Preparation method of polyphenol-aluminum nanoparticles

Abstract

The invention discloses a preparation method of polyphenol-aluminum nanoparticles, which comprises the following steps: dissolving polyphenol in deionized water to obtain a polyphenol solution; dissolving trivalent aluminum salt in deionized water to obtain a trivalent aluminum salt solution; under the condition of stirring at room temperature, slowly dropwise adding a trivalent aluminum salt solution into the polyphenol solution to obtain a mixed solution; a NaOH solution is added into the mixed solution under the ultrasonic and / or microwave condition until turbidity appears, the turbidity of the solution is gradually increased along with the prolonging of the reaction time, and the reaction is stopped when the turbidity of the solution is not increased any more; and carrying out post-treatment to obtain the polyphenol-aluminum nanoparticles. The polyphenol-aluminum nanoparticles disclosed by the invention are simple and easy to obtain and high in biological safety, and have great potential value in biological application.

Description

Technical Field

[0001] The invention belongs to the technical field of functional nano materials, and in particular relates to a method for preparing polyphenol-aluminum nano particles. Background Art

[0002] Aluminum adjuvants, as an important component of commercial vaccines, account for more than 80% of the total number of vaccines that have been marketed and under development in my country. Aluminum adjuvants can strongly adsorb protein antigens, form an antigen reservoir after vaccination, slowly release antigens, prolong the time of antigen action, and enhance the immune efficacy of vaccines (Liu Chao et al., Chinese Journal of Pharmaceutical Industry, 2023, 54: 719-725; Barbateskovic M, etc. Vaccines, 2023, 11: 1763). Aluminum adjuvants mainly include aluminum hydroxide, aluminum phosphate and alum, among which aluminum hydroxide is more widely used. Although aluminum adjuvants have been used clinically for many years, there are still large differences between batches, difficult to control quality during use, and local adverse reactions to the body after vaccination, such as erythema, subcutaneous nodules, granulomas, etc. (Iijima S, etc. PNAS, 2016, 11: 11759-11764; He Peng et al., Chinese Journal of Biological Products, 2016, 29: 654-659). Therefore, there is still much room for performance improvement of aluminum adjuvants.

[0003] At present, the improvement of traditional aluminum adjuvants is mostly focused on nano-ization (Sun B, etc. Encyclopedia of Nanotechnology, 2016: 117-122; Zhao J, etc. Advanced Science, 2024, 11: 2307389) to enhance their immune-enhancing efficacy, but not enough attention is paid to improving their biosafety.

[0004] Polyphenols are a class of compounds naturally found in plants that have strong antioxidant properties and can remove free radicals in the body, thereby reducing oxidative stress and protecting cells from damage. In addition, polyphenols also have anti-inflammatory effects, regulate the immune system, and enhance the body's immune function (Ge Tianrui et al., Journal of Food Safety and Quality, 2024, 15: 176-184). However, there are currently no reports on polyphenol aluminum adjuvants. Summary of the invention

[0005] In order to solve the problems in the existing aluminum adjuvants, the present invention provides a method for preparing polyphenol-aluminum nanoparticles. The trivalent aluminum salt in the polyphenol-aluminum nanomaterial as a component of the traditional aluminum adjuvant can activate the Th2 immune response and activate a higher antibody response level; and the polyphenol has an anti-oxidative stress effect to reduce the side effects at the injection site. The nanomaterial has good stability and safety as a vaccine adjuvant, is easy to operate and obtain, and is easy to mass produce.

[0006] In a first aspect, the present invention provides a method for preparing polyphenol-aluminum nanoparticles, comprising:

[0007] Step 1, dissolving polyphenols in deionized water to obtain a polyphenol solution;

[0008] Step 2, dissolving a trivalent aluminum salt in deionized water to obtain a trivalent aluminum salt solution;

[0009] Step 3, slowly adding a trivalent aluminum salt solution to the polyphenol solution under stirring at room temperature to obtain a mixed solution;

[0010] Step 4: Under ultrasonic and / or microwave conditions, add NaOH solution to the mixed solution until turbidity appears and then stop adding. As the reaction time increases, the turbidity of the solution gradually increases. When the turbidity of the solution no longer increases, stop the reaction and obtain polyphenol-aluminum nanoparticles after purification.

[0011] In some embodiments, in step 1, the polyphenol is one of tannic acid, gallic acid, and chlorogenic acid.

[0012] In some embodiments, in step 2, the trivalent aluminum salt is one of aluminum sulfate, aluminum nitrate, and aluminum chloride.

[0013] In some embodiments, in step 3, the polyphenols and Al in the mixed solution 3+ The molar ratio is 100:1 to 1:100, preferably 1:1.4.

[0014] In some embodiments, in step 4, the ultrasonic treatment conditions are: frequency 20kHz-1000kHz, power 0.05W-500W, and time greater than 1 minute, preferably 40kHz, 60W, and 20 minutes.

[0015] In some embodiments, in step 4, the microwave treatment conditions are: power 1 mW to 2000 W, temperature 0° C. to 100° C., and time greater than 1 minute, preferably 200 W, temperature 30° C., and 30 min.

[0016] In some embodiments, in step 4, ultrasonic treatment and microwave treatment are performed simultaneously.

[0017] In some embodiments, in step 4, the concentration of the NaOH solution is 0.01M to 10M.

[0018] In some embodiments, in step 4, the purification includes washing with deionized water and centrifugation; the centrifugation parameters are 12000 rpm and centrifugation for 5 min.

[0019] In a second aspect, the present invention provides polyphenol-aluminum nanoparticles obtained by the above preparation method.

[0020] In some embodiments, the polyphenol-aluminum nanoparticles have a Zeta potential of -21.1 mV.

[0021] In some embodiments, the hydrated particle size distribution of the polyphenol-aluminum nanoparticles is between 5 nm and 300 nm.

[0022] In a third aspect, the present invention provides the use of the above-mentioned polyphenol-aluminum nanoparticles as a vaccine adjuvant.

[0023] Technical Effects

[0024] The invention provides a method for preparing polyphenol-aluminum nanoparticles. The method comprises the following steps: dissolving polyphenol and trivalent aluminum salt in water respectively and then mixing them. Under ultrasonic and / or microwave conditions, adding NaOH solution to the mixed solution, reacting and purifying to obtain polyphenol-aluminum nanoparticles, and obtaining the polyphenol-aluminum nanoparticles with a yield of more than 85%. The hydrated particle size of the polyphenol-aluminum nanoparticles obtained by the invention is mainly distributed between 200 and 300 nm. Particles larger than 200 nm may stay longer at the injection site or in the body circulation, providing sustained antigen release, while other methods are difficult to obtain nanoparticles of this size. For example, nanoparticles cannot be obtained without ultrasonic and / or microwave treatment. In addition, the Zeta potential of the polyphenol-aluminum nanoparticles obtained by the invention is about -21.1 mV, and the particles are negatively charged, which is a high Zeta potential, meaning that the particle surface carries a relatively strong charge, which will result in a relatively strong electrostatic repulsion between the particles. The repulsion can prevent the particles from aggregating, thereby improving the stability of the suspension.

[0025] The polyphenol-aluminum nanoparticles of the invention have the effect of an adjuvant and reduce the side effects at the injection site. The polyphenol-aluminum nanoparticles are simple and easy to obtain, can be prepared at room temperature, have high biological safety, and have great potential value for biological applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a hydrated particle size distribution diagram of polyphenol-aluminum nanoparticles prepared in a preferred embodiment of the present invention;

[0027] Figure 2 is a Zeta potential diagram of polyphenol-aluminum nanoparticles prepared in a preferred embodiment of the present invention;

[0028] Figure 3 This is a diagram showing the cytotoxicity results of polyphenol-aluminum nanoparticles prepared in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0029] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0030] Example 1

[0031] Dissolve 1g of tannic acid completely in 50mL of deionized water, and the solution becomes yellow and transparent;

[0032] 0.198 g AlCl3·6H2O was completely dissolved in 20 mL deionized water, and the solution was colorless and transparent;

[0033] Under stirring at room temperature, slowly add aluminum chloride solution dropwise to the tannic acid solution;

[0034] Under ultrasonic conditions (frequency 40kHz, power 60W, reaction time 20min), 1M NaOH solution was added to the mixed solution of polyphenol and trivalent aluminum salt until turbidity appeared, and then the addition of NaOH solution was stopped. As the reaction time increased, the turbidity of the solution gradually increased, and the reaction was stopped when the turbidity of the solution no longer increased;

[0035] The mixture was filtered, washed with deionized water, and centrifuged (centrifugation parameters were 12000 rpm, 5 min) to obtain polyphenol-aluminum nanoparticles with a yield of more than 85%.

[0036] Example 2

[0037] Dissolve 1g of tannic acid completely in 50mL of deionized water, and the solution becomes yellow and transparent;

[0038] 0.198 g AlCl3·6H2O was completely dissolved in 20 mL deionized water, and the solution was colorless and transparent;

[0039] Under stirring at room temperature, slowly add aluminum chloride solution dropwise to the tannic acid solution;

[0040] Under microwave conditions (heating time of 6 min, target reaction temperature of 30° C., holding time of 15 min), 1 M NaOH solution was added to the mixed solution of polyphenol and trivalent aluminum salt until turbidity appeared, and then the addition of NaOH solution was stopped. As the reaction time increased, the turbidity of the solution gradually increased, and the reaction was stopped when the turbidity of the solution no longer increased.

[0041] The mixture was filtered, washed with deionized water, and centrifuged (centrifugation parameters were 12000 rpm, 5 min) to obtain polyphenol-aluminum nanoparticles with a yield of more than 85%.

[0042] Example 3

[0043] Dissolve 1g of tannic acid completely in 50mL of deionized water, and the solution becomes yellow and transparent;

[0044] 0.198 g AlCl3·6H2O was completely dissolved in 20 mL deionized water, and the solution was colorless and transparent;

[0045] Under stirring at room temperature, slowly add aluminum chloride solution dropwise to the tannic acid solution;

[0046] Under the conditions of ultrasound (frequency 40kHz, power 60W) and microwave (heating time 6min, target reaction temperature 30°C, holding time 10min), 1M NaOH solution was added to the mixed solution of polyphenol and trivalent aluminum salt until turbidity appeared, then the addition of NaOH solution was stopped, and the turbidity of the solution gradually increased with the extension of reaction time, and the reaction was stopped when the turbidity of the solution no longer increased;

[0047] The mixture was filtered, washed with deionized water, and centrifuged (centrifugation parameters were 12000 rpm, 5 min) to obtain polyphenol-aluminum nanoparticles with a yield of more than 85%.

[0048] Example 4

[0049] The polyphenol-aluminum nanoparticles in Example 1 were characterized by measuring the hydrated particle size and Zeta potential. Figure 1 and 2 As shown, it can be seen that:

[0050] From the hydrated particle size distribution and Zeta potential diagram of the nanoparticles, the hydrated particle size is mainly distributed between 200-300nm, and the Zeta potential of the polyphenol aluminum nanoparticles is about -21.1mV, which is negatively charged.

[0051] Example 5

[0052] C2C12, RAW264.7, and DC2.4 cells were used as cell models to evaluate the cytotoxicity of polyphenol-aluminum nanoparticles. C2C12, RAW264.7, and DC2.4 cells were cultured at a rate of 1×10 4 The cells were seeded at a density of 100 cells per well in a 96-well plate and incubated at 5% CO2 and 37°C for 24 h. Experimental groups: negative group (100 μL culture medium), blank group (100 μL culture medium), adjuvant group, five different concentration gradients of 25, 50, 100, 200, 400 μg . mL -1(100 μL culture medium + adjuvant of Example 1), 6 replicates were set for each gradient, and for the 96-well cell culture plate, incubated for 24 hours under 5% CO2 and 37°C. Washed three times with PBS, and then C2C12 and RAW264.7 were added with a DMEM culture medium (100 μL / well) solution containing 10% (v / v) CCK-8 (10 μL), and cultured in the incubator for 4 hours. DC2.4 was added with a RPMI 1640 culture medium (100 μL / well) solution containing 10% (v / v) CCK-8 (10 μL), and cultured in the incubator for 4 hours. Finally, the absorbance of each well was tested at a wavelength of 450nm using an enzyme marker. The cells treated with DMEM and RPMI 1640 culture media were used as blank controls, and the cell viability was marked as 100%. The results are as follows Figure 3 shown.

[0053] From the cytotoxicity test graph of polyphenol-aluminum nanoparticles, it can be seen that polyphenol-aluminum has no obvious cytotoxicity when the concentration is as high as 100 mg / mL, and can be used as a vaccine adjuvant.

[0054] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for preparing polyphenol-aluminum nanoparticles, comprising: Step 1, dissolving polyphenols in deionized water to obtain a polyphenol solution; Step 2, dissolving a trivalent aluminum salt in deionized water to obtain a trivalent aluminum salt solution; Step 3, slowly adding a trivalent aluminum salt solution to the polyphenol solution under stirring at room temperature to obtain a mixed solution; Step 4: adding NaOH solution to the mixed solution under ultrasonic and / or microwave conditions until turbidity appears and then stopping the addition; as the reaction time increases, the turbidity of the solution gradually increases; and stopping the reaction when the turbidity of the solution no longer increases; and obtaining polyphenol-aluminum nanoparticles after purification.

2. The preparation method according to claim 1, wherein In step 1, the polyphenol is one of tannic acid, gallic acid and chlorogenic acid.

3. The preparation method according to claim 1, wherein In step 2, the trivalent aluminum salt is one of aluminum sulfate, aluminum nitrate and aluminum chloride.

4. The preparation method according to claim 1, wherein In step 3, the polyphenols and Al in the mixed solution 3+ The molar ratio is 100:1 to 1:

100.

5. The preparation method according to claim 1, wherein In step 4, at least one of the following conditions must be met: Ultrasonic treatment: frequency 20kHz~1000kHz, power 0.05W~500W, time greater than 1 minute; Microwave treatment: power 1mW~2000W, temperature 0℃~100℃, time greater than 1 minute.

6. The preparation method according to claim 1, wherein In step 4, the concentration of the NaOH solution is 0.01M to 10M.

7. The polyphenol-aluminum nanoparticles obtained according to the preparation method described in any one of claims 1 to 6.

8. The polyphenol-aluminum nanoparticles according to claim 7, wherein The zeta potential of the polyphenol-aluminum nanoparticles is 0 to -40 mV.

9. The polyphenol-aluminum nanoparticles according to claim 7, wherein The hydrated particle size of the polyphenol-aluminum nanoparticles is distributed between 5nm and 300nm.

10. Use of the polyphenol-aluminum nanoparticles according to any one of claims 7 to 9 as a vaccine adjuvant.

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