Piezoelectric tetrapeptide PZE201-loaded cartilage targeted penetrating liposome as well as preparation method and application thereof

Abstract

The invention discloses a cartilage targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201 as well as a preparation method and application of the cartilage targeted penetrating liposome. The method comprises the following steps: preparing PZE201 tetrapeptide by using a chemical synthesis method; the preparation method comprises the following steps: synthesizing DSPE-PEG-CAQK by adopting a sulfydryl-maleimide Michael addition reaction; dissolving DSPE-PEG-CAQK, soya bean lecithin, DOTAP and cholesterol in ethanol, heating, stirring, and carrying out ultrasonic dispersion to obtain an organic phase; the preparation method comprises the following steps: dissolving PZE201 in a PBS (Phosphate Buffer Solution), heating, violently stirring, and dispersing under a vortex and ultrasonic quadruple process to prepare a water phase; respectively loading the organic phase solution and the water phase solution into an injector, and preparing a crude liposome solution through a double-phase micro-channel mixing process; the liposome constructed by the invention is simple and convenient in preparation method and mild in preparation condition, realizes accurate, stable, efficient and safe electrical stimulation on cartilage in vivo and in vitro, relieves cartilage injury, and effectively improves osteoarthritis symptoms.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and specifically relates to a cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201, and a preparation method and application thereof. Background Art

[0002] Osteoarthritis (OA) is a common chronic degenerative joint disease that mainly affects middle-aged and elderly people. The disease is characterized by the gradual wear and degeneration of articular cartilage, accompanied by changes in the surrounding bone and synovium. OA can affect multiple joints in the body, but the most common sites include weight-bearing joints such as the knee and hip. As the disease progresses, patients may experience symptoms such as joint pain, stiffness, limited movement and swelling, which seriously affect the quality of daily life. Although the exact cause of OA has not been fully elucidated, it is known that a variety of factors may increase its risk of development, including age, obesity, history of joint injury, genetic factors and long-term overuse of joints. Currently, OA treatment is still difficult, and treatment mainly focuses on symptom management. Therefore, designing a highly active biomaterial is of great significance for OA treatment.

[0003] Current studies have found that electrical stimulation promotes cartilage to secrete extracellular matrix, but electrical stimulation requires additional implantation of electrodes, requiring secondary surgery, high risk of infection, and cannot precisely stimulate cartilage tissue. In the past, inorganic metal-based nanoparticles such as barium titanate and zinc oxide were used for in vivo electrical stimulation, which can replace electrodes and avoid surgical operations, but there are problems such as difficult delivery, poor biocompatibility, and difficulty in degradation, which can cause immune responses and are difficult to meet the actual repair needs of osteoarthritis cartilage. Summary of the invention

[0004] In order to solve the problems in the background technology and address the difficulties of the prior art in electrical stimulation and efficient delivery of cartilage in vivo, the present invention aims to provide a cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201 and a preparation method and application thereof.

[0005] The present invention adopts the following technical scheme as follows, comprising the following steps:

[0006] Step S1, firstly synthesizing the tetrapeptide PZE201 by chemical synthesis, and separating and purifying the synthesized tetrapeptide PZE201;

[0007] Step S2, synthesizing DSPE-PEG-CAQK (phospholipid polyethylene glycol polypeptide CAQK) by thiol-maleimide Michael addition reaction;

[0008] Step S3, preparing an organic phase: after equilibration to room temperature, sequentially dissolve the DSPE-PEG-CAQK, soybean lecithin, (2,3-dioleyloxypropyl)trimethylammonium chloride DOTAP, and cholesterol in step S2 in ethanol, and heat, stir, and ultrasonically disperse to obtain an organic phase solution for later use;

[0009] Step S4, preparing an aqueous phase: dissolving the tetrapeptide PZE201 prepared in step S1 in a PBS solution having a pH of 7.2 to 7.5, and dispersing the solution under a four-fold process of heating, vigorous stirring, vortexing and ultrasound to obtain an aqueous phase solution for standby use;

[0010] Step S5, loading the organic phase solution prepared in step S3 and the aqueous phase solution prepared in step S4 into two syringes respectively, connecting the outlets of the two syringes to the two inlets of the microfluidic chip through pipelines respectively, and connecting the syringes to a microfluidic pressure pump outside the syringes, and under the control of the microfluidic pressure pump, mixing the organic phase solution and the aqueous phase solution in the microfluidic chip in a preset ratio, and collecting the original liposome solution at the outlet of the microfluidic chip;

[0011] Step S6: remove ethanol from the original liposome solution through a dialysis bag and filter it through a filter membrane for sterilization to obtain the final cartilage-targeted penetrating liposomes.

[0012] The step S2 is specifically as follows: dissolving phospholipid polyethylene glycol maleimide DSPE-PEG-Mal and polypeptide CAQK in polybutylene succinate PBS solution, stirring at a certain reaction temperature, protecting with nitrogen, and dialyzing with deionized water, and finally freeze-drying to obtain a DSPE-PEG-CAQK sample.

[0013] In the step S1, PZE201 is a tetrapeptide consisting of the amino acid sequence of Hyp-Hyp-Phe-Phe, and the chemical synthesis method adopts one of a solid phase synthesis method, a liquid phase synthesis method or a solid-liquid combination method.

[0014] Here, Hyp represents hydroxyproline, and Phe represents phenylalanine. Hyp uses one of 3-hydroxyproline (3Hyp) and 4-hydroxyproline (4Hyp).

[0015] In the step S2, the molar ratio of DSPE-PEG-Mal to CAQK is 1:1-1:2, the reaction temperature is 0-10°C, and the stirring time is 12-24 hours.

[0016] In the step S3, the molar ratio of DSPE-PEG-CAQK, soybean lecithin, DOTAP and cholesterol is 1-5%: 20-70%: 5-10%: 30-50%, the total lipid concentration is 2-20 mg / ml, the heating temperature is 40-60° C., the stirring rate is greater than 300 rpm, the stirring time is 1-10 min, the ultrasonic power is greater than 20 W, and the ultrasonic time is 1-10 min.

[0017] Lipids included DSPE-PEG-CAQK, soybean lecithin, DOTAP, and cholesterol.

[0018] In step S4, the concentration of tetrapeptide PZE201 is 0.5-30 mg / ml, the heating temperature is 50-90° C., the heating time is 1-10 min, the vigorous stirring refers to a stirring rate greater than 1000 rpm, a stirring time of 1-10 min, a vortex time of 0.1-1 min, an ultrasonic power greater than 20 W, and an ultrasonic time of 1-10 min.

[0019] In the step S5, the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:1-1:20, the total flow rate of the mixed solution is 5-20 ml / min, and the microfluidic chip is herringbone-shaped or Y-shaped.

[0020] A cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201: the cartilage-targeted penetrating liposome is prepared by the above-mentioned preparation method. The application of the cartilage-targeted penetrating liposome in the preparation of a drug for preventing and / or treating osteoarthritis.

[0021] The present invention innovatively designs and uses chemical synthesis to prepare the Hyp-Hyp-Phe-Phe (PZE201) tetrapeptide. The Hyp-Hyp-Phe-Phe tetrapeptide is deformed after being squeezed by mechanical force and generates voltage at both ends of the crystal. The introduction of Hyp-Hyp improves the piezoelectric properties of the polypeptide. The Phe-Phe structure is used as a self-assembly recognition template. After being encapsulated, the Hyp-Hyp-Phe-Phe tetrapeptide self-assembles in the liposome to form a collagen-like fibrous spiral assembly, and forms a core-shell structure with the liposome. In the present invention, the liposome uses four functional components such as DOTAP, soybean lecithin, cholesterol, and DSPE-PEG-CAQK, wherein soybean lecithin is the main framework of the liposome, cholesterol stabilizes the liposome structure, and DOTAP, as a permanent cationic lipid, increases the zeta potential of the liposome and interacts with the negative charge in the cartilage. The introduction of DSPE-PEG-CAQK enhances the active targeting of the liposome to the cartilage CSPGs. The liposome shell controls the size of Hyp-Hyp-Phe-Phe while maintaining its excellent piezoelectric properties, avoiding direct contact with enzymes and uncontrolled degradation after entering the body.

[0022] The liposome preparation can be directly injected into the joint cavity. After reaching the cartilage, it relies on the DOTAP cationic charge and CAQK to perform dual active and passive targeting of the cartilage, penetrate into the deep cartilage, and after Hyp-Hyp-Phe-Phe is subjected to mechanical force, it can achieve precise electrical stimulation of chondrocytes, activate downstream signaling pathways, and promote chondrocytes to secrete type II collagen. Through synergistic effects, it meets the requirements for osteoarthritis cartilage repair.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] 1. The liposomes prepared by the present invention have piezoelectric properties, injectability, cartilage permeability, cartilage targeting, biocompatibility, and biodegradability. The above functions complement each other. The liposome preparation can be directly injected into the joint cavity, and can accurately, stably, efficiently, and safely electrically stimulate the cartilage, reduce cartilage damage, and effectively improve the symptoms of osteoarthritis.

[0025] 2. The liposome preparation conditions are mild. The liposomes are prepared using a combination of processes such as microfluidics, self-assembly, and nanocomposites. The process route is formulated according to the actual preparation needs of piezoelectric liposomes. The liposome size is uniform and controllable, which is original.

[0026] 3. The present invention combines the independently designed Hyp-Hyp-Phe-Phe with multifunctional liposomes, which can accurately, stably, efficiently and safely electrically stimulate cartilage both in vivo and in vitro, reduce cartilage damage and promote extracellular matrix synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a TEM image of the liposome obtained in Example 1 of the present invention;

[0028] Figure 2 This is the in vivo SO staining image obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and examples. The following specific examples are used to illustrate the present invention, but should not be construed as limiting the present invention. Within the spirit of the present invention and the protection scope of the claims, any modifications and changes made to the present invention fall within the protection scope of the present invention. The reagents used in all examples are not less than analytically pure standards.

[0030] Example 1

[0031] A preparation method of cartilage-targeted penetrating liposomes loaded with piezoelectric tetrapeptide PZE201 comprises the following steps:

[0032] Step S1, firstly synthesizing the tetrapeptide PZE201 by solid phase synthesis method (amino acid condensation reaction), and separating and purifying the synthesized tetrapeptide PZE201;

[0033] PZE201 is a tetrapeptide composed of the amino acid sequence of Hyp-Hyp-Phe-Phe, wherein Hyp represents hydroxyproline, Phe represents phenylalanine, and Hyp is 4-hydroxyproline (4Hyp).

[0034] Step S2, synthesizing DSPE-PEG-CAQK (phospholipid polyethylene glycol polypeptide CAQK) by thiol-maleimide Michael addition reaction: dissolving phospholipid polyethylene glycol maleimide DSPE-PEG-Mal and polypeptide CAQK in polybutylene succinate PBS solution, the molar ratio of DSPE-PEG-Mal to CAQK being 1:1.5, stirring at a reaction temperature of 4°C for 24 hours, protecting with nitrogen, and dialyzing with deionized water, and finally lyophilizing to obtain a DSPE-PEG-CAQK sample

[0035] Step S3, preparing an organic phase: after equilibration to room temperature, sequentially dissolve the DSPE-PEG-CAQK, soybean lecithin, (2,3-dioleyloxypropyl)trimethylammonium chloride DOTAP, and cholesterol in step S2 in ethanol, heat to 50°C, stir at 350 r / min for 1 min, and ultrasonically disperse at 30W for 1 min to obtain an organic phase solution for standby use; wherein the molar ratio of DSPE-PEG-CAQK, soybean lecithin, DOTAP, and cholesterol is 3:52:5:40, and the total lipid concentration is 8 mg / ml;

[0036] Step S4, preparing the aqueous phase: dissolving the tetrapeptide PZE201 in step S1 in a PBS solution with a pH of 7.4 at a concentration of 10 mg / ml, heating at 60° C. for 1 min, vigorously stirring at 1500 r / min for 1 min, vortexing for 0.1 min, and ultrasonicating at 25 W for 1 min, dispersing under the quadruple process to obtain an aqueous phase solution for standby use;

[0037] Step S5, loading the organic phase solution prepared in step S3 and the aqueous phase solution prepared in step S4 into two syringes respectively, connecting the outlets of the two syringes to the two inlets of the microfluidic chip through pipelines respectively, and simultaneously connecting the syringes to a microfluidic pressure pump outside, and under the control of the microfluidic pressure pump, mixing the aqueous phase and the organic phase solution in the microfluidic chip at an organic phase: aqueous phase flow rate ratio of 1:5 and a total flow rate of 10 ml / min, and collecting the original liposome solution at the outlet of the microfluidic chip;

[0038] In a specific implementation, the organic phase solution / aqueous phase solution in the syringe flows into the microfluidic chip under the control of a microfluidic pressure pump, and undergoes a mixed reaction in the microfluidic chip, and then flows out from the outlet of the microfluidic chip after the mixed reaction.

[0039] Step S6, removing ethanol from the original liposome solution through a dialysis bag and filtering and sterilizing with a filter membrane to obtain the final liposome solution, namely, the cartilage-targeted penetrating liposome.

[0040] The liposomes prepared in this Example 1 are spherical nano-sized vesicles, such as Figure 1 As shown, CCK8 test showed that chondrocyte proliferation increased by 1.24 times, and chondrocyte type II collagen mRNA expression increased by 2.13 times. Figure 2 Figure 2 is an in vivo SO staining image without injection of cartilage-targeted penetrating liposomes (blank control) and after injection of cartilage-targeted penetrating liposomes (PZE201@cLipo). Figure 2 As shown, cartilage repair was good 28 days after injection in the rat knee OA model.

[0041] Example 2

[0042] The preparation method of the cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201 comprises the following steps:

[0043] Step S1, firstly synthesizing the tetrapeptide PZE201 by solid phase synthesis method (amino acid condensation reaction), and separating and purifying the synthesized tetrapeptide PZE201;

[0044] Step S2, synthesizing DSPE-PEG-CAQK (phospholipid polyethylene glycol polypeptide CAQK) by thiol-maleimide Michael addition reaction: dissolving phospholipid polyethylene glycol maleimide DSPE-PEG-Mal and polypeptide CAQK in polybutylene succinate PBS solution, the molar ratio of DSPE-PEG-Mal to CAQK being 1:1.5, stirring at a reaction temperature of 4°C for 24 hours, protecting with nitrogen, and dialyzing with deionized water, and finally lyophilizing to obtain a DSPE-PEG-CAQK sample

[0045] Step S3, preparing an organic phase: after equilibration to room temperature, sequentially dissolve the DSPE-PEG-CAQK, soybean lecithin, (2,3-dioleyloxypropyl)trimethylammonium chloride DOTAP, and cholesterol in step S2 in ethanol, heat to 50°C, stir at 350 r / min for 1 min, and ultrasonically disperse at 30W for 1 min to obtain an organic phase solution for standby use; wherein the molar ratio of DSPE-PEG-CAQK, soybean lecithin, DOTAP, and cholesterol is 1:52:5:40, and the total lipid concentration is 8 mg / ml;

[0046] Step S4, preparing the aqueous phase: dissolving the tetrapeptide PZE201 in step S1 in a PBS solution with a pH of 7.4 at a concentration of 10 mg / ml, heating at 60° C. for 1 min, vigorously stirring at 1500 r / min for 1 min, vortexing for 0.1 min, and ultrasonicating at 25 W for 1 min, dispersing under the quadruple process to obtain an aqueous phase solution for standby use;

[0047] Step S5, loading the organic phase solution prepared in step S3 and the aqueous phase solution prepared in step S4 into two syringes respectively, connecting the outlets of the two syringes to the two inlets of the microfluidic chip through pipelines respectively, and simultaneously connecting the syringes to a microfluidic pressure pump outside, and under the control of the microfluidic pressure pump, mixing the aqueous phase and the organic phase solution in the microfluidic chip at an organic phase: aqueous phase flow rate ratio of 1:5 and a total flow rate of 10 ml / min, and collecting the original liposome solution at the outlet of the microfluidic chip;

[0048] Step S6, removing ethanol from the original liposome solution through a dialysis bag and filtering and sterilizing with a filter membrane to obtain the final liposome solution, namely, the cartilage-targeted penetrating liposome.

[0049] Compared with Example 1, the DSPE-PEG-CAQK content of the liposomes was reduced. The liposomes were spherical nano-sized vesicles. The CCK8 test showed that the proliferation of chondrocytes increased by 1.03 times, and the expression of type II collagen mRNA in chondrocytes increased by 1.26 times. After 28 days of injection in the rat knee OA model, the cartilage repair was good.

[0050] Example 3

[0051] The preparation method of the cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201 comprises the following steps:

[0052] Step S1, firstly synthesizing the tetrapeptide PZE201 by solid phase synthesis method (amino acid condensation reaction), and separating and purifying the synthesized tetrapeptide PZE201;

[0053] Step S2, synthesizing DSPE-PEG-CAQK (phospholipid polyethylene glycol polypeptide CAQK) by thiol-maleimide Michael addition reaction: dissolving phospholipid polyethylene glycol maleimide DSPE-PEG-Mal and polypeptide CAQK in polybutylene succinate PBS solution, the molar ratio of DSPE-PEG-Mal to CAQK being 1:1.5, stirring at a reaction temperature of 4°C for 24 hours, protecting with nitrogen, and dialyzing with deionized water, and finally lyophilizing to obtain a DSPE-PEG-CAQK sample

[0054] Step S3, preparing an organic phase: after equilibration to room temperature, sequentially dissolve the DSPE-PEG-CAQK, soybean lecithin, (2,3-dioleyloxypropyl)trimethylammonium chloride DOTAP, and cholesterol in step S2 in ethanol, heat to 50°C, stir at 350 r / min for 1 min, and ultrasonically disperse at 30W for 1 min to obtain an organic phase solution for standby use; wherein the molar ratio of DSPE-PEG-CAQK, soybean lecithin, DOTAP, and cholesterol is 3:52:5:40, and the total lipid concentration is 8 mg / ml;

[0055] Step S4, preparing an aqueous phase: dissolving the tetrapeptide PZE201 in step S1 in a PBS solution with a pH of 7.4 at a concentration of 1 mg / ml, heating at 60° C. for 1 min, vigorously stirring at 1500 r / min for 1 min, vortexing for 0.1 min, and ultrasonicating at 25 W for 1 min, dispersing under a quadruple process to obtain an aqueous phase solution for standby use;

[0056] Step S5, loading the organic phase solution prepared in step S3 and the aqueous phase solution prepared in step S4 into two syringes respectively, connecting the outlets of the two syringes to the two inlets of the microfluidic chip through pipelines respectively, and simultaneously connecting the syringes to a microfluidic pressure pump outside, and under the control of the microfluidic pressure pump, mixing the aqueous phase and the organic phase solution in the microfluidic chip at an organic phase: aqueous phase flow rate ratio of 1:5 and a total flow rate of 10 ml / min, and collecting the original liposome solution at the outlet of the microfluidic chip;

[0057] Step S6, removing ethanol from the original liposome solution through a dialysis bag and filtering and sterilizing with a filter membrane to obtain the final liposome solution, namely, the cartilage-targeted penetrating liposome.

[0058] Compared with Example 1, the Hyp-Hyp-Phe-Phe concentration of the liposomes was reduced. The liposomes were spherical nanoscale vesicles. The CCK8 test showed that the proliferation of chondrocytes increased by 1.05 times, and the expression of type II collagen mRNA in chondrocytes increased by 1.08 times. After 28 days of injection in the rat knee OA model, the cartilage repair was good.

[0059] Example 4

[0060] The preparation method of the cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201 comprises the following steps:

[0061] The preparation method of the cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201 comprises the following steps:

[0062] Step S1, firstly synthesizing the tetrapeptide PZE201 by solid phase synthesis method (amino acid condensation reaction), and separating and purifying the synthesized tetrapeptide PZE201;

[0063] Step S2, synthesizing DSPE-PEG-CAQK (phospholipid polyethylene glycol polypeptide CAQK) by thiol-maleimide Michael addition reaction: dissolving phospholipid polyethylene glycol maleimide DSPE-PEG-Mal and polypeptide CAQK in polybutylene succinate PBS solution, the molar ratio of DSPE-PEG-Mal to CAQK being 1:1.5, stirring at a reaction temperature of 4°C for 24 hours, protecting with nitrogen, and dialyzing with deionized water, and finally lyophilizing to obtain a DSPE-PEG-CAQK sample

[0064] Step S3, preparing an organic phase: after equilibration to room temperature, sequentially dissolve the DSPE-PEG-CAQK, soybean lecithin, (2,3-dioleyloxypropyl)trimethylammonium chloride DOTAP, and cholesterol in step S2 in ethanol, heat to 50°C, stir at 350 r / min for 1 min, and ultrasonically disperse at 30W for 1 min to obtain an organic phase solution for standby use; wherein the molar ratio of DSPE-PEG-CAQK, soybean lecithin, DOTAP, and cholesterol is 3:52:5:40, and the total lipid concentration is 8 mg / ml;

[0065] Step S4, preparing an aqueous phase: dissolving the tetrapeptide PZE201 in step S1 in a PBS solution with a pH of 7.4 at a concentration of 1 mg / ml, heating at 60° C. for 1 min, vigorously stirring at 1500 r / min for 1 min, vortexing for 0.1 min, and ultrasonicating at 25 W for 1 min, dispersing under a quadruple process to obtain an aqueous phase solution for standby use;

[0066] Step S5, loading the organic phase solution prepared in step S3 and the aqueous phase solution prepared in step S4 into two syringes respectively, connecting the outlets of the two syringes to the two inlets of the microfluidic chip through pipelines respectively, and simultaneously connecting the syringes to a microfluidic pressure pump outside, and under the control of the microfluidic pressure pump, mixing the aqueous phase and the organic phase solution in the microfluidic chip at an organic phase: aqueous phase flow rate ratio of 1:5 and a total flow rate of 1 ml / min, and collecting the original liposome solution at the outlet of the microfluidic chip;

[0067] Step S6, removing ethanol from the original liposome solution through a dialysis bag and filtering and sterilizing with a filter membrane to obtain the final liposome solution, namely, the cartilage-targeted penetrating liposome.

[0068] Compared with Example 1, the total flow rate of the liposomes during preparation was reduced. The liposomes were spherical nanoscale vesicles. The CCK8 test showed that chondrocyte proliferation was increased by 1.06 times, and the expression of type II collagen mRNA in chondrocytes was increased by 1.22 times. After 28 days of injection in the rat knee OA model, the cartilage repair was good.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201 and a preparation method thereof, characterized in that: The following steps are involved: Step S1, firstly synthesizing the tetrapeptide PZE201 by chemical synthesis; Step S2, synthesizing DSPE-PEG-CAQK by thiol-maleimide Michael addition reaction; Step S3, preparing an organic phase: after equilibration to room temperature, sequentially dissolve the DSPE-PEG-CAQK, soybean lecithin, (2,3-dioleyloxypropyl)trimethylammonium chloride DOTAP, and cholesterol in step S2 in ethanol, and heat, stir, and ultrasonically disperse to obtain an organic phase solution for later use; Step S4, preparing an aqueous phase: dissolving the tetrapeptide PZE201 prepared in step S1 in a PBS solution and dispersing the solution under a four-step process of heating, stirring, vortexing and ultrasound to prepare an aqueous phase solution for later use; Step S5, loading the organic phase solution prepared in step S3 and the aqueous phase solution prepared in step S4 into two syringes respectively, connecting the outlets of the two syringes to the two inlets of the microfluidic chip through pipelines respectively, and connecting the syringes to a microfluidic pressure pump outside the syringes, and under the control of the microfluidic pressure pump, mixing the organic phase solution and the aqueous phase solution in the microfluidic chip in a preset ratio, and collecting the original liposome solution at the outlet of the microfluidic chip; Step S6: remove ethanol from the original liposome solution through a dialysis bag and filter it through a filter membrane for sterilization to obtain the final cartilage-targeted penetrating liposomes.

2. The cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201 and the preparation method thereof according to claim 1, characterized in that: The step S2 is specifically as follows: dissolving phospholipid polyethylene glycol maleimide DSPE-PEG-Mal and polypeptide CAQK in polybutylene succinate PBS solution, stirring at a certain reaction temperature, protecting with nitrogen, and dialyzing with deionized water, and finally freeze-drying to obtain a DSPE-PEG-CAQK sample.

3. The cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201 and the preparation method thereof according to claim 1, characterized in that: In the step S1, PZE201 is a tetrapeptide consisting of the amino acid sequence of Hyp-Hyp-Phe-Phe, and the chemical synthesis method adopts one of a solid phase synthesis method, a liquid phase synthesis method or a solid-liquid combination method.

4. The cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201 and the preparation method thereof according to claim 2, characterized in that: In the step S2, the molar ratio of DSPE-PEG-Mal to CAQK is 1:1-1:2, the reaction temperature is 0-10°C, and the stirring time is 12-24 hours.

5. The cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201 and the preparation method thereof according to claim 1, characterized in that: In the step S3, the molar ratio of DSPE-PEG-CAQK, soybean lecithin, DOTAP and cholesterol is 1-5%: 20-70%: 5-10%: 30-50%, the total lipid concentration is 2-20 mg / ml, the heating temperature is 40-60° C., the stirring rate is greater than 300 rpm, the stirring time is 1-10 min, the ultrasonic power is greater than 20 W, and the ultrasonic time is 1-10 min.

6. The cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201 and the preparation method thereof according to claim 1, characterized in that: In step S4, the concentration of tetrapeptide PZE201 is 0.5-30 mg / ml, the heating temperature is 50-90° C., the heating time is 1-10 min, the stirring rate is greater than 1000 rpm, the stirring time is 1-10 min, the vortex time is 0.1-1 min, the ultrasonic power is greater than 20 W, and the ultrasonic time is 1-10 min.

7. The cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201 and the preparation method thereof according to claim 1, characterized in that: In the step S5, the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:1-1:20, the total flow rate of the mixed solution is 5-20 ml / min, and the microfluidic chip is herringbone-shaped or Y-shaped.

8. A cartilage-targeted penetrating liposome loaded with piezoelectric tetrapeptide PZE201, characterized in that: The cartilage-targeted penetrating liposomes are prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the cartilage-targeting penetrating liposomes prepared by the method according to any one of claims 1 to 7 in the preparation of a drug for preventing and / or treating osteoarthritis.

Images