Zwitterionic polymer-curcumin nano-micelle as well as preparation method and application thereof

Abstract

The invention relates to a zwitterionic polymer-curcumin nano-micelle as well as a preparation method and application thereof, and belongs to the technical field of nano-drugs. The technical problems that existing curcumin is low in bioavailability, poor in lipophilicity, low in solubility and the like are solved. According to the zwitterionic polymer-curcumin nano-micelle disclosed by the invention, the zwitterionic polymer is used as a carrier, and the curcumin is used as a loaded drug. In the zwitterionic polymer-curcumin nano-micelle, curcumin has wide anti-inflammatory, anti-oxidation and osteogenesis promoting effects, the zwitterionic polymer has the characteristics of low toxicity and high cell penetrability, and the zwitterionic polymer-curcumin nano-micelle formed by the zwitterionic polymer and curcumin has the advantages of the zwitterionic polymer and curcumin and has the advantages of low toxicity and high cell penetrability. The traditional Chinese medicine composition has good anti-inflammatory, anti-oxidation and osteogenesis promoting effects, can relieve local inflammation, and has a relatively good treatment effect on periodontitis. And the zwitterionic polymer can improve the defect of poor solubility of curcumin, curcumin can be slowly released, and meanwhile, the curcumin has relatively high biological safety.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomedicines, and specifically relates to a zwitterionic polymer-curcumin nanomicelle and a preparation method and application thereof, and in particular relates to the application of the zwitterionic polymer-curcumin nanomicelle in preparing a drug for treating periodontitis. Background Art

[0002] Periodontitis is a chronic inflammatory disease induced by bacterial aggregation, affecting more than 20% of the world's population. Periodontitis leads to progressive destruction of periodontal tissues, including loss of connective tissue attachment and alveolar bone resorption, resulting in tooth loss, pain, and functional limitations. Inflammation is the main feature of periodontitis. Bacterial antigens activate immune cells in periodontal tissues to produce inflammatory cytokines, such as the release of proinflammatory factors interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), which promote inflammation, osteoclastogenesis, extracellular matrix degradation, and accelerate alveolar bone resorption. Macrophages play a role in defense against bacterial infection, antigen presentation and mobilization, and serve as effective regulators of immune response homeostasis. The inflammatory response will significantly increase the number of proinflammatory macrophage M1 phenotypes and hinder the polarization to the anti-inflammatory macrophage M2 phenotype.

[0003] Existing treatments for periodontitis mainly include antibiotic therapy, mechanical debridement, and regenerative surgery. Although mechanical debridement is the mainstay of periodontal therapy, it cannot completely remove irritants. Therefore, systemic antibiotics are widely used as an adjuvant therapy. However, this brings with it a new problem, namely the development of bacterial resistance. The most important work at present is to screen a suitable non-antibiotic drug model that can serve as a long-term medium to alternately inhibit the polarization of the pro-inflammatory macrophage M1 phenotype and simultaneously activate the polarization of the anti-inflammatory macrophage M2 phenotype. Local drug delivery (LDD) is an alternative to systemic antibiotics and has been shown to be effective against subgingival microbial communities.

[0004] As a natural plant extract, curcumin can prevent the progression of many inflammatory diseases. Its anti-inflammatory effect is manifested by inhibiting the activity of lipoxygenase and cyclooxygenase in the human body. For example, it can reduce the levels of proinflammatory factors interleukin-6 and tumor necrosis factor-α in the culture supernatant of rat gingival fibroblasts stimulated by lipopolysaccharide (LPS), inhibit the activation of the nuclear factor-κ-light chain-enhanced (NF-κB) signaling pathway of activated B cells, and thus inhibit the infiltration and activation of inflammatory cells. At present, it has gradually been used to treat a variety of oral diseases such as oral mucosal diseases, oral cancer and periodontal disease. However, curcumin in non-nanoformulation products will stain teeth and mucosa when used topically, and curcumin has low bioavailability, poor lipophilicity and low solubility, which limits its application effect. Summary of the invention

[0005] In view of this, the present invention provides a zwitterionic polymer-curcumin nano-micelle and a preparation method and application thereof to solve the technical problems of low bioavailability, poor lipophilicity and low solubility of curcumin in the prior art.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows.

[0007] In a first aspect, the present invention provides a zwitterionic polymer-curcumin nanomicelle (OPDEA-PGED@Cur), wherein the nanomicelle uses a zwitterionic polymer as a carrier and curcumin (Cur) as a drug carrier; The zwitterionic polymer is poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate-glycidyl methacrylate] (OPDEA-PGED) ring-opened with ethylenediamine.

[0008] The full English name of the zwitterionic polymer of the present invention is: Poly[2-(N-oxide-N,N-diethylamino)methyl acrylate-glycidyl methacrylate] derived from ethylenediamine ring-opening. The zwitterionic polymer is synthesized with reference to the existing literature (A pH-triggered N-oxide polyzwitterionicnano-drug loaded system for the anti-tumor immunity activation research, Zhao et al. Journal of Nanobiotechnology (2024) 22:420, https: / / doi.org / 10.1186 / s12951-024-02677-0), and the specific synthesis part is detailed in the supplementary information section of the literature.

[0009] Preferably, the average particle size of the zwitterionic polymer-curcumin nanomicelles is 88.69-91.31 nm.

[0010] Preferably, the drug loading of curcumin is 15-20wt%, and the encapsulation efficiency is 50-70%.

[0011] In a second aspect, the present invention provides a method for preparing the above-mentioned zwitterionic polymer-curcumin nanomicelles, comprising the following steps:

[0012] Step 1, dissolving the zwitterionic polymer in tetrahydrofuran to obtain a zwitterionic polymer solution;

[0013] Step 2, dissolving curcumin in methanol to obtain a curcumin solution;

[0014] Step 3, adding curcumin solution and 4-dimethylaminopyridine (DMAP) to the zwitterionic polymer solution, and then adding pyridine, the obtained reaction solution is reacted in an oil bath pot, and after the reaction is completed, the obtained mixed solution is added dropwise to an ultrasonic ice water bath to obtain a light yellow emulsion;

[0015] Step 4: dialyze the light yellow emulsion with deionized water to remove methanol, 4-dimethylaminopyridine and pyridine, and filter to obtain zwitterionic polymer-curcumin nanomicelles.

[0016] Preferably, in step 1, the zwitterionic polymer is dissolved in tetrahydrofuran by stirring at room temperature for 18-36 hours to obtain a zwitterionic polymer solution.

[0017] Preferably, in step 2, curcumin is dissolved in methanol by stirring at room temperature for 3-8 minutes.

[0018] Preferably, in step 3, the ratio of the zwitterionic polymer, curcumin, 4-dimethylaminopyridine (DMAP) and pyridine is 10-15 mg: 3-7 mg: 5 mg: 2 ml.

[0019] Preferably, in step three, the reaction temperature is 30-40° C. and the reaction time is 18-36 h.

[0020] Preferably, in step 3, the mixed solution is added dropwise to the ultrasonic ice-water bath at a speed of 6-8 L / min.

[0021] Preferably, in step 4, the molecular weight cutoff (MWCO) of the dialysis is 3000-3500Da, and the dialysis time is 3-8h.

[0022] Preferably, in step 4, the pore size of the filtration pores is 250 nm or 450 nm.

[0023] In a third aspect, the present invention also provides the use of the above-mentioned zwitterionic polymer-curcumin nano-micelles in the preparation of a drug for treating periodontitis.

[0024] The principle of the present invention is:

[0025] Periodontitis is the sixth most common disease in the world. It mainly refers to the immune inflammatory destruction of the alveolar bone that supports the teeth, and this destruction is irreversible. During the occurrence and development of periodontitis, bacterial LPS, as its main virulence factor, can induce host defense cells to produce immune inflammatory responses to release inflammatory factors such as IL-1β and TNF-α, thereby causing periodontal tissue destruction.

[0026] Zwitterionic polymers are macromolecular compounds composed of positive and negative ions, including organic anionic macromolecular polymers and organic cationic macromolecular polymers. Zwitterionic polymers have good hydrophilicity, polarity and solubility, can be dissolved in water, and have better stability and dispersibility. The present invention utilizes zwitterionic polymers to improve the water solubility of the drug (curcumin) and reduce the toxicity of the drug. At the same time, the acid responsiveness of zwitterionic nanomedicines is utilized to release the drug in the acidic environment of periodontitis, thereby reducing the release of inflammatory factors and the generation of reactive oxygen species (ROS), thereby achieving the purpose of reducing inflammation and promoting osteogenesis, and finding a new direction for the treatment of periodontitis.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] In the zwitterionic polymer-curcumin nano-micelles of the present invention, curcumin has extensive anti-inflammatory, anti-oxidation and osteopromoting effects, and the zwitterionic polymer has the characteristics of low toxicity and strong cell permeability. The zwitterionic polymer-curcumin nano-micelles formed by the two have the advantages of both, have low toxicity, good anti-inflammatory, anti-oxidation and osteopromoting effects, reduce local inflammation, and have a good effect on the treatment of periodontitis.

[0029] In the zwitterionic polymer-curcumin nano-micelles of the present invention, the zwitterionic polymer has good hydrophilicity and is very soluble in water, and can be used as a carrier to improve the disadvantage of poor solubility of curcumin. The formed zwitterionic polymer-curcumin nano-micelles can be slowly released and have high biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0031] Figure 1 It is the standard curve of curcumin in Example 2 of the present invention.

[0032] Figure 2 This is the particle size distribution of OPDEA-PGED@Cur in Example 3 of the present invention.

[0033] Figure 3 These are the cytotoxicity test results in Example 4 of the present invention, wherein a is the cytotoxicity test result of OPDEA-PGED@Cur, b is the cytotoxicity test result of Cur, and c is the cytotoxicity test result of OPDEA-PGED.

[0034] Figure 4It is the real-time fluorescence quantitative PCR (RT-qPCR) detection result in Example 5 of the present invention, wherein a is the real-time fluorescence quantitative PCR detection result of IL-6, and b is the real-time fluorescence quantitative PCR detection result of TNF-α. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with embodiments.

[0036] In the following examples, various processes and methods not described in detail are conventional methods known in the art. Unless otherwise specified, the materials, reagents, devices, instruments, equipment, etc. used in the following examples can be obtained from commercial sources.

[0037] Example 1

[0038] Step 1: Take 10 mg of OPDEA-PGED in a 25 ml brown single-necked bottle, add 2 ml of tetrahydrofuran, and stir at room temperature for 24 h to dissolve to obtain an OPDEA-PGED solution, i.e., the first solution.

[0039] Step 2: Take another 4 mg of Cur and dissolve it in a brown single-necked bottle containing 2 ml of methanol, and stir it at room temperature for 5 minutes to obtain a Cur solution, i.e., the second solution.

[0040] Step 3: Add the second solution and 5 mg of DMAP to the first solution, then add 2 ml of pyridine. After stirring in an oil bath at 37°C for 24 h, add the obtained mixed solution dropwise into 8 ml of ice water at a rate of 8 L / min under ultrasound to obtain a light yellow emulsion, i.e., the third solution.

[0041] Step 4: The third solution was dialyzed with deionized water for 6 h (MWCO: 3500 Da) to remove methanol, DMAP and pyridine, and then filtered with a 450 nm organic filter to obtain zwitterionic polymer-curcumin nanomicelles, recorded as OPDEA-PGED@Cur micelles.

[0042] Example 2

[0043] In vitro detection of drug loading of OPDEA-PGED@Cur micelles. Weigh 10 mg of curcumin accurately, dissolve it in methanol and dilute it to 10 ml, and prepare a 1 mg / ml curcumin solution as the mother solution. Dilute the mother solution step by step to the set mass concentration gradient, measure the absorbance value of each mass concentration of curcumin solution at 426 nm, and establish a standard curve: Y=61.628x-0.0286, R 2 =0.9993, e.g. Figure 1 shown.

[0044] Take 1 mg of OPDEA-PGED@Cur micelles prepared in Example 1, adjust the pH value to 6, and destroy the micelle structure after ultrasonication with 1 ml of methanol, so that curcumin is dissolved in dichloromethane. After high-speed centrifugation, the dichloromethane layer is transferred to a cuvette, and the ultraviolet absorption peak of curcumin is observed at 25°C, and the encapsulation efficiency (EE) and drug loading (DL) are obtained according to the established standard curve.

[0045] The drug loading and encapsulation efficiency were calculated according to the following formula:

[0046] The calculated results are: curcumin loading: 18.62wt%, encapsulation efficiency: 65.18%.

[0047] Example 3

[0048] Testing the particle size and zeta potential of OPDEA-PGED@Cur

[0049] 1 mg of OPDEA-PGED@Cur micelles in Example 1 was taken and diluted ten times. The average particle size of OPDEA-PGED@Cur micelles was measured by dynamic light scattering (DLS), and the polymer dispersibility index (PDI) and zeta potential were recorded. The measurement was repeated three times and finally expressed as the average value ± standard deviation.

[0050] The test results are: average particle size: (90.00±1.31) nm, such as Figure 2 As shown, zeta potential: (-2.97±0.24) mV, polymer dispersion index: 0.228±0.016.

[0051] Example 4

[0052] The cell viability of mouse mononuclear macrophage leukemia cells (RAW264.7) was detected using the CCK8 method. RAW264.7 cells were suspended in DMEM medium containing 10% fetal bovine serum (FBS) (hereinafter referred to as DMEM medium) and cultured at 1×10 4 Cells were seeded at a density of 100 μg / well in a 96-well plate and incubated at 37°C and 5% CO. 2The cells were cultured overnight under the conditions of 4% paraformaldehyde and 1% paraformaldehyde. After the culture medium was discarded, 100 μL of DMEM culture medium containing different concentrations of Cur (0 μg / ml, 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, with DMSO as solvent and the volume ratio of DMSO in the culture medium was less than 0.1%), OPDEA-PGED@Cur micelles (0 μg / ml, 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, based on the content of Cur) and OPDEA-PGED (0 μg / ml, 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml) were added respectively and incubated at 37°C and 5% CO 2 After discarding the culture medium, wash twice with PBS, add 100 μL of working solution (DMEM medium without FBS: CCK8 solution volume ratio of 10:1) in dark, incubate at 37℃ in dark for 2 hours, use a microplate reader to detect the absorbance of each well at 450nm, and use the following formula to evaluate the effect of the material on cell viability:

[0053] In the formula, OD S is the absorbance of OPDEA-PGED@Cur micelles, OD C is the absorbance of the blank control (DMEM medium without FBS), OD b is the absorbance of cell-free DMEM medium without FBS. The results of the effects of OPDEA-PGED@Cur micelles, Cur and OPDEA-PGED on the activity of RAW264.7 cells are shown in Figure 2. Figure 3 The results show that after incubation of RAW264.7 with different concentrations of OPDEA-PGED@Cur micelles for 24 hours, the cell survival rate was higher than 85% when the OPDEA-PGED@Cur micelle concentration (based on Cur content) was ≤5μg / ml, showing good cell compatibility. Therefore, the OPDEA-PGED@Cur micelle concentration of 5μg / ml was selected for subsequent experiments.

[0054] Example 5

[0055] RT-qPCR experiments were used to detect the regulatory effect of OPDEA-PGED@Cur micelles on inflammatory factors in RAW264.7 cells. 5 Cells were seeded in 6-well plates and incubated at 37°C and 5% CO 2After culturing for 24 h under the same conditions, the cells were replaced with DMEM medium containing LPS (1 mg / mL), LPS (1 mg / mL) + OPDEA-PGED (17.4 μg / mL), LPS (1 mg / mL) + Cur (5 μg / mL), and LPS (1 mg / mL) + OPDEA-PGED@Cur (calculated as Cur 5 μg / mL) for 6 h. The culture medium was discarded and washed twice with PBS. Total RNA was extracted using Trizol reagent. RNA concentration was measured using a Nanodrop2000 fiber optic spectrophotometer (NanodrodropND-1000, Thermo). cDNA was synthesized using the Hifair®Ш 1st StrandcDNA Synthesis Kit according to the manufacturer's description. Quantification was performed using Hieff® qPCR SYBR Green Master Mix. The PCR reaction conditions were 95°C for 5 min, 95°C for 10 s, and 60°C for 30 s, for a total of 40 cycles. β-actin was used as the internal reference gene. 2 -ΔΔct The data were calculated by the method of IL-6 and TNF-α. Figure 4 As shown in a and b, it can be seen that compared with other drug-treated groups, OPDEA-PGED@Cur micelles can significantly reduce the mRNA levels of cellular proinflammatory factors IL-6 and TNF-α, indicating that OPDEA-PGED@Cur micelles play a good role in reducing the release of inflammatory factors.

[0056] Obviously, the above embodiments are merely examples for clear explanation, and are not intended to limit the embodiments. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. The obvious changes or modifications derived from them are still within the protection scope of the invention.

Claims

1. Zwitterionic polymer-curcumin nano-micelles, characterized in that: The nanomicelle uses a zwitterionic polymer as a carrier and curcumin as a drug carrier; The zwitterionic polymer is poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate-glycidyl methacrylate] ring-opened with ethylenediamine.

2. The zwitterionic polymer-curcumin nano-micelle according to claim 1, characterized in that The average particle size of the zwitterionic polymer-curcumin nano-micelles is 88.69-91.31 nm.

3. The zwitterionic polymer-curcumin nano-micelle according to claim 1, characterized in that The drug loading of the curcumin is 15-20wt%, and the encapsulation efficiency is 50-70%.

4. The method for preparing the zwitterionic polymer-curcumin nano-micelles according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1, dissolving the zwitterionic polymer in tetrahydrofuran to obtain a zwitterionic polymer solution; Step 2, dissolving curcumin in methanol to obtain a curcumin solution; Step 3, adding the curcumin solution and 4-dimethylaminopyridine to the zwitterionic polymer solution, and then adding pyridine, the obtained reaction solution is reacted in an oil bath, and after the reaction is completed, the obtained mixed solution is added dropwise to an ultrasonic ice-water bath to obtain a light yellow emulsion; Step 4: dialyze the light yellow emulsion with deionized water to remove methanol, 4-dimethylaminopyridine and pyridine, and filter to obtain zwitterionic polymer-curcumin nanomicelles.

5. The method for preparing zwitterionic polymer-curcumin nano-micelles according to claim 4, characterized in that: In step 1, the zwitterionic polymer is dissolved in tetrahydrofuran by stirring at room temperature for 18-36 hours to obtain a zwitterionic polymer solution.

6. The method for preparing zwitterionic polymer-curcumin nano-micelles according to claim 4, characterized in that: In step 2, curcumin was dissolved in methanol by stirring at room temperature for 3-8 min.

7. The method for preparing zwitterionic polymer-curcumin nano-micelles according to claim 4, characterized in that: In step 3, the ratio of the zwitterionic polymer, curcumin, 4-dimethylaminopyridine and pyridine is 10-15 mg: 3-7 mg: 5 mg: 2 ml.

8. The method for preparing zwitterionic polymer-curcumin nano-micelles according to claim 4, characterized in that: In step 3, the reaction temperature is 30-40°C and the reaction time is 18-36h; The mixed solution is added dropwise into the ultrasonic ice-water bath at a speed of 6-8 L / min.

9. The method for preparing zwitterionic polymer-curcumin nano-micelles according to claim 4, characterized in that: In step 4, the molecular weight cut-off of the dialysis is 3000-3500Da, and the dialysis time is 3-8h; The pore size of the filtering pores is 250nm or 450nm.

10. Use of the zwitterionic polymer-curcumin nanomicelles according to any one of claims 1 to 3 in the preparation of a medicament for treating periodontitis.

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