposomes containing drugs; Step 6. Prepare unilamellar liposomes containing drugs: Use a liposome extruder to repeatedly extrude the mixed solution A2 prepared in Step 5 to obtain a mixed solution B2, and the mixed solution B2 contains unilamellar liposomes containing drugs; Step 7. Prepare multilamellar vesicles containing drugs: Add a divalent ion solution and a dithiothreitol solution to the mixed solution B2, incubate with stirring, after the incubation is completed, centrifuge, discard the supernatant to obtain a solid C2, wash the solid C2 with bis-tris buffer solution and then centrifuge again to obtain a solid D2, which is the multilamellar vesicles containing drugs; Step 8. Prepare multilamellar cross-linked liposomes: The obtained multilamellar vesicles containing drugs are mixed with a ligand and incubated, and the ligand is covalently conjugated to the multilamellar vesicles containing drugs to obtain multilamellar cross-linked liposomes.

10. A drug, characterized in that, the drug comprises the multilayer cross-linked liposomes described in claims 6-9.

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