Preparation method of polydopamine nanoparticles loaded with antioxidant active substances

Abstract

The invention belongs to the field of medicines. The embodiment of the invention provides a preparation method of polydopamine nanoparticles loaded with antioxidant active substances. According to the present invention, the polydopamine nano-particles have active oxygen responsiveness, the polydopamine nano-particles are formed through the self-polymerization of the dopamine hydrochloride, and the nano-particles loaded with the antioxidant active substance EGCG can be obtained through the pi-pi conjugation interaction with the antioxidant active substance EGCG, such that the anti-oxidation activity of the polydopamine nano-particles can be significantly improved, and the anti-oxidation activity of the polydopamine nano-particles can be significantly improved. The nanoparticle reaches a damaged kidney through in-vivo circulation and releases EGCG in a pathological microenvironment, so that the stability of EGCG is improved, side effects possibly caused by repeated administration are avoided, and the compliance of a patient is improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, and specifically relates to a method for preparing polydopamine nanoparticles loaded with antioxidant active substances. Background Technology

[0002] Acute kidney injury (AKI) is a severe syndrome characterized by the sudden loss of kidney function, clinically manifested as elevated serum creatinine levels and decreased urine output. The global burden of mortality associated with AKI far exceeds that of breast cancer, heart failure, or diabetes; therefore, AKI treatment and medical research have significant social and economic value.

[0003] Currently, various antioxidants are used to treat acute kidney injury. This involves alleviating kidney damage by eliminating high levels of reactive oxygen species in the kidney area.

[0004] However, the poor stability and low bioavailability of some antioxidant substances (such as EGCG) limit their clinical application in the treatment of acute kidney injury.

[0005] Polydopamine (PDA) is a novel organic nanomaterial with strong viscosity and other excellent biological properties. Due to its superior biocompatibility, it is widely used in bioimaging, drug loading and release, photothermal therapy, and integrated diagnostic and therapeutic platforms. Furthermore, studies have found that PDA exhibits sensitivity to reactive oxygen species (ROS), and its degradation rate accelerates under high ROS conditions, making it a promising candidate for use as a drug delivery platform with ROS-responsive characteristics. Summary of the Invention

[0006] In view of this, this application provides polydopamine nanoparticles loaded with antioxidant active substances and a method for preparing the same, in order to at least partially solve the above-mentioned problems.

[0007] To achieve the above objectives, this application provides the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing polydopamine nanoparticles loaded with antioxidant active substances, the method comprising the steps of:

[0009] S1: Disperse dopamine hydrochloride in water and mix by vortexing or sonication to obtain a dopamine hydrochloride solution;

[0010] S2: Add the dopamine hydrochloride solution to a mixed solution of ammonia, 90 mL distilled water, and 40 mL anhydrous ethanol, stir in the dark for 12-48 h, centrifuge at 13,000 rpm for 15 min, wash, and obtain polydopa.

[0011] Amine nanoparticles;

[0012] S3: The polydopamine nanoparticles are added to a solution of epigallocatechin gallate, an antioxidant active substance, and stirred for 4-12 hours. After centrifugation at 13,000 rpm for 15 minutes, the nanoparticles are washed to obtain polydopamine nanoparticles loaded with antioxidant active substances.

[0013] Regarding the mass ratio of dopamine hydrochloride to antioxidant active substances, 1:25-50 refers to any value within the range of 1:25 to 1:50, such as 1:25.5, 1:27, 1:29.5, 1:43.5, 1:46.5, 1:49.8, 1:49.9, and 1:50.

[0014] Regarding the volume ratio of water to the mass ratio of dopamine hydrochloride, 1:25-100 refers to any value within the range of 1:25 to 1:100, such as 1:26, 1:29, 1:50, 1:68, 1:82, 1:99, and 1:100.

[0015] Regarding the volume ratio of ammonia to the mass ratio of dopamine hydrochloride, 1:100-500 refers to any value within the range of 1:100-1:500, such as 1:100, 1:101, 1:120, 1:150, 1:190, 1:200, 1:350, 1:450, and 1:500.

[0016] In some preferred embodiments, in step S1, the volume ratio of the water to the mass ratio of dopamine hydrochloride is 1:50-100, for example, 1:80.

[0017] In some preferred embodiments, in step S2, the volume ratio of the ammonia water to the mass ratio of dopamine hydrochloride is 1:125-250, for example, 1:150.

[0018] In some preferred embodiments, the stirring time in step S2 is 24-36 hours, for example, 24 hours.

[0019] In some preferred embodiments, the stirring time in step S3 is 5-10 hours, for example, 10 hours.

[0020] In a third aspect, the present invention provides the above-mentioned polydopamine nanoparticles loaded with antioxidant active substances to improve the stability of the antioxidant active substances.

[0021] A third aspect of the present invention provides the application of the above-described polydopamine nanoparticles loaded with antioxidant active substances in the treatment of acute kidney injury.

[0022] Beneficial effects of the present invention

[0023] This method, by preparing polydopamine nanoparticles loaded with antioxidant active substances, can specifically address the following shortcomings:

[0024] (1) Polydopamine has a simple and universal reaction process. Its surface has catechol, amine and imine groups, which can be used as a delivery platform for antioxidant active substances such as EGCG through π-π conjugation interaction, thus avoiding its oxidation under physiological conditions and improving its stability.

[0025] (2) Polydopamine can release the antioxidant active substance EGCG in response to ROS, thus avoiding its premature leakage in the blood circulation and reducing the side effects of EGCG on other organs and tissues. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 These are the transmission electron microscopy results provided in Example 1 of this application;

[0028] Figure 2 This is the result of the investigation on scavenging ABTS free radicals provided in Example 1 of this application;

[0029] Figure 3 This is the result of the DPPH free radical scavenging investigation provided in Example 1 of this application;

[0030] Figure 4 This is the result of the investigation on the inhibition of oxidative stress cell damage provided in Example 1 of this application;

[0031] Figure 5 This is the result of the investigation on the relief of folic acid-induced acute kidney injury provided in Example 1 of this application; Detailed Implementation

[0032] To enable those skilled in the art to better understand the technical solutions in the embodiments of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments provided in this application, all other embodiments obtained by those skilled in the art should fall within the protection scope of the embodiments of this application.

[0033] Terminology Explanation

[0034] PDA stands for: Polydopamine nanoparticles;

[0035] EGCG refers to epigallocatechin gallate;

[0036] EGCG@PDA refers to polydopamine nanoparticles loaded with the antioxidant active substance epigallocatechin gallate;

[0037] The present application will be further described below with reference to specific embodiments.

[0038] Example 1

[0039] (1) Preparation of polydopamine nanoparticles

[0040] 0.5 g of dopamine hydrochloride was weighed and dispersed in 10 mL of water, and mixed until completely dissolved. Then, the solution was added to a mixture of 4 mL ammonia, 90 mL distilled water, and 40 mL anhydrous ethanol, and stirring was continued for 24 hours. Finally, the product was collected by centrifugation (13,000 rpm, 15 min), washed several times with water and ethanol, and vacuum dried to obtain polydopamine nanoparticle powder (PDA).

[0041] (2) Preparation and encapsulation efficiency detection of polydopamine nanoparticles loaded with antioxidant active substance EGCG

[0042] To construct an EGCG standard curve: Accurately weigh 10 mg of EGCG into a 100 mL volumetric flask, dissolve and dilute to the mark with ethanol, and mix well. Measure 0.75, 1.25, 2.5, 5, and 10 mL of this solution into 25 mL volumetric flasks, add water to the mark, and mix well to obtain standard solutions with concentrations of 2.5, 5, 10, 20, 25, and 40 μg / mL, respectively. Measure the absorbance at 273 nm for each solution and construct an EGCG standard curve based on the results.

[0043] Weigh 1 mg of polydopamine nanoparticle powder sample and add it dropwise to a 0.5 mg / mL EGCG solution, stirring for 10 h. Then centrifuge (13,000 rpm, 15 min), wash with water, collect, and vacuum dry to obtain polydopamine nanoparticle powder sample loaded with the antioxidant EGCG (EGCG@PDA). Collect the washing liquid and supernatant together and measure the absorbance at 273 nm. Calculate the drug loading and encapsulation efficiency of EGCG using the EGCG standard curve, using the following formula:

[0044]

[0045] (3) Characterization of polydopamine nanoparticles loaded with the antioxidant EGCG

[0046] ① Morphological observation under transmission electron microscopy

[0047] Take a PDA suspension, dilute it to a suitable concentration, disperse the suspension using an ultrasonic disruptor (50%, 2s working, 2s intermittent, 5min), measure 10μL with a pipette, transfer it to a copper grid covered with a carbon film, let it dry, and then observe its shape and appearance characteristics with a transmission electron microscope (TEM), and take pictures at appropriate angles and orientations to record the results.

[0048] ② Determine the free radical scavenging ability

[0049] 2.1 Total Antioxidant Capacity Assay Kit - Spectrophotometric Method (ABTS Method)

[0050] Take the Merrill ABTS kit and, following the instructions, take one vial of Reagent II, add 25 mL of Reagent I, vortex to mix for 20 min, let stand, and discard the supernatant before use. Add 50 μL of extraction buffer and 950 μL of working solution to one tube as a blank control. Then, dilute the PDA suspension with distilled water to the appropriate concentration.

[0051] Substitute the data into the formula to calculate: ABTS free radical scavenging rate (%) = (A blank - A determination) ÷ A blank × 100%

[0052] 2.2 Total Antioxidant Capacity Assay Kit - Micro-method (DPPH Method)

[0053] Using the Merrill DPPH kit, following the instructions, take a 1.5 mL centrifuge tube and add 20 μL of extraction buffer. Use 380 μL of reagent one as a blank control. Weigh 1 mg of EGCG standard using an analytical balance and add it to distilled water to prepare a 1 mg / mL solution. Then, take 0.5 mL of this solution and mix it with PDA suspension at the same concentration to obtain a 1 mg / mL PDA@EGCG solution. Take 20 μL of the PDA suspension. Now, designate 380 μL of mixed reagent one as sample one. Next, take 20 μL of the 1 mg / mL EGCG solution and add it to 380 μL of mixed reagent one to form sample two. Finally, take 20 μL of the 1 mg / mL PDA@EGCG solution and mix it with 380 μL of mixed reagent one to form sample three. After thoroughly mixing the reagents, react at room temperature in the dark for 20 minutes. Then, accurately spot 200 μL of the mixture into a 96-well plate, ensuring that each sample group is spotted three times. Finally, the absorbance of each well was measured at a wavelength of 515 nm. The data was then input into plotting software to calculate the standard concentration curve. Finally, the sample absorbance was substituted into the curve to calculate the sample concentration and obtain the result.

[0054] Calculate using the data: DPPH free radical scavenging rate (%) = (A blank - A determination) ÷ A blank × 100%

[0055] (4) The therapeutic effect of polydopamine nanoparticles loaded with the antioxidant EGCG on cells damaged by oxidative stress

[0056] NRK-52E cells were seeded in 96-well plates at 1×10⁵ cells / mL and cultured for 24 h (5% CO₂, 37°C). PBS and different concentrations of EGCG@PDA solution were added, followed by 30 min incubation in an oven. Immediately afterward, 300 μM hydrogen peroxide solution was added to each well, and the plates were incubated for another 24 h. The culture medium was then removed, and the plates were washed twice with PBS. For cell viability assays, a 10% CCK-8 solution was prepared by mixing CCK-8 reagent with PBS. 200 μL of the CCK-8 solution was added to each well, and the plates were cultured at 37°C, 5% CO₂ for 30 min. Finally, the OD value at 450 nm was measured in each well using a microplate reader, and cell viability was calculated.

[0057]

[0058] (5) The therapeutic effect of polydopamine nanoparticles loaded with the antioxidant EGCG on folic acid-induced acute kidney injury.

[0059] Healthy male C57BL / 6J mice, 6-8 weeks old and weighing 20±2g, were acclimatized for one week before modeling was initiated. On the day of modeling, the mice were weighed first, and then administered folic acid (5mg / mL, dissolved in 0.3mmol / L NaHCO3) via intraperitoneal injection (0.2mL) and tail vein injection of polydopamine nanoparticles loaded with the antioxidant EGCG (10mg / kg) for treatment. This was done once, with a total administration volume of 0.2mL.

[0060] One day after drug administration, the following procedures were performed to detect serum CRE and BUN levels: Blood samples were collected from the eyeballs of mice in each group, and the whole blood samples were incubated in a 37°C water bath for 30 minutes. Subsequently, the samples were centrifuged at 3000 rpm for 15 minutes to effectively separate the serum. Finally, the levels of blood urea nitrogen (BUN) and serum creatinine (CRE) in the separated serum samples were measured.

[0061] Comparative Example 1

[0062] The difference from Example 1 is that Comparative Example 1 contains only the polydopamine nanoparticles prepared in step 1.

[0063] Comparative Example 2

[0064] The difference from Example 1 is that Comparative Example 2 only contains the EGCG aqueous solution prepared in step 2.

[0065] A comparison of Example 1 and Comparative Example 1 shows that after loading the antioxidant EGCG, the entire nanoparticle exhibits stronger antioxidant stress resistance and therapeutic effects on acute kidney injury. A comparison of Example 1 and Comparative Example 2 shows that, compared to EGCG, polydopamine encapsulation provides stronger antioxidant stress resistance and therapeutic effects on acute kidney injury.

[0066] Finally, it should be noted that the above descriptions are merely embodiments of this application, used only to illustrate the technical solutions of the present invention, and are not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

1. A method for preparing polydopamine nanoparticles loaded with antioxidant active substances, characterized in that, include: S1: Disperse dopamine hydrochloride in water, mix well, and obtain a dopamine hydrochloride solution; S2: Add the dopamine hydrochloride solution to a mixed solution of ammonia, distilled water, and anhydrous ethanol. Stirring in the dark, centrifuging, and washing yielded polydopamine nanoparticles. S3: The polydopamine nanoparticles are added to the antioxidant active substance solution, stirred, centrifuged, and washed to obtain polydopamine nanoparticles loaded with antioxidant active substances.

2. The method for preparing polydopamine nanoparticles loaded with antioxidant active substances according to claim 1, characterized in that, In step S1, the mixing method includes vortexing and ultrasound.

3. The method for preparing polydopamine nanoparticles loaded with antioxidant active substances according to claim 1, characterized in that, In step S1, the mass ratio of dopamine hydrochloride to the antioxidant active substance is 1:25-50; In step S1, the volume ratio of water to the mass ratio of dopamine hydrochloride is 1:25-100.

4. The method for preparing polydopamine nanoparticles loaded with antioxidant active substances according to claim 1, characterized in that, In step S2, the volume ratio of the ammonia water to the mass ratio of dopamine hydrochloride is 1:100-500.

5. The method for preparing polydopamine nanoparticles loaded with antioxidant active substances according to claim 1, characterized in that, In step S2, the volume ratio of distilled water to anhydrous ethanol is 90:

40.

6. The method for preparing polydopamine nanoparticles loaded with antioxidant active substances according to claim 1, characterized in that, In step S2, the stirring time is 12-48 hours; In step S2, the centrifugation conditions are 13,000 rpm for 15 min.

7. The method for preparing polydopamine nanoparticles loaded with antioxidant active substances according to claim 1, characterized in that, In step S3, the antioxidant active substance is epigallocatechingallate (EGCG).

8. The method for preparing polydopamine nanoparticles loaded with antioxidant active substances according to claim 1, characterized in that, In step S3, the stirring time is 4-12 hours; In step S3, the centrifugation conditions are 13,000 rpm for 15 min.

9. A polydopamine nanoparticle loaded with antioxidant active substances prepared by the method according to any one of claims 1-8.

10. The application of the polydopamine nanoparticles loaded with antioxidant active substances as described in claim 9 in acute kidney injury.

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