PH response nanogel for natural polyphenol delivery and preparation method thereof

Abstract

The invention discloses a preparation method of pH-responsive nanogel for natural polyphenol delivery, which comprises the following steps: respectively dissolving a phenylboronic acid derivative and polyphenol in a dimethyl sulfoxide solution, adding a polyphenol solution into a phenylboronic acid derivative solution, mixing, standing for a period of time at room temperature, filtering, washing, and drying to obtain the pH-responsive nanogel for natural polyphenol delivery. Respectively adding the phenylboronic acid-polyphenol compound, a dimethyl sulfoxide solution and a condensing agent into a hyaluronic acid solution, after all the reagents are dissolved, adjusting the pH value of the solution by using a 2-morpholine ethanesulfonic acid buffer solution, and stirring a reaction mixture; and dialyzing and purifying the obtained mixture, and freeze-drying the dialyzed solution by using a freeze dryer to obtain the HPE nanogel. The invention also discloses a pH response nanogel for natural polyphenol delivery. The phenylboronic acid derivative is added to carry out structural modification on the pH response nanogel, and the pH response nanogel is loaded in the carrier to improve the bioavailability of the pH response nanogel, so that better anti-inflammatory and anti-oxidation effects are realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical nano-drug carriers, and particularly relates to a pH-responsive nanogel for natural polyphenol delivery and a preparation method thereof. Background Art

[0002] The pathophysiology of many serious diseases is related to inflammation and inflammation-induced oxidative stress. The excessive accumulation and activation of inflammatory cells can lead to the overproduction of a variety of bioactive substances, including pro-inflammatory cytokines and reactive oxygen species (ROS). The dysregulation of inflammation and the imbalance of reactive oxygen species may lead to chronic tissue damage and organ dysfunction. Recent studies have shown that markers of inflammation and oxidative stress are increased in many different diseases, including cancer, cardiovascular disease, metabolic disorders (including diabetes), chronic kidney disease, and neurodegenerative diseases.

[0003] Understanding the fundamental role of inflammation and oxidative stress in the occurrence and development of the above diseases has prompted people to successfully use various anti-inflammatory and antioxidants to treat these diseases. Polyphenols are a class of compounds composed of two or more phenolic structural units, which are widely distributed in plants and marine organisms. In recent years, polyphenols, as natural bioactive substances, have received extensive attention due to their significant antioxidant, anti-inflammatory, and anti-cancer properties.

[0004] However, in the actual application process, these bioactive substances are easily affected by environmental factors (such as temperature, light, pH value, and salt ion concentration), resulting in the loss of their functional properties, such as poor stability and rapid release. For example, alone epigallocatechin gallate (EGCG) has its bioactivity reduced due to easy oxidation during use, and then loses its original efficacy, which limits its application scope (Colloids and Surfaces B: Biointerfaces, DOI: 10.1016 / j.colsurfb.2020.110802). Therefore, it is particularly important to modify the structure of natural polyphenols to improve their bioavailability. Summary of the Invention

[0005] In order to overcome the above deficiencies of the prior art, the purpose of the present invention is to provide a preparation method of a pH-responsive nanogel for natural polyphenol delivery, which improves its bioavailability by adding a phenylboronic acid derivative for structural modification and loading it on a carrier, so as to achieve better anti-inflammatory and antioxidant effects.

[0006] Another purpose of the present invention is to provide a pH-responsive nanogel for natural polyphenol delivery.

[0007] The purpose of the present invention is achieved by the following technical solutions: A preparation method of a pH-responsive nanogel for natural polyphenol delivery, comprising the following steps: (1) Dissolve a phenylboronic acid derivative and a polyphenol in dimethyl sulfoxide solution respectively to obtain a polyphenol solution and a phenylboronic acid derivative solution; (2) After adding the polyphenol solution to the phenylboronic acid derivative solution, mix and let stand at room temperature for a period of time to obtain a phenylboronic acid-polyphenol complex, and the molar ratio of phenylboronic acid to polyphenol is 6:1 to 10:1; (3) Dissolve hyaluronic acid in deionized water to prepare a solution with a certain concentration. Next, add the phenylboronic acid-polyphenol complex, dimethyl sulfoxide solution and a condensing agent to the hyaluronic acid solution respectively. After all the reagents are dissolved, adjust the pH of the solution with 2-morpholinoethanesulfonic acid buffer, and then stir the reaction mixture at room temperature; (4) Purify the obtained mixture by dialysis, dialyze with deionized water at room temperature to remove dimethyl sulfoxide, change the water twice a day, and lyophilize the dialyzed solution with a lyophilizer to obtain HPE nanogel, and the HPE nanogel is stored in the dark before use.

[0008] Preferably, the molar ratio of phenylboronic acid to polyphenol is 8:1.

[0009] Preferably, the molecular weight of the hyaluronic acid is 3 to 800 kDa.

[0010] Preferably, the phenylboronic acid derivative in step (1) is one or more of 2-aminophenylboronic acid, 3-aminophenylboronic acid, 4-aminophenylboronic acid, 3-acrylaminophenylboronic acid, 2-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 4-carboxyphenylboronic acid.

[0011] Preferably, the polyphenol in step (1) includes one or more of baicalin, apigenin, luteolin, myricetin, gallic acid, catechin, epicatechin, tannic acid, ellagic acid, quercetin, rosmarinic acid, proanthocyanidin, genistein, naringenin, carminic acid, rutin.

[0012] Preferably, in step (1), the phenylboronic acid derivative is dissolved in dimethyl sulfoxide solution with a concentration of 1 M.

[0013] Preferably, in step (1), the polyphenol is dissolved in dimethyl sulfoxide solution with a concentration of 0.125 M.

[0014] Preferably, in step (2), the mixing time is 5 to 20 min, and the standing time is 30 to 60 min.

[0015] Preferably, in step (3), the concentration of the hyaluronic acid solution is 0.1% to 5%.

[0016] Preferably, the mass ratio of the amount of the phenylboronic acid-polyphenol complex described in step (3) to the hyaluronic acid is 1:20 to 1:5, and the mass ratio of the condensing agent to the hyaluronic acid is 1:100 to 1:50.

[0017] Preferably, the condensing agent described in step (3) includes one or more of dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, O-(7-azabenzotriazol-1-yl)-bis(dimethylamino)carbenium hexafluorophosphate, O-(benzotriazol-1-yl)-bis(dimethylamino)carbenium hexafluorophosphate, O-(5-chlorobenzotriazol-1-yl)-bis(dimethylamino)carbenium hexafluorophosphate, O-(benzotriazol-1-yl)-bis(dimethylamino)carbenium tetrafluoroborate, O-(N-succinimido)-bis(dimethylamino)carbenium tetrafluoroborate, O-(N-endo-5-norbornene-2,3-dicarboximide)-bis(dimethylamino)carbenium tetrafluoroborate, diphenylphosphoryl chloride, diethyl cyanophosphonate, diphenylphosphoryl azide (DPPA), thiodimethylphosphoryl azide, and bis(2-oxo-3-oxazolidinyl)phosphoryl chloride.

[0018] Preferably, the total concentration of the 2-morpholinoethanesulfonic acid (MES) buffer solution is 1 mol / L and pH = 5.5.

[0019] Preferably, in step (3), the pH of the solution is adjusted to 4.5 to 6.5.

[0020] More preferably, in step (3), the pH of the solution is adjusted to 5.

[0021] Preferably, after dialysis purification in step (4), the molecular weight cut-off is 10 kDa.

[0022] A pH-responsive nanogel for natural polyphenol delivery, with a pH-responsive range of 4.5 to 6.5.

[0023] The present invention has the following advantages and beneficial effects compared with the prior art: (1) The present invention combines a phenylboronic acid derivative with a polyphenol substance to prepare a phenylboronic acid-polyphenol complex, uses hyaluronic acid as the nanogel matrix material, and uses a condensing agent to cause a chemical reaction between the phenylboronic acid-polyphenol complex and hyaluronic acid to form a hyaluronic acid-phenylboronic acid-polyphenol complex. A nanogel is prepared by an emulsification method to improve the stability of natural polyphenols, and the anti-inflammatory and antioxidant effects of polyphenols are improved by the controlled release effect of the nanogel.

[0024] (2) The present invention utilizes the property that phenylboronic acid derivatives combine with polyols and diphenol compounds to reversibly form cyclic borate esters, enabling phenylboronic acid and polyphenols to form borate esters in the drug delivery system to improve their stability; meanwhile, the borate ester bond is stable in a physiologically neutral environment and dissociates in an inflammatory microenvironment (pH 5.0 - 6.5), thereby achieving targeted release.

[0025] (3) The present invention utilizes polyphenols to achieve anti-inflammatory and antioxidant functions. The polyphenol nanogel has excellent biocompatibility and a long-lasting release time, and can continuously and effectively capture excess reactive oxygen species, thereby protecting cells from reactive oxygen species-mediated death and bioactivity inhibition.

[0026] (4) The preparation method of the present invention has universality, the preparation process is simple, green, and suitable for industrial production; the prepared nanogel has a complete structure and good dispersibility, and has potential application value in the protection, release, and delivery of active substances for sensitive cosmetics, medicinal active substances, and edible nutrients, etc. Brief Description of the Drawings

[0027] Figure 1 It is a schematic structural diagram of a pH-responsive nanogel for natural polyphenol delivery.

[0028] Figure 2 It is a scanning electron micrograph of the nanogels of Examples 1 - 4 and Comparative Examples 1 - 4.

[0029] Figure 3 It is an in vitro release curve of EGCG in the nanogels of Examples 1, 4 and Comparative Example 1.

[0030] Figure 4 It is the DPPH radical scavenging rate of the nanogels of Examples 1, 4 and Comparative Example 1.

[0031] Figure 5 It is the effect of the nanogels of Examples 1, 4 and Comparative Example 1 on the concentration of TNF-α. Detailed Description of the Specific Embodiments

[0032] The following further describes the invention object of the present invention in detail with reference to the drawings and specific embodiments. The embodiments cannot be elaborated one by one here, but the implementation manners of the present invention are not limited to the following embodiments.

[0033] Example 1 Preparation of phenylboronic acid-polyphenol complex: Dissolve 0.279 g of 3-aminophenylboronic acid (PBA) in dimethyl sulfoxide solution at a concentration of 1 M. Dissolve 0.1134 g of epigallocatechin gallate (EGCG) in dimethyl sulfoxide solution at a concentration of 0.125 M, and make the molar ratio of PBA to EGCG 8:1. Add the EGCG solution to the PBA solution, vortex for 15 min with a vortex mixer, and let it stand at room temperature for 30 min to promote the formation of the PBA-EGCG complex.

[0034] Preparation of hyaluronic acid-phenylboronic acid-polyphenol nanogel: Dissolve 1.5 g of hyaluronic acid (HA, 3 kDa) in 500 ml of deionized water. Next, add the prepared PBA-EGCG complex, 2 ml of dimethyl sulfoxide solution, and the condensing agent 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride to the hyaluronic acid solution respectively. After all the reagents are dissolved, adjust the pH of the solution to 5.0 with MES buffer (1 M, pH = 5.5), and stir at room temperature for 3 days for the reaction. The resulting mixture is purified by dialysis (molecular weight cut-off 10 kDa), dialyzed against deionized water at room temperature for 3 days to remove dimethyl sulfoxide and unreacted raw materials, and the water is changed three times a day. The dialyzed solution is freeze-dried using a freeze dryer to obtain nanogel. The nanogel is stored in the dark before use.

[0035] Example 2 Preparation of phenylboronic acid-polyphenol complex: Dissolve 0.279 g of 3-aminophenylboronic acid (PBA) in dimethyl sulfoxide solution at a concentration of 1 M. Dissolve 0.1134 g of epigallocatechin gallate (EGCG) in dimethyl sulfoxide solution at a concentration of 0.125 M, and make the molar ratio of PBA to EGCG 8:1. Add the EGCG solution to the PBA solution, vortex for 15 min with a vortex mixer, and let it stand at room temperature for 30 min to promote the formation of the PBA-EGCG complex.

[0036] Preparation of hyaluronic acid-phenylboronic acid-polyphenol nanogel: Dissolve 1.5 g of hyaluronic acid (HA, 30 - 45 kDa) in 500 ml of deionized water. Next, add the prepared PBA - EGCG complex, 2 ml of dimethyl sulfoxide solution, and the condensing agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride to the hyaluronic acid solution respectively. After all the reagents are dissolved, adjust the pH of the solution to 5.0 using MES buffer (1 M, pH = 5.5), and stir at room temperature for 3 days for the reaction. The resulting mixture is purified by dialysis (molecular weight cut-off is 10 kDa), dialyze against deionized water at room temperature for 3 days to remove dimethyl sulfoxide and unreacted raw materials, changing the water three times a day. The dialyzed solution is freeze-dried using a freeze dryer to obtain the nanogel. The nanogel is stored in the dark before use.

[0037] Example 3 Preparation of phenylboronic acid - polyphenol complex: Dissolve 0.279 g of 3-aminophenylboronic acid (PBA) in dimethyl sulfoxide solution at a concentration of 1 M, and dissolve 0.1134 g of epigallocatechin gallate (EGCG) in dimethyl sulfoxide solution at a concentration of 0.125 M, so that the molar ratio of PBA to EGCG is 8:1. Add the EGCG solution to the PBA solution, vortex for 15 min using a vortex mixer, and let stand at room temperature for 30 min to promote the formation of the PBA - EGCG complex.

[0038] Preparation of hyaluronic acid - phenylboronic acid - polyphenol nanogel: Dissolve 1.5 g of hyaluronic acid (HA, 100 - 200 kDa) in 500 ml of deionized water. Next, add the prepared PBA - EGCG complex, 2 ml of dimethyl sulfoxide solution, and the condensing agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride to the hyaluronic acid solution respectively. After all the reagents are dissolved, adjust the pH of the solution to 5.0 using MES buffer (1 M, pH = 5.5), and stir at room temperature for 3 days for the reaction. The resulting mixture is purified by dialysis (molecular weight cut-off is 10 kDa), dialyze against deionized water at room temperature for 3 days to remove dimethyl sulfoxide and unreacted raw materials, changing the water three times a day. The dialyzed solution is freeze-dried using a freeze dryer to obtain the nanogel. The nanogel is stored in the dark before use.

[0039] Example 4 Preparation of phenylboronic acid - polyphenol complex: Dissolve 0.279 g of 3-aminophenylboronic acid (PBA) in dimethyl sulfoxide solution at a concentration of 1 M. Dissolve 0.1134 g of epigallocatechin gallate (EGCG) in dimethyl sulfoxide solution at a concentration of 0.125 M, and make the molar ratio of PBA to EGCG 8:1. Add the EGCG solution to the PBA solution, vortex for 15 min using a vortex mixer, and let it stand at room temperature for 30 min to promote the formation of the PBA-EGCG complex.

[0040] Preparation of hyaluronic acid-phenylboronic acid-polyphenol nanogel: Dissolve 1.5 g of hyaluronic acid (HA, 400 - 800 kDa) in 500 ml of deionized water. Next, add the prepared PBA-EGCG complex, 2 ml of dimethyl sulfoxide solution, and the condensing agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride to the hyaluronic acid solution respectively. After all the reagents are dissolved, adjust the pH of the solution to 5.0 using MES buffer (1 M, pH = 5.5), and stir at room temperature for 3 days for the reaction. The resulting mixture is purified by dialysis (molecular weight cut-off is 10 kDa), dialyzed against deionized water at room temperature for 3 days to remove dimethyl sulfoxide and unreacted raw materials, and change the water three times a day. The dialyzed solution is freeze-dried using a freeze dryer to obtain the nanogel. The nanogel is stored in the dark before use.

[0041] Comparative Example 1 (physical adsorption of EGCG by nanogel) Dissolve 1.5 g of hyaluronic acid (HA, 3000 Da) in 500 ml of deionized water. Dissolve 0.1134 g of epigallocatechin gallate (EGCG) in dimethyl sulfoxide solution at a concentration of 0.125 M. Next, add the dimethyl sulfoxide solution of EGCG and the condensing agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride to the hyaluronic acid solution respectively. After all the reagents are dissolved, adjust the pH of the solution to 5.0 using MES buffer (1 M, pH = 5.5), and stir at room temperature for 3 days for the reaction. The resulting mixture is purified by dialysis (molecular weight cut-off is 10 kDa), dialyzed against deionized water at room temperature for 3 days to remove dimethyl sulfoxide and unreacted raw materials, and change the water three times a day. The dialyzed solution is freeze-dried using a freeze dryer to obtain the nanogel. The nanogel is stored in the dark before use.

[0042] Comparative Example 2 Dissolve 1.5 g of hyaluronic acid (HA, 30 - 45 kDa) in 500 ml of deionized water. Dissolve 0.1134 g of epigallocatechin gallate (EGCG) in dimethyl sulfoxide solution at a concentration of 0.125 M. Next, add the dimethyl sulfoxide solution of EGCG and the condensing agent 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride to the hyaluronic acid solution respectively. After all the reagents are dissolved, adjust the pH of the solution to 5.0 using MES buffer (1 M, pH = 5.5), and stir at room temperature for 3 days for reaction. The resulting mixture is purified by dialysis (molecular weight cut-off is 10 kDa), and dialyzed against deionized water at room temperature for 3 days to remove dimethyl sulfoxide and unreacted raw materials, changing the water three times a day. The dialyzed solution is freeze-dried using a freeze dryer to obtain nano-gel. The nano-gel is stored in the dark before use.

[0043] Comparative Example 3 Dissolve 1.5 g of hyaluronic acid (HA, 100 - 200 kDa) in 500 ml of deionized water. Dissolve 0.1134 g of epigallocatechin gallate (EGCG) in dimethyl sulfoxide solution at a concentration of 0.125 M. Next, add the dimethyl sulfoxide solution of EGCG and the condensing agent 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride to the hyaluronic acid solution respectively. After all the reagents are dissolved, adjust the pH of the solution to 5.0 using MES buffer (1 M, pH = 5.5), and stir at room temperature for 3 days for reaction. The resulting mixture is purified by dialysis (molecular weight cut-off is 10 kDa), and dialyzed against deionized water at room temperature for 3 days to remove dimethyl sulfoxide and unreacted raw materials, changing the water three times a day. The dialyzed solution is freeze-dried using a freeze dryer to obtain nano-gel. The nano-gel is stored in the dark before use.

[0044] Comparative Example 4 Dissolve 1.5 g of hyaluronic acid (HA, 400 - 800 kDa) in 500 ml of deionized water. Dissolve 0.1134 g of epigallocatechin gallate (EGCG) in dimethyl sulfoxide solution at a concentration of 0.125 M. Next, add the dimethyl sulfoxide solution of EGCG and the condensing agent 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride to the hyaluronic acid solution respectively. After all the reagents are dissolved, adjust the pH of the solution to 5.0 using MES buffer (1 M, pH = 5.5), and stir at room temperature for 3 days for reaction. The resulting mixture is purified by dialysis (molecular weight cut-off is 10 kDa), and dialyzed against deionized water at room temperature for 3 days to remove dimethyl sulfoxide and unreacted raw materials, changing the water three times a day. The dialyzed solution is freeze-dried using a freeze dryer to obtain nano-gel. The nano-gel is stored in the dark before use.

[0045] Performance Characterization: (1)Scanning Electron Microscope The morphology of the nanogels of Examples 1-4 and Comparative Examples 1-4 was observed using a high-resolution field emission scanning electron microscope (Merlin, Zeiss, Germany). After freeze-drying the nanogels, they were adhered to the sample stage with conductive glue, and after sputtering with gold for 1 min, the surface morphology of the nanogels was observed using a scanning electron microscope.

[0046] (2)Particle Size Measurement The nanogels of Examples 1-4 and Comparative Examples 1-4 were suspended in PBS buffer at a concentration of 1 mg / mL, and vortexed for 1 minute to ensure complete dispersion of the nanogels and minimize potential aggregates. The particle size and particle size distribution (PDI) of the samples were measured at room temperature using a multi-angle particle size and high-sensitivity Zeta potential analyzer (NanoBrook Omni, Brookhaven, USA).

[0047] (3)Encapsulation Efficiency and Drug Loading Measurement The nanogels prepared in Examples 1-4 and Comparative Examples 1-4 were dissolved in deionized water to make solutions of different concentrations. The absorbance of all samples was detected at 273 nm using a UV spectrophotometer (UV-2600, Shimadzu, Japan). The free EGCG content in the samples was calculated by the ultrafiltration method. The samples were placed in ultrafiltration concentration centrifugal tubes and centrifuged at 4000 rpm for 20 minutes in a high-speed refrigerated centrifuge at 4°C. The supernatant was collected to detect the free content of EGCG. The values of encapsulation efficiency (EE) and drug loading (DL) were determined by the following formulas:

[0048] Where, Wa is the total mass of the initially added drug; Wb is the mass of EGCG in the nanogel; Ws is the total mass of the nanogel.

[0049] (4)In Vitro Drug Release Solutions of a certain concentration were prepared from the nanogels of Examples 1, 4 and Comparative Example 1. 0.5 mL of the nanogel dispersion was taken and placed in a dialysis bag (MWCO: 14 kDa) and immersed in 5 mL of PBS solution (pH = 7.4) at 37°C; another 0.5 mL of the dispersion of the nanogel of Example 1 was placed in a dialysis bag (MWCO: 14 kDa) and then immersed in 5 mL of PBS solution with pH = 5.0. The experiment was carried out in a shaker at 37°C. At each preset time point, 2 mL of the buffer solution was taken out, and the corresponding volume of the buffer solution was replenished to keep the volume of the buffer solution constant. The content of EGCG in the release solution was determined by a UV-visible spectrophotometer at an absorption wavelength of 275 nm. All experiments were repeated three times to obtain Figure 3 .

[0050] (5)Antioxidant performance test The antioxidant activities of the nanogels of Example 1, Example 4 and Comparative Example 1 were evaluated by scavenging the content of 1,1-diphenyl-2-picrylhydrazyl (DPPH). First, a 100 mM ethanol solution of DPPH was prepared. The nanogel samples of Example 1, Example 4 and Comparative Example 1 were dissolved in deionized water to form a 0.1 mg / ml solution. Then, 3 mL of the DPPH solution was added to the nanogel samples and stirred in the dark for half an hour. After the reaction, the mixture was centrifuged at 2000 rpm for five minutes, and the supernatant was aspirated. The DPPH solution without adding nanogel was used as a control group. The absorbance of the supernatant at 517 nm was detected using a microplate reader (Varioskan Flash 3001, Thermo-Fisher), and the DPPH scavenging rate was calculated. The calculation formula is as follows:

[0051] where Ab and Ah are the absorbances of the DPPH solution without adding nanogel and the DPPH solution adding nanogel at 517 nm, respectively.

[0052] (6)Anti-inflammatory performance test An in vitro macrophage inflammation model was constructed, with lipopolysaccharide (LPS) as an inflammatory inducer. By monitoring the release of tumor necrosis factor α (TNF-α) by macrophages, the anti-inflammatory effect of the nanogel was quantitatively evaluated. Raw264.7 macrophages were selected as the experimental subjects, and the cells were inoculated into a 12-well cell culture plate at a density of about 1×10 5 cells per well and cultured overnight. The next day, the original culture medium was removed, and 500 μL of complete medium containing 0.5 mg / mL of the nanogels of Example 1, Example 4 and Comparative Example 1 was added to each well and incubated for 5 hours. After incubation, 500 μL of complete medium was added to the cells and cultured for another 19 hours. After the culture was completed, the cells were washed three times with PBS. After washing, 500 μL of LPS solution with a concentration of 0.5 μg / mL was added to each well of the positive control group (PC) and the experimental group, and the negative control group (NC) was added with an equal volume of complete medium as a reference for normal cell growth. After the addition, the culture plate was placed in an incubator and co-incubated for 3 hours. After co-incubation, the cell supernatant was collected and centrifuged, and the TNF-α in the supernatant was quantitatively analyzed using an ELISA kit.

[0053]

[0054]

[0055] Table 1 shows the particle size and polydispersity coefficient of the nano-gels in the examples and comparative examples. From Table 1 and Figure 2 it can be seen that the particle size of the pH-responsive nano-gels of the present invention and the nano-gels of the comparative examples does not differ much, and the particle size of the nano-gels is greatly affected by the molecular weight of the matrix material hyaluronic acid.

[0056] Table 2 shows the encapsulation efficiency (%) and drug loading (%) of the nano-gels in the examples and comparative examples. From Table 2, it can be seen that compared with the comparative examples, the pH-responsive nano-gels added with phenylboronic acid derivatives have higher encapsulation efficiency and drug loading than the physical adsorption of EGCG by nano-gels. When the molecular weight of hyaluronic acid is 3 kDa, the encapsulation efficiency is as high as 79.31% and the drug loading is 7.87%.

[0057] Figure 3 is the in vitro release curve of EGCG in the nano-gels of Example 1, Example 4 and Comparative Example 1. From Figure 3 it can be seen that the nano-gels prepared by the technical solutions protected by the present invention have pH responsiveness. The drug release rate of Example 1 under acidic conditions (pH = 5.0) is significantly increased compared with that under neutral conditions (pH = 7.4).

[0058] Figure 4 and Figure 5 are the DPPH radical scavenging rates of the nano-gels of Example 1, Example 4 and Comparative Example 1 respectively. From Figure 4 it can be seen that the pH-responsive nano-gels prepared in Example 1 have a higher scavenging rate of free radicals than Example 4 and Comparative Example 1, and have strong antioxidant ability.

[0059] Figure 5 is the influence diagram of the nano-gels of Example 1, Example 4 and Comparative Example 1 on the TNF-α concentration. From Figure 5 it can be seen that the pH-responsive nano-gels prepared in Example 1 have a high inhibition rate on the TNF-α concentration and have excellent anti-inflammatory effects.

[0060] The above specific embodiments are the preferred embodiments of the present invention and cannot limit the present invention. Any other changes or other equivalent replacement methods made without departing from the technical solutions of the present invention are included in the protection scope of the present invention.

Claims

1. A preparation method of a pH-responsive nanogel for natural polyphenol delivery, characterized in that, It includes the following steps: (1) Dissolve the phenylboronic acid derivative and polyphenol in dimethyl sulfoxide solution respectively to obtain a polyphenol solution and a phenylboronic acid derivative solution; (2) After adding the polyphenol solution to the phenylboronic acid derivative solution, mix and let stand at room temperature for a period of time to obtain a phenylboronic acid-polyphenol complex, and the molar ratio of phenylboronic acid to polyphenol is 6:1 to 10:1; (3) Dissolve hyaluronic acid in deionized water to prepare a solution with a certain concentration. Next, add the phenylboronic acid-polyphenol complex, dimethyl sulfoxide solution and condensing agent to the hyaluronic acid solution respectively. After all the reagents are dissolved, adjust the pH of the solution with 2-morpholinoethanesulfonic acid buffer, and then stir the reaction mixture at room temperature; (4) Purify the obtained mixture by dialysis. Dialyze with deionized water at room temperature to remove dimethyl sulfoxide, change the water twice a day, and freeze-dry the dialyzed solution with a freeze dryer to obtain HPE nanogel, and the HPE nanogel is stored in the dark before use.

2. The preparation method of the pH-responsive nanogel for natural polyphenol delivery according to claim 1, characterized in that, The molecular weight of the hyaluronic acid is 3 to 800 kDa.

3. The preparation method of the pH-responsive nanogel for natural polyphenol delivery according to claim 1, characterized in that, The phenylboronic acid derivative in step (1) is one or more of 2-aminophenylboronic acid, 3-aminophenylboronic acid, 4-aminophenylboronic acid, 3-acrylaminophenylboronic acid, 2-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 4-carboxyphenylboronic acid.

4. The preparation method of the pH-responsive nanogel for natural polyphenol delivery according to claim 1, characterized in that, The polyphenol in step (1) includes one or more of baicalin, apigenin, luteolin, myricetin, gallic acid, catechin, epicatechin, tannic acid, ellagic acid, quercetin, rosmarinic acid, procyanidin, genistein, naringenin, carminic acid, rutin.

5. The preparation method of the pH-responsive nanogel for natural polyphenol delivery according to claim 1, characterized in that, The mixing time in step (2) is 5 to 20 min, and the standing time is 30 to 60 min.

6. The preparation method of the pH-responsive nanogel for natural polyphenol delivery according to claim 1, wherein, The concentration of the hyaluronic acid solution in step (3) is 0.1% to 5%.

7. The preparation method of the pH-responsive nanogel for natural polyphenol delivery according to claim 1, wherein, The mass ratio of the dosage of the phenylboronic acid-polyphenol complex to the hyaluronic acid in step (3) is 1:20 to 1:5, and the mass ratio of the condensing agent to the hyaluronic acid is 1:100 to 1:

50.

8. The preparation method of the pH-responsive nanogel for natural polyphenol delivery according to claim 1, characterized in that, The condensing agent in step (3) includes one or more of dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, O-(7-azabenzotriazol-1-yl)-bis(dimethylamino)carbenium hexafluorophosphate, O-(benzotriazol-1-yl)-bis(dimethylamino)carbenium hexafluorophosphate, O-(5-chlorobenzotriazol-1-yl)-bis(dimethylamino)carbenium hexafluorophosphate, O-(benzotriazol-1-yl)-bis(dimethylamino)carbenium tetrafluoroborate, O-(N-succinimido)-bis(dimethylamino)carbenium tetrafluoroborate, O-(N-endo-5-norbornene-2,3-dicarboximide)-bis(dimethylamino)carbenium tetrafluoroborate, diphenylphosphoryl chloride, diethyl cyanophosphonate, diphenylphosphoryl azide (DPPA), thiodimethylphosphoryl azide, bis(2-oxo-3-oxazolidinyl)phosphoryl chloride.

9. The preparation method of the pH-responsive nanogel for natural polyphenol delivery according to claim 1, characterized in that, The molecular weight cut-off after dialysis purification in step (4) is 10 kDa.

10. A pH-responsive nanogel for natural polyphenol delivery prepared by any one of claims 1-9, characterized in that, The pH response range is 4.5 to 6.5.

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