A sodium pyruvate sustained-release microsphere preparation based on enzyme response and its preparation method and application

Abstract

The present invention relates to the field of biomedicine, and discloses a sodium pyruvate sustained-release microsphere preparation based on enzyme response, and a preparation method and application thereof, including: (1) adding matrix metalloproteinase sensitive peptide and triethylamine to a hyaluronic acid solution, stirring the reaction, and after the reaction is completed, dialyzing and drying the reaction solution to obtain hyaluronic acid grafted with matrix metalloproteinase sensitive peptide; (2) adding phosphatidylserine, 4-dimethylaminopyridine and N,N-dicyclohexylcarbodiimide to a gelatin solution for reaction, and the reaction solution after the reaction is post-treated to obtain gelatin grafted with phosphatidylserine; (3) dispersing the hyaluronic acid grafted with matrix metalloproteinase sensitive peptide and the gelatin grafted with phosphatidylserine in water to obtain a mixed solution; adding sodium pyruvate and a cross-linking agent thereto, stirring the reaction, and preparing a sustained-release microsphere preparation by spray drying the reaction solution after the reaction. The microsphere preparation of the present invention can slowly release sodium pyruvate and enhance its therapeutic effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a sodium pyruvate sustained-release microsphere preparation based on enzyme response, and a preparation method and application thereof. Background Art

[0002] Sodium pyruvate is the sodium salt form of pyruvate and plays a vital role in human metabolism. Sodium pyruvate is not only involved in energy supply, but also has multiple physiological functions such as regulating metabolism, anti-oxidation, anti-inflammation and cell protection. It can remove free radicals in the body and reduce the damage of oxidative stress to cells. Studies have shown that sodium pyruvate can upregulate the expression of antioxidant enzymes and improve the resistance of cells to oxidative stress. In addition, sodium pyruvate can also inhibit cell apoptosis and protect cells from damage. In recent years, with the deepening of scientific research, the pharmacological effects of sodium pyruvate have gradually received widespread attention, and it has a wide range of applications in pharmaceuticals, scientific research, food and cosmetics.

[0003] Although sodium pyruvate currently has the above advantages and can be used to treat lung inflammation, the traditional method of administration may cause it to degrade or deteriorate during storage and use, making it impossible to maintain a relatively stable drug concentration in the lungs. Fluctuations in drug concentrations may lead to unstable treatment effects and even adverse reactions. In addition, the traditional method of administration is unable to accurately control the release rate of the drug according to the severity and progression of the lung disease, which may cause the drug to act in the lungs for an insufficient or excessive time, affecting the treatment effect.

[0004] At present, drugs can be slowly released at the lesion site through drug delivery carriers. Hyaluronic acid is considered to be one of the ideal drug carrier materials because of its good biocompatibility and biodegradability. It can reduce the rejection reaction in the body and can also be compounded with other biocompatible materials (such as chitosan, gelatin, etc.) to prepare carriers with excellent performance to regulate the release behavior of drugs. Gelatin, as a collagen extracted from animal bones and cartilage, also has good biocompatibility and biodegradability. The composite carrier of hyaluronic acid and gelatin has good biocompatibility, drug loading capacity and controlled release ability, and can achieve drug delivery. However, the functionality of the carrier is relatively single and does not have responsiveness, and cannot effectively achieve the slow release and intelligent response of drugs at the lesion site.

[0005] Therefore, in view of the shortcomings of the existing technology, it is very necessary to design a drug preparation that can specifically release drugs for treating lung diseases to increase the local concentration. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, one of the objects of the present invention is to provide a method for preparing a sodium pyruvate sustained-release microsphere preparation based on enzyme response, which is simple, efficient and easy to operate.

[0007] The second object of the present invention is to provide a sodium pyruvate sustained-release microsphere preparation based on enzyme response prepared according to the above preparation method. The prepared sodium pyruvate sustained-release microsphere preparation has good enzyme responsiveness, can directly target lung lesions, slowly release sodium pyruvate under the joint action of matrix metalloproteinase-9 (MMP-9) and phospholipase C, increase its concentration in lung lesions, prolong the action time and improve the therapeutic effect.

[0008] The third object of the present invention is to provide the application of the above-mentioned sodium pyruvate sustained-release microsphere preparation based on enzyme response.

[0009] One of the purposes of the present invention is achieved by the following technical solution:

[0010] A method for preparing a sodium pyruvate sustained-release microsphere preparation based on enzyme response comprises the following steps:

[0011] (1) adding a matrix metalloproteinase sensitive peptide and triethylamine to a hyaluronic acid solution, stirring for reaction, and after the reaction is completed, dialyzing and drying the reaction solution to obtain a hyaluronic acid grafted with a matrix metalloproteinase sensitive peptide;

[0012] (2) adding phosphatidylserine, 4-dimethylaminopyridine and N,N-dicyclohexylcarbodiimide to a gelatin solution for reaction, and after the reaction is completed, the reaction solution is post-treated to obtain gelatin grafted with phosphatidylserine;

[0013] (3) Dispersing hyaluronic acid grafted with matrix metalloproteinase sensitive peptide and gelatin grafted with phosphatidylserine in water to obtain a mixed solution; adding sodium pyruvate and a cross-linking agent to the mixed solution, stirring for reaction, and after the reaction is completed, spray drying the reaction solution to obtain the enzyme-responsive sodium pyruvate sustained-release microsphere preparation.

[0014] Furthermore, in step (1), the matrix metalloproteinase-sensitive peptide is PLGLWA, and the amino acid sequence of PLGLWA is Pro-Leu-Gly-Leu-Trp-Ala; the molecular weight of the hyaluronic acid is 1000-2000 KDa, and the concentration of the hyaluronic acid solution is 10-50 mg / mL; and the hyaluronic acid solution is an N,N-dimethylformamide solution of hyaluronic acid.

[0015] Furthermore, in step (1), the mass ratio of hyaluronic acid, matrix metalloproteinase sensitive peptide and triethylamine is 1:(0.1-0.2):(0.01-0.03), and the stirring reaction time is 10-15 hours.

[0016] Furthermore, the gelatin solution in step (2) is a gelatin N,N-dimethylformamide solution, and the concentration of the gelatin solution is 30-70 mg / mL; the mass ratio of the gelatin, phosphatidylserine, 4-dimethylaminopyridine and N,N-dicyclohexylcarbodiimide is 1:(0.05-0.2):(0.1-0.5):(1-2), and the reaction time is 3-5 h.

[0017] Furthermore, in step (3), the mass ratio of hyaluronic acid grafted with matrix metalloproteinase sensitive peptide, gelatin grafted with phosphatidylserine, sodium pyruvate, and cross-linking agent is 1:(1-2):(0.4-0.81):(0.01-0.1).

[0018] Furthermore, the total concentration of hyaluronic acid grafted with matrix metalloproteinase sensitive peptide and gelatin grafted with phosphatidylserine in the mixed solution of step (3) is 1-5wt%.

[0019] Furthermore, the cross-linking agent is glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or N-hydroxysuccinimide.

[0020] Furthermore, the stirring reaction time in step (3) is 2-4 hours, the spray drying inlet temperature of the spray drying method is 120-180°C, and the outlet temperature is 60-90°C.

[0021] The second object of the present invention is achieved by adopting the following technical solution:

[0022] The sodium pyruvate sustained-release microsphere preparation based on enzyme response of the present invention is prepared according to the above preparation method.

[0023] The third object of the present invention is achieved by adopting the following technical solution:

[0024] The present invention provides application of the sodium pyruvate sustained-release microsphere preparation based on enzyme response in preparing medicines for treating lung diseases.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention provides a method for preparing a sodium pyruvate sustained-release microsphere preparation based on enzyme response. In view of the high expression of matrix metalloproteinase-9 and phospholipase C in the lungs of pneumonia patients, matrix metalloproteinase sensitive peptides and phosphatidylserine are selected to modify sustained-release microspheres. The present invention firstly reacts the carboxyl group in hyaluronic acid with the amino group in the matrix metalloproteinase sensitive peptide PLGLWA to graft the matrix metalloproteinase sensitive peptide on hyaluronic acid; and then reacts the carboxyl group in gelatin with the amino group of phosphatidylserine to graft the gelatin with phosphatidylserine. Under the action of a cross-linking agent, composite microspheres of hyaluronic acid grafted with matrix metalloproteinase sensitive peptide and gelatin grafted with phosphatidylserine are prepared, and the drug sodium pyruvate is added at the same time, and finally a sodium pyruvate sustained-release microsphere preparation with enzyme response is prepared. The preparation method is simple and easy to operate, and the prepared microsphere preparation has a small particle size and uniform morphology.

[0027] 2. The enzyme-responsive sodium pyruvate sustained-release microsphere preparation prepared by the present invention has a targeting effect, and can target the microsphere preparation to the lung lesions, increase its concentration in the lung lesions, thereby better exerting antioxidant and anti-inflammatory effects and enhancing the therapeutic effect. Secondly, the release system based on enzyme response can adjust the release rate of sodium pyruvate as needed, thereby optimizing its therapeutic effect.

[0028] 3. The present invention applies the prepared enzyme-responsive sodium pyruvate sustained-release microsphere preparation to the treatment of lung diseases, so that the distribution of sodium pyruvate in the lungs is more uniform and the delivery efficiency is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a morphology diagram of the sustained-release microsphere preparation obtained in Example 1 of the present invention;

[0030] Figure 2 The in vitro cytotoxicity test results of the sustained-release microsphere preparations of Examples 1-3 and Comparative Examples 1-3 of the present invention are shown;

[0031] Figure 3 The results of the IL-1β content in the supernatant of human bronchial epithelial cells in each group are shown in FIG.

[0032] Figure 4 The graph shows the results of TNF-α content in the supernatant of human bronchial epithelial cells in each group. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0034] Example 1

[0035] A method for preparing a sodium pyruvate sustained-release microsphere preparation based on enzyme response, the method comprising the following steps:

[0036] (1) Weigh 3 g of hyaluronic acid and add it to 100 mL of DMF. Add 0.45 g of matrix metalloproteinase-sensitive peptide PLGLWA (amino acid sequence: Pro-Leu-Gly-Leu-Trp-Ala) and 0.06 g of triethylamine (Et 3 N), stirring and reacting at room temperature for 12 hours; after the reaction, the reaction solution was put into a dialysis bag, dialyzed at 4°C for 48 hours, and then freeze-dried to obtain hyaluronic acid grafted with matrix metalloproteinase sensitive peptide;

[0037] (2) Weigh 5 g of gelatin and add it to 100 mL of DMF to obtain a gelatin solution, to which 0.5 g of phosphatidylserine, 1.25 g of 4-dimethylaminopyridine and 7.5 g of N,N-dicyclohexylcarbodiimide were added in sequence, and the mixture was reacted at room temperature for 3 h. After the reaction, the reaction solution was placed in a dialysis bag, dialyzed at 4°C for 48 h, and then freeze-dried to obtain gelatin grafted with phosphatidylserine.

[0038] (3) Weigh 1.2 g of hyaluronic acid grafted with matrix metalloproteinase sensitive peptide and 1.8 g of gelatin grafted with phosphatidylserine, dissolve the two in 97 mL of deionized water to obtain a mixed solution, add 0.75 g of sodium pyruvate and 0.06 g of glutaraldehyde to the mixed solution, and stir at 400 rpm at room temperature for 3 h. After the reaction, the reaction solution is spray dried to obtain microspheres. Specifically, the spray drying inlet temperature is 150 °C and the outlet temperature is 75 °C. The microspheres are collected and dried in a vacuum drying oven for 36 h to obtain an enzyme-responsive sodium pyruvate sustained-release microsphere preparation.

[0039] A sodium pyruvate sustained-release microsphere preparation based on enzyme response is prepared by the above preparation method.

[0040] Example 2

[0041] A method for preparing a sodium pyruvate sustained-release microsphere preparation based on enzyme response comprises the following steps:

[0042] (1) Weigh 1 g of hyaluronic acid and add it to 100 mL of DMF. Add 0.1 g of matrix metalloproteinase-sensitive peptide PLGLWA (amino acid sequence: Pro-Leu-Gly-Leu-Trp-Ala) and 0.01 g of triethylamine (Et 3 N), stirring and reacting at room temperature for 10 hours; after the reaction, the reaction solution was put into a dialysis bag, dialyzed at 4°C for 48 hours, and then freeze-dried to obtain hyaluronic acid grafted with matrix metalloproteinase sensitive peptide;

[0043] (2) 3 g of gelatin was added to 100 mL of DMF to obtain a gelatin solution, to which 0.15 g of phosphatidylserine, 0.3 g of 4-dimethylaminopyridine and 3 g of N,N-dicyclohexylcarbodiimide were added in sequence, and the mixture was reacted at room temperature for 4 h. After the reaction, the reaction solution was placed in a dialysis bag and dialyzed at 4°C for 48 h, followed by freeze-drying to obtain gelatin grafted with phosphatidylserine;

[0044] (3) Weigh 0.5 g of hyaluronic acid grafted with matrix metalloproteinase sensitive peptide and 0.5 g of gelatin grafted with phosphatidylserine, dissolve the two in 99 mL of deionized water to obtain a mixed solution, add 0.2 g of sodium pyruvate and 0.005 g of glutaraldehyde to the mixed solution, and stir at 400 rpm for 3 h at room temperature; after the reaction, the reaction solution is spray dried to obtain microspheres. Specifically, the spray drying inlet temperature is 120 °C and the outlet temperature is 60 °C. The microspheres are collected and dried in a vacuum drying oven for 36 h to obtain an enzyme-responsive sodium pyruvate sustained-release microsphere preparation.

[0045] A sodium pyruvate sustained-release microsphere preparation based on enzyme response is prepared by the above preparation method.

[0046] Example 3

[0047] A method for preparing a sodium pyruvate sustained-release microsphere preparation based on enzyme response comprises the following steps:

[0048] (1) Weigh 5 g of hyaluronic acid and add it to 100 mL of DMF. Add 1 g of matrix metalloproteinase-sensitive peptide PLGLWA (amino acid sequence: Pro-Leu-Gly-Leu-Trp-Ala) and 0.15 g of triethylamine (Et 3 N), stirring and reacting at room temperature for 15 hours; after the reaction, the reaction solution was put into a dialysis bag, dialyzed at 4°C for 48 hours, and then freeze-dried to obtain hyaluronic acid grafted with matrix metalloproteinase sensitive peptide;

[0049] (2) Weigh 7 g of gelatin and add it to 100 mL of DMF to obtain a gelatin solution, to which 1.4 g of phosphatidylserine, 3.5 g of 4-dimethylaminopyridine and 14 g of N,N-dicyclohexylcarbodiimide are added in sequence, and the mixture is reacted at room temperature for 5 h. After the reaction, the reaction solution is placed in a dialysis bag, dialyzed at 4°C for 48 h, and then freeze-dried to obtain gelatin grafted with phosphatidylserine.

[0050] (3) Weigh 1.67 g of hyaluronic acid grafted with matrix metalloproteinase-sensitive peptide and 3.33 g of gelatin grafted with phosphatidylserine, dissolve the two in 95 mL of deionized water to obtain a mixed solution, add 1.35 g of sodium pyruvate and 0.167 g of glutaraldehyde to the mixed solution, and stir at 400 rpm at room temperature for 3 h. After the reaction, the reaction solution is spray dried to obtain microspheres. Specifically, the spray drying inlet temperature is 180°C and the outlet temperature is 90°C. The microspheres are collected and dried in a vacuum drying oven for 36 h to obtain an enzyme-responsive sodium pyruvate sustained-release microsphere preparation.

[0051] A sodium pyruvate sustained-release microsphere preparation based on enzyme response is prepared by the above preparation method.

[0052] Comparative Example 1

[0053] The preparation method of the sodium pyruvate sustained-release microsphere preparation based on enzyme response in this comparative example is different from that in Example 1 in that step (1) is omitted and the hyaluronic acid grafted with the matrix metalloproteinase sensitive peptide in step (3) is replaced by hyaluronic acid.

[0054] Comparative Example 2

[0055] The difference between the preparation method of the sodium pyruvate sustained-release microsphere preparation based on enzyme response in this comparative example and that in Example 1 is that step (2) is omitted, and the gelatin grafted with phosphatidylserine in step (3) is replaced by gelatin.

[0056] Comparative Example 3

[0057] The preparation method of the sodium pyruvate sustained-release microsphere preparation based on enzyme response in this comparative example is different from that in Example 1 in that steps (1) and (2) are omitted, and the hyaluronic acid grafted with matrix metalloproteinase sensitive peptide in step (3) is replaced by hyaluronic acid, and the gelatin grafted with phosphatidylserine is replaced by gelatin.

[0058] Test Example 1

[0059] The microspheres obtained in Example 1 were diluted with deionized water at a volume ratio of 1:5, and the diluted suspension was dropped onto a glass slide, which was placed under an optical microscope to observe its microscopic morphology.

[0060] like Figure 1 As shown, it can be seen that the morphology of the microspheres obtained in Example 1 of the present invention is relatively uniform, all of which are spherical structures, and the particle size range is small, which is conducive to expanding its release efficiency in the lung lesion site.

[0061] Test Example 2

[0062] In vitro cytotoxicity test: Mouse fibroblast L929 cells were selected as test cells, and the microsphere preparations obtained in Examples 1-3 and Comparative Examples 1-3 were used for cytotoxicity study. The cells in the de-logarithmic phase were resuspended in complete medium (90% DMEM medium + 10% FBS) and the concentration was 2×10 4 The cells were inoculated at a density of 100 mg / mL in a 96-well plate and cultured in a 37°C incubator for 24 h. The sustained-release microsphere preparations obtained in Examples 1-3 and Comparative Examples 1-3 were added at different concentrations (20 mg / mL, 80 mg / mL, 160 mg / mL). Three replicate wells were set for each group, and a blank control group was set. After 48 h of culture, the culture medium was discarded, and CCK-8 solution was added to each well. The cells were incubated in the dark for 1 h. The absorbance value A of each well at 450 nm was measured by an ELISA instrument. The average value was selected as the final result, and the cell survival rate was calculated. The results are shown in FIG. Figure 2 shown.

[0063] Depend on Figure 2 The results show that the microsphere preparations obtained in Examples 1-3 and Comparative Examples 1-3 have no cytotoxicity to normal mouse fibroblasts and have good cell compatibility.

[0064] Test Example 3

[0065] Encapsulation efficiency test: The reaction solution of step (3) in Example 1-3 and Comparative Example 1-3 was centrifuged at 5000 rpm for 10 min, and the absorbance of the supernatant at a wavelength of 255 nm was measured using an ultraviolet spectrophotometer. Based on the measured free sodium pyruvate concentration in the supernatant, the encapsulation efficiency of the sustained-release nanosystem for sodium pyruvate was calculated using the following formula. The results are shown in Table 1.

[0066] Encapsulation efficiency (%) = (initial drug input - free drug content in the supernatant) / initial drug input × 100%.

[0067] Table 1

[0068]

[0069] It can be seen from Table 1 that the enzyme-responsive sodium pyruvate sustained-release microsphere preparation prepared in the present invention has a good encapsulation rate.

[0070] Test Example 4

[0071] Sustained release performance test: Hepes buffer, 50 ng / mL matrix metalloproteinase-9 (MMP-9) and 50 ng / mL phospholipase C mixed solution (Hepes buffer as solvent) were selected as release media, and the sustained-release microsphere preparations prepared in Examples 1-3 and Comparative Examples 1-3 were placed in the above two release media, respectively. Three parallel tests were set up for each group, and they were placed in a constant temperature shaker at 37°C and 100 rpm for incubation. At 0h, 1h, 2h, 4h, 8h, 12h, 24h and 48h of culture, 200 μL of the supernatant of each group was sampled for sodium pyruvate detection. Each group was sampled 3 times, and then the corresponding release medium of the same volume and temperature was added. The absorbance value of the taken solution was detected at a wavelength of 255nm, and the cumulative release rate was statistically analyzed. The results are shown in Tables 2 and 3. The drug release behavior of the prepared sustained-release microsphere preparation in the presence and absence of enzymes was analyzed.

[0072] Table 2 Sustained release effect of sustained-release microsphere preparation in Hepes buffer

[0073]

[0074] Table 3 Sustained release effect of sustained-release microsphere preparation in mixed solution of MMP-9 and phospholipase C

[0075]

[0076] From the experimental results in Table 2, it can be seen that in Hepes buffer, the cumulative release rate of sodium pyruvate in Examples 1-3 and Comparative Examples 1-3 is low, and the cumulative release rate in 48h is only about 40%. From the results in Table 3, it can be seen that compared with the sustained release effect of the sustained release microsphere preparation in the absence of enzyme, in the presence of the mixed solution of MMP-9 and phospholipase C, the cumulative release rate of the sustained release microsphere preparation obtained in Examples 1-3 and Comparative Examples 1-2 is significantly increased, while the cumulative release rate of sodium pyruvate in Comparative Example 3 is similar to the effect in the absence of enzyme, indicating that the sustained release microsphere preparations obtained in Examples 1-3 and Comparative Examples 1-2 are both enzyme responsive, because the sustained release microsphere preparations obtained in Examples 1-3 and Comparative Examples 1-2 are grafted with substrates with enzyme responsiveness, while the sustained release microsphere preparation in Comparative Example 3 contains neither matrix metalloproteinase sensitive peptide (PLGLWA) nor phosphatidylserine. This allows the sustained-release microsphere preparations obtained in Examples 1-3 to be in the release medium. As the action time increases, MMP enzyme and phospholipase C can specifically cleave the matrix metalloproteinase-sensitive peptide PLGLWA in the sustained-release microsphere preparations, respectively. The sustained-release microsphere preparations are enzymatically hydrolyzed by MMP-9 and phospholipase C, and the sodium pyruvate loaded inside is also slowly released.

[0077] As shown in Table 3, the cumulative release rate of Example 1-3 in the first 12 hours is about 75%, and the cumulative release rate in 48 hours reaches about 90%. This shows that the sodium pyruvate sustained-release microspheres of the present invention have a longer drug release time, still have a slow release effect within 48 hours, and have the potential for long-term sustained release. Compared with Examples 1-3, the drug cumulative release rate of Comparative Examples 1-2 is lower, because Comparative Examples 1 and 2 are only enzyme responsive to phospholipase C or MMP-9, respectively, and the enzyme cleavage sites are less than those of Examples 1-3, so the release effect is poor.

[0078] In summary, the enzyme-responsive sodium pyruvate sustained-release preparation obtained in Examples 1-3 has a higher efficiency in releasing sodium pyruvate in an enzyme medium than a release medium without an enzyme, and has good enzyme responsiveness, so that sodium pyruvate can be rapidly released at the site of inflammatory lesions, thereby prolonging its action time at the lesions and improving the therapeutic effect.

[0079] Test Example 5

[0080] Human bronchial epithelial cells (16HBE) growing in the logarithmic phase were selected and divided into the control group: 16HBE were cultured with complete medium only; the model group: human bronchial epithelial cells were incubated with complete medium containing 50μg / mL lipopolysaccharide (LPS) for 12h, and then cultured with complete medium for 24h; the positive drug group: human bronchial epithelial cells were incubated with complete medium containing 50μg / mL lipopolysaccharide (LPS) for 12h, and then cultured with complete medium containing 1μmol / L dexamethasone. Incubate in the culture medium for 3 h. Example 1 group: incubate human bronchial epithelial cells with complete culture medium containing 50 μg / mL lipopolysaccharide (LPS) for 12 h, and then incubate with complete culture medium (200 μg / mL) containing the sustained-release microsphere preparation obtained in Example 1 for 24 h. Example 2 group: incubate human bronchial epithelial cells with complete culture medium containing 50 μg / mL lipopolysaccharide (LPS) for 12 h, and then incubate with complete culture medium (200 μg / mL) containing the sustained-release microsphere preparation obtained in Example 2 for 24 h. / mL) and incubated for 24h, Example 3 group: human bronchial epithelial cells were incubated with a complete medium containing 50µg / mL lipopolysaccharide (LPS) for 12h, and then incubated with a complete medium containing the sustained-release microsphere preparation obtained in Example 3 (200µg / mL) for 24h, Comparative Example 1 group: human bronchial epithelial cells were incubated with a complete medium containing 50µg / mL lipopolysaccharide (LPS) for 12h, and then incubated with a complete medium containing the sustained-release microsphere preparation obtained in Comparative Example 1 (200µg / mL) for 24h, Comparative Example 2 group: human bronchial epithelial cells were incubated with a complete medium containing 50µg / mL lipopolysaccharide (LPS) for 12h, and then incubated with a complete medium containing the sustained-release microsphere preparation obtained in Comparative Example 2 (200µg / mL) for 24h.

[0081] The content of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the supernatant after centrifugation was detected by enzyme-linked immunosorbent assay (ELISA): 16HBE cells in the logarithmic growth phase were cultured at 2×10 4 The cells were inoculated into 6-well plates at a density of 100 cells / well, and specific operations were performed according to the above grouping. After the culture was completed, the cell culture fluid of each group was collected and centrifuged at 4000 rpm for 10 min. The supernatant was taken and the expression levels of IL-1β and TNF-α were determined according to the specific operations in the instructions. The absorbance values ​​at a wavelength of 450 nm were determined. The results are shown in FIG. Figure 3 and 4 shown.

[0082] Depend on Figure 3 and 4 It can be seen that: compared with the control group, the content of IL-1β and TNF-α in the model group was significantly increased, indicating that the bronchial epithelial cell inflammation model was successfully constructed. Compared with the model group, the levels of IL-1β and TNF-α in the positive drug group and the Example 1-3 group showed a significant downward trend, while the IL-1β and TNF-α content of the comparative examples 1 and 2 showed the second downward trend. It is shown that the sustained-release microsphere preparations prepared in Examples 1-3 can release sodium pyruvate in the inflammatory cell model, enrich it in the lesion, thereby reducing cell inflammation, while there is only a single enzyme responsiveness in the comparative examples 1 and 2, and the cumulative release of the drug in the same time is low, so the therapeutic effect of the comparative examples 1 and 2 on inflammatory cells is poor.

[0083] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for preparing a sodium pyruvate sustained-release microsphere preparation based on enzyme response, characterized in that: The steps include: (1) adding a matrix metalloproteinase sensitive peptide and triethylamine to a hyaluronic acid solution, stirring for reaction, and after the reaction is completed, dialyzing and drying the reaction solution to obtain a hyaluronic acid grafted with a matrix metalloproteinase sensitive peptide; (2) adding phosphatidylserine, 4-dimethylaminopyridine and N,N-dicyclohexylcarbodiimide to a gelatin solution for reaction. After the reaction is completed, the reaction solution is dialyzed and freeze-dried to obtain gelatin grafted with phosphatidylserine; (3) dispersing hyaluronic acid grafted with matrix metalloproteinase sensitive peptide and gelatin grafted with phosphatidylserine in water to obtain a mixed solution; adding sodium pyruvate and a cross-linking agent to the mixed solution, stirring for reaction, and after the reaction is completed, spray drying the reaction solution to obtain the sodium pyruvate sustained-release microsphere preparation based on enzyme response; In step (1), the matrix metalloproteinase-sensitive peptide is PLGLWA, and the amino acid sequence of PLGLWA is Pro-Leu-Gly-Leu-Trp-Ala; the cross-linking agent is glutaraldehyde.

2. The method for preparing the sodium pyruvate sustained-release microsphere preparation based on enzyme response according to claim 1, characterized in that: The molecular weight of the hyaluronic acid in step (1) is 1000-2000 KDa, and the concentration of the hyaluronic acid solution is 10-50 mg / mL; the hyaluronic acid solution is an N,N-dimethylformamide solution of hyaluronic acid.

3. The method for preparing the sodium pyruvate sustained-release microsphere preparation based on enzyme response according to claim 1, characterized in that: In step (1), the mass ratio of hyaluronic acid, matrix metalloproteinase sensitive peptide and triethylamine is 1:(0.1-0.2):(0.01-0.03), and the stirring reaction time is 10-15 hours.

4. The method for preparing the sodium pyruvate sustained-release microsphere preparation based on enzyme response according to claim 1, characterized in that: The gelatin solution in step (2) is a gelatin N,N-dimethylformamide solution, and the concentration of the gelatin solution is 30-70 mg / mL; the mass ratio of the gelatin, phosphatidylserine, 4-dimethylaminopyridine and N,N-dicyclohexylcarbodiimide is 1:(0.05-0.2):(0.1-0.5):(1-2), and the reaction time is 3-5 h.

5. The method for preparing the sodium pyruvate sustained-release microsphere preparation based on enzyme response according to claim 1, characterized in that: In step (3), the mass ratio of hyaluronic acid grafted with matrix metalloproteinase sensitive peptide, gelatin grafted with phosphatidylserine, sodium pyruvate and cross-linking agent is 1:(1-2):(0.4-0.81):(0.01-0.1).

6. The method for preparing the sodium pyruvate sustained-release microsphere preparation based on enzyme response according to claim 1, characterized in that: The total concentration of hyaluronic acid grafted with matrix metalloproteinase sensitive peptide and gelatin grafted with phosphatidylserine in the mixed solution of step (3) is 1-5wt%.

7. The method for preparing the sodium pyruvate sustained-release microsphere preparation based on enzyme response according to claim 1, characterized in that: The stirring reaction time in step (3) is 2-4 hours, the spray drying inlet temperature of the spray drying method is 120-180°C, and the outlet temperature is 60-90°C.

8. A sodium pyruvate sustained-release microsphere preparation based on enzyme response, characterized in that: The enzyme-responsive sodium pyruvate sustained-release microsphere preparation is prepared according to the preparation method according to any one of claims 1-7.

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