A kind of sodium pyruvate sustained-release gel nanoparticle based on redox response and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a sodium pyruvate sustained-release gel nanoparticle based on redox response, and a preparation method and application thereof. The preparation method comprises: adding modified gellan gum containing thiol and catechol groups, gellan gum modified by phenylboronic acid and sodium pyruvate into water to obtain a mixed solution; under the protection of an inert gas, adding a divalent metal salt and potassium persulfate to the mixed solution, stirring for 3-5 h, and after the reaction is completed, centrifuging, collecting the precipitate, washing, and drying, to obtain. Experimental results show that the sodium pyruvate sustained-release gel nanoparticles prepared by the present invention can achieve controlled release of drugs, reduce the toxic and side effects caused by sudden release of drugs, improve drug efficacy, and improve bioavailability.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a sodium pyruvate sustained-release gel nanoparticle based on redox response, and a preparation method and application thereof. Background Art

[0002] As a multifunctional compound, sodium pyruvate is often used as an alternative carbon source in cell culture, showing good biocompatibility and low toxic side effects. With the deepening of scientific research, the application areas of sodium pyruvate are constantly expanding, including the treatment of chronic obstructive pulmonary disease, asthma, as a free radical inhibitor, and potential applications in the treatment of rhinitis and hemorrhagic shock. Especially in the treatment of lung diseases, sodium pyruvate has gradually shown its unique advantages and has become one of the candidate drugs for the prevention and treatment of lung diseases.

[0003] Although sodium pyruvate has the above advantages and can be used to treat pneumonia, the traditional method of sodium pyruvate administration has limitations, such as the degradation or deterioration of the drug during storage and use, making it difficult to maintain a relatively stable drug concentration in the lungs, thus affecting the therapeutic effect. In order to overcome these challenges, researchers began to explore the possibility of encapsulating sodium pyruvate in nano drug carriers in order to improve the stability and bioavailability of the drug.

[0004] Nano drug carriers generally refer to particles with a size of 10-1000 nm, composed of natural or synthetic polymers, and have become a research hotspot in the field of drug delivery. These nanoparticles can significantly enhance the targeting and permeability of drugs through their unique size effects and surface properties, while protecting drugs from the influence of the external environment. In order to further improve the intelligence level of nano drug carriers, researchers have developed a variety of stimuli-responsive nanocarriers, including redox-responsive nanocarriers. The redox state in lung diseases and tumor microenvironments is significantly different from that in normal tissues, and is rich in reducing substances (such as glutathione GSH). Redox-responsive nanocarriers can intelligently sense changes in these microenvironments and adjust drug release behavior accordingly to achieve precise drug release.

[0005] Therefore, it is necessary to design a highly stable drug carrier that can utilize the redox state in lung diseases and tumor microenvironments as a means of stimulation to achieve efficient intracellular entry while rapidly releasing sodium pyruvate into the cell to improve efficacy. Summary of the invention

[0006] One of the purposes of the present invention is to provide a method for preparing sodium pyruvate sustained-release gel nanoparticles based on redox response.

[0007] To achieve one of the purposes of the present invention, the technical solution adopted by the present invention is:

[0008] The preparation method of sodium pyruvate sustained-release gel nanoparticles based on redox response comprises the following steps: adding modified gellan gum containing thiol and catechol groups, gellan gum modified by phenylboronic acid and sodium pyruvate into water to obtain a mixed solution; adding divalent metal salt and potassium persulfate into the mixed solution under the protection of inert gas, stirring for 3-5 hours, centrifuging after the reaction is completed, collecting the precipitate, washing and drying, and obtaining the sodium pyruvate sustained-release gel nanoparticles based on redox response.

[0009] Furthermore, the mass ratio of the modified gellan gum containing thiol and catechol groups, the gellan gum modified by phenylboric acid and sodium pyruvate is (1-1.5):1:(1-2), and the mass ratio of the gellan gum modified by phenylboric acid, the divalent metal salt and potassium persulfate is 1:(0.2-0.5):(0.005-0.01).

[0010] Furthermore, the preparation method of the modified gellan gum containing thiol and catechol groups comprises:

[0011]

[0012] EDC is added to the gellan gum aqueous solution, and the mixture is stirred to obtain a mixed solution; NHS, β-mercaptoethylamine and dopamine are added to the mixed solution, and the pH is adjusted to 4.0-4.5 for reaction; after the reaction is completed, the reaction solution is treated to obtain the modified gellan gum containing thiol and catechol groups.

[0013] Furthermore, the mass ratio of the gellan gum, β-mercaptoethylamine and dopamine is 1:(0.5-1):(0.5-1); the temperature for adjusting the pH to 4.0-4.5 and conducting the reaction is 25-30°C and the reaction time is 45-55 h.

[0014] Furthermore, the specific operation of the treatment is: adjusting the pH of the reaction solution to 5.0-5.5, adding ethanol to the reaction solution to precipitate the product, collecting the product for washing and freeze-drying to obtain the modified gellan gum containing thiol and catechol groups; the freeze-drying temperature is -55~-50°C, and the freeze-drying time is 25-30 h.

[0015] Furthermore, the preparation method of the phenylboronic acid modified gellan gum comprises:

[0016]

[0017] Tetrabutylammonium bromide is added to an organic solution of 4-chlorophenylboric acid to obtain an organic phase; the organic phase is added to a gellan gum aqueous solution, and stirred for reaction at a pH of 8.0-8.5; after the reaction is completed, the reaction solution is post-treated to obtain the phenylboric acid-modified gellan gum.

[0018] Furthermore, the mass ratio of the 4-chlorophenylboric acid, gellan gum and tetrabutylammonium bromide is 1:(1-2):(0.1-0.2).

[0019] Furthermore, the stirring reaction temperature is 40-50° C., and the stirring reaction time is 6-10 h. The specific operation of the post-treatment is: adding ethanol to the reaction solution to precipitate the product, collecting the product and washing and freeze-drying it to obtain the phenylboronic acid-modified gellan gum.

[0020] Furthermore, the organic solution of 4-chlorophenylboronic acid is a DMSO solution of 4-chlorophenylboronic acid.

[0021] Furthermore, the divalent metal salt is Ca-containing 2+ Mg 2+ 、Zn 2+ One or more salts of.

[0022] The second object of the present invention is to provide a sodium pyruvate sustained-release gel nanoparticle based on redox response, which can achieve a controlled slow release of sodium pyruvate.

[0023] To achieve the second purpose of the present invention, the technical solution adopted by the present invention is:

[0024] Sodium pyruvate sustained-release gel nanoparticles based on redox response prepared according to the above preparation method.

[0025] The third object of the present invention is to provide a method for applying sodium pyruvate sustained-release gel nanoparticles based on redox response in a controlled drug release system.

[0026] To achieve the third purpose of the present invention, the technical solution adopted by the present invention is:

[0027] Application of sodium pyruvate sustained-release gel nanoparticles based on redox response in controlled drug release system.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention first prepares modified gellan gum containing thiol and catechol groups and gellan gum modified by phenylboronic acid, and then 2+ As a chelating agent, KPS as an oxidant, the two modified gellan gums form disulfide bonds and phenylboronic acid ester bonds through cross-linking. The formation of disulfide bonds and phenylboronic acid ester bonds endows the final nanoparticles with redox sensitivity. 2+ Chelating with the carboxyl group of gellan gum makes the structure of the nanoparticles more stable. The drug sodium pyruvate is added during the cross-linking process, and finally the gel nanoparticles encapsulating sodium pyruvate are formed.

[0030] 2. The sodium pyruvate sustained-release gel nanoparticles prepared by the present invention have redox-responsive disulfide bonds and phenylboronic acid ester bonds in their structure. The nanoparticles can utilize the unique redox state in the simulated tumor microenvironment as a stimulus to achieve controlled release of the drug sodium pyruvate, reduce toxic side effects caused by sudden drug release, and improve drug efficacy and bioavailability. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a SEM image of the nanoparticles prepared in Comparative Example 2 of the present invention;

[0032] Figure 2 This is a SEM image of the nanoparticles prepared in Comparative Example 1 of the present invention;

[0033] Figure 3 This is a SEM image of the sodium pyruvate sustained-release gel nanoparticles prepared in Example 1 of the present invention;

[0034] Figure 4 This is a SEM image of the sodium pyruvate sustained-release gel nanoparticles prepared in Example 2 of the present invention;

[0035] Figure 5 This is a SEM image of the sodium pyruvate sustained-release gel nanoparticles prepared in Example 3 of the present invention;

[0036] Figure 6 This is a graph showing the relative activity results of each group of cells in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further explained below in conjunction with specific embodiments, comparative examples and test examples.

[0038] In the following examples, comparative examples and test examples, the raw materials and preparation methods used are conventional materials and techniques in the art unless otherwise specified.

[0039] Example 1

[0040] A sodium pyruvate sustained-release gel nanoparticle based on redox response, comprising the following steps:

[0041] (1) Preparation of modified gellan gum containing thiol and catechol groups

[0042] 5.0 g of gellan gum (molecular weight 50 WDa) was dispersed in 500 mL of deionized water, stirred at 85°C until completely dissolved, and cooled to room temperature to obtain a gellan gum solution; 5.0 g of EDC was added to the gellan gum solution and stirred at room temperature for 1 h to activate the carboxyl groups in the gellan gum to obtain a mixed solution; 3.7 g of NHS, 2.5 g of β-mercaptoethylamine and 5.0 g of dopamine were added to the mixed solution, and the pH of the solution was adjusted to 4.0 by adding HCl (1M), and the reaction solution continued to react at 25°C for 55 h. After the reaction was completed, NaOH (1M) was added to the reaction solution to adjust the pH to 5.0, and 500 mL of ethanol was added to the reaction solution to precipitate the product, which was collected and washed with ethanol to remove unreacted raw materials and by-products. The washed product was freeze-dried to obtain a modified gellan gum containing thiol and catechol groups.

[0043] The specific reaction formula is as follows:

[0044]

[0045] (2) Preparation of modified gellan gum of phenylboronic acid

[0046] Disperse 5.0 g of gellan gum in 500 mL of deionized water, stir at 80°C until completely dissolved, and cool to room temperature to obtain a gellan gum solution. Slowly drop NaOH (1 M) solution into the gellan gum solution to adjust the pH to 8.5 to increase the reactivity of the hydroxyl groups on the gellan gum, and set aside.

[0047] Dissolve 2.5 g of 4-chlorophenylboronic acid in 100 mL of DMSO to form an organic phase solution. Add 0.5 g of tetrabutylammonium bromide as a phase transfer catalyst to the 4-chlorophenylboronic acid solution and mix thoroughly. Slowly add the activated chlorophenylboronic acid solution to the gellan gum solution and mix evenly under stirring. Stir the mixture at 40°C for 6 hours, keep the pH at about 8.5, and add NaOH solution if necessary. After the reaction, cool the reaction solution to room temperature, add 500 mL of ethanol to the reaction solution to precipitate the modified gellan gum. Collect the product by centrifugation or filtration, and wash the product with ethanol and deionized water alternately for 3-5 times to remove unreacted raw materials and by-products. The purified product is freeze-dried to obtain phenylboronic acid-modified gellan gum.

[0048] The specific reaction formula is as follows:

[0049]

[0050] (3) Preparation of sodium pyruvate sustained-release gel nanoparticles

[0051] The modified gellan gum containing thiol and catechol groups prepared in step (1), the gellan gum modified with phenylboronic acid prepared in step (2) and sodium pyruvate are added into water in a mass ratio of 1:1:1, stirred at 85°C until completely dissolved so that the concentrations of the two modified gellan gums are both 5% (w / w), cooled to room temperature, and a mixed solution is obtained; under a nitrogen atmosphere, a 2 wt% CaCl2 solution (the mass ratio of the gellan gum modified with phenylboronic acid to CaCl2 is 5:1) and a 75 mmol / L potassium persulfate (KPS) aqueous solution (the mass ratio of the gellan gum modified with phenylboronic acid to KPS is 100:0.5) are slowly added into the mixed solution, and after magnetic stirring for 3 h, the sodium pyruvate sustained-release gel nanoparticles are obtained by centrifugation, washing and freeze-drying.

[0052] Example 2

[0053] A sodium pyruvate sustained-release gel nanoparticle based on redox response, comprising the following steps:

[0054] (1) Preparation of modified gellan gum containing thiol and catechol groups

[0055] 5.0 g of gellan gum (molecular weight 100 WDa) was dispersed in 1000 mL of deionized water, stirred at 80 °C until completely dissolved, and cooled to room temperature to obtain a gellan gum solution. Then, 5.5 g of EDC was added to the gellan gum solution and stirred at room temperature for 1.5 h to activate the carboxyl groups in the gellan gum to obtain a mixed solution. 4.2 g of NHS, 5 g of β-mercaptoethylamine and 2.5 g of dopamine were added to the mixed solution, and the pH of the solution was adjusted to 4.5 by adding HCl (1 M). The reaction solution continued to react at 30 °C for 50 h. After the reaction was completed, the reaction solution was cooled to room temperature, and NaOH (1 M) was added to the reaction solution to adjust the pH to 5.5. Then, 500 mL of ethanol was added to the reaction solution to precipitate the product. The product was collected and washed with ethanol to remove unreacted raw materials and by-products. The washed product was freeze-dried to obtain a modified gellan gum containing thiol and catechol groups.

[0056] The specific reaction formula is as follows:

[0057]

[0058] (2) Preparation of phenylboronic acid modified gellan gum

[0059] Disperse 5.0 g of gellan gum in 1000 mL of deionized water, stir at 80°C until completely dissolved, and cool to room temperature to obtain a gellan gum solution. Slowly drop NaOH (1 M) solution into the gellan gum solution to adjust the pH to 8.0 to increase the reactivity of the hydroxyl groups on the gellan gum, and set aside.

[0060] Dissolve 5.0 g of 4-chlorophenylboronic acid in 100 mL of DMSO to form an organic phase solution. Add 0.5 g of tetrabutylammonium bromide to the 4-chlorophenylboronic acid solution as a phase transfer catalyst and mix thoroughly. Slowly add the activated chlorophenylboronic acid solution to the gellan gum solution and mix evenly under stirring. Stir the reaction mixture at 50°C for 10 hours, keeping the pH at about 8.0 during the reaction, and add NaOH solution if necessary. After the reaction, cool the reaction solution to room temperature, add 500 mL of ethanol to the reaction solution to precipitate the modified gellan gum. Collect the product by centrifugation or filtration, and wash the product alternately with ethanol and deionized water 3-5 times to remove unreacted raw materials and by-products. The purified product is freeze-dried to obtain phenylboronic acid modified gellan gum. The specific reaction formula is as follows:

[0061]

[0062] (3) Preparation of sodium pyruvate sustained-release gel nanoparticles

[0063] The modified gellan gum containing thiol and catechol groups prepared in step (1), the phenylboric acid modified gellan gum prepared in step (2) and sodium pyruvate are added into water in a mass ratio of 1.5:1:2, stirred at 85°C until completely dissolved so that the concentration of the phenylboric acid modified gellan gum is 5% (w / w), cooled to room temperature, and a mixed solution is obtained; under a nitrogen atmosphere, a CaCl2 solution (the mass ratio of the phenylboric acid modified gellan gum to CaCl2 is 2:1) and a 75 mmol / L KPS aqueous solution (the mass ratio of the phenylboric acid modified gellan gum to KPS is 100:1) are slowly added into the mixed solution, and after magnetic stirring for 3 hours, the drug-loaded nanoparticles are collected by centrifugation, washing and freeze-drying to obtain the sodium pyruvate sustained-release gel nanoparticles.

[0064] Example 3

[0065] A sodium pyruvate sustained-release gel nanoparticle based on redox response, comprising the following steps:

[0066] (1) Preparation of modified gellan gum containing thiol and catechol groups

[0067] 5.0 g of gellan gum (molecular weight 80 WDa) was dispersed in 500 mL of deionized water, stirred at 85°C until completely dissolved, and cooled to room temperature to obtain a gellan gum solution; 4.0 g of EDC was added to the gellan gum solution and stirred at room temperature for 1 h to activate the carboxyl groups in the gellan gum to obtain a mixed solution; 3.0 g of NHS, 2.5 g of β-mercaptoethylamine and 2.5 g of dopamine were added to the mixed solution, and the pH of the solution was adjusted to 4.0 by adding HCl (1 M). The reaction solution continued to react at 30°C for 45 h. After the reaction was completed, the reaction solution was cooled to room temperature, NaOH (1 M) was added to the reaction solution to adjust the pH to 5.0, and 500 mL of ethanol was added to the reaction solution to precipitate the product, which was collected and washed with ethanol to remove unreacted raw materials and by-products. The washed product was freeze-dried to obtain a modified gellan gum containing thiol and catechol groups.

[0068] The specific reaction formula is as follows:

[0069]

[0070] (2) Preparation of phenylboronic acid modified gellan gum

[0071] Disperse 5.0 g of gellan gum in 500 mL of deionized water, stir at 80°C until completely dissolved, and cool to room temperature to obtain a gellan gum solution. Slowly drop NaOH (1 M) solution into the gellan gum solution to adjust the pH to 8.5 to increase the reactivity of the hydroxyl groups on the gellan gum, and set aside.

[0072] Dissolve 4 g of 4-chlorophenylboronic acid in 100 mL of DMSO to form an organic phase solution. Add 0.5 g of tetrabutylammonium bromide to the 4-chlorophenylboronic acid solution as a phase transfer catalyst and mix thoroughly. Slowly add the activated 4-chlorophenylboronic acid solution to the gellan gum solution and mix evenly under stirring. Stir the mixture at 45°C for 8 hours, keep the pH at about 8.5, and add NaOH solution if necessary. After the reaction, cool the reaction solution to room temperature, add 500 mL of ethanol to the reaction solution to precipitate the modified gellan gum. Collect the product by centrifugation or filtration, and wash the product alternately with ethanol and deionized water for 3-5 times to remove unreacted raw materials and by-products. The purified product is freeze-dried to obtain phenylboronic acid modified gellan gum.

[0073] The specific reaction formula is as follows:

[0074]

[0075] (3) Preparation of sodium pyruvate sustained-release gel nanoparticles

[0076] The modified gellan gum containing thiol and catechol groups prepared in step (1), the phenylboronic acid modified gellan gum prepared in step (2) and sodium pyruvate are added into water in a mass ratio of 1:1:2, stirred at 85°C until completely dissolved so that the concentrations of the two modified gellan gums are both 5% (w / w), cooled to room temperature, and a mixed solution is obtained; under a nitrogen atmosphere, 5 wt% CaCl2 solution (the mass ratio of phenylboronic acid modified gellan gum to CaCl2 is 3:1) and 75 mmol / L KPS aqueous solution (the mass ratio of phenylboronic acid modified gellan gum to KPS is 100:0.8) are slowly added into the mixed solution, and after magnetic stirring for 3 h, the drug-loaded nanoparticles are collected by centrifugation, washing and freeze-drying to obtain the sodium pyruvate sustained-release gel nanoparticles.

[0077] Comparative Example 1

[0078] A sodium pyruvate-loaded gellan gum nanoparticle, the preparation method of which is as follows: adding gellan gum (molecular weight of 50 WDa) and sodium pyruvate to water at a mass ratio of 2:1, stirring at 85°C until completely dissolved so that the concentration of the gellan gum is 10% (w / w), cooling to room temperature, and obtaining a mixed solution; slowly adding a 2 wt% CaCl2 solution (the mass ratio of gellan gum to CaCl2 is 10:1) and a 75 mmol / L KPS aqueous solution (the mass ratio of gellan gum to KPS is 200:0.5) to the mixed solution under a nitrogen atmosphere, and collecting the drug-loaded nanoparticles by centrifugation, washing and freeze-drying after magnetic stirring for 3 hours, so as to obtain the sodium pyruvate-loaded gellan gum nanoparticles.

[0079] Comparative Example 2

[0080] A gellan gum nanoparticle, the preparation method is as follows: adding gellan gum (molecular weight of 50 WDa) into water, stirring at 85°C until completely dissolved so that the concentration of the gellan gum is 10% (w / w), and cooling to room temperature; slowly adding 2 wt% CaCl2 solution (the mass ratio of gellan gum to CaCl2 is 10:1) and 75 mmol / L KPS aqueous solution (the mass ratio of gellan gum to KPS is 200:0.5) into the gellan gum solution under a nitrogen atmosphere, magnetically stirring for 3 hours, and then collecting the drug-loaded nanoparticles by centrifugation, washing and freeze-drying to obtain the gellan gum nanoparticles.

[0081] Product structure characterization

[0082] The samples prepared in Examples 1-3 and Comparative Examples 1-2 were subjected to morphological tests. The scanning electron microscope (SEM) images of the samples in Examples 1-3 and Comparative Examples 1-2 are shown in FIG. Figure 1-5 As shown, Figure 1-5 They correspond to comparative example 2, comparative example 1, embodiment 1, embodiment 2 and embodiment 3 respectively. Figure 1-5It can be seen that the sodium pyruvate sustained-release nanoparticles prepared in Examples 1-3 of the present invention have uniform morphology and an average particle size of 400-600 nm ( Figure 3-5 ). Comparative Example 2 prepared gellan gum nanoparticles without adding sodium pyruvate drug, and obtained gellan gum nanoparticles with regular morphology ( Figure 1 ), while the gellan gum nanoparticles prepared in Comparative Example 1 added the drug sodium pyruvate, resulting in the inability to form regular spherical nanoparticles ( Figure 2 ), its actual drug loading rate is 25.81%, but the encapsulation rate is only 20.70%. This shows that the presence of sodium pyruvate raw material not only inhibits the formation of regular microspheres, but also makes it difficult to be evenly encapsulated in the microspheres during the microsphere formation process, but mainly accumulates on the surface of the microspheres in the form of attachment. This extremely low encapsulation rate is not only not conducive to extending the release cycle of the drug, but also easily forms a burst release phenomenon at the initial stage of administration. The nanoparticles prepared in Examples 1-3 of the present application modified gellan gum and Ca 2+ Chelation with gellan gum allows sodium pyruvate to be encapsulated into nanoparticles.

[0083] Test Example 1

[0084] In this test example, the loading efficiency of the products of Examples 1-3 and Comparative Example 1 was tested by the following method: 255 nm was selected as the detection wavelength of sodium pyruvate, its content was determined by UV method, and a standard curve of sodium pyruvate was established; the reaction solution of modified gellan gum or gellan gum cross-linked in Examples 1-3 and Comparative Example 1 was taken, centrifuged and the volume of the supernatant was measured, and the absorbance was substituted into the standard curve to obtain the content of free sodium pyruvate in the supernatant, and the encapsulation rate and drug loading of sodium pyruvate were calculated. The calculation formula was: drug loading rate = [(initial addition amount of sodium pyruvate - mass of free sodium pyruvate in supernatant) / mass of sodium pyruvate sustained-release gel nanoparticles] × 100%, encapsulation rate = [(initial addition amount of sodium pyruvate - mass of free sodium pyruvate in supernatant) / initial addition amount of sodium pyruvate] × 100%, and the specific results are shown in Table 1.

[0085] Table 1

[0086]

[0087] It can be seen from Table 1 that the sodium pyruvate nanoparticles of Examples 1-3 of the present invention have good drug loading rate and encapsulation rate. In addition, the drug loading amount and encapsulation rate of Comparative Example 1 are lower than those of the embodiment, which also shows that after grafting thiol and catechol on gellan gum and modifying it with phenylboronic acid, the drug loading is significantly improved.

[0088] Test Example 2

[0089] In order to study the drug release behavior of sodium pyruvate sustained-release gel nanoparticles, this test example selected a PBS solution with a pH of 7.4 for the experiment, and added 50 mL of glutathione (GSH) of different concentrations as a release medium to simulate the drug release under the tumor microenvironment. The nanoparticles prepared in Examples 1-3 and Comparative Example 1 were placed in the release medium, and three parallel tests were set up for each group. 200 μL of the supernatant of each group was sampled for sodium pyruvate detection at 1 h, 10 h, 20 h, 30 h, 40 h, 50 h, 60 h, and 70 h, respectively. Each group was sampled 3 times, and then the release medium of the same volume and temperature was added. The absorbance value of the taken solution was detected at a wavelength of 255 nm, and the cumulative release rate was statistically analyzed. The results of the cumulative release rate of drugs in each embodiment and comparative example are shown in Table 2. In Table 2, the cumulative release rate of drugs = cumulative release amount of drugs / total amount of drugs in the carrier × 100%.

[0090] Table 2

[0091]

[0092] As can be seen from Table 2, in a buffer solution of pH = 7.4, the cumulative drug release rate of each group in the first 10 hours was about 12-20%, and the cumulative drug release rate was about 25-35% at 70 hours. However, after adding 10 mM glutathione (GSH), the drug release rate of Examples 1-3 increased significantly, and was 73-77% after 70 hours. After adding 20 mM glutathione (GSH), the drug release rate increased to 95-98% after 70 hours. Compared with Examples 1-3, the cumulative drug release rate of Comparative Example 1 is significantly poor, and there is no redox response effect. The above results show that the sodium pyruvate sustained-release gel nanoparticles of the present invention have the performance of sustained drug release under physiological conditions, accelerate the drug release rate under the action of glutathione, and increase the glutathione concentration. The drug release rate can be increased, so that sodium pyruvate can be quickly released at the inflammatory site of the lesion, prolong its action time at the lesion, and improve the therapeutic effect.

[0093] Test Example 3

[0094] This test example explores the effect of the sodium pyruvate sustained-release gel nanoparticles of the present invention on the activity of human bronchial epithelial cells (BEAS-2B), and the specific method is as follows:

[0095] Human bronchial epithelial cells BEAS-2B growing in the logarithmic phase were selected for grouping:

[0096] Blank control group: only complete medium was added; model group: human bronchial epithelial cells were cultured with complete medium containing 50 μg / mL lipopolysaccharide (LPS) for 12 h, and then cultured with DMEM complete medium for 48 h; positive control group: human bronchial epithelial cells were incubated with complete medium containing 50 ug / mL lipopolysaccharide (LPS) for 12 h, and then cultured with complete medium containing 1umoL / L dexamethasone for 48 h; Example 1 group: human bronchial epithelial cells were cultured with complete medium containing 50 μg / mL lipopolysaccharide for 12 h, and then cultured with complete medium supplemented with sustained-release gel nanoparticles (200 μg / mL) obtained in Example 1 for 48 h; Example 2 group: human bronchial epithelial cells were cultured with complete medium containing 50 μg / mL lipopolysaccharide for 12 h, and then cultured with complete medium supplemented with sustained-release gel nanoparticles (200 μg / mL) obtained in Example 2 for 48 h; Example 3 group: human bronchial epithelial cells were cultured with complete medium containing 50 μg / mL lipopolysaccharide for 12 h, and then cultured with complete medium supplemented with sustained-release gel nanoparticles (200 μg / mL) obtained in Example 2 for 48 h. The human bronchial epithelial cells were cultured in a complete medium containing 50 μg / mL lipopolysaccharide for 12 h, and then cultured in a complete medium supplemented with the sustained-release gel nanoparticles (200 μg / mL) obtained in Example 3 for 48 h; Comparative Example 1 group: human bronchial epithelial cells were cultured in a complete medium containing 50 μg / mL lipopolysaccharide for 12 h, and then cultured in a complete medium supplemented with the nanoparticles (200 μg / mL) obtained in Comparative Example 1 for 48 h.

[0097] The cell activity was detected by CCK-8 method. 4 The cells were inoculated at a density of 100 / well in a 96-well plate, with 5 replicate wells in each group. After performing the corresponding operations according to the above grouping, 10 μL of CCK-8 reagent was added to each well, and the cells were returned to the incubator for further incubation for 3 h. The absorbance (OD) value at 450 nm was measured by a spectrophotometer, and the average value was selected as the final result. The relative activity of cells in each group was calculated. The results are shown in Figure 6 .

[0098] from Figure 6 It can be seen that compared with the blank control group, the cell activity of the model group was significantly reduced; compared with the model group, the examples 1-3 groups could increase the cell activity, while the effects of the comparative example 1 group and the positive control group were slightly worse than those of the example group.

[0099] The above are only preferred embodiments of the present invention, and are not limited to the above examples. For those skilled in the art, various changes and variations are possible under the principle of the present invention. Any modifications and improvements made should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing sodium pyruvate sustained-release gel nanoparticles based on redox response, characterized in that: The following steps are involved: Adding modified gellan gum containing thiol and catechol groups, gellan gum modified by phenylboronic acid and sodium pyruvate into water to obtain a mixed solution; adding divalent metal salt and potassium persulfate into the mixed solution under the protection of inert gas, stirring for 3-5 hours, centrifuging after the reaction, collecting the precipitate, washing and drying, and obtaining the sodium pyruvate sustained-release gel nanoparticles based on redox response; The preparation method of the modified gellan gum containing thiol and catechol groups comprises: Adding EDC to the gellan gum aqueous solution and stirring to obtain a mixed solution; adding NHS, β-mercaptoethylamine and dopamine to the mixed solution, adjusting the pH to 4.0-4.5 for reaction; after the reaction is completed, treating the reaction solution to obtain the modified gellan gum containing thiol and catechol groups; The preparation method of the phenylboronic acid modified gellan gum comprises: Tetrabutylammonium bromide is added to an organic solution of 4-chlorophenylboric acid to obtain an organic phase; the organic phase is added to a gellan gum aqueous solution, and stirred for reaction at a pH of 8.0-8.5; after the reaction is completed, the reaction solution is post-treated to obtain the phenylboric acid-modified gellan gum.

2. The method for preparing sodium pyruvate sustained-release gel nanoparticles based on redox response according to claim 1, characterized in that: The mass ratio of the modified gellan gum containing thiol and catechol groups, the gellan gum modified by phenylboric acid and sodium pyruvate is (1-1.5):1:(1-2), and the mass ratio of the gellan gum modified by phenylboric acid, the divalent metal salt and potassium persulfate is 1:(0.2-0.5):(0.005-0.01).

3. The method for preparing sodium pyruvate sustained-release gel nanoparticles based on redox response according to claim 1, characterized in that: When preparing modified gellan gum containing thiol and catechol groups, the mass ratio of the gellan gum, β-mercaptoethylamine and dopamine is 1:(0.5-1):(0.5-1); the reaction temperature is 25-30°C, and the reaction time is 45-55 h.

4. The method for preparing the redox-responsive sodium pyruvate sustained-release gel nanoparticles according to claim 1, characterized in that: When preparing the modified gellan gum containing thiol and catechol groups, the specific operation of the treatment is: adjusting the pH of the reaction solution to 5.0-5.5, adding ethanol to the reaction solution to precipitate the product, collecting the product for washing and freeze-drying to obtain the modified gellan gum containing thiol and catechol groups.

5. The method for preparing sodium pyruvate sustained-release gel nanoparticles based on redox response according to claim 1, characterized in that: When preparing phenylboronic acid-modified gellan gum, the mass ratio of 4-chlorophenylboric acid, gellan gum and tetrabutylammonium bromide is 1:(1-2):(0.1-0.2).

6. The method for preparing sodium pyruvate sustained-release gel nanoparticles based on redox response according to claim 1, characterized in that: When preparing phenylboronic acid-modified gellan gum, the stirring reaction temperature is 40-50° C., and the stirring reaction time is 6-10 h. The specific operation of the post-treatment is: adding ethanol to the reaction solution to precipitate the product, collecting the product and washing and freeze-drying it to obtain the phenylboronic acid-modified gellan gum.

7. Sodium pyruvate sustained-release gel nanoparticles based on redox response prepared according to any one of the preparation methods described in claims 1-6.

8. Use of the redox-responsive sodium pyruvate sustained-release gel nanoparticles according to claim 7 in the preparation of a controlled drug release system.

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