Preparation and application of ring brush polymer drug-loaded micelle based on polysulfobetaine

Abstract

The invention provides preparation and application of ring brush polymer drug-loaded micelles based on polysulfobetaine, a hydrophobic drug resveratrol (RSV) is dissolved in a dimethyl sulfoxide solution, a ring brush polymer (CP) is dissolved in deionized water, the two solutions are mixed and stirred for 12 hours, then the mixed solution is transferred into a dialysis bag, the dialysis bag is sealed, and the ring brush polymer drug-loaded micelles based on polysulfobetaine are obtained. And dialyzing in deionized water for 48 hours, and finally freeze-drying the liquid in a dialysis bag to obtain a white solid, namely the polysulfobetaine-based ring brush polymer drug-loaded nano-micelle CP (at) RSV. The ring-brush-shaped polymer is formed by densely grafting a ring-shaped hydrophobic core and a radial polymer brush, stable nano-micelles can be formed, the drug loading rate is increased, the polysulfobetaine serving as a brush body endows the micelles with low toxicity and drug sustained release characteristics, and the drug loading rate is increased. And the micromolecular hydrophobic drug resveratrol can achieve a better treatment effect when being injected into the joint, so that the development of early osteoarthritis is inhibited.

Description

Technical Field

[0001] The invention belongs to the field of biomedical polymer materials, and in particular relates to the preparation and application of a ring-brush polymer drug-loaded micelle based on polybetaine sulfonate. Background Art

[0002] Osteoarthritis is one of the most common joint diseases, affecting 151 million people worldwide, and the number of patients is increasing with longer life expectancy, higher rates of obesity and lack of exercise. The joints have poor availability of systemically administered drugs, and the drug clearance rate is very fast after intra-articular injection. Therefore, there is an urgent need to prepare a drug delivery vehicle to deliver drugs to the inflamed joints for treatment, in order to reduce the risk of adverse side effects and ultimately improve the overall treatment effect.

[0003] Resveratrol (RSV) is a natural polyphenol found in grapes, red wine, grape juice and several berries. It is considered to be an antioxidant, anti-inflammatory, anti-apoptotic and anti-cancer drug. A large number of experiments have shown that resveratrol has a certain protective effect on articular cartilage, which is regulated by various mechanisms such as anti-inflammatory, anti-apoptotic or regulation of different signaling pathways. However, due to the poor chemical stability and water solubility of resveratrol, its bioavailability after oral administration is low. Therefore, local administration can improve its therapeutic effect.

[0004] Ring brush polymers are composed of a ring core and a dense graft of radial polymer brushes, which can form single-molecule nanoparticles. Compared with bottle brush polymers with the same molecular weight, they have higher stability in blood and body fluids, so they are very desirable as drug delivery carriers. Zwitterionic polymers refer to polyelectrolytes that contain both anions and cations in the side chains but are electrically neutral as a whole. Methacryloylethylsulfonate betaine has a unique zwitterionic structure and can form a stable hydration layer through hydrogen bonds and ion-dipole interactions with water molecules, so it has high hydrophilicity. In addition, methacryloylethylsulfonate betaine also has good biocompatibility, weak immune stimulation to the body, low toxicity, efficient transcytosis and deep cell penetration.

[0005] Therefore, a ring-shaped brush polymer drug-loaded nanomicelle with betaine methacryloylethylsulfonate as the brush body can effectively improve the solubility of resveratrol, prolong the circulation time in the body, reduce side effects, and thus inhibit the further development of osteoarthritis. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and solve the problem that small molecule drugs are limited in intra-articular injection due to their poor solubility, short half-life, poor stability and other shortcomings, and to provide a ring-shaped brush polymer drug-loaded micelle based on methyl acryloylethyl sulfonic acid betaine, which can enhance the stability of the drug in body fluids, improve the therapeutic effect of the drug, prolong the treatment time of the drug, and provide an innovative solution for the treatment of osteoarthritis.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention first discloses a method for preparing a ring-brush polymer drug-loaded micelle based on polybetaine sulfonate, comprising:

[0009] Dissolving a hydrophobic drug resveratrol (RSV) in a dimethyl sulfoxide solution to obtain a first solution;

[0010] The ring-brush polymer CP is dissolved in deionized water to obtain a second solution;

[0011] The first solution and the second solution are mixed and stirred, and then placed in deionized water for dialysis, and finally the liquid obtained in the dialysis bag is freeze-dried to obtain the product.

[0012] Furthermore, the mass ratio of resveratrol to the cyclic brush polymer CP is 8:50.

[0013] Furthermore, the ring-brush polymer CP is prepared by the following method:

[0014] The initiator cP(HEMA-Br) 30 The monomer methyl acryloylethyl sulfonic acid betaine and the ligand 2,2'-bipyridine are dissolved in 2,2,2-trifluoroethanol solvent, and three freeze-pump-thaw cycles are carried out in the polymerization tube. Then, the catalyst cuprous bromide is added under nitrogen protection, followed by two freeze-pump cycles. Then, the polymerization tube is placed in a preheated 60°C oil bath to react for 3 hours. After the reaction stops, the polymerization tube is opened and exposed to the air for quenching. Ice ether is added to precipitate the product and then centrifuged. The precipitate is collected and dissolved in a small amount of deionized water. Then, the solution is transferred to a dialysis bag, dialyzed with deionized water for 48 hours, and finally freeze-dried to obtain a white solid product, which is the ring brush polymer CP.

[0015] Furthermore, the methacryloylethylsulfonic acid betaine, cP(HEMA-Br) 30 The molar ratio of 2,2'-bipyridine and cuprous bromide is 60:1:2:1.

[0016] Furthermore, the monomer concentration of the methacryloylethylsulfonate betaine in the 2,2,2-trifluoroethanol solvent is 0.5 mmol / ml.

[0017] Furthermore, the molecular weight cut-off of the dialysis bag is 1000Da.

[0018] Furthermore, the first solution and the second solution were stirred and mixed for 12 hours, and then dialyzed in deionized water for 48 hours.

[0019] Furthermore, the molecular weight cut-off of the dialysis bag is 1000Da.

[0020] The invention also discloses a ring-brush polymer drug-loaded micelle prepared according to any of the above preparation methods.

[0021] The invention also discloses an application of the annular brush polymer drug-loaded micelles in preparing drugs for treating osteoarthritis.

[0022] The beneficial effects of the present invention are:

[0023] The present invention provides a drug-loaded ring-brush polymer micelle based on polysulfonated betaine, and the small molecule hydrophobic drug resveratrol is loaded by the ring-brush polymer nano-micelle, thereby overcoming the shortcomings of poor solubility, short half-life and low bioavailability of resveratrol after oral administration. The unique topological structure of the ring-brush polymer also improves the stability of the material in body fluids. At the same time, methacryloylethyl sulfonated betaine as a brush body gives it better drug solubility and increases the loading amount of resveratrol. Therefore, the ring-brush polymer nano-micelle based on polysulfonated betaine prepared in the present invention provides a new idea and method for delivering poorly soluble drugs and then treating osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the synthesis route of the polysulfonated betaine-based cyclic brush polymer CB in Example 1.

[0025] Figure 2 This is the NMR characterization of the polysulfonated betaine-based cyclic brush polymer CB in Example 1.

[0026] Figure 3 This is the particle size distribution diagram of the polybetaine sulfonate-based ring-brush polymer drug-loaded nanomicelles CB@RSV in Example 2.

[0027] Figure 4 This is a scanning electron microscope image of the polysulfonated betaine-based ring-brush polymer drug-loaded nanomicelles CB@RSV in Example 2.

[0028] Figure 5 This is the standard curve of resveratrol in Experimental Example 1.

[0029] Figure 6This is the in vitro drug release curve of the ring-brush polymer drug-loaded nanomicelles CB@RSV in polybetaine sulfonate in Experimental Example 1.

[0030] Figure 7 The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows:

[0031] Figure 8 The cell proliferation of mouse chondrocytes co-cultured with resveratrol and the ring-brush polymer nano-micelles before and after drug loading in Experiment 3 for 1 and 3 days. Among them: 8(a) is the live and dead staining of chondrocytes on days 1 and 3, and 8(b) is the absorbance value of CCK-8 of chondrocytes on days 1 and 3.

[0032] Fig. 9 This is the cell morphology of resveratrol and the ring-brush polymer nanomicelles before and after drug loading in Experimental Example 4 co-cultured with mouse chondrocytes. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0034] Example 1

[0035] Preparation of cyclic brush polymers based on poly(betaine sulfonate)

[0036] Accurately weigh 4 mg of initiator cP(HEMA-Br) 30 235.57 mg of monomer methacryloylethylsulfonic acid betaine and 4.39 mg of ligand 2,2'-bipyridine were dissolved in 1.687 ml of 2,2,2-trifluoroethanol solvent, and then the mixed solution was quickly transferred to a polymerization reaction tube. After three freeze-pump-thaw cycles, in the fourth frozen state, 2.02 mg of catalyst cuprous bromide was quickly added under nitrogen protection, followed by two freeze-pump cycles. After the last thawing, the polymerization tube was placed in a preheated 60°C oil bath for three hours with magnetic stirring. After the reaction stops, open the polymerization tube and expose it to the air for quenching. Slowly drop the product into 20 ml of ice ether, then centrifuge at 6000 rpm. The collected precipitate is the crude product. Add a small amount of deionized water to dissolve the crude product, then transfer the solution to a dialysis bag with a molecular weight cutoff of 1000 Da, dialyze with deionized water for 48 hours, replace the dialysis water every 3-4 hours, and finally freeze-dry to obtain a white solid product CP. The reaction equation is as follows: Figure 1 As shown, the NMR results of the ring-shaped brush polymer based on polysulfonated betaine prepared in this example are as follows Figure 2 shown.

[0037] Example 2

[0038] Preparation of drug-loaded ring-brush polymer micelles based on poly(betaine sulfonate)

[0039] 8 mg of hydrophobic drug resveratrol (RSV) was dissolved in 1 ml of dimethyl sulfoxide solvent, and 50 mg of ring-brush polymer CP was dissolved in 5 ml of deionized water. The two solutions were mixed and stirred for 12 hours for drug encapsulation. In order to remove the unencapsulated drug and organic solvent, the mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, dialyzed in deionized water for 24 hours, and finally the solution in the dialysis bag was freeze-dried to obtain a white solid, which is the ring-brush polymer drug-loaded nanomicelles CP@RSV.

[0040] 1 mg of RSV-encapsulated ring-brush polymer nanomicelles was weighed and dissolved in 1 ml of deionized water to obtain a 1 mg / ml nanomicelle solution.

[0041] The particle size distribution of the 1 mg / ml nanomicelle solution prepared in this example is shown in Figure 3 The transmission electron microscopy image of the nanomicelles is shown in Figure 4 shown.

[0042] Test Example 1

[0043] In vitro drug release profile and drug loading determination

[0044] Resveratrol was dissolved in deionized water, and a series of RSV solutions with different concentrations were prepared. The absorbance at the maximum absorption wavelength (304 nm) was measured using a UV-visible spectrophotometer. The concentration was used as the horizontal coordinate and the corresponding absorbance was used as the vertical coordinate to obtain its standard curve. According to the standard curve, the corresponding RSV concentration can be obtained from the absorbance of the drug-loaded micelle solution. The standard curve is shown in FIG. Figure 5 shown.

[0045] 1 mL of drug-loaded micelle solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and suspended in PBS at pH 7.4. The total volume of each sample solution was 20 mL. The whole was placed in a shaker at 37 ° C and a stirring speed of 120 rpm. 2 mL of sample was collected at specific time intervals (0.5, 1, 2, 4, 8, 12, 24, 48, 72 h) and supplemented with corresponding fresh PBS. The absorbance at different time points was measured by UV-visible spectrophotometer and then Figure 5 The standard curve is used to obtain the corresponding RSV release amount, and finally the RSV drug release curve is obtained, such as Figure 6 shown.

[0046] 1 mg of the drug-loaded nanomicelles prepared in Example 2 was dissolved in 5 ml of deionized water to obtain a 0.2 mg / ml nanomicelle solution. The absorbance of the nanomicelle solution at 304 nm was measured using a UV-visible spectrophotometer. The drug loading rate and encapsulation rate of the drug-loaded nanomicelles were calculated based on the RSV standard curve. The calculation formula is as follows:

[0047]

[0048]

[0049] Calculation showed that the drug loading rate of the nanomicelles was 5.72% and the encapsulation efficiency was 35.75%.

[0050] Test Example 2

[0051] In vitro cytotoxicity assay

[0052] CCK-8 was used to detect the cytotoxicity of free resveratrol and the ring-brush polymer nanoemulsions before and after drug loading on mouse chondrocytes and macrophages Raw 264.7. First, the two cells were cultured at 3×10 3 The cells / well were inoculated in a 96-well plate at a density of 100 μL and cultured at 37°C and 5% CO2 for 24 hours until the cells were completely attached to the wall. Then, 100 μL of RSV, CP and CP@RSV solutions of different concentrations prepared with complete culture medium as solvent were added for co-culture. After 24 hours, the old culture medium was removed, washed 2-3 times with PBS solution, and 100 μL of CCK-8 reagent prepared with culture medium as solvent (the volume ratio of CCK-8 to culture medium was 1:10) was added under light-proof conditions. The incubation continued for 3 hours, and the absorbance value at 450 nm was detected with an ELISA reader. The corresponding blank group was set to deduct the absorbance deviation caused by the material.

[0053] The formula for calculating cell viability is as follows:

[0054]

[0055] Among them, OD experimental group represents the absorbance value of the cell wells under co-culture of different experimental groups; OD experimental blank represents the absorbance value of the solution after the experimental material (without cells) reacts with the CCK-8 reagent; OD control group represents the absorbance value of the pure cell group; OD control blank represents the absorbance value of the solution after the culture medium (without cells) reacts with the CCK-8 reagent.

[0056] The results show that if Figure 7As shown in the figure, within the set concentration range, the three material groups have almost no adverse effects on the cell activity of chondrocytes and macrophages, and the cell survival rate at each concentration is almost greater than 80%, indicating that the material has good biocompatibility, but compared with RSV, the cell survival rate of CP@RSV is significantly higher, indicating that CP@RSV can play a role in reducing RSV cytotoxicity. Since the cell survival rate of CP@RSV at a concentration of 125μg / ml is the highest, the 125μg / ml nanomicelle solution was selected for the subsequent cell and animal experiments.

[0057] Test Example 3

[0058] In vitro cell proliferation assay

[0059] Live-dead cell staining was used to observe the effect of ring-brush polymer nanomicelles CP@RSV on chondrocyte proliferation. First, chondrocytes were plated at 6×10 3 The cells were inoculated in a 48-well plate at a density of 100 cells / well and cultured at 37°C and 5% CO2 for 24 hours until the cells were completely attached to the wall. Then, 200 μL of RSV, CP, and CP@RSV solution were added and co-cultured for 1 and 3 days. After 1 and 3 days of co-culture, 10 μg / mL of FDA (live cells, green) and 1 μg / mL of PI (dead cells, red) staining solution were prepared with PBS as solvent under light-proof conditions. 200 μL of staining solution was added to each well. After incubation for 15 minutes, the cells were washed 5 times with PBS and then immediately observed and photographed with a 10x fluorescence microscope.

[0060] The results are as follows Figure 8 As shown in (a), there are almost no dead cells in the chondrocytes co-cultured with 125 μg / ml CP@RSV nanomicelles, and the green fluorescence is more. From the first day to the third day, the number of cells increases with time, proving that CP@RSV nanomicelles have almost no effect on the cell proliferation of chondrocytes.

[0061] CCK-8 was used to detect the cell proliferation of mouse chondrocytes co-cultured with ring-brush polymer nanoparticles for 1 and 3 days. First, chondrocytes were cultured at 3×10 3 The cells were seeded at a density of 100 μL in a 96-well plate and cultured at 37°C and 5% CO2 for 24 h until the cells were completely attached to the wall. Then, 100 μL of RSV, CP, and CP@RSV solutions were added and cultured for 1.3 days. The culture medium was removed and 100 μL of CCK-8 reagent prepared with culture medium as solvent was added in a light-proof condition (the volume ratio of CCK-8 to culture medium was 1:10). The culture was continued for 3 h and the absorbance at 450 nm was detected with an enzyme reader.

[0062] The results are as follows Figure 8As shown in (b), the number of cells increases with time, which also proves that CP@RSV nanomicelles do not affect the proliferation behavior of chondrocytes.

[0063] Test Example 4

[0064] In vitro cell morphology assay

[0065] Morphological staining was used to observe the effect of ring-brush polymer nanomicelles CP@RSV on chondrocyte morphology. First, chondrocytes were plated at 6×10 3 The cells were inoculated in a 48-well plate at a density of 10 cells / well and cultured at 37°C and 5% CO2 for 24 hours until the cells were completely attached to the wall. Then 200 μL of RSV, CP and CP@RSV solution were added and cultured for 24 hours. The old culture medium was then removed, the cells were rinsed 2-3 times with PBS solution, fixed with 4% paraformaldehyde (PFA) solution at room temperature for 15 minutes, and then rinsed 4-5 times with PBS solution. Then rhodamine-labeled phalloidin was added to stain the cells for 30 minutes. After staining, the cells were rinsed 4-5 times with PBS solution. Finally, 2-(4-aminophenyl)-6-indolecarbamidine (DAPI) was used to counterstain the cell nuclei for 10 minutes, and then rinsed 4-5 times with PBS solution. The cells were immediately observed and photographed under a 10x fluorescence microscope.

[0066] The results are as follows Fig. 9 As shown, all cells were healthy in morphology, with prominent pseudopodia and spindle shape, indicating that CP@RSV nanomicelles had no effect on the cell morphology of chondrocytes.

[0067] The above in vitro cell experimental results jointly confirmed that CP@RSV nanomicelles have good biocompatibility and are non-toxic to chondrocytes, which is a prerequisite for in vivo experiments.

[0068] The specification and drawings of the present invention are considered to be illustrative rather than restrictive. On the basis of the present invention, those skilled in the art can make some substitutions and modifications to some of the technical features according to the disclosed technical content without creative labor, all of which are within the protection scope of the present invention.

Claims

1. A method for preparing a ring-brush polymer drug-loaded micelle based on polybetaine sulfonate, comprising: Dissolving the hydrophobic drug resveratrol in a dimethyl sulfoxide solution to obtain a first solution; The ring-brush polymer CP is dissolved in deionized water to obtain a second solution; The first solution and the second solution are mixed and stirred, and then placed in deionized water for dialysis, and finally the liquid obtained in the dialysis bag is freeze-dried to obtain the product.

2. The preparation method according to claim 1, wherein: The mass ratio of the resveratrol to the cyclic brush polymer CP is 8:

50.

3. The preparation method according to claim 1, wherein: The ring-brush polymer CP is prepared by the following method: The initiator cP(HEMA-Br) 30 The monomer methyl acryloylethyl sulfonic acid betaine and the ligand 2,2'-bipyridine are dissolved in 2,2,2-trifluoroethanol solvent, and three freeze-pump-thaw cycles are carried out in the polymerization tube. Then, the catalyst cuprous bromide is added under nitrogen protection, followed by two freeze-pump cycles. Then, the polymerization tube is placed in a preheated 60°C oil bath to react for 3 hours. After the reaction stops, the polymerization tube is opened and exposed to the air for quenching. Ice ether is added to precipitate the product and then centrifuged. The precipitate is collected and dissolved in a small amount of deionized water. Then, the solution is transferred to a dialysis bag, dialyzed with deionized water for 48 hours, and finally freeze-dried to obtain a white solid product, which is the ring brush polymer CP.

4. The preparation method according to claim 3, wherein: The methacryloylethylsulfonic acid betaine, cP (HEMA-Br) 30 The molar ratio of 2,2'-bipyridine and cuprous bromide is 60:1:2:

1.

5. The preparation method according to claim 3, wherein: The monomer concentration of the methacryloylethylsulfonate betaine in the 2,2,2-trifluoroethanol solvent is 0.5 mmol / ml.

6. The preparation method according to claim 3, wherein: The molecular weight cut-off of the dialysis bag is 1000Da.

7. The preparation method according to claim 1, wherein: The first solution and the second solution were stirred and mixed for 12 hours, and then dialyzed in deionized water for 48 hours.

8. The preparation method according to claim 1, wherein: The molecular weight cut-off of the dialysis bag is 1000Da.

9. A drug-loaded ring-brush polymer micelle prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the annular brush polymer drug-loaded micelle according to claim 9 in preparing a drug for treating osteoarthritis.

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