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

Abstract

The invention relates to a zwitterionic polymer double-drug nano-micelle as well as a preparation method and application thereof, and belongs to the technical field of nano-drugs. According to the zwitterionic polymer double-drug nano-micelle disclosed by the invention, the zwitterionic polymer is taken as a carrier, and curcumin and naringenin are taken as carried drugs. In the zwitterionic polymer double-drug nano-micelle disclosed by the invention, the curcumin and the naringenin both have anti-inflammatory, antioxidant and antibacterial effects, and the bioavailability of the zwitterionic polymer double-drug nano-micelle can be improved by slowly releasing the curcumin and the naringenin. Besides, the zwitterionic polymer double-drug nano-micelle has the characteristics of low zwitterionic toxicity and high cell penetrability, so that the zwitterionic polymer double-drug nano-micelle has good anti-inflammatory and anti-oxidation effects and has a good treatment effect on oral ulcer.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomedicine, and specifically relates to a zwitterionic polymer dual-drug nano-micelle and a preparation method and application thereof, and in particular to an application of the zwitterionic polymer dual-drug nano-micelle in preparing a drug for treating oral ulcers. Background Art

[0002] Oral ulcers are a common inflammatory disease, with an incidence rate of up to 25% among young people. Oral ulcers can destroy the integrity of the mucosal barrier, induce microbial invasion and infection of the oral mucosa, and cause cells to release a large number of inflammatory factors and reactive oxygen species (ROS), ultimately leading to increased tissue damage.

[0003] Oral ulcers are characterized by epithelial and lamina propria destruction and inflammatory cell infiltration. Improving wound healing capacity is one of the main principles of oral ulcer management. Epithelial cells play a vital role in wound healing. Effective oral ulcer management will accelerate clinical improvement and prevent complications. Optimal wound healing involves multiple aspects, such as appropriate angiogenesis, appropriate inflammation, cell proliferation and migration to the wound site, and promotion of re-epithelialization and tissue remodeling. Angiogenesis provides nutrients and oxygen, and growth factors secreted by the edges and base of oral ulcers promote epithelial cell migration and proliferation, leading to the final re-epithelialization, reconstruction, and maturation of the mucosal defect.

[0004] In the prior art, drugs for oral ulcers include corticosteroids, nonsteroidal anti-inflammatory / antiseptic agents, antibiotics, hyaluronic acid, local anesthetics and natural topical drugs. However, the therapeutic effect needs to be further improved.

[0005] Curcumin (Cur) has been shown to reduce inflammatory responses and adhesion molecule expression, promote endothelial cell proliferation and migration for angiogenesis, and accelerate tissue growth, organ regeneration, and wound healing. It can also effectively treat gastrointestinal ulcers and colitis by inhibiting inflammatory cell infiltration and activation and enhancing epithelial cell defense. Naringenin (NGN) can promote the healing process by inhibiting inflammation and enhancing cell proliferation and angiogenesis. It also plays a role in angiogenesis and anticoagulation. It can promote angiogenesis, inhibit inflammation, and accelerate skin wound healing. However, the effect of the combined use of the two on oral mucosal healing remains largely unknown, and the low solubility of the two also limits their application. Summary of the invention

[0006] In view of this, the present invention provides a zwitterionic polymer dual-drug nano-micelle and a preparation method and application thereof based on the design concept of reducing inflammation, relieving pain and shortening the duration of oral ulcers to promote wound healing and then treat ulcers.

[0007] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows.

[0008] In a first aspect, the present invention provides a zwitterionic polymer dual-drug nano-micelle (OPDEA-PGED@CN), wherein the dual-drug nano-micelle uses a zwitterionic polymer (OPDEA-PGED) as a carrier and uses curcumin (Cur) and naringenin (NGN) as drug carriers;

[0009] The zwitterionic polymer is poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate-glycidyl methacrylate] ring-opened with ethylenediamine.

[0010] Preferably, the average particle size of the zwitterionic polymer dual-drug nano-micelles is 92-102.12 nm.

[0011] Preferably, the drug loading of naringenin is 10-20wt%, and the encapsulation efficiency is 45-60%.

[0012] Preferably, the drug loading of curcumin is 10-20wt%, and the encapsulation efficiency is 65-75%.

[0013] 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.

[0014] In a second aspect, the present invention provides a method for preparing the above-mentioned zwitterionic polymer dual-drug nano-micelles, comprising the following steps:

[0015] Step 1: dissolving the zwitterionic polymer in tetrahydrofuran to obtain a yellow transparent solution;

[0016] Step 2, dissolving naringenin, curcumin and 4-dimethylaminopyridine (DMAP) in methanol respectively to obtain naringenin solution, curcumin solution and 4-dimethylaminopyridine solution;

[0017] Step 3: After the naringenin solution, curcumin solution and 4-dimethylaminopyridine solution are uniformly mixed, the mixture is added to the yellow transparent solution, and then pyridine is added. The obtained reaction solution is reacted in an oil bath. After the reaction is completed, the obtained mixed solution is added dropwise to an ultrasonic ice-water bath to obtain a light yellow emulsion;

[0018] Step 4: dialyze the light yellow emulsion with deionized water to remove methanol, 4-dimethylaminopyridine and pyridine, and filter to obtain zwitterionic polymer dual-drug nano-micelles.

[0019] Preferably, in step 1, the zwitterionic polymer is dissolved in tetrahydrofuran by stirring at room temperature for 18-36 hours.

[0020] Preferably, in step 2, naringenin, curcumin and 4-dimethylaminopyridine are dissolved in methanol by stirring at room temperature for 1-1.5 hours respectively.

[0021] Preferably, in step 3, the mass ratio of the zwitterionic polymer, naringenin, curcumin, and 4-dimethylaminopyridine is 10-15:4-8:4-8:4-8.

[0022] Preferably, in step three, the reaction temperature is 30-40° C. and the reaction time is 18-36 h.

[0023] Preferably, in step 4, the molecular weight cutoff (MWCO) of the dialysis is 3000-3500Da, and the dialysis time is 6-8h.

[0024] Preferably, in step 4, the pore size of the filtration pores is 250 nm or 450 nm.

[0025] In a third aspect, the present invention also provides the use of the above-mentioned zwitterionic polymer dual-drug nano-micelles in the preparation of a drug for treating oral ulcers.

[0026] The principle of the present invention is:

[0027] Oral ulcer is a common inflammatory disease of the oral mucosa, manifested as inflammation and oral mucosal damage. The massive release of inflammatory factors activates organelles such as mitochondria and endoplasmic reticulum, and produces a large amount of reactive oxygen species (ROS) in the cells. ROS directly causes tissue damage through membrane lipid peroxidation, initiation of inflammatory cascade reactions and system dysfunction. Therefore, inflammatory factors and ROS play a very important role in the pathophysiological process of oral ulcer. The present invention utilizes the acidic environment of oral ulcer as a target site, so that the pH response releases a large amount of drugs at the lesion, thereby reducing the release of inflammatory factors and reducing the generation of ROS, thereby alleviating inflammation, while solving the disadvantage of poor water solubility of drugs, significantly improving the anti-inflammatory and antioxidant effects, and finding a new breakthrough for the treatment of oral ulcer.

[0028] Curcumin can exert anti-inflammatory effects by reducing the expression of endothelial cell adhesion molecules induced by inflammatory cytokines and inhibiting the activation of nuclear factor (NF-κB), inflammatory cytokines, and nitric oxide synthase (iNOS) genes.

[0029] Naringenin can promote the healing process by inhibiting inflammation and enhancing cell proliferation and angiogenesis. It also plays a role in angiogenesis and anticoagulation. It can promote angiogenesis, inhibit inflammation, and accelerate skin wound healing.

[0030] Zwitterionic polymers are macromolecular polymers composed of positive and negative ions, and have good water solubility, polarity and hydrophilicity, high stability and easy dispersion. The present invention uses zwitterionic polymers as drug carriers to improve the toxicity and release of drugs, and improve the bioavailability, biological properties and solubility of drugs.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The preparation method of the zwitterionic polymer dual-drug nano-micelles of the present invention utilizes the characteristics of good water solubility, polarity and hydrophilicity of the zwitterionic polymer to solve the problem of poor solubility of the combined use of curcumin and naringenin, and forms zwitterionic polymer dual-drug nano-micelles for use in the treatment of oral ulcers.

[0033] The zwitterionic polymer dual-drug nano-micelle of the invention has the effects of low toxicity, promoting wound healing and alleviating local inflammation.

[0034] In the zwitterionic polymer dual-drug nano-micelles of the present invention, both curcumin and naringenin have anti-inflammatory, anti-oxidative and antibacterial effects, and the zwitterionic polymer dual-drug nano-micelles can improve the bioavailability of curcumin and naringenin by sustained-release. In addition, the zwitterionic polymer dual-drug nano-micelles have the characteristics of low zwitterionic toxicity and strong cell permeability, so the zwitterionic polymer dual-drug nano-micelles have good anti-inflammatory and antioxidant effects, and have a good therapeutic effect on oral ulcers. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] 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.

[0036] Figure 1 This is the standard curve of naringenin in Example 2 of the present invention.

[0037] Figure 2 It is the standard curve of curcumin in Example 2 of the present invention.

[0038] Figure 3 This is the particle size distribution of OPDEA-PGED@CN in Example 3 of the present invention.

[0039] Figure 4 These are the cell viability detection results in Example 4 of the present invention, wherein a is the cell viability detection result of OPDEA-PGED@CN, b is the cell viability detection result of naringenin and curcumin (CN), and c is the cell viability detection result of OPDEA-PGED.

[0040] Figure 5 It 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 for releasing the pro-inflammatory factor interleukin-6 (IL-6), and b is the real-time fluorescence quantitative PCR for tumor necrosis factor-α (TNF-α).

[0041] Figure 6is the wound healing effect in Example 6 of the present invention, wherein a is the wound closure picture of the model group on the 1st day, b is the wound closure picture of the OPDEA-PGED group on the 1st day, c is the wound closure picture of the CN group on the 1st day, d is the wound closure picture of the OPDEA-PGED@CN group on the 1st day, e is the wound closure picture of the model group on the 7th day, f is the wound closure picture of the OPDEA-PGED group on the 7th day, g is the wound closure picture of the CN group on the 7th day, h is the wound closure picture of the OPDEA-PGED@CN group on the 7th day, and i is the wound closure rate of the model group, OPDEA-PGED group, CN group and OPDEA-PGED@CN group. DETAILED DESCRIPTION

[0042] 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.

[0043] 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.

[0044] Example 1

[0045] Step 1: Take 10 mg of OPDEA-PGED in a 10 mL single-mouth bottle, add 2 mL of tetrahydrofuran, and stir at room temperature for 24 h until there are no turbid small particles to obtain a yellow transparent solution.

[0046] Step 2: Take 4 mg of NGN, Cur and DMAP respectively and dissolve them in a single-necked bottle containing 1 mL of methanol, and stir at room temperature for 1 h to obtain NGN solution, Cur solution and DMAP solution.

[0047] Step 3: Mix the NGN solution, Cur solution and DMAP solution, add them to the yellow transparent solution, stir at 37°C for 24 hours, and then add the obtained mixed solution dropwise into 10 mL of ice water under ultrasound to obtain a light yellow solution.

[0048] Step 4: dialyze the light yellow solution with deionized water for 6 h (MWCO: 3500 Da) to remove methanol, DMAP and pyridine, and finally filter it with a 450 nm organic filter to obtain zwitterionic polymer dual-drug nano-micelles, recorded as OPDEA-PGED@CN micelles.

[0049] Example 2

[0050] Determination of drug loading in vitro. Weigh naringenin and curcumin accurately and fully dissolve them in anhydrous ethanol, prepare naringenin reference substance mother solution (concentration 0.1 mg / mL) and curcumin reference substance mother solution (concentration 0.1 mg / mL), dilute them step by step with anhydrous ethanol to the set mass concentration gradient, shake well, and use ultraviolet spectrophotometry to detect the absorbance of naringenin solution and curcumin solution at various concentrations, and establish standard curves: naringenin: Y=0.0634x+0.0324, R 2 =0.9991, e.g. Figure 1 , Curcumin: Y=61.628x-0.0286, R 2 =0.9993, e.g. Figure 2 shown.

[0051] 1 mg of the OPDEA-PGED@CN micelles prepared in Example 1 was taken, 2 ml of methanol was added to destroy the micelle structure, 2 mL of dichloromethane was added for centrifugation, and the supernatant was taken. Under the same conditions as those for the standard curve determination, the absorbance values ​​were respectively measured, and the contents of naringenin and curcumin in the OPDEA-PGED@CN micelles prepared in Example 1 were calculated by the standard curve, and the encapsulation efficiency (EE) and drug loading (DL) were calculated by the formula.

[0052] The drug loading and encapsulation efficiency were calculated according to the following formula:

[0053] The calculated results are as follows: the drug loading of naringenin is 11.19wt%, the encapsulation efficiency is 50.34%, the drug loading of curcumin is 15.94wt%, and the encapsulation efficiency is 71.73%.

[0054] Example 3

[0055] Particle size and zeta potential of OPDEA-PGED@CN. Take 1 mg of OPDEA-PGED@CN micelles, dilute them, and measure the average particle size of OPDEA-PGED@CN micelles using a dynamic light scattering (DLS) instrument. Record the polymer dispersibility index (PDI) and zeta potential. Repeat the measurement three times and finally express it as the average value ± standard deviation.

[0056] The test results are: average particle size: 93.56±1.56nm, such as Figure 3 As shown, the potential is -16.76±3.28 mV and the polymer dispersion index is 0.279±0.013.

[0057] Example 4

[0058] The cell viability of mouse mononuclear macrophage leukemia cells (RAW 264.7) was detected by CCK8 method. Cur and NGN were mixed at a mass ratio of 16:11 and dissolved in dimethyl sulfoxide (DMSO), and diluted to 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL (based on Cur content) with DMEM medium containing 10% fetal bovine serum (FBS), i.e., complete medium, and the volume ratio of DMSO in the medium was ensured to be less than 0.1%. The cell density was 1 × 10 4 Cells / well were added and cultured in complete medium overnight at 37 °C and 5% CO2. After discarding the original medium, 100 μL of complete medium containing different concentrations of CN (0 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL), OPDEA-PGED@CN micelles (0 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, based on the Cur content in micelles) and OPDEA-PGED (0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL) were added and co-cultured for 24 h. After discarding the old culture medium, wash with PBS, add 100 μL of the prepared working solution (DMEM culture medium without FBS: CCK8 volume ratio is 10:1), culture for 2 hours in the dark, use a microplate reader to obtain the absorbance of each well at 450 nm, and use the following formula to evaluate the effect of the material on cell viability:

[0059] In the formula, OD S and OD C are the absorbance of OPDEA-PGED@CN micelles and blank control (DMEM medium). OD b is the absorbance of the cell-free DMEM medium. The results of the effects of OPDEA-PGED@CN micelles, CN and OPDEA-PGED on the activity of RAW 264.7 cells are shown in Figure 4 As shown in a, b, and c. The results showed that after incubation of RAW 264.7 with different concentrations of OPDEA-PGED@CN micelles for 24 h, it was found that when the concentration of OPDEA-PGED@CN micelles (based on the content of Cur) did not exceed 5 μg / mL, the cell survival rate was higher than 80%, showing good cell compatibility.

[0060] Example 5

[0061] RT-qPCR experiments were used to detect the regulatory effect of OPDEA-PGED@CN micelles on inflammatory factors in RAW264.7 cells. 5 Cells / well were seeded in 6-well plates and cultured in complete medium at 37°C and 5% CO2 for 24 h. Complete medium containing LPS (1 mg / mL), LPS (1 mg / mL) + OPDEA-PGED (17.4 μg / mL), LPS (1 mg / mL) + CN (in terms of Cur 5 μg / mL), and LPS (1 mg / mL) + OPDEA-PGED@CN micelles (in terms of Cur 5 μg / mL) was replaced and incubated for 6 h. The medium was discarded and the cells were washed twice with PBS. 1 mL of Trizol reagent was added to each well and allowed to stand at room temperature for 10 min to rupture the cells. The mixture was collected after being fully blown, and 200 μL of chloroform was added and mixed by inversion for 10 s. After standing for 10 min, the mixture was centrifuged at 4°C and 15,000 rpm for 20 min. The supernatant was collected, an equal amount of isopropanol was added, and the mixture was centrifuged at 4°C and 15,000 rpm for 20 min after standing at room temperature. The supernatant was discarded. Add 1 mL of anhydrous ethanol to wash the precipitate, centrifuge at 4°C and 15,000 rpm for 5 min, discard the supernatant, and repeat this process once. Dry the RNA precipitate in air, add 20 μL of enzyme-free water to dissolve the precipitate, measure the RNA concentration using a Nanodrop 2000 fiber optic spectrophotometer, and synthesize cDNA using the Hifair® Ⅲ 1st Strand cDNA 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. The test results are as follows: Figure 5 As shown, it can be seen that the mRNA levels of pro-inflammatory factors IL-6 and TNF-α in cells treated with OPDEA-PGED@CN micelles were significantly reduced, indicating that OPDEA-PGED@CN micelles have a good anti-inflammatory effect.

[0062] Example 6

[0063] The ability of OPDEA-PGED@CN micelles to promote ulcer healing was evaluated in animals. Male SD rats (weight 180-200g) were selected and anesthetized by intraperitoneal injection of 2% sodium pentobarbital (0.2 mL / 100 g), and an ulcer model was established in the rat buccal mucosa using a spherical dental drill with a diameter of 2 mm. The model was successfully established one day later, and the wound size and appearance were recorded by taking photos. PBS (model group), OPDEA-PGED (17.4μg / mL), CN (5μg / mL, based on Cur content) and OPDEA-PGED@CN micelles (5μg / mL, based on Cur content) were injected into the ulcer site 10μL each. On the 7th day of administration, photos were taken and the wound size and appearance were recorded, and the wound closure rate was calculated as follows: Wound closure rate (%) = (A0-A) / A0×100%, where A0 is the ulcer area after the model is successfully established, and A is the ulcer area on the 7th day of administration. The healing photos are shown in Figure 6 As shown in a~h, the healing rate is Figure 6 As shown in Figure i, it can be seen that the oral ulcers of rats in the OPDEA-PGED@CN micelle group were basically healed 7 days after administration, while the ulcers in the other treatment groups still had obvious defects. In addition, compared with the other treatment groups, the OPDEA-PGED@CN micelle group had a higher wound healing rate (86.25%).

[0064] 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 dual-drug nano-micelles, characterized in that: The dual-drug nano-micelles use zwitterionic polymers as carriers and curcumin and naringenin as drug carriers; The zwitterionic polymer is poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate-glycidyl methacrylate] ring-opened with ethylenediamine.

2. The zwitterionic polymer dual-drug nano-micelle according to claim 1, characterized in that: The average particle size of the zwitterionic polymer dual-drug nano-micelles is 92-102.12 nm.

3. The zwitterionic polymer dual-drug nano-micelle according to claim 1, characterized in that: The drug loading amount of naringenin is 10-20wt%, and the encapsulation efficiency is 45-60%.

4. The zwitterionic polymer dual-drug nano-micelle according to claim 1, characterized in that: The drug loading of the curcumin is 10-20wt%, and the encapsulation efficiency is 65-75%.

5. The method for preparing zwitterionic polymer dual-drug nano-micelles according to any one of claims 1 to 4, characterized in that: The following steps are involved: Step 1: dissolving the zwitterionic polymer in tetrahydrofuran to obtain a yellow transparent solution; Step 2: dissolving naringenin, curcumin and 4-dimethylaminopyridine in methanol respectively to obtain a naringenin solution, a curcumin solution and a 4-dimethylaminopyridine solution; Step 3: After the naringenin solution, curcumin solution and 4-dimethylaminopyridine solution are uniformly mixed, the mixture is added to the yellow transparent solution, and then pyridine is added. The obtained reaction solution is reacted in an oil bath. 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 dual-drug nano-micelles.

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

7. The method for preparing zwitterionic polymer dual-drug nano-micelles according to claim 5, characterized in that: In step 2, naringenin, curcumin and 4-dimethylaminopyridine were dissolved in methanol by stirring at room temperature for 1-1.5 hours respectively.

8. The method for preparing zwitterionic polymer dual-drug nano-micelles according to claim 5, characterized in that: In step 3, the mass ratio of the zwitterionic polymer, naringenin, curcumin, and 4-dimethylaminopyridine is 10-15:4-8:4-8:4-8; The reaction temperature is 30-40°C, and the reaction time is 18-36h.

9. The method for preparing zwitterionic polymer dual-drug nano-micelles according to claim 5, characterized in that: In step 4, the molecular weight cut-off of the dialysis is 3000-3500Da, and the dialysis time is 6-8h; The pore size of the filtering pores is 250nm or 450nm.

10. Use of the zwitterionic polymer dual-drug nano-micelles according to any one of claims 1 to 4 in the preparation of a drug for treating oral ulcers.

Images