Preparation method and application of polydopamine drug-loaded nanoparticles

Abstract

The invention discloses a preparation method and application of a polydopamine drug-loaded nanoparticle. The preparation method comprises the following steps: firstly, performing primary wrapping on quercetin (Que) and 2, 4-dinitrophenol (Dnp) by dopamine hydrochloride (DA.HCl) under an alkaline environment through an oxidation self-polymerization effect of the dopamine hydrochloride (DA.HCl); the preparation method comprises the following steps: adding DA.HCl into a reaction kettle, reacting for 8 hours, adding arginine (Arg), and doping the arginine into a PDA shell in the process of polymerizing DA.HCl to generate PDA. And further reacting for 16 hours to obtain nano particles (QRDP) with the particle size of about 180nm. The method is simple to operate, short in time consumption and mild and controllable in synthesis condition. The synthesized nano particles are good in biocompatibility; good antioxidant activity is shown inside and outside cells; meanwhile, the functions of down-regulating cell proinflammatory factors and up-regulating anti-inflammatory factors are realized; moreover, lipid excessively accumulated in cells can be effectively reduced, and multiple treatment effects of oxidation resistance, inflammation resistance and lipid accumulation resistance are achieved for fatty liver diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical nanomaterials and their preparation, and more specifically, relates to a preparation method and application of a polydopamine-loaded nanoparticle multifunctional preparation having the functions of anti-oxidation, anti-inflammation and anti-lipid accumulation. Background Art

[0002] In recent years, with the improvement of living standards, the incidence of obesity and diabetes has continued to rise worldwide. It is estimated that by 2030, the prevalence of nonalcoholic steatohepatitis (NASH), which is mainly characterized by abnormal accumulation of lipids in the liver, will increase by 63%, affecting more than 100 million people (Hepatology 2018, 67, 123). Despite the high medical needs, there is currently no treatment for NASH. Clinically, patients are usually recommended to lose weight and change their lifestyle, but long-term exercise or dietary restrictions are difficult to achieve, so the treatment effect is not ideal; there are not many treatment options using antioxidants such as vitamin E and metabolic drugs such as glitazones, because vitamin E has a potential risk of increasing mortality, and long-term safety is controversial, while pioglitazone has the risk of multiple organ failure and acute myocardial infarction (ActaPharm.Sin.B2024, 14, 2228). The complex biological interactions between oxidative stress, inflammation, metabolism and extracellular matrix deposition also limit the development and application of drugs. Therefore, improving the treatment of NASH is urgent and of great significance.

[0003] A large amount of evidence shows that oxidative stress mediated by reactive oxygen species (ROS) produced by fatty acid oxidation in hepatocyte mitochondria is a key factor in the progression of NASH, and oxidative stress can further lead to inflammation and fibrosis (Adv. Mater. 2024, 36, e2305830). Therefore, we consider using polydopamine (PDA), which has its own antioxidant function and is widely used in drug delivery, as a carrier to encapsulate effective natural anti-inflammatory drugs in it to significantly improve drug stability and bioavailability. Protect the encapsulated anti-inflammatory drugs from degradation during the circulation process in the body, and accumulate in the liver through the passive targeting characteristics of nanoparticles, ensuring the continuous release of drugs in the liver and providing a lasting therapeutic effect, so that nanoparticles have great potential as a drug delivery system in combating NASH.

[0004] Although nanotechnology is a promising strategy for addressing oxidative stress-related liver diseases, the high toxicity, unclear role and single function have made nanomaterials a considerable challenge in practical applications (Cell Commun. Signal. 2024, 22, 18). It is crucial to address the safety and biocompatibility of nanomaterials. The design of nanomaterials should prioritize ensuring that the preparations do not induce hepatotoxicity and aggravate liver damage, and then further consider long-term efficacy. Therefore, multifunctional preparations with good biocompatibility and multi-pronged antioxidant, anti-inflammatory and anti-lipid accumulation that can target multiple characteristics of NASH are promising strategies for the treatment of NASH.

[0005] In the present invention, we synthesized a multifunctional polydopamine drug-loaded nanoparticle for the treatment of NASH. First, dopamine hydrochloride (DA·HCl) preliminarily encapsulates quercetin (Que) and 2,4-dinitrophenol (Dnp) through its own oxidative self-polymerization in an alkaline environment. Arginine (Arg) is then added to be doped in the PDA shell during the polymerization of DA·HCl to generate PDA, and finally nanoparticles (QRDP) with a particle size of about 180nm are obtained. This method is simple to operate, time-saving, and the synthesis conditions are mild and controllable. The synthesized nanoparticles have good biocompatibility; they show good antioxidant activity both inside and outside the cell; they also have the function of downregulating cellular proinflammatory factors and upregulating anti-inflammatory factors; and they can effectively reduce the excessive accumulation of lipids in the cells, achieving multiple therapeutic effects of anti-oxidation, anti-inflammation, and anti-lipid accumulation for NASH. Summary of the invention

[0006] The purpose of the present invention is to provide a multifunctional polydopamine drug-loaded nanoparticle, which can achieve efficient treatment of NASH through multiple antioxidant activities, synergistic anti-inflammatory and anti-lipid accumulation effects. That is, Arg-doped PDA is used as a carrier to load Que and Dnp to achieve a multi-pronged treatment of NASH.

[0007] A method for preparing polydopamine drug-loaded nanoparticles, characterized by comprising the following steps:

[0008] An alkaline buffer solution is prepared by disodium hydrogen phosphate dodecahydrate Na2HPO4·12H2O, sodium dihydrogen phosphate dihydrate NaH2PO4·2H2O and sodium hydroxide; dopamine hydrochloride DA·HCl, quercetin Que, arginine Arg and 2,4-dinitrophenol Dnp react in a water and ethanol reaction system with an alkaline buffer solution added to obtain polydopamine drug-loaded nanoparticles QRDP; the details are as follows:

[0009] 1) Dissolve Na2HPO4·12H2O and NaH2PO4·2H2O in water to prepare two solutions respectively, blend the two solutions, add sodium hydroxide, adjust the pH to 8.5, and obtain an alkaline buffer solution;

[0010] 2) Add DA·HCl aqueous solution, Que ethanol solution, Dnp ethanol solution and the above alkaline buffer solution to the water and ethanol system and react for 8 hours;

[0011] 3) Add Arg aqueous solution, continue the reaction for 16 hours, and remove the supernatant by centrifugation to obtain QRDP;

[0012] The concentrations of the Na2HPO4·12H2O solution and the NaH2PO4·2H2O solution are both 0.2M, the molar ratio of Na2HPO4·12H2O:NaH2PO4·2H2O is 4:1, the volume ratio of water to ethanol in the water and ethanol system is 3:1, and the molar ratio of DA·HCl:Que:Arg:Dnp is 50:3:55:5.

[0013] The centrifugal speed is 13000-14000 rpm and the centrifugal time is 5-10 min;

[0014] The preparation method can prepare polydopamine drug-loaded nanoparticles.

[0015] The product containing the polydopamine drug-loaded nanoparticles is used as a drug for treating non-alcoholic fatty liver disease.

[0016] A method for preparing polydopamine drug-loaded nanoparticles, characterized in that the preparation method of QRDP comprises the following steps: dissolving 3.58g Na2HPO4·12H2O and 1.56g NaH2PO4·2H2O in 50mL water respectively to prepare two 0.2M aqueous solutions; mixing 40.5mL Na2HPO4·12H2O solution with 9.5mL NaH2PO4·2H2O solution, adding NaOH solution, adjusting the pH to 8.5, and obtaining an alkaline buffer solution; mixing 8mL ultrapure water with 2.6mL anhydrous ethanol, and adding 250μL, 2mg mL -1 Que ethanol solution, 250 μL, 16 mg mL -1 DA·HCl aqueous solution, 250 μL, 2 mg mL -1 ethanol solution of Dnp and 1.25 mL of alkaline buffer were reacted for 8 h; then 250 μL of 20 mg mL -1 The Arg aqueous solution was added and the reaction was continued at room temperature for 16 h. The supernatant was removed by centrifugation at 14000 rpm for 5 min to obtain QRDP.

[0017] The obtained nanoparticles have the following characteristics:

[0018] (1) It has a uniform size, with a particle size of about 180 nm;

[0019] (2) It has good extracellular antioxidant activity, has SOD-like enzyme activity and can effectively remove ·OH;

[0020] (3) It has good intracellular antioxidant activity and can remove excess ROS in macrophages;

[0021] (4) It has good anti-inflammatory activity, downregulating pro-inflammatory cytokines secreted by macrophages and upregulating anti-inflammatory cytokines;

[0022] (5) It has a good function of regulating lipid homeostasis and effectively reduces the excessive accumulation of lipids in human hepatoma cells (HepG2); BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Particle size diagram of nano-microspheres of the present invention.

[0024] Figure 2 : The SOD-like enzyme activity diagram of the nano-microspheres of the present invention.

[0025] Figure 3 : Schematic diagram of the OH scavenging activity of the nano-microspheres of the present invention.

[0026] Figure 4 : The nanoparticles of the present invention remove ROS in macrophages.

[0027] Figure 5 :The nanospheres of the present invention down-regulate pro-inflammatory cytokines in macrophages.

[0028] Figure 6 :The nanospheres of the present invention upregulate anti-inflammatory cytokines in macrophages.

[0029] Figure 7 : The nanospheres of the present invention regulate lipid homeostasis in HepG2 cells. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the following examples, but the present invention is not limited to the following examples. For example, the concentration and volume of the solution used can be adjusted as required.

[0031] Example 1

[0032] (1) Weigh 3.58 g of Na2HPO4·12H2O, dissolve it in 50 mL of water, and shake thoroughly until it is completely dissolved to obtain a 0.2 M Na2HPO4·12H2O solution.

[0033] (2) Weigh 1.56 g of NaH2PO4·2H2O, dissolve it in 50 mL of water, and shake thoroughly until it is completely dissolved to obtain a 0.2 M NaH2PO4·2H2O solution.

[0034] (3) Take 40.5 mL of Na2HPO4·12H2O solution and 9.5 mL of NaH2PO4·2H2O solution and mix them evenly in a 100 mL reaction bottle. After the pH meter is calibrated, place the pH meter probe in the mixed solution, add a magnet, and add NaOH solution dropwise under stirring to adjust the pH to 8.5 to obtain an alkaline buffer solution.

[0035] (4) Prepare 2 mg mL -1 Que ethanol solution, 16 mg mL -1 of DA·HCl aqueous solution, and 2 mg mL -1 Ethanol solution of Dnp.

[0036] (5) 8 mL of ultrapure water and 2.6 mL of anhydrous ethanol were mixed in a 20 mL reaction bottle, and 250 μL of Que solution, 250 μL of DA·HCl solution, 250 μL of Dnp solution, and 1.25 mL of alkaline buffer were added in sequence. The mixture was stirred at 500 rpm at room temperature for 8 h.

[0037] (6) After 8 hours, prepare 20 mg mL -1 Arg aqueous solution was added, and 250 μL was added to the reaction system. The reaction was continued at 500 rpm and stirred at room temperature for 16 h. The supernatant was removed by centrifugation at 14000 rpm for 5 min, and the supernatant was washed twice with ultrapure water to obtain QRDP nanoparticles.

[0038] The hydrated particle size was measured using Mastersize 3000 dynamic light scattering (DLS) (Malvern Instruments, UK) to be 182.9 nm and the PDI was 0.090. Figure 1 The data showed that the particle size of the obtained QRDP nanospheres was about 180 nm.

[0039] (7) Detection of SOD-like enzyme activity by pyrogallol autooxidation method: Prepare 50 mM hydrochloric acid and Tris-HCl (50 mM) buffer solution at pH 8.2, and dissolve pyrogallol in hydrochloric acid to a concentration of 1.5 mg mL -1 Then, 150 μL of Tris-HCl buffer, 40 μL of an aqueous solution (200 μg mL ) of the nanoparticles to be tested (PDA nanoparticles (P), PDA-encapsulated Que nanoparticles (QP), PDA-doped Arg-encapsulated Que nanoparticles (QRP), and QRDP nanoparticles) were added sequentially in a 96-well plate. -1) and 10 μL of pyrogallol hydrochloric acid solution, and the absorbance at 325 nm of each well was measured every 30 seconds using a microplate reader. The total reaction time was 20 minutes. In the SOD enzyme activity assay, P, QP, QRP, and QRDP nanoparticles were measured. Figure 2 The absorption-time curves of pyrogallol auto-oxidation under four kinds of nanoparticles show that QRDP has good SOD-like enzyme activity, which can significantly inhibit the process of pyrogallol auto-oxidation.

[0040] (8) Salicylic acid method to detect ·OH scavenging activity: prepare 9 mM salicylic acid ethanol solution, 9 mM FeSO4·H2O aqueous solution, 8.8 mM H2O2 solution, and 200 μg mL -1 The aqueous solution of the nanoparticles (P, QP, QRP, and QRDP) to be tested was set up as a control group (A0), an experimental group (A x ), and background group (A x′ ), FeSO4 solution, salicylic acid solution, H2O2 solution, and ultrapure water were added to the control group; in the experimental group, in addition to the above solutions, an additional aqueous solution of the nanoparticles to be tested was added; the same reagents as the experimental group were added to the background group, but no H2O2 solution was added, and the volume of ultrapure water was adjusted to ensure that the total volume of each group was the same. After heating each group in a 37°C water bath for 15 minutes, the absorbance at 510nm was measured. The property of salicylic acid that can react with ·OH to generate 2,3-dihydroxybenzoic acid with characteristic absorption at 510nm was used to measure the absorbance [OD A0 -(OD Ax -OD Ax′ )] / OD A0 · 100% calculation· OH removal rate. Figure 3 The ·OH scavenging rates of P, QP, QRP, and QRDP were analyzed. The data results showed that QRDP had good ·OH scavenging activity.

[0041] (9) Determination of ROS content in macrophages: 10,000 macrophages were seeded in a black 96-well plate and incubated for 24 h. Then, 100 μL of 100 ng mL ROS was added to each well. -1 The inflammatory model was constructed by incubating the lipopolysaccharide (LPS) solution for 24 h. After the model was constructed, 100 μL, 200 μg mL -1 The nanoparticles (P, QP, QRP, and QRDP) were co-incubated for 24 h, and the fluorescence intensity of intracellular ROS was measured using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE), respectively. Figure 4The relative content of ROS remaining after treatment with each material was calculated by the ratio of the fluorescence intensity of each experimental group to the LPS group. The data results showed that QRDP could remove more than 85% of the excessive intracellular ROS caused by LPS, showing excellent intracellular antioxidant activity.

[0042] (10) Detection of pro-inflammatory cytokine levels in macrophages: 200,000 macrophages were seeded in a 6-well plate and incubated for 24 h. Then, 1 mL of 100 ng mL -1 The inflammatory model was constructed by incubating the LPS solution for 24 h. After the model was constructed, 1 mL and 200 μg mL -1 The nanoparticles (P, QP, and QRDP) were co-incubated for 24 h, and the cell culture supernatant was collected to detect the levels of pro-inflammatory factors using an ELISA kit. Figure 5 The data show that QRDP has a significant effect on downregulating the expression level of pro-inflammatory factors secreted by macrophages and has excellent anti-inflammatory activity.

[0043] (11) Detection of anti-inflammatory factor levels in macrophages: 200,000 macrophages were seeded in a 6-well plate and incubated for 24 h. Then, 1 mL of 100 ng mL -1 The inflammatory model was constructed by incubating the LPS solution for 24 h. After the model was constructed, 1 mL and 200 μg mL -1 The nanoparticles (P, QP, and QRDP) were co-incubated for 24 h, and the cell culture supernatant was collected to detect the levels of anti-inflammatory factors using an ELISA kit. Figure 6 The data show that QRDP has a significant effect on restoring the expression level of anti-inflammatory factors secreted by macrophages and can effectively reduce inflammatory damage.

[0044] (12) Detection of lipid levels in HepG2 cells: 100,000 HepG2 cells were seeded in a 6-well plate and incubated for 24 h. Then, 1 mL of a mixed solution of oleic acid and palmitic acid (the final concentrations of oleic acid and palmitic acid were 0.66 mM and 0.33 mM, respectively) was added to each well and incubated for 24 h to construct a lipid accumulation model. After the model was constructed, 1 mL of 200 μg mL -1 Nanoparticles (P, QP, QRP, and QRDP) were co-incubated for 24 h, and the intracellular lipid levels were observed by staining the intracellular lipids with Oil Red O. Figure 7 The lipid content in HepG2 cells after different treatments. The data results show that QRDP effectively reduces the excessive accumulation of lipids in HepG2 cells and regulates cellular lipid homeostasis.

Claims

1. A method for preparing polydopamine drug-loaded nanoparticles, characterized in that The steps include: An alkaline buffer solution is prepared by disodium hydrogen phosphate dodecahydrate Na2HPO4·12H2O, sodium dihydrogen phosphate dihydrate NaH2PO4·2H2O and sodium hydroxide; dopamine hydrochloride DA·HCl, quercetin Que, arginine Arg and 2,4-dinitrophenol Dnp react in a water and ethanol reaction system with an alkaline buffer solution added to obtain polydopamine drug-loaded nanoparticles QRDP; the details are as follows: 1) Dissolve Na2HPO4·12H2O and NaH2PO4·2H2O in water to prepare two solutions respectively, blend the two solutions, add sodium hydroxide, adjust the pH to 8.5, and obtain an alkaline buffer solution; 2) Add DA·HCl aqueous solution, Que ethanol solution, Dnp ethanol solution and the above alkaline buffer solution to the water and ethanol system and react for 8 hours; 3) Add Arg aqueous solution, continue the reaction for 16 hours, and remove the supernatant by centrifugation to obtain QRDP; The concentrations of the Na2HPO4·12H2O solution and the NaH2PO4·2H2O solution are both 0.2M, the molar ratio of Na2HPO4·12H2O:NaH2PO4·2H2O is 4:1, the volume ratio of water to ethanol in the water and ethanol system is 3:1, and the molar ratio of DA·HCl:Que:Arg:Dnp is 50:3:55:

5.

2. The preparation method according to claim 1, characterized in that: The centrifugal speed is 13000-14000rpm, and the centrifugal time is 5-10min.

3. Polydopamine drug-loaded nanoparticles are prepared by the preparation method described in claim 1.

4. A product containing the polydopamine drug-loaded nanoparticles according to claim 3 as a drug for treating non-alcoholic fatty liver disease.

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