CN118986934B - Sodium pyruvate sustained-release nanosystem based on targeted modification and its preparation method and application - Google Patents
Sodium pyruvate sustained-release nanosystem based on targeted modification and its preparation method and application Download PDFInfo
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Abstract
The invention belongs to the field of biological medicine, and in particular relates to a sodium pyruvate sustained-release nano system based on targeted modification, and a preparation method and application thereof. The preparation method of the slow-release nano system comprises the following steps of a, preparing modified silk fibroin, b, adding a mixed aqueous solution of sodium phytate and sodium pyruvate into the modified silk fibroin aqueous solution under a homogenizing condition, obtaining a mixed solution after homogenization, stirring the mixed solution for reaction, centrifuging the reaction solution after the reaction is completed, collecting a precipitate, washing and dialyzing to obtain the sodium pyruvate slow-release nano system based on targeted modification. The slow release nano system has good biocompatibility, can be specifically enriched in lung lesion sites and realize slow release of sodium pyruvate, has obvious targeting and slow release effects, and is suitable for preparing medicines for treating lung diseases.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a sodium pyruvate sustained-release nano system based on targeted modification, and a preparation method and application thereof.
Background
With the deep research on the effects of resisting oxidation, resisting inflammation and the like of sodium pyruvate, the sodium pyruvate has wide development prospect in the fields of medicine, health care products, cosmetics and the like. Although sodium pyruvate has the advantages of reducing inflammatory cytokines, effectively clearing active oxygen free radicals, protecting nerve cells from being damaged by oxidative stress, and has wide application prospects in medical science, diagnostic reagents and medical instruments, the administration mode of the sodium pyruvate needs to be improved in the use process to improve the curative effect of the medicament.
Conventional modes of administration of sodium pyruvate are generally oral formulations, injectable administration and topical administration. These traditional administration modes often have certain drawbacks, such as low drug utilization rate and poor targeting, and in addition, the drugs can be dispersed throughout the whole 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 dosage of the drug by an order of magnitude, directly deliver the drug to lung tissues and reach high concentration, thereby achieving higher therapeutic index and reducing systemic side effects.
Targeted drug delivery systems can deliver drugs precisely to diseased tissues, organs or sites by selecting the appropriate drug carrier or drug delivery system. The accurate performance greatly improves the treatment effect of the medicine and enhances the curative effect of the medicine.
Silk fibroin has good biocompatibility and biodegradability with human tissues, so that the silk fibroin is suitable for being used as a drug carrier, but has certain defects, such as lower drug loading capacity, and influences the release and treatment effects of drugs in vivo. However, silk fibroin has strong plasticity, and has the property of being modified by chemical modification, so that a functional slow-release nano system is obtained, and the medicine is effectively targeted to a focus. At present, the research of preparing nano particles by using folic acid derivatives, 5-carboxypentyl-triphenyl phosphorus bromide modified silk fibroin and sodium phytate and simultaneously coating sodium pyruvate as a nano slow-release system is not reported.
Therefore, developing the sodium pyruvate sustained-release nano system based on targeted modification has important significance for improving the effect of sodium pyruvate on anti-inflammatory and antioxidant aspects.
Disclosure of Invention
The invention aims to provide a preparation method of a sodium pyruvate sustained-release nano system based on targeted modification, which is simple and easy for industrial production, and the prepared sustained-release nano system has good targeting property.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The preparation method of the sodium pyruvate sustained-release nano system based on targeted modification comprises the following steps:
a. preparing modified silk fibroin;
b. Under the condition of homogenization, adding a mixed aqueous solution of sodium phytate and sodium pyruvate into 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 a precipitate, washing and dialyzing to obtain the sodium pyruvate slow-release nano system based on targeted modification.
Further, the preparation steps of the modified silk fibroin are as follows:
(1) Adding NaH into N, N-dimethylformamide solution of folic acid at 0 ℃, stirring for 15-30min, adding 4-methoxybenzyl chloride, stirring at room temperature for reaction for 3-5h, concentrating the reaction solution after the reaction is finished, and separating by column chromatography to obtain folic acid derivative 1B;
the structural formulas of the folic acid and the folic acid derivative 1B are as follows:
、
;
(2) Adding silk fibroin, N-diisopropylethylamine and glycidol trimethyl ammonium chloride into water, heating for reaction, dialyzing and freeze-drying after the reaction is finished to obtain a compound 1;
the structural formulas of the silk fibroin and the compound 1 are as follows:
、
;
(3) Dicyclohexylcarbodiimide, 4-dimethylaminopyridine and 5-carboxypentyl-triphenylphosphine bromide are added into dimethyl sulfoxide solution containing a compound 1 to react for 2-5 hours, so as to obtain a reaction solution 1;
(4) Adding dicyclohexylcarbodiimide, 1-hydroxybenzotriazole and folic acid derivative 1B into the reaction liquid 1, reacting for 20-24 hours to obtain reaction liquid 2, centrifuging, washing and drying the reaction liquid 2 to obtain a compound 2;
The structural formula of the compound 2 is as follows:
;
(5) Adding 2, 3-dichloro-5, 6-dicyanobenzoquinone into the solution of the compound 2, stirring, centrifuging, washing and drying the stirred reaction solution to obtain modified silk fibroin, wherein the modified silk fibroin has the structural formula:
。
Further, in the step (1), the concentration of folic acid in the N, N-dimethylformamide solution is 87.5-116.7mg/mL, the mass ratio of folic acid to NaH is 1 (0.28-0.58), and the mass ratio of folic acid to 4-methoxychlorobenzyl is 1 (0.3-0.5).
Further, the mass ratio of the silk fibroin to the N, N-diisopropylethylamine to the glycidol trimethyl ammonium chloride in the step (2) is 1 (0.06-0.18) (0.05-0.15), the temperature of the heating reaction in the step (2) is 40-60 ℃, and the heating reaction time is 2-4 hours.
Further, the concentration of the dimethyl sulfoxide solution of the compound 1 in the step (3) is 10-20mg/mL, and the mass ratio of the compound 1 to dicyclohexylcarbodiimide to 4-dimethylaminopyridine to 5-carboxypentyl-triphenylphosphine bromide is 1 (3.3-9.9): (2.69-8.06): (9.14-27.44).
Further, the mass ratio of dicyclohexylcarbodiimide, 1-hydroxybenzotriazole and folic acid derivative 1B in the step (4) is (17.32-51.99): 11.35-34.05): 11.23-33.69.
Further, the solvent of the solution of the compound 2 in the step (5) is a dichloromethane/water mixed solvent, the dosage ratio of the compound 2 to the dichloromethane/water mixed solvent is 1g (8-12) mL, the mass ratio of the compound 2 to the 2, 3-dichloro-5, 6-dicyanobenzoquinone is 1 (90.8-272.4), and the stirring time is 0.5-3h.
Further, the concentration of the modified silk fibroin aqueous solution in the step b is 1-5wt%, the concentration of sodium phytate in the mixed aqueous solution of sodium phytate and sodium pyruvate is 1-5wt%, 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 homogenizing time is 20-30min, and the stirring reaction time is 20-24h.
The second object of the invention is to provide a sodium pyruvate sustained release nano system based on targeted modification.
The second purpose of the invention is realized by adopting the following technical scheme:
the sodium pyruvate sustained-release nano system based on targeted modification is prepared according to the preparation method.
The invention further aims to provide application of the sodium pyruvate sustained-release nano system based on targeted modification.
The third purpose of the invention is realized by adopting the following technical scheme:
Application of a sodium pyruvate sustained release nano system based on targeted modification in preparing a medicament for treating chronic obstructive pulmonary disease.
Compared with the prior art, the invention has the beneficial effects that:
1. The invention provides a sodium pyruvate sustained-release nano system based on targeted modification, which takes silk fibroin as a raw material, and prepares modified silk fibroin with lung targeting property through chemical modification, wherein 5-carboxypentyl-triphenyl phosphorus bromide has three benzene rings, the molecular surface area can be increased, delocalized positive charge is formed, and the modification of folic acid derivative 1B enables the modified silk fibroin to target focus. And then adopting an ion gel method to prepare a slow-release nano system, wherein the method is also called ion induction assembly, utilizes the electrostatic interaction of modified silk fibroin and sodium phytate to form a nano carrier, and simultaneously wraps sodium pyruvate to finally form the targeted slow-release nano system.
2. The invention also provides a preparation method of the sodium pyruvate sustained-release nano system based on targeted modification, which is simple and easy for industrial production, and the prepared sustained-release nano system has good targeting property.
3. The invention provides application of a sodium pyruvate sustained-release nano system based on targeted modification in preparation of a medicament for treating lung diseases, and the sustained-release nano system can also enable the medicament to be selectively accumulated at lung inflammation positions, so that toxic and side effects are reduced, and side effects are reduced.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a folic acid derivative 1B of the present invention;
FIG. 2 is a topography of the product obtained in example 1 of the present invention;
FIG. 3 is a graph showing the results of a biocompatibility experiment between the slow release nano-system and lung cells;
FIG. 4 is a graph showing the results of cell uptake rate of the slow release nanosystems.
Detailed Description
The present invention will be further described with reference to the following specific embodiments, and it should be noted that, on the premise of no conflict, new embodiments may be formed by any combination of the embodiments or technical features described below. The specific conditions not specified in the examples were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used, unless otherwise specified, are all conventional products obtained from commercial sources.
Example 1
The preparation method of the sodium pyruvate sustained-release nano system based on targeted modification comprises the following steps:
a. Preparation of modified silk fibroin:
(1) Adding 7g of folic acid into 65mL of N, N-Dimethylformamide (DMF) to obtain a DMF solution of folic acid, adding 3g of NaH (60% purity) into the DMF solution of folic acid under the inert atmosphere of nitrogen at 0 ℃, stirring for 20min, adding 2.8g of 4-methoxychlorobenzyl chloride (PMBCl), stirring at room temperature for reaction for 4h, quenching the reaction solution with water, adjusting the pH=6.2 of the reaction solution with 1M HCl, concentrating the reaction solution, and separating by column chromatography to obtain folic acid derivative 1B;
The specific reaction formula is as follows:
The nuclear magnetic resonance hydrogen spectrum of the folic acid derivative 1B is shown in figure 1, the structural characterization result is 1H NMR (400 MHz, 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)., and the obtained product is proved to be a target product.
(2) Adding 1g of silk fibroin (soluble silk fibroin freeze-dried raw material with a molecular weight of 5-10 kilodaltons), 0.1g of N, N-diisopropylethylamine and 0.12g of glycidol trimethyl ammonium chloride into 5mL of water to obtain a mixed solution, heating at 50 ℃ for reaction for 3 hours, filling the reaction solution into a dialysis bag (14000 Da) after the reaction is completed, dialyzing for 48 hours, and freeze-drying the dialyzate to obtain a compound 1;
The specific reaction formula is as follows:
(3) 1g of compound 1 is placed in 75mL of dimethyl sulfoxide (DMSO), 6.2g of Dicyclohexylcarbodiimide (DCC), 5.375g of 4-Dimethylaminopyridine (DMAP) and 18.295 g of 5-carboxypentyl-triphenylphosphine bromide are added in turn, and the mixture is reacted for 3.5 hours at room temperature to obtain a reaction solution 1;
(4) 30.945g of Dicyclohexylcarbodiimide (DCC), 20.268g of 1-hydroxybenzotriazole (HOBt) and 22.462g of folic acid derivative 1B are added into the reaction liquid 1 to react for 22 hours at room temperature to obtain a reaction liquid 2, the reaction liquid 2 is centrifuged to obtain a crude product, the crude product is collected and washed with deionized water for 5 times in an ultrasonic water bath, each washing is carried out for 5 minutes, and the product is dried in a vacuum oven at 30 ℃ for 5 hours to obtain a compound 2;
(5) 1g of compound 2 was placed in 10mL of a mixed solvent of methylene chloride/water (methylene chloride/water volume ratio: 1:1), 181.6g of 2, 3-dichloro-5, 6-dicyanobenzoquinone (DDQ) was added at room temperature and stirred at room temperature for 2 hours, the reaction solution 2 was centrifuged to obtain a crude product, the crude product was collected and washed with deionized water 5 times in an ultrasonic water bath at room temperature for 5 minutes each time, and then the product was placed in a vacuum oven for drying at 30℃for 5 hours to obtain a modified silk fibroin.
The specific reaction formulas of the steps (3), (4) and (5) are as follows:
b. preparing a target modified sodium pyruvate slow release nano system:
Placing a modified silk fibroin aqueous solution with the concentration of 2.5wt% into a homogenizer, setting the rotating speed of the homogenizer to 5000rpm, slowly adding a mixed aqueous solution of sodium phytate and sodium pyruvate (wherein the concentration of sodium phytate is 2.5wt% and the concentration of sodium pyruvate is 7.5 wt%) in the homogenizing process, wherein the volume ratio of the modified silk fibroin aqueous solution to the mixed solution of sodium phytate and sodium pyruvate is 1:1.25, homogenizing for 25min, stirring the mixed solution at 25 ℃ for 22h after homogenizing is finished, centrifuging the reaction solution (8000 rpm,10 min), washing the precipitate obtained by centrifugation with deionized water for 3 times, and dialyzing with a dialysis bag with the molecular weight cutoff of 3500Da to obtain the sodium pyruvate slow-release nano system based on targeted modification.
Example 2
The preparation method of the sodium pyruvate sustained-release nano system based on targeted modification comprises the following steps:
a. Preparation of modified silk fibroin:
(1) Adding 7g of folic acid into 60mL of DMF to obtain a folic acid DMF solution, adding 2g of NaH (60% purity) into the folic acid DMF solution in an inert nitrogen atmosphere at 0 ℃, stirring for 15min, adding 2.1g PMBCl again, stirring at room temperature for reaction for 3h, quenching the reaction solution with water, adjusting the pH of the reaction solution with 1M HCl to be=6, concentrating the reaction solution, and separating by column chromatography to obtain folic acid derivative 1B;
The specific reaction formula is as follows:
(2) Adding 1g of silk fibroin (soluble silk fibroin freeze-dried raw material with a molecular weight of 5-10 kilodaltons), 0.06g of N, N-diisopropylethylamine and 0.05g of glycidol trimethyl ammonium chloride into 5mL of water to obtain a mixed solution, heating at 40 ℃ for reaction for 4 hours, filling the reaction solution into a dialysis bag (14000 Da) after the reaction is completed, dialyzing for 48 hours, and freeze-drying the dialyzate to obtain a compound 1;
The specific reaction formula is as follows:
(3) 1g of compound 1 is placed in 50mL of DMSO, 3.3g of DCC, 2.69g of DMAP and 9.14g of 5-carboxypentyl-triphenylphosphine bromide are sequentially added to react for 2h at room temperature, so as to obtain a reaction solution 1;
(4) Adding 17.32g of DCC, 11.35g of HOBt and 11.23g of folic acid derivative 1B into the reaction liquid 1, reacting for 20 hours at room temperature to obtain a reaction liquid 2, centrifuging the reaction liquid 2 to obtain a crude product, collecting the crude product, washing the crude product with deionized water in an ultrasonic water bath for 5 times, washing for 5 minutes each time, and drying the product in a vacuum oven at 30 ℃ for 5 hours to obtain a compound 2;
(5) 1g of compound 2 was placed in 8mL of a mixed solvent of methylene chloride/water (methylene chloride/water volume ratio: 1:1), 90.8g of DDQ was added at room temperature and stirred at room temperature for 0.5h, the reaction solution 2 was centrifuged to obtain a crude product, and the crude product was collected and washed with deionized water 5 times in an ultrasonic water bath at room temperature for 5min each time. The product was then dried in a vacuum oven at 30 ℃ for 5h to give the modified silk fibroin.
The specific reaction formulas of the steps (3), (4) and (5) are as follows:
b. preparing a target modified sodium pyruvate slow release nano system:
Placing a modified silk fibroin aqueous solution with the concentration of 1wt% into a homogenizer, setting the rotating speed of the homogenizer to be 5000rpm, slowly adding 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 5 wt%) in the homogenizing process, wherein the volume ratio of the modified silk fibroin aqueous solution to the mixed solution of sodium phytate and sodium pyruvate is 1:1, homogenizing for 20min, stirring the mixed solution at 25 ℃ for 20h after homogenizing is finished, centrifuging (8000 rpm,10 min), washing a precipitate obtained by centrifugation with deionized water for 3 times, and dialyzing with a dialysis bag with the molecular weight cutoff of 3500Da to obtain the sodium pyruvate slow-release nano system based on targeted modification.
Example 3
The preparation method of the sodium pyruvate sustained-release nano system based on targeted modification comprises the following steps:
a. Preparation of modified silk fibroin:
(1) Adding 7g of folic acid into 80mL of DMF to obtain a folic acid DMF solution, adding 4g of NaH (60% purity) into the folic acid DMF solution in an inert nitrogen atmosphere at 0 ℃, stirring for 30min, adding 3.5g PMBCl again, stirring at room temperature for reaction for 5h, quenching the reaction solution with water, adjusting the pH of the reaction solution with 1M HCl to be=6.5, concentrating the reaction solution, and separating by column chromatography to obtain folic acid derivative 1B;
The specific reaction formula is as follows:
(2) Adding 1g of silk fibroin (soluble silk fibroin freeze-dried raw material with a molecular weight of 5-10 kilodaltons), 0.18g of N, N-diisopropylethylamine and 0.15g of glycidol trimethyl ammonium chloride into 5mL of water to obtain a mixed solution, heating at 60 ℃ for reaction for 2 hours, filling the reaction solution into a dialysis bag (14000 Da) after the reaction is completed, dialyzing for 48 hours, and freeze-drying the dialyzate to obtain a compound 1;
The specific reaction formula is as follows:
(3) 1g of compound 1 is placed in 100mL of DMSO, 9.9gDCC g of DMAP, 8.06g of DMAP and 27.44g of 5-carboxypentyl-triphenylphosphine bromide are sequentially added for reaction for 5h at room temperature, so as to obtain a reaction solution 1;
(4) Adding 51.99g DCC, 34.05g HOBt and 33.69g folic acid derivative 1B into the reaction liquid 1, reacting for 24 hours at room temperature to obtain a reaction liquid 2, centrifuging the reaction liquid 2 to obtain a crude product, collecting the crude product, washing the crude product with deionized water in an ultrasonic water bath for 5 times, washing for 5 minutes each time, and drying the product in a vacuum oven at 30 ℃ for 5 hours to obtain a compound 2;
(5) 1g of compound 2 was placed in 12mL of a mixed solvent of methylene chloride/water (methylene chloride/water volume ratio: 1:1), 272.4g of DDQ was added at room temperature and stirred at room temperature for 3 hours, the reaction solution 2 was centrifuged to obtain a crude product, and the crude product was collected and washed with deionized water 5 times in an ultrasonic water bath at room temperature for 5 minutes each time. The product was then dried in a vacuum oven at 30 ℃ for 5h to give the modified silk fibroin.
The specific reaction formulas of the steps (3), (4) and (5) are as follows:
b. preparing a target modified sodium pyruvate slow release nano system:
placing a modified silk fibroin aqueous solution with the concentration of 5wt% into a homogenizer, setting the rotating speed of the homogenizer to be 5000rpm, slowly adding a mixed aqueous solution of sodium phytate and sodium pyruvate (wherein the concentration of sodium phytate is 5wt% and the concentration of sodium pyruvate is 10 wt%) in the homogenizing process, wherein the volume ratio of the modified silk fibroin aqueous solution to the mixed solution of sodium phytate and sodium pyruvate is 1:1.5, homogenizing for 30min, stirring the mixed solution at 25 ℃ after homogenizing is finished, centrifuging (8000 rpm,10 min), washing a precipitate obtained by centrifugation with deionized water for 3 times, and dialyzing with a dialysis bag with the molecular weight cutoff of 3500Da to obtain the sodium pyruvate slow-release nano system based on targeted modification.
Comparative example 1
The preparation method of the sodium pyruvate sustained-release nano system based on targeted modification is different from the embodiment 1 in that the addition of 5-carboxypentyl-triphenylphosphine bromide is omitted when preparing modified silk fibroin.
Comparative example 2
The preparation method of the sodium pyruvate sustained-release nano system based on targeted modification is different from the embodiment 1 in that folic acid derivative 1B is omitted when modified silk fibroin is prepared.
Comparative example 3
The preparation method of the sodium pyruvate sustained-release nano system is different from the embodiment 1 in that the modified silk fibroin aqueous solution is replaced by silk fibroin aqueous solution.
Test example 1
The product obtained in example 1 and deionized water were diluted in a volume ratio of 1:5, and the diluted suspension was dropped on a glass slide, and the microscopic morphological characteristics were observed under an optical microscope, and the results are shown in fig. 2.
As shown in FIG. 2, the products obtained in example 1 of the present invention are all spherical structures, and the particle size ranges are smaller, which is beneficial to expanding the release efficiency at the focus of lung.
Test example 2
Biocompatibility test first, 100. Mu.L of lung cells HULEC-5a (1X 10 5/mL) in the logarithmic phase were added to a 96-well plate and the cells were cultured using DMEM complete medium. The 96-well plates inoculated with cells were then placed in a 37 ℃ 5% co 2 cell incubator for 12 hours. After the completion of the culture, the cell culture medium in each well was discarded, and 200. Mu.L of the DMEM complete medium containing the slow release nanosystem of example 1at a concentration of 50. Mu.g/mL was added to the 96-well plate, and the culture was continued under the above conditions for 24 hours. After the completion of the culture, the cell morphology was observed under a microscope. The invention uses DMEM serum-free culture medium as a negative control group and DMEM complete culture medium added with 5% phenol as a positive control group, and the experimental result is shown in figure 3.
FIG. 3 is a graph showing the results of the biocompatibility of the slow release nanosystems of example 1 with lung cells HULEC-5 a. As shown in fig. 3, the cell morphology of the experimental group (the slow-release nano system of example 1) is similar to that of the negative control group, and is a normal lung cell morphology, which indicates that the slow-release nano system of example 1 does not damage the growth morphology of lung cells, while the cell morphology of the positive control group is degraded, so that the slow-release nano system prepared in example 1 has good biocompatibility.
Test example 3
Cell quantitative uptake experiments cells from LA795 mice in logarithmic growth phase were divided into example 1 and comparative examples 1-3, inoculated into 96-well plates at a density of 1X 10 5 cells/mL, added with 0.2mL per well, and cultured at 37℃in 5% CO 2 for 24 hours. After 24h incubation, the original complete medium was discarded, then 0.2mL of the slow release nanosystems obtained in example 1 and comparative examples 1-3 containing stearylamine-isothiocyanato fluorescein (ODA-FITC) at a concentration of 2mg/mL was added to each well, after 6h incubation, the cells were washed with PBS and digested with trypsin, centrifuged, and the pellet was resuspended in PBS buffer, and the fluorescence intensity was measured in each group of cells at different time points using a flow cytometer, as shown in FIG. 4.
As can be seen from fig. 4, the cell uptake rate of the example 1 group is always maintained at a higher level and is far higher than that of the comparative example 3 group, wherein the cell uptake rate of the example 1 is close to 100% at 6h, which demonstrates that the slow release nano system of the present invention can carry sodium pyruvate into cells, and improve the targeting of drugs.
The group 3 of comparative example did not carry out quaternary ammonium salt modification, folic acid derivative 1B modification and 5-carboxypentyl-triphenylphosphine bromide modification on silk fibroin, and had the lowest cell uptake rate, the slow release nano system described in the group 1 of comparative example did not add 5-carboxypentyl-triphenylphosphine bromide, the cell uptake rate at 6 hours was 82.38%, the group 2 of comparative example did not add folic acid derivative 1B, and the cell uptake rate at 6 hours was 70.56%. The uptake rate was reduced in the groups 1 to 3 compared with example 1. Therefore, the targeting of the slow-release nano system can be improved by modifying the silk fibroin by quaternary ammonium salt, folic acid derivative and 5-carboxypentyl-triphenyl phosphonium bromide.
In conclusion, the slow-release nano system prepared in the embodiment 1 is prepared from silk fibroin serving as a raw material through quaternary ammonium modification, folic acid derivative modification and 5-carboxyamyl-triphenylphosphine bromide modification, and then a nano carrier is formed by utilizing electrostatic interaction of the modified silk fibroin and sodium phytate, so that sodium pyruvate wrapped in the modified silk fibroin can be effectively delivered to the lung of a mouse, and the delivery efficiency of a drug is greatly improved.
Test example 4
Rat plasma and pulmonary bronchoalveolar lavage (BALF) drug concentration assay:
(1) 70 healthy SD rats are selected, the weight of the healthy SD rats is 180g to 220g at 6-8 weeks, the healthy SD rats are randomly divided into free groups (sodium pyruvate is directly administered), and 10 healthy SD rats are selected from the groups of examples 1 to 3 and the groups of comparative examples 1 to 3 (the corresponding slow release nano system is given in the example 1, and the rest is the same). Each of the above groups was administered by tracheal instillation at a dose of 300. Mu.g/kg. 24h after administration, blood was taken through the orbit using heparin sodium as an anticoagulant. The collected blood was centrifuged at 4000rpm for 5min to separate plasma, triamcinolone acetonide was selected as an internal standard, and then measured by HPLC to calculate the concentration of sodium pyruvate in the plasma.
(2) After the rats were bled, the lung bronchoalveolar lavage fluid was collected. Specifically, 2mL of DMSO is injected into the lung of a rat, sucked out after 30 seconds, lung bronchoalveolar lavage fluid (BALF) cell suspension is obtained, each group is repeated 3 times, the collected BALF supernatant is mixed with methanol according to the volume ratio of 1:2, the mixture is centrifuged at 1000rpm for 10 minutes, the centrifuged BALF supernatant is analyzed by High Performance Liquid Chromatography (HPLC), and the sodium pyruvate content in the BALF is determined, and the experimental results are shown in Table 1.
As shown in Table 1, the concentration of sodium pyruvate in the plasma of the free group and the comparative example 1-3 is obviously higher than that of BALF, and the sodium pyruvate of the example 1-3 is mainly distributed in BALF, which shows that the slow release nano system prepared by the invention can reduce the distribution of the medicine in the whole body range and increase the availability of the medicine in lung tissues.
Wherein, the nano system of the comparative example 1 does not adopt 5-carboxypentyl-triphenyl phosphorus bromide to modify the silk fibroin in the preparation process, the nano system of the comparative example 2 does not adopt folic acid derivative 1B to modify the silk fibroin, the nano system of the comparative example 3 adopts unmodified silk fibroin, and the concentration of sodium pyruvate of the comparative example 1-3 in plasma is higher, which also shows that the finally prepared slow release nano system can better improve the enrichment of sodium pyruvate in the lung and further improve the targeting of the slow release nano carrier through the modification of the silk fibroin.
Test example 5
Drug loading rate and encapsulation rate test of sodium pyruvate sustained release nano system based on targeted modification:
In the preparation of the targeted modified sodium pyruvate sustained-release nano system in the step b, 1mL of the reaction solution after the reaction of the examples 1-3 and the comparative examples 1-3 is completed is taken, supernatant fluid is taken after centrifugation, absorbance value of the supernatant fluid at 255nm wavelength is measured by an ultraviolet spectrophotometer, and according to the concentration of free sodium pyruvate in the measured supernatant fluid, the drug loading rate and encapsulation rate of the sustained-release nano system on sodium pyruvate are calculated by using the following formula, and the result is shown in Table 2.
Drug loading (%) = (initial input of drug-free drug content)/total mass of sustained release nanosystems x 100%;
Encapsulation efficiency (%) = (initial input amount of drug-free drug content)/initial input amount of drug×100%.
As can be seen from the test data in Table 2, the drug loading of each of examples 1 to 3 of the present invention was maintained at 24% or more and the encapsulation efficiency was maintained at 93% or more, but the drug loading and encapsulation efficiency of comparative examples 1 to 3 were reduced. The modified silk fibroin modified slow-release nano system has higher encapsulation efficiency, the higher the encapsulation efficiency is, the more controllable the release behavior of the medicine in vivo is, and the bioavailability and the treatment effect of the medicine can be improved.
Test example 5
In order to explore the in vitro slow release performance of the slow release nano system prepared in the embodiments 1-3 and the comparative examples 1-3, the in vitro release effect of the slow release nano system is studied by simulating the lung environment with mucus. Firstly preparing artificial lung fluid (SLF), then weighing 30mg of the slow release nano system prepared in the examples 1-3 and the comparative examples 1-3, filling the slow release nano system into a dialysis bag, placing the dialysis bag into an artificial lung fluid (50 mL) release medium, then carrying out shaking culture in a constant-temperature shaking incubator at 37 ℃ and 50rpm, sucking 1mL of suspension in the culture 1h, 2h, 4h, 8h, 12h, 18h and 24h, then adding 1mL of corresponding release medium with the same volume and temperature, centrifuging the suspension taken out for 5min at 10000rpm, taking the supernatant, measuring the absorbance value at 255nm, and calculating the cumulative release rate according to the following formula, wherein the final measurement result is shown in Table 3.
Wherein M t is the mass of sodium pyruvate released at the sampling time point, and M 0 is the total mass of sodium pyruvate in the nano system.
The results of the observation of table 3 show that the cumulative release rate of the slow release nano system of the embodiments 1-3 for 24h can reach about 90%, so that the slow release nano system of the embodiments 1-3 can stably and continuously release sodium pyruvate within 24h and continuously and effectively reduce lung inflammation. Comparative example 1 omits modification of silk fibroin by 5-carboxypentyl-triphenylphosphine bromide, which had a cumulative release rate of 10.9% over 1 hour, 25.5% over 2 hours, and an accumulated release rate of 82.3% over 24 hours, but was still lower than example 1. Comparative example 2 and comparative example 3 also have relatively poor slow release effects due to incomplete or no modification of the silk fibroin.
In conclusion, the sustained-release nano system obtained in the embodiments 1-3 can stably encapsulate the drug, can be specifically enriched in the lesion parts of the lung and realize the sustained release of sodium pyruvate, has obvious targeting and sustained-release effects, and is suitable for preparing the drug for treating the lung diseases. Therefore, the slow release nano system prepared by the invention has potential application value in the aspect of targeted treatment of lung diseases.
The foregoing is illustrative of the preferred embodiments of the present invention and is not to be construed as limiting thereof, and various modifications and variations may be made by those skilled in the art without departing from the principles of the invention. Any modification, improvement, etc. should be considered as being within the scope of the present invention.
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