Anti-inflammatory lipid Phoyunene C nano material, preparation method and application

Abstract

The invention discloses an anti-inflammatory lipid Phoyunene C nano material as well as a preparation method and application of the anti-inflammatory lipid Phoyunene C nano material. The nano material is mainly prepared from a Phoyunbene C nano dot core, DSPE-PEG2000 (Distearoyl Phosphate Polyethylene-Polyethylene Glycol 2000), soya bean lecithin and cholesterol, the preparation method comprises the following steps: dissolving Phoyunbene C in a phosphate buffer solution, heating and stirring to obtain a water phase; dSPE-PEG2000, soybean lecithin and cholesterol are dissolved in ethyl alcohol, heating, stirring and ultrasonic dispersion are conducted, and an organic phase is obtained; and respectively loading the organic phase solution and the water phase solution into an injector, and carrying out a two-phase micro-channel mixing process to obtain the lipid Phoyunbene C nanodot solution. The preparation method of the lipid Phoyunene C nano material provided by the invention is simple and convenient, the preparation condition is mild, a stronger anti-inflammatory effect in vivo and in vitro is realized, and the symptom of osteoarthritis is effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and specifically relates to an anti-inflammatory lipid Phoyunbene C nanomaterial, a preparation method and an application thereof. Background Art

[0002] Osteoarthritis (OA) is a common chronic degenerative joint disease that occurs frequently in the elderly, and its incidence rate increases significantly with age. The disease is characterized by the gradual wear and degeneration of articular cartilage, accompanied by changes in the surrounding bone tissue (such as osteophyte formation), structural changes in the synovium (such as synovial hyperplasia), and increased levels of inflammation (such as the release of proinflammatory cytokines IL-1β and TNF-α). OA can affect multiple joints, among which weight-bearing joints such as the knee and hip are the most common, but it may also affect small joints in the hands and spinal joints. As the disease progresses, patients often experience symptoms such as joint pain (especially after activity), stiffness (morning stiffness usually lasts no more than 30 minutes), limited mobility, and swelling, which seriously impair their quality of life.

[0003] Although the exact cause of OA is not yet fully understood, multiple factors have been shown to be associated with its risk of developing. These factors include aging (decreased cartilage repair capacity), obesity (increased joint mechanical load and inflammation levels), history of joint injury (such as ligament tear or fracture), genetic predisposition (such as COL2A1 gene mutation), and long-term overuse of joints (such as occupational or sports-related injuries). In addition, metabolic abnormalities (such as diabetes) and gender (women are more susceptible to the disease) are also considered potential risk factors.

[0004] At present, the treatment of OA is still mainly to relieve symptoms, and there is a lack of radical cure. Conventional treatments include non-drug treatments (such as exercise therapy, weight management) and drug treatments (such as non-steroidal anti-inflammatory drugs, intra-articular injection of hyaluronic acid or glucocorticoids). In recent years, the development of regenerative medicine and biomaterials has provided new ideas for the treatment of OA, such as stem cell therapy, tissue engineering cartilage, and nanomaterial delivery systems. However, these methods still face many challenges, such as the long-term stability of biomaterials, immune rejection reactions, and the controllability of therapeutic effects. Therefore, the development of an efficient and safe biomaterial to promote cartilage repair, inhibit inflammatory responses, and improve joint function has important scientific significance and clinical value for the treatment of OA. Summary of the invention

[0005] In order to solve the problems in the background technology and address the difficulty of the prior art in maintaining intra-articular anti-inflammatory effects, the present invention aims to provide an anti-inflammatory lipid Phoyunbene C nanodot and a preparation method and application thereof.

[0006] The present invention adopts the following technical solution:

[0007] 1. A lipid Phoyunbene C nanomaterial:

[0008] It comprises a lipid spherical shell and a Phoyunbene C nanodot core encapsulated in the lipid spherical shell.

[0009] The lipid spherical shell includes 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 (DSPE-PEG 2000), soybean lecithin and cholesterol.

[0010] Soybean lecithin is the main framework of the lipid shell, cholesterol stabilizes the lipid structure, and the introduction of DSPE-PEG2000 increases the hydrophilicity.

[0011] The mass ratio of the Phoyunbene C nanodot core, DSPE-PEG2000, soybean lecithin and cholesterol is: 8-12: 1-3: 30-40: 5-10.

[0012] The lipid Phoyunbene C nanomaterial is in the form of a particle solution and can be a preparation, such as a solution or an injection.

[0013] 2. A method for preparing lipid Phoyunbene C nanomaterials:

[0014] (1) preparing an aqueous phase: dispersing the original Phoyunbene C monomer in a phosphate buffer, vortexing and sonicating until completely dissolved to obtain a Phoyunbene C solution, and then heating the Phoyunbene C solution to 60-70° C., keeping the temperature for 5-8 hours while stirring to obtain an aqueous phase solution;

[0015] The monomer here refers to the original untreated Phoyunbene C molecule, and the Phoyunbene C nanodot core refers to the original Phoyunbene C molecules that polymerize when heated and self-assemble to form nanodots of nanometer size.

[0016] (2) preparing an organic phase: dissolving the required mass of DSPE-PEG2000, soybean lecithin and cholesterol balanced to room temperature in anhydrous ethanol in sequence according to the mass ratio, heating to 40-60° C. and stirring for a period of time, and then ultrasonically dispersing to obtain an organic phase solution;

[0017] (3) preparing liposomes: loading the aqueous solution described in step (1) and the organic solution described in step (2) into two syringes respectively, and then connecting the outlets of the two syringes to the two inlets of a microfluidic chip respectively through pipes, mixing the organic solution and the aqueous solution in the microfluidic chip at a preset flow rate ratio under the control of a microfluidic pressure pump, and collecting the mixed solution at the outlet of the microfluidic chip to obtain an original liposome solution;

[0018] (4) Dialysis filtration: Add the original liposome solution described in step (3) into a dialysis bag, immerse the dialysis bag in the dialysis solution, remove the ethanol, and filter and sterilize through a filter membrane to obtain the final lipid Phoyunbene C nanomaterial.

[0019] In the step (1), the concentration of the Phoyunbene C solution is 5-30 mg / ml, the pH of the phosphate buffer is 7.3-7.5, the vortex time is 0.1-1 min, the ultrasonic power is greater than 20 W, the ultrasonic time is 1-10 min, and the stirring rate is greater than 1000 rpm.

[0020] In the step (2), the total lipid concentration of the organic phase solution is 2-20 mg / ml, 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.

[0021] In the step (3), the preset flow rate ratio is organic phase solution flow rate ratio: aqueous phase solution flow rate ratio = 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.

[0022] In the step (4), the molecular weight cutoff of the dialysis bag is 1000-10000Da, the dialysate uses a phosphate buffer solution with a pH of 7.3-7.5, the dialysis time is 48-72h, and the micropore diameter of the filter membrane is mainly 0.22μm.

[0023] 3. Application of lipid Phoyunbene C nanomaterials in the preparation of osteoarthritis therapeutic drugs. Lipid Phoyunbene C nanodots have anti-inflammatory effects. The lipid Phoyunbene C nanomaterial is a solution or an injection.

[0024] The lipid Phoyunbene C nanomaterial is prepared into a solution or an injection for treating osteoarthritis, and then injected into the joint cavity.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. The present invention innovatively designs and uses π-π stacking and thermal oxidation polymerization to prepare Phoyunbene C nanodots. Compared with the monomer form of Phoyunbene C, the nanodots have improved anti-inflammatory efficacy and form a core-shell structure together with the outer lipid layer.

[0027] 2. The lipid Phoyunbene C nanodots prepared by the present invention have injectability, cartilage permeability, biocompatibility, biodegradability, and anti-inflammatory effects. The above-mentioned multiple functions complement each other. The lipid Phoyunbene C nanodot preparation can be directly injected into the joint cavity. The lipid shell protects the internal core Phoyunbene C nanodots to avoid uncontrolled degradation due to direct contact with enzymes, increase the residence time in the joint cavity, inhibit the expression of inflammatory factors in chondrocytes, enhance the anti-inflammatory effect, thereby reducing cartilage damage and effectively improving the symptoms of osteoarthritis.

[0028] 3. The preparation conditions of lipid Phoyunbene C nanodots are mild. The lipid Phoyunbene C nanodots are prepared by using a combination of processes such as microfluidics, self-assembly, self-polymerization, and nanocomposite. The process route is formulated according to the actual preparation needs of lipid Phoyunbene C nanodots. The size of lipid Phoyunbene C nanodots is controllable and original.

[0029] 4. The present invention combines the independently designed Phoyunbene C nanodots with multifunctional lipids, which can accurately, stably and efficiently treat cartilage both in vivo and in vitro, reduce cartilage damage and inhibit inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a TEM image of lipid Phoyunbene C nanodots obtained in Example 1 of the present invention.

[0031] Figure 2 This is a graph showing the protein content of inflammatory factors G1-G7 in the examples and comparative examples of the present invention.

[0032] Figure 3 This is a TEM image of lipid Phoyunbene C nanodots obtained in Example 2 of the present invention.

[0033] Figure 4 This is a TEM image of lipid Phoyunbene C nanodots obtained in Example 3 of the present invention.

[0034] Figure 5 This is a TEM image of lipid Phoyunbene C nanodots obtained in Example 4 of the present invention.

[0035] Figure 6This is a TEM image of the blank lipid spherical shell without Phoyunbene C nanodots obtained in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0036] 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.

[0037] Embodiments of the present invention are as follows:

[0038] Example 1

[0039] Step S1, preparing the aqueous phase: dispersing the Phoyunbene C monomer in PBS, with a Phoyunbene C concentration of 5 mg / ml, vortexing for 1 min, and ultrasonicating at 20W for 1 min. The solution was heated at 60°C and stirred at 1000 rpm for 5 h to prepare an aqueous phase solution for use;

[0040] Step S2, preparing an organic phase: after equilibration to room temperature, dissolve DSPE-PEG2000, soybean lecithin and cholesterol in anhydrous ethanol in turn, heat to 50°C, stir at 350r / min for 1min, and ultrasonically disperse at 30W for 1min to obtain an organic phase solution for standby use; wherein the molar ratio of DSPE-PEG2000, soybean lecithin and cholesterol is 1:35:5, and the total lipid concentration is 5mg / ml;

[0041] Step S3, loading the aqueous solution prepared in step S1 and the organic solution prepared in step S2 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 the syringes, and under the control of the microfluidic pressure pump, mixing the organic phase solution and the aqueous phase solution in the herringbone-shaped 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;

[0042] Step S4: The solution was filtered through a dialysis bag for 48 hours to remove ethanol, and the final lipid Phoyunbene C nanodots were obtained after sterilization by filtration with a molecular weight cutoff of 1000 Da and a 0.22 μm filter membrane.

[0043] The lipid Phoyunbene C nanodots prepared in Example 1 are spherical nanoscale vesicles (such as Figure 1As shown in Figure 2, CCK8 test showed that chondrocyte proliferation increased by 1.05 times, and chondrocyte type II collagen mRNA expression increased by 1.07 times. After 28 days of injection in rat knee OA model, compared with injection of PBS (G1) and monomer Phoyunbene C solution (G2), the expression of inflammatory factors in Example 1 (G3) decreased ( Figure 2 ).

[0044] Example 2

[0045] Step S1, preparing the aqueous phase: dispersing the Phoyunbene C monomer in PBS, with a Phoyunbene C concentration of 5 mg / ml, vortexing for 1 min, and ultrasonicating at 20W for 1 min. The solution was heated at 60°C and stirred at 1000 rpm for 8 h to prepare an aqueous phase solution for use;

[0046] Step S2, preparing an organic phase: after equilibration to room temperature, dissolve DSPE-PEG2000, soybean lecithin and cholesterol in anhydrous ethanol in turn, heat to 50°C, stir at 350r / min for 1min, and ultrasonically disperse at 30W for 1min to obtain an organic phase solution for standby use; wherein the molar ratio of DSPE-PEG2000, soybean lecithin and cholesterol is 1:35:5, and the total lipid concentration is 5mg / ml;

[0047] Step S3, loading the aqueous solution prepared in step S1 and the organic solution prepared in step S2 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 the syringes, and under the control of the microfluidic pressure pump, mixing the organic phase solution and the aqueous phase solution in the herringbone-shaped 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 S4: The solution was filtered through a dialysis bag for 48 hours to remove ethanol, and the final lipid Phoyunbene C nanodots were obtained after sterilization by filtration with a molecular weight cutoff of 1000 Da and a 0.22 μm filter membrane.

[0049] Compared with Example 1, the heating and stirring time of Phoyunbene C was prolonged, and the lipid Phoyunbene C nanodots (G4) were spherical nanoscale vesicles ( Figure 3 ), CCK8 test showed that chondrocyte proliferation increased by 1.1 times, chondrocyte type II collagen mRNA expression increased by 1.96 times, and the expression of inflammatory factors decreased after 28 days of injection in rat knee OA model ( Figure 2 ).

[0050] Example 3

[0051] Step S1, preparing the aqueous phase: dispersing the Phoyunbene C monomer in PBS, with a Phoyunbene C concentration of 10 mg / ml, vortexing for 1 min, and ultrasonicating at 20W for 1 min. The solution was heated at 60°C and stirred at 1000 rpm for 5 h to prepare an aqueous phase solution for use;

[0052] Step S2, preparing an organic phase: after equilibration to room temperature, dissolve DSPE-PEG2000, soybean lecithin and cholesterol in anhydrous ethanol in turn, heat to 50°C, stir at 350r / min for 1min, and ultrasonically disperse at 30W for 1min to obtain an organic phase solution for standby use; wherein the molar ratio of DSPE-PEG2000, soybean lecithin and cholesterol is 1:35:5, and the total lipid concentration is 5mg / ml;

[0053] Step S3, loading the aqueous solution prepared in step S1 and the organic solution prepared in step S2 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 the syringes, and under the control of the microfluidic pressure pump, mixing the organic phase solution and the aqueous phase solution in the herringbone-shaped 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;

[0054] Step S4: The solution was filtered through a dialysis bag for 48 hours to remove ethanol, and the final lipid Phoyunbene C nanodots were obtained after sterilization by filtration with a molecular weight cutoff of 1000 Da and a 0.22 μm filter membrane.

[0055] Compared with Example 1, the concentration of Phoyunbene C was increased, and the liposome (G5) was a spherical nano-scale vesicle ( Figure 4 ), CCK8 test showed that chondrocyte proliferation increased by 1.05 times, chondrocyte type II collagen mRNA expression increased by 1.08 times, and the expression of inflammatory factors decreased after 28 days of injection in rat knee OA model ( Figure 2 ).

[0056] Example 4

[0057] Step S1, preparing the aqueous phase: dispersing the Phoyunbene C monomer in PBS, with a Phoyunbene C concentration of 5 mg / ml, vortexing for 1 min, and ultrasonicating at 20W for 1 min. The solution was heated at 60°C and stirred at 1000 rpm for 5 h to prepare an aqueous phase solution for use;

[0058] Step S2, preparing an organic phase: after equilibration to room temperature, dissolve DSPE-PEG2000, soybean lecithin and cholesterol in anhydrous ethanol in turn, heat to 50°C, stir at 350r / min for 1min, and ultrasonically disperse at 30W for 1min to obtain an organic phase solution for standby use; wherein the molar ratio of DSPE-PEG2000, soybean lecithin and cholesterol is 1:35:5, and the total lipid concentration is 5mg / ml;

[0059] Step S3, loading the aqueous solution prepared in step S1 and the organic solution prepared in step S2 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 the syringes, and under the control of the microfluidic pressure pump, mixing the organic phase solution and the aqueous phase solution in the herringbone-shaped microfluidic chip at an organic phase: aqueous phase flow rate ratio of 1:5 and a total flow rate of 5 ml / min, and collecting the original liposome solution at the outlet of the microfluidic chip;

[0060] Step S4: The solution was filtered through a dialysis bag for 48 hours to remove ethanol, and the final lipid Phoyunbene C nanodots were obtained after sterilization by filtration with a molecular weight cutoff of 1000 Da and a 0.22 μm filter membrane.

[0061] Compared with Example 1, the total flow rate of lipids during preparation was reduced, and the liposome (G6) was a spherical nano-sized vesicle ( Figure 5 ), CCK8 test showed that chondrocyte proliferation increased by 1.06 times, chondrocyte type II collagen mRNA expression increased by 1.22 times, and the expression of inflammatory factors decreased after 28 days of injection in rat knee OA model ( Figure 2 ).

[0062] Comparative Example 1: Only lipid spherical shell

[0063] Step S1, preparing an aqueous phase: taking PBS as an aqueous phase solution for standby use;

[0064] Step S2, preparing an organic phase: after equilibration to room temperature, dissolve DSPE-PEG2000, soybean lecithin and cholesterol in anhydrous ethanol in turn, heat to 50°C, stir at 350r / min for 1min, and ultrasonically disperse at 30W for 1min to obtain an organic phase solution for standby use; wherein the molar ratio of DSPE-PEG2000, soybean lecithin and cholesterol is 1:35:5, and the total lipid concentration is 5mg / ml;

[0065] Step S3, loading the aqueous phase solution of step S1 and the organic phase solution prepared in step S2 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 the syringes, and under the control of the microfluidic pressure pump, mixing the organic phase solution and the aqueous phase solution in the herringbone-shaped 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;

[0066] Step S4: The solution was filtered through a dialysis bag for 48 hours to remove ethanol, and then sterilized by filtration with a molecular weight cutoff of 1000 Da and a 0.22 μm filter membrane to obtain a blank lipid spherical shell without Phoyunbene C nanodots.

[0067] The blank lipid spherical shell (G7) without Phoyunbene C nanodots prepared in this comparative example 1 is a spherical nanoscale vesicle ( Figure 6 ), 28 days after injection of rat knee OA model, there was no significant difference in the expression of inflammatory factors between Comparative Example 1 and Example 1 ( Figure 2 ).

[0068] 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 lipid Phoyunbene C nanomaterial, characterized in that: It comprises a lipid spherical shell and a Phoyunbene C nanodot core encapsulated in the lipid spherical shell.

2. A lipid Phoyunbene C nanomaterial according to claim 1, characterized in that: The lipid spherical shell includes 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 (DSPE-PEG 2000), soybean lecithin and cholesterol.

3. A lipid Phoyunbene C nanomaterial according to claim 2, characterized in that: The mass ratio of the Phoyunbene C nanodot core, DSPE-PEG2000, soybean lecithin and cholesterol is: 8-12: 1-3: 30-40: 5-10.

4. The method for preparing the lipid Phoyunbene C nanomaterial according to any one of claims 1 to 3, characterized in that: The method process is as follows: (1) preparing an aqueous phase: dispersing the original Phoyunbene C in a phosphate buffer, vortexing and sonicating until dissolved to obtain a Phoyunbene C solution, and then heating the Phoyunbene C solution to 60-70° C., keeping the temperature for 5-8 hours while stirring to obtain an aqueous phase solution; (2) preparing an organic phase: dissolving the required mass of DSPE-PEG2000, soybean lecithin and cholesterol in anhydrous ethanol in sequence according to the mass ratio, heating to 40-60° C. and stirring for a period of time, and then ultrasonically dispersing to obtain an organic phase solution; (3) preparing liposomes: loading the aqueous solution described in step (1) and the organic solution described in step (2) into two syringes respectively, and then connecting the outlets of the two syringes to the two inlets of a microfluidic chip respectively through pipes, mixing the organic solution and the aqueous solution in the microfluidic chip at a preset flow rate ratio under the control of a microfluidic pressure pump, and collecting the mixed solution at the outlet of the microfluidic chip to obtain an original liposome solution; (4) Dialysis filtration: Add the original liposome solution described in step (3) into a dialysis bag, immerse the dialysis bag in the dialysis solution, and filter and sterilize through a filter membrane to obtain the lipid Phoyunbene C nanomaterial.

5. The method for preparing the lipid Phoyunbene C nanomaterial according to claim 4, characterized in that: In the step (1), the concentration of the Phoyunbene C solution is 5-30 mg / ml, the pH of the phosphate buffer is 7.3-7.5, the vortex time is 0.1-1 min, the ultrasonic power is greater than 20 W, the ultrasonic time is 1-10 min, and the stirring rate is greater than 1000 rpm.

6. The method for preparing the lipid Phoyunbene C nanomaterial according to claim 4, characterized in that: In the step (2), the total lipid concentration of the organic phase solution is 2-20 mg / ml, 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.

7. The method for preparing the lipid Phoyunbene C nanomaterial according to claim 4, characterized in that: In the step (3), the preset flow rate ratio is organic phase solution flow rate ratio: aqueous phase solution flow rate ratio = 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. The method for preparing the lipid Phoyunbene C nanomaterial according to claim 4, characterized in that: In the step (4), the molecular weight cutoff of the dialysis bag is 1000-10000Da, the dialysate uses a phosphate buffer solution with a pH of 7.3-7.5, the dialysis time is 48-72h, and the micropore diameter of the filter membrane is mainly 0.22μm.

9. Use of the lipid Phoyunbene C nanomaterial according to any one of claims 1 to 3 or the lipid Phoyunbene C nanomaterial prepared by the preparation method according to any one of claims 4 to 8 in the preparation of a drug for treating osteoarthritis.

10. A use according to claim 9, characterized in that: The lipid Phoyunbene C nanomaterial is prepared into a solution or an injection for treating osteoarthritis.

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