Sodium pyruvate sustained-release nanosystem based on targeted modification and preparation method and application thereof

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a sodium pyruvate sustained-release nanosystem based on targeted modification, and a preparation method and application thereof. The preparation method of the sustained-release nanosystem comprises the following steps: a. preparing modified silk fibroin; b. under homogenization conditions, adding a mixed aqueous solution of sodium phytate and sodium pyruvate to a modified silk fibroin aqueous solution, obtaining a mixed solution after homogenization, stirring the mixed solution for reaction, centrifuging the reaction solution after the reaction is completed, collecting the precipitate, washing, and dialyzing to obtain the sodium pyruvate sustained-release nanosystem based on targeted modification. The sustained-release nanosystem has good biocompatibility, can be specifically enriched in lung lesions and realize the sustained release of sodium pyruvate, has significant targeting and sustained-release effects, and is suitable for preparing drugs for treating lung diseases.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a sodium pyruvate sustained-release nanosystem based on targeted modification, and a preparation method and application thereof. Background Art

[0002] With the in-depth study of the antioxidant and anti-inflammatory effects of sodium pyruvate, it has broad development prospects in the fields of medicine, health products, cosmetics, etc. Although sodium pyruvate has many advantages such as reducing inflammatory cytokines, effectively clearing reactive oxygen free radicals, and protecting nerve cells from oxidative stress, it has broad application prospects in medicine, diagnostic reagents, and medical devices, but its administration method still needs to be improved during use to improve the efficacy of the drug.

[0003] The conventional administration methods of sodium pyruvate are usually oral preparations, injections and topical administration. These traditional administration methods often have certain disadvantages, such as low drug utilization and poor targeting. In addition, the drug will be dispersed throughout the body and have certain side effects on other organs of the body. Compared with systemic administration by oral or injection, the targeted administration route can reduce the drug dose by an order of magnitude and directly deliver the drug to the lung tissue and achieve high concentrations, thereby achieving a higher therapeutic index and reducing systemic side effects.

[0004] Targeted drug delivery systems can deliver drugs precisely to diseased tissues, organs or parts by selecting appropriate drug carriers or drug delivery systems. This precision performance can greatly improve the therapeutic effect of drugs and enhance their efficacy.

[0005] Silk fibroin has good biocompatibility and biodegradability with human tissues, so it is suitable for use as a drug carrier, but silk fibroin has certain defects, for example, its drug loading is low, which affects the release and therapeutic effect of drugs in vivo. However, silk fibroin has strong plasticity and can be modified by chemical modification to obtain a functional sustained-release nanosystem, so that drugs can be effectively targeted to the lesion. At present, there is no report on the preparation of nanoparticles with silk fibroin and sodium phytate modified with folic acid derivatives and 5-carboxypentyl-triphenylphosphonium bromide, and the research on the simultaneous encapsulation of sodium pyruvate as a nano sustained-release system.

[0006] Therefore, the development of a sodium pyruvate sustained-release nanosystem based on targeted modification is of great significance for improving the anti-inflammatory and antioxidant effects of sodium pyruvate. Summary of the invention

[0007] One of the purposes of the present invention is to provide a method for preparing a sodium pyruvate sustained-release nanosystem based on targeted modification. The preparation method is simple and easy for industrial production, and the prepared sustained-release nanosystem has good targeting.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The preparation method of the sodium pyruvate sustained-release nanosystem based on targeted modification comprises the following steps:

[0010] a. Preparation of modified silk fibroin;

[0011] b. Under homogenization conditions, a mixed aqueous solution of sodium phytate and sodium pyruvate is added to the modified silk fibroin aqueous solution to obtain a mixed solution after homogenization. The mixed solution is stirred for reaction. After the reaction is completed, the reaction solution is centrifuged, and the precipitate is collected, washed, and dialyzed to obtain the sodium pyruvate sustained-release nanosystem based on targeted modification.

[0012] Furthermore, the preparation steps of the modified silk fibroin are as follows:

[0013] (1) At 0°C, add NaH to a solution of folic acid in N,N-dimethylformamide in an inert atmosphere, stir for 15-30 min, then add 4-methoxybenzyl chloride, stir at room temperature for 3-5 h. After the reaction is completed, concentrate the reaction solution, separate by column chromatography, and obtain folic acid derivative 1B;

[0014] The structural formula of the folic acid and folic acid derivative 1B is:

[0015] , ;

[0016] (2) adding silk fibroin, N,N-diisopropylethylamine and glycidyl trimethylammonium chloride into water, heating for reaction, and after the reaction is completed, dialyzing and freeze-drying to obtain compound 1;

[0017] The structural formulas of the silk fibroin and compound 1 are:

[0018] ,

[0019] ;

[0020] (3) adding dicyclohexylcarbodiimide, 4-dimethylaminopyridine and 5-carboxypentyl-triphenylphosphonium bromide to a dimethyl sulfoxide solution containing compound 1 and reacting for 2-5 hours to obtain a reaction solution 1;

[0021] (4) Add dicyclohexylcarbodiimide, 1-hydroxybenzotriazole and folic acid derivative 1B to reaction solution 1, and react for 20-24 hours to obtain reaction solution 2. Centrifuge, wash and dry reaction solution 2 to obtain compound 2;

[0022] The structural formula of the compound 2 is:

[0023] ;

[0024] (5) Adding 2,3-dichloro-5,6-dicyanobenzoquinone to the solution of compound 2 and stirring, centrifuging, washing, and drying the stirred reaction solution to obtain modified silk fibroin, wherein the structural formula of the modified silk fibroin is:

[0025] .

[0026] Furthermore, in step (1), the concentration of folic acid in the N,N-dimethylformamide solution is 87.5-116.7 mg / mL, the mass ratio of folic acid to NaH is 1:(0.28-0.58), and the mass ratio of folic acid to 4-methoxybenzyl chloride is 1:(0.3-0.5).

[0027] Furthermore, the mass ratio of the silk fibroin, N,N-diisopropylethylamine and glycidyl trimethylammonium chloride in step (2) is 1:(0.06-0.18):(0.05-0.15), the temperature of the heating reaction in step (2) is 40-60° C., and the heating reaction time is 2-4 h.

[0028] Furthermore, the concentration of the dimethyl sulfoxide solution of compound 1 in step (3) is 10-20 mg / mL; the mass ratio of compound 1, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and 5-carboxypentyl-triphenylphosphonium bromide is 1:(3.3-9.9):(2.69-8.06):(9.14-27.44).

[0029] Furthermore, in step (4), the mass ratio of dicyclohexylcarbodiimide, 1-hydroxybenzotriazole and folic acid derivative 1B is (17.32-51.99):(11.35-34.05):(11.23-33.69).

[0030] Furthermore, the solvent of the solution of compound 2 in step (5) is a dichloromethane / water mixed solvent, the dosage ratio of compound 2 to dichloromethane / water mixed solvent is 1 g:(8-12) mL, the mass ratio of compound 2 to 2,3-dichloro-5,6-dicyanobenzoquinone is 1(90.8-272.4), and the stirring time is 0.5-3 h.

[0031] Furthermore, in step b, the concentration of the modified silk fibroin aqueous solution is 1-5wt%, the concentration of sodium phytate in the mixed aqueous solution of sodium phytate and sodium pyruvate is 1-5wt%, and the concentration of sodium pyruvate is 5-10wt%, the volume ratio of the modified silk fibroin aqueous solution to the mixed solution of sodium phytate and sodium pyruvate is 1:(1-1.5), the homogenization time is 20-30min, and the stirring reaction time is 20-24h.

[0032] The second purpose of the present invention is to provide a sodium pyruvate sustained-release nanosystem based on targeted modification.

[0033] The second object of the present invention is achieved by adopting the following technical solution:

[0034] The sodium pyruvate sustained-release nanosystem based on targeted modification is prepared according to the above preparation method.

[0035] The third object of the present invention is to provide an application of a sodium pyruvate sustained-release nanosystem based on targeted modification.

[0036] The third object of the present invention is achieved by adopting the following technical solution:

[0037] Application of sodium pyruvate sustained-release nanosystem based on targeted modification in the preparation of drugs for the treatment of chronic obstructive pulmonary disease.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The present invention provides a sodium pyruvate sustained-release nanosystem based on targeted modification. The sustained-release nanosystem uses silk fibroin as a raw material and is chemically modified to prepare a modified silk fibroin with lung targeting, wherein 5-carboxypentyl-triphenylphosphonium bromide has three benzene rings, which can increase the molecular surface area and form a delocalized positive charge, and the modification of the folic acid derivative 1B enables the modified silk fibroin to target the lesion. Then, the sustained-release nanosystem is prepared by an ion gel method, which is also called ion-induced assembly. The electrostatic interaction between the modified silk fibroin and sodium phytate is used to form a nanocarrier, and sodium pyruvate is simultaneously wrapped therein, finally forming a targeted sustained-release nanosystem; the sustained-release nanosystem can effectively improve the bioavailability of the drug, control the release rate of the drug, and release the drug slowly and evenly in the body, thereby enhancing the therapeutic effect of the drug.

[0040] 2. The present invention also provides a method for preparing a sodium pyruvate sustained-release nanosystem based on targeted modification. The preparation method is simple and easy for industrial production, and the prepared sustained-release nanosystem has good targeting properties.

[0041] 3. The present invention provides the use of a sodium pyruvate sustained-release nanosystem based on targeted modification in the preparation of drugs for treating lung diseases. The sustained-release nanosystem can also selectively accumulate drugs in the site of lung inflammation, thereby reducing the occurrence of toxic and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the folic acid derivative 1B of the present invention;

[0043] Figure 2This is a morphology diagram of the product obtained in Example 1 of the present invention;

[0044] Figure 3 This is a diagram showing the biocompatibility test results of the sustained-release nanosystem and lung cells;

[0045] Figure 4 This is a graph showing the cellular uptake rate results of the sustained-release nanosystem. DETAILED DESCRIPTION

[0046] Below, in conjunction with specific embodiments, the present invention is further described. It should be noted that, under the premise of no conflict, the embodiments described below or the technical features can be arbitrarily combined to form new embodiments. The specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0047] Example 1

[0048] The preparation method of the sodium pyruvate sustained-release nanosystem based on targeted modification comprises the following steps:

[0049] a. Preparation of modified silk fibroin:

[0050] (1) 7 g of folic acid was added to 65 mL of N,N-dimethylformamide (DMF) to obtain a DMF solution of folic acid. 3 g of NaH (60% purity) was added to the DMF solution of folic acid at 0°C in an inert nitrogen atmosphere, and the mixture was stirred for 20 min. 2.8 g of 4-methoxybenzyl chloride (PMBCl) was then added and the mixture was stirred at room temperature for 4 h. The reaction solution was then quenched with water, and the pH of the reaction solution was adjusted to 6.2 with 1 M HCl. The reaction solution was concentrated and separated by column chromatography to obtain folic acid derivative 1B.

[0051] The specific reaction formula is as follows:

[0052]

[0053] The nuclear magnetic resonance hydrogen spectrum of the folic acid derivative 1B is as follows Figure 1As shown, the structural characterization results are 1H NMR (400MHz, DMSO) δ 12.68(s,1H), 12.02(s,1H), 10.27(s,1H),9.04(s,1H), 8.89(s,1H),7.15(d,2H), 7.60(d,2H), 6.86-6.90(m,4H), 6.29(s,1H), 4.56-4.64(m,3H), 4.41(d,2H), 3.82(s,3H), 2.51(t, 1H), 2.35(t,2H), 2.11(m,2H). The above results confirm that the obtained product is the target product.

[0054] (2) 1 g of silk fibroin (soluble silk fibroin freeze-dried raw material, molecular weight 50,000-100,000 Daltons), 0.1 g of N,N-diisopropylethylamine and 0.12 g of glycidyl trimethylammonium chloride were added to 5 mL of water to obtain a mixed solution, which was heated at 50°C for 3 h. After the reaction was completed, the reaction solution was placed in a dialysis bag (14000 Da) and dialyzed for 48 h. The dialyzate was then freeze-dried to obtain compound 1;

[0055] The specific reaction formula is as follows:

[0056]

[0057] (3) 1 g of compound 1 was placed in 75 mL of dimethyl sulfoxide (DMSO), and then 6.2 g of dicyclohexylcarbodiimide (DCC), 5.375 g of 4-dimethylaminopyridine (DMAP) and 18.294 g of 5-carboxypentyl-triphenylphosphonium bromide were added in sequence, and the mixture was reacted at room temperature for 3.5 h to obtain reaction solution 1;

[0058] (4) 30.945 g of dicyclohexylcarbodiimide (DCC), 20.268 g of 1-hydroxybenzotriazole (HOBt), and 22.462 g of folic acid derivative 1B were added to reaction solution 1, and the mixture was reacted at room temperature for 22 h to obtain reaction solution 2. Reaction solution 2 was centrifuged to obtain a crude product, which was collected and washed with deionized water in an ultrasonic water bath for 5 times, each time for 5 min, and the product was dried in a vacuum oven at 30 °C for 5 h to obtain compound 2;

[0059] (5) 1 g of compound 2 was placed in 10 mL of a dichloromethane / water mixed solvent (dichloromethane / water volume ratio of 1:1), 181.6 g of 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) was added at room temperature and stirred for 2 h. The reaction solution 2 was centrifuged to obtain a crude product. The crude product was collected and washed with deionized water in an ultrasonic water bath at room temperature for 5 times, each washing for 5 min. The product was then placed in a vacuum oven and dried at 30 °C for 5 h to obtain modified silk fibroin.

[0060] The specific reaction formulas of steps (3), (4) and (5) are as follows:

[0061]

[0062] b. Preparation of targeted modified sodium pyruvate sustained-release nanosystem:

[0063] A modified silk fibroin aqueous solution with a concentration of 2.5wt% was placed in a homogenizer, and the speed of the homogenizer was set to 5000rpm. During the homogenization process, a mixed aqueous solution of sodium phytate and sodium pyruvate (wherein the concentration of sodium phytate was 2.5wt% and the concentration of sodium pyruvate was 7.5wt%) was slowly added, wherein the volume ratio of the modified silk fibroin solution to the mixed solution of sodium phytate and sodium pyruvate was 1:1.25, and homogenization was performed for 25 minutes. After the homogenization, the mixed solution was stirred at 25°C for 22 hours; then the reaction solution was centrifuged (8000rpm, 10min), and the precipitate obtained by centrifugation was washed 3 times with deionized water, and then dialyzed with a dialysis bag with a molecular weight cutoff of 3500Da to obtain a sodium pyruvate sustained-release nanosystem based on targeted modification.

[0064] Example 2

[0065] The preparation method of the sodium pyruvate sustained-release nanosystem based on targeted modification comprises the following steps:

[0066] a. Preparation of modified silk fibroin:

[0067] (1) 7 g of folic acid was added to 60 mL of DMF to obtain a DMF solution of folic acid. 2 g of NaH (60% purity) was added to the DMF solution of folic acid at 0°C in an inert nitrogen atmosphere, and the mixture was stirred for 15 min. 2.1 g of PMBCl was then added and the mixture was stirred at room temperature for 3 h. The reaction solution was then quenched with water, and the pH of the reaction solution was adjusted to 6 with 1 M HCl. The reaction solution was concentrated and separated by column chromatography to obtain folic acid derivative 1B.

[0068] The specific reaction formula is as follows:

[0069]

[0070] (2) 1 g of silk fibroin (soluble silk fibroin freeze-dried raw material, molecular weight 50,000-100,000 Daltons), 0.06 g of N,N-diisopropylethylamine and 0.05 g of glycidyl trimethylammonium chloride were added to 5 mL of water to obtain a mixed solution, which was heated at 40°C for 4 h. After the reaction was completed, the reaction solution was placed in a dialysis bag (14000 Da) and dialyzed for 48 h. The dialyzate was then freeze-dried to obtain compound 1;

[0071] The specific reaction formula is as follows:

[0072]

[0073] (3) 1 g of compound 1 was placed in 50 mL of DMSO, and then 3.3 g of DCC, 2.69 g of DMAP and 9.14 g of 5-carboxypentyl-triphenylphosphonium bromide were added in sequence and reacted at room temperature for 2 h to obtain reaction solution 1;

[0074] (4) 17.32 g DCC, 11.35 g HOBt and 11.23 g folic acid derivative 1B were added to the reaction solution 1 and reacted at room temperature for 20 h to obtain a reaction solution 2. The reaction solution 2 was centrifuged to obtain a crude product, which was collected and washed with deionized water in an ultrasonic water bath for 5 times, each washing for 5 min. The product was dried in a vacuum oven at 30 °C for 5 h to obtain compound 2.

[0075] (5) 1 g of compound 2 was placed in 8 mL of a dichloromethane / water mixed solvent (dichloromethane / water volume ratio of 1:1), 90.8 g of DDQ was added at room temperature and stirred for 0.5 h. The reaction solution 2 was centrifuged to obtain a crude product, which was collected and washed with deionized water in an ultrasonic water bath at room temperature for 5 times, each washing for 5 min. The product was then placed in a vacuum oven and dried at 30 °C for 5 h to obtain modified silk fibroin.

[0076] The specific reaction formulas of steps (3), (4) and (5) are as follows:

[0077]

[0078] b. Preparation of targeted modified sodium pyruvate sustained-release nanosystem:

[0079] A modified silk fibroin aqueous solution with a concentration of 1wt% is placed in a homogenizer, and the speed of the homogenizer is set to 5000rpm. During the homogenization process, a mixed aqueous solution of sodium phytate and sodium pyruvate (wherein the concentration of sodium phytate is 1wt% and the concentration of sodium pyruvate is 5wt%) is slowly added, wherein the volume ratio of the modified silk fibroin solution to the mixed solution of sodium phytate and sodium pyruvate is 1:1, and homogenization is performed for 20 minutes. After the homogenization, the mixed solution is stirred at 25°C for 20 hours; then the reaction solution is centrifuged (8000rpm, 10min), the precipitate obtained by centrifugation is washed 3 times with deionized water, and then dialyzed with a dialysis bag with a molecular weight cutoff of 3500Da to obtain a sodium pyruvate sustained-release nanosystem based on targeted modification.

[0080] Example 3

[0081] The preparation method of the sodium pyruvate sustained-release nanosystem based on targeted modification comprises the following steps:

[0082] a. Preparation of modified silk fibroin:

[0083] (1) 7 g of folic acid was added to 80 mL of DMF to obtain a DMF solution of folic acid. 4 g of NaH (60% purity) was added to the DMF solution of folic acid at 0°C in an inert nitrogen atmosphere, and the mixture was stirred for 30 min. 3.5 g of PMBCl was then added, and the mixture was stirred at room temperature for 5 h. The reaction solution was then quenched with water, and the pH of the reaction solution was adjusted to 6.5 with 1 M HCl. The reaction solution was concentrated and separated by column chromatography to obtain folic acid derivative 1B.

[0084] The specific reaction formula is as follows:

[0085]

[0086] (2) 1 g of silk fibroin (soluble silk fibroin freeze-dried raw material, molecular weight 50,000-100,000 Daltons), 0.18 g of N,N-diisopropylethylamine and 0.15 g of glycidyl trimethylammonium chloride were added to 5 mL of water to obtain a mixed solution, which was heated at 60°C for 2 h. After the reaction was completed, the reaction solution was placed in a dialysis bag (14000 Da) and dialyzed for 48 h. The dialyzate was then freeze-dried to obtain compound 1;

[0087] The specific reaction formula is as follows:

[0088]

[0089] (3) 1 g of compound 1 was placed in 100 mL of DMSO, and then 9.9 g of DCC, 8.06 g of DMAP and 27.44 g of 5-carboxypentyl-triphenylphosphonium bromide were added in sequence and reacted at room temperature for 5 h to obtain reaction solution 1;

[0090] (4) 51.99 g DCC, 34.05 g HOBt and 33.69 g folic acid derivative 1B were added to the reaction solution 1 and reacted at room temperature for 24 h to obtain a reaction solution 2. The reaction solution 2 was centrifuged to obtain a crude product. The crude product was collected and washed with deionized water in an ultrasonic water bath for 5 times, each washing for 5 min. The product was dried in a vacuum oven at 30 °C for 5 h to obtain compound 2.

[0091] (5) 1 g of compound 2 was placed in 12 mL of a dichloromethane / water mixed solvent (dichloromethane / water volume ratio of 1:1), 272.4 g of DDQ was added at room temperature and stirred for 3 h. The reaction solution 2 was centrifuged to obtain a crude product, which was collected and washed with deionized water in an ultrasonic water bath at room temperature for 5 times, each washing for 5 min. The product was then placed in a vacuum oven and dried at 30 °C for 5 h to obtain modified silk fibroin.

[0092] The specific reaction formulas of steps (3), (4) and (5) are as follows:

[0093]

[0094] b. Preparation of targeted modified sodium pyruvate sustained-release nanosystem:

[0095] A modified silk fibroin aqueous solution with a concentration of 5wt% was placed in a homogenizer, and the speed of the homogenizer was set to 5000rpm. During the homogenization process, a mixed aqueous solution of sodium phytate and sodium pyruvate (wherein the concentration of sodium phytate was 5wt% and the concentration of sodium pyruvate was 10wt%) was slowly added, wherein the volume ratio of the modified silk fibroin solution to the mixed solution of sodium phytate and sodium pyruvate was 1:1.5, and homogenization was performed for 30 minutes. After the homogenization, the mixed solution was stirred at 25°C for 24 hours; then the reaction solution was centrifuged (8000rpm, 10min), and the precipitate obtained by centrifugation was washed 3 times with deionized water, and then dialyzed with a dialysis bag with a molecular weight cutoff of 3500Da to obtain a sodium pyruvate sustained-release nanosystem based on targeted modification.

[0096] Comparative Example 1

[0097] The preparation method of the sodium pyruvate sustained-release nanosystem based on targeted modification is different from that of Example 1 in that when preparing the modified silk fibroin, the addition of 5-carboxypentyl-triphenylphosphonium bromide is omitted.

[0098] Comparative Example 2

[0099] The method for preparing the sodium pyruvate sustained-release nanosystem based on targeted modification is different from that of Example 1 in that the folic acid derivative 1B is omitted when preparing the modified silk fibroin.

[0100] Comparative Example 3

[0101] The preparation method of the sodium pyruvate sustained-release nanosystem is different from that of Example 1 in that the modified silk fibroin aqueous solution is replaced by a silk fibroin aqueous solution.

[0102] Test Example 1

[0103] The product obtained in Example 1 was diluted with deionized water at a volume ratio of 1:5, and the diluted suspension was dropped onto a glass slide and observed under an optical microscope. The results are as follows: Figure 2 shown.

[0104] like Figure 2 As shown, it can be seen that the products obtained in Example 1 of the present invention are all spherical in structure and have a small particle size range, which is conducive to expanding the release efficiency in the lung lesion site.

[0105] Test Example 2

[0106] Biocompatibility test: First, 100 μL of HULEC-5a lung cells (1×10 5 / mL), and cultured the cells using DMEM complete medium. The 96-well plate inoculated with the cells was then placed in a cell culture incubator at 37°C and 5% CO2 for 12 hours. After the culture was completed, the cell culture medium in each well was discarded, and 200 μL of DMEM complete medium containing the sustained-release nanosystem of Example 1 at a concentration of 50 μg / mL was added to the 96-well plate, and the culture was continued under the above conditions for 24 hours. After the culture was completed, the cell morphology was observed under a microscope. The present invention uses DMEM serum-free medium as the negative control group, and DMEM complete medium supplemented with 5% phenol as the positive control group. The experimental results are shown in the figure. Figure 3 shown.

[0107] Figure 3 The figure is the biocompatibility result of the sustained-release nanosystem in Example 1 and lung cells HULEC-5a. Figure 3 It can be seen that the cell morphology of the experimental group (sustained-release nanosystem of Example 1) is similar to that of the negative control group, which is a normal lung cell morphology, indicating that the sustained-release nanosystem of Example 1 does not damage the growth morphology of lung cells; while the cell morphology of the positive control group is deteriorated. Therefore, the sustained-release nanosystem prepared in Example 1 has good biocompatibility.

[0108] Test Example 3

[0109] Cell quantitative uptake experiment: LA795 mouse lung adenocarcinoma cells in logarithmic growth phase were divided into Example 1 group and Comparative Examples 1-3 groups. The cells were plated at 1×10 5 The cells were inoculated into a 96-well plate at a density of 100 / mL, 0.2 mL was added to each well, and cultured for 24 hours at 37°C and 5% CO2. After 24 hours of culture, the original complete culture medium was discarded, and then 0.2 mL of the sustained-release nanosystem obtained in Example 1 and Comparative Examples 1-3 labeled with stearylamine-isothiocyanate fluorescein (ODA-FITC) at a concentration of 2 mg / mL was added to each well. After 6 hours of culture, the cells were washed with PBS, digested with trypsin, centrifuged, and the precipitate was resuspended with PBS buffer. The fluorescence intensity of each group of cells at different time points was detected by flow cytometry. The results are shown in FIG. Figure 4 shown.

[0110] Depend on Figure 4 It can be seen that the cellular uptake rate of Example 1 group is always maintained at a high level and is much higher than that of Comparative Example 3 group, wherein the cellular uptake rate of Example 1 at 6 h is close to 100%, which indicates that the sustained-release nanosystem of the present invention can carry sodium pyruvate into cells and improve the targeting of the drug.

[0111] Comparative Example 3 did not modify the silk protein with quaternary ammonium salt, folic acid derivative 1B, and 5-carboxypentyl-triphenylphosphonium bromide, and its cell uptake rate was the lowest. The sustained-release nanosystem described in Comparative Example 1 omitted the addition of 5-carboxypentyl-triphenylphosphonium bromide, and the cell uptake rate at 6h was 82.38%, and the addition of folic acid derivative 1B was omitted in Comparative Example 2, and the cell uptake rate at 6h was 70.56%. Compared with Example 1, the cell uptake rates of Comparative Examples 1-3 groups all decreased. It can be seen that the modification of silk protein with quaternary ammonium salt, folic acid derivative, and 5-carboxypentyl-triphenylphosphonium bromide can improve the targeting of the sustained-release nanosystem.

[0112] In summary, the sustained-release nanosystem prepared in Example 1 uses silk fibroin as raw material, and is modified by quaternary ammonium salt modification, folic acid derivative modification and 5-carboxypentyl-triphenylphosphonium bromide modification to prepare modified silk fibroin with lung targeting; then the electrostatic interaction between the modified silk fibroin and sodium phytate is utilized to form a nanocarrier, which can effectively deliver the sodium pyruvate encapsulated therein to the lungs of mice, greatly improving the drug delivery efficiency.

[0113] Test Example 4

[0114] Determination of drug concentration in rat plasma and bronchoalveolar lavage fluid (BALF):

[0115] (1) 70 healthy SD rats, 6-8 weeks old and weighing 180g-220g, were randomly divided into a free group (sodium pyruvate was directly administered), Example 1-3 groups and Comparative Example 1-3 groups (administered with the corresponding sustained-release nanosystem as in Example 1, and the rest was the same), with 10 rats in each group. All the above groups were administered by tracheal instillation at a dose of 300 μg / kg. 24 hours after administration, blood was collected from the eye sockets, and sodium heparin was used as an anticoagulant. The collected blood was centrifuged at 4000rpm for 5min to separate the plasma, triamcinolone acetonide was selected as the internal standard, and then HPLC was used to determine the concentration of sodium pyruvate in the plasma.

[0116] (2) After blood was drawn from the rats, their lung bronchoalveolar lavage fluid was collected. Specifically, 2 mL of DMSO was injected into the rat lungs and aspirated after 30 seconds to obtain a lung bronchoalveolar lavage fluid (BALF) cell suspension. Each group was repeated 3 times. The collected BALF supernatant was mixed with methanol at a volume ratio of 1:2, centrifuged at 1000 rpm for 10 minutes, and the centrifuged BALF supernatant was analyzed by high performance liquid chromatography (HPLC) to determine the content of sodium pyruvate in BALF. The above experimental results are shown in Table 1.

[0117]

[0118] As shown in Table 1, the concentration of sodium pyruvate in the plasma of the free group and the comparative example 1-3 groups was significantly higher than that in the BALF, while the sodium pyruvate in the example 1-3 groups was mainly distributed in the BALF, which indicates that the sustained-release nanosystem prepared by the present invention can reduce the distribution of the drug throughout the body and increase the availability of the drug in the lung tissue.

[0119] Among them, the nanosystem of comparative example 1 group did not use 5-carboxypentyl-triphenylphosphonium bromide to modify the silk fibroin during the preparation process, the nanosystem of comparative example 2 group did not use folic acid derivative 1B to modify the silk fibroin during the preparation process, and the nanosystem of comparative example 3 group used unmodified silk fibroin. The concentration of sodium pyruvate in plasma of comparative examples 1-3 groups was higher, which also shows that the present invention, through the modification of silk fibroin, enables the finally prepared sustained-release nanosystem to better improve the enrichment of sodium pyruvate in the lungs, and further improve the targeting of the sustained-release nanocarrier.

[0120] Test Example 5

[0121] Drug loading rate and encapsulation efficiency test of sodium pyruvate sustained-release nanosystem based on targeted modification:

[0122] In step b, in preparing the targeted modified sodium pyruvate sustained-release nanosystem, 1 mL of the reaction solution after the reaction of Example 1-3 and Comparative Example 1-3 was taken, the supernatant was taken after centrifugation, and the absorbance value of the supernatant at a wavelength of 255 nm was measured by an ultraviolet spectrophotometer. According to the measured free sodium pyruvate concentration in the supernatant, the drug loading amount and encapsulation efficiency of the sustained-release nanosystem for sodium pyruvate were calculated using the following formula. The results are shown in Table 2.

[0123] Drug loading rate (%) = (initial drug input - free drug content) / total mass of sustained-release nanosystem × 100%;

[0124] Encapsulation efficiency (%) = (initial drug input - free drug content) / initial drug input × 100%.

[0125]

[0126] From the test data in Table 2, it can be seen that the drug loading of Examples 1-3 of the present invention is maintained at more than 24%, and the encapsulation rate is maintained at more than 93%, but the drug loading and encapsulation rate of Comparative Examples 1-3 are reduced. This shows that the sustained-release nanosystem prepared by modifying the silk fibroin of the present invention has a high encapsulation rate. The higher the encapsulation rate, the more controllable the release behavior of the drug in the body, which can improve the bioavailability and therapeutic effect of the drug.

[0127] Test Example 5

[0128] In order to explore the in vitro sustained release performance of the sustained-release nanosystems prepared in Examples 1-3 and Comparative Examples 1-3 of the present invention, the in vitro release effect of the sustained-release nanosystems was studied by simulating the lung environment with mucus. First, artificial lung fluid (SLF) was prepared, and then 30 mg of the sustained-release nanosystems prepared in Examples 1-3 and Comparative Examples 1-3 were weighed and loaded into a dialysis bag, which was placed in an artificial lung fluid (50 mL) release medium, and then shaken and cultured in a constant temperature shaking incubator at 37°C and 50 rpm. 1 mL of the suspension was drawn at 1h, 2h, 4h, 8h, 12h, 18h, and 24h of the culture, and then 1 mL of the corresponding release medium of the same volume and temperature was added, and the taken suspension was centrifuged at 10000 rpm for 5 min, and the supernatant was taken, and its absorbance at 255 nm was measured; the cumulative release rate was calculated according to the following formula, and the final measurement results are shown in Table 3.

[0129] , where M t is the mass of sodium pyruvate released at the sampling time point, and M0 is the total mass of sodium pyruvate in the nanosystem.

[0130]

[0131] Observe the results in Table 3 and find that the cumulative release rate of the sustained-release nanosystem described in Examples 1-3 can reach about 90% in 24 hours. It can be concluded that the sustained-release nanosystem obtained in Examples 1-3 can stably and continuously release sodium pyruvate within 24 hours, and continuously and effectively reduce lung inflammation. Comparative Example 1 omits the modification of silk fibroin by 5-carboxypentyl-triphenylphosphonium bromide, and its cumulative release rate in 1 hour is 10.9%, and the cumulative release rate in 2 hours is 25.5%. As time increases, its cumulative release amount in 24 hours reaches 82.3%, but it is still lower than Example 1. Comparative Examples 2 and 3 have relatively poor sustained-release effects due to incomplete or no modification of silk fibroin.

[0132] In summary, the sustained-release nanosystems obtained in Examples 1-3 can stably encapsulate drugs, can be specifically enriched in lung lesions and achieve sustained release of sodium pyruvate, have significant targeting and sustained-release effects, and are suitable for preparing drugs for treating lung diseases. Therefore, the sustained-release nanosystems prepared by the present invention have potential application value in targeted treatment of lung diseases.

[0133] 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 a sodium pyruvate sustained-release nanosystem based on targeted modification, characterized in that: The following steps are involved: a. Preparation of modified silk fibroin; b. Under homogenization conditions, a mixed aqueous solution of sodium phytate and sodium pyruvate is added to the modified silk fibroin aqueous solution, and a mixed solution is obtained after homogenization, and the mixed solution is stirred for reaction. After the reaction is completed, the reaction solution is centrifuged, and the precipitate is collected and washed and dialyzed to obtain the sodium pyruvate sustained-release nanosystem based on targeted modification; Wherein, the preparation steps of the modified silk fibroin are as follows: (1) At 0°C, NaH was added to a solution of folic acid in N,N-dimethylformamide in an inert atmosphere, and the mixture was stirred for 15-30 min. Then, 4-methoxybenzyl chloride was added and the mixture was stirred at room temperature for 3-5 h. After the reaction was completed, the reaction solution was concentrated and separated by column chromatography to obtain folic acid derivative 1B. The structural formulas of the folic acid and folic acid derivative 1B are respectively: 、 ； (2) adding silk fibroin, N,N-diisopropylethylamine and glycidyl trimethylammonium chloride into water, heating for reaction, and after the reaction is completed, dialyzing and freeze-drying are performed to obtain compound 1; The structural formulas of the silk fibroin and compound 1 are respectively: 、 ； (3) adding dicyclohexylcarbodiimide, 4-dimethylaminopyridine and 5-carboxypentyl-triphenylphosphonium bromide to a dimethyl sulfoxide solution containing compound 1 and reacting for 2-5 hours to obtain a reaction solution 1; (4) Add dicyclohexylcarbodiimide, 1-hydroxybenzotriazole and folic acid derivative 1B to reaction solution 1, react for 20-24 hours to obtain reaction solution 2, and centrifuge, wash and dry reaction solution 2 to obtain compound 2; The structural formula of the compound 2 is: ； (5) Adding 2,3-dichloro-5,6-dicyanobenzoquinone to the solution of compound 2 and stirring, centrifuging, washing and drying the stirred reaction solution to obtain modified silk fibroin, wherein the structural formula of the modified silk fibroin is: 。 2. The method for preparing the sodium pyruvate sustained-release nanosystem based on targeted modification according to claim 1, characterized in that: In step (1), the concentration of folic acid in the N,N-dimethylformamide solution is 87.5-116.7 mg / mL, the mass ratio of folic acid to NaH is 1:(0.28-0.58), and the mass ratio of folic acid to 4-methoxybenzyl chloride is 1:(0.3-0.5).

3. The method for preparing the sodium pyruvate sustained-release nanosystem based on targeted modification according to claim 1, characterized in that: The mass ratio of the silk fibroin, N,N-diisopropylethylamine and glycidyl trimethylammonium chloride in step (2) is 1:(0.06-0.18):(0.05-0.15), the temperature of the heating reaction in step (2) is 40-60° C., and the heating reaction time is 2-4 h.

4. The method for preparing the sodium pyruvate sustained-release nanosystem based on targeted modification according to claim 1, characterized in that: The concentration of the dimethyl sulfoxide solution of compound 1 in step (3) is 10-20 mg / mL; the mass ratio of compound 1, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and 5-carboxypentyl-triphenylphosphonium bromide is 1:(3.3-9.9):(2.69-8.06):(9.14-27.44).

5. The method for preparing the sodium pyruvate sustained-release nanosystem based on targeted modification according to claim 1, characterized in that: The mass ratio of dicyclohexylcarbodiimide, 1-hydroxybenzotriazole and folic acid derivative 1B in step (4) is (17.32-51.99):(11.35-34.05):(11.23-33.69).

6. The method for preparing the sodium pyruvate sustained-release nanosystem based on targeted modification according to claim 1, characterized in that: The solvent of the solution of compound 2 in step (5) is a dichloromethane / water mixed solvent, the dosage ratio of compound 2 to the dichloromethane / water mixed solvent is 1 g:(8-12) mL, the mass ratio of compound 2 to 2,3-dichloro-5,6-dicyanobenzoquinone is 1:(90.8-272.4), and the stirring time is 0.5-3 h.

7. The method for preparing the sodium pyruvate sustained-release nanosystem based on targeted modification according to claim 1, characterized in that: In step b, the concentration of the modified silk fibroin aqueous solution is 1-5wt%, the concentration of sodium phytate in the mixed aqueous solution of sodium phytate and sodium pyruvate is 1-5wt% and the concentration of sodium pyruvate is 5-10wt%, the volume ratio of the modified silk fibroin aqueous solution: the mixed solution of sodium phytate and sodium pyruvate is 1:(1-1.5), the homogenization time is 20-30min, and the stirring reaction time is 20-24h.

8. Sodium pyruvate sustained-release nanosystem based on targeted modification, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 7.

9. Use of the sodium pyruvate sustained-release nanosystem based on targeted modification according to claim 8 in the preparation of a drug for treating chronic obstructive pulmonary disease.

Images