Dexamethasone-linolenic acid prodrug self-assembled nanoparticles for synergistic treatment of inflammation and application thereof

Abstract

The present invention discloses a dexamethasone-linolenic acid prodrug self-assembled nanoparticle for synergistic treatment of inflammation and its application, which belongs to the field of new excipients and new dosage forms of pharmaceutical preparations. The present invention designs and synthesizes a dexamethasone-linolenic acid small molecule prodrug, which is connected by a disulfide bond or a thioether bond to promote the rapid release of drugs in inflammatory sites or inflammatory cells with high reactive oxygen and glutathione levels, and dexamethasone and linolenic acid are released simultaneously to exert synergistic therapeutic effects. On this basis, a small molecule prodrug self-assembled nano drug delivery system is prepared. With the sensitivity of the chemical linker arm to the redox microenvironment, dexamethasone and linolenic acid are specifically released at the inflammatory site, improving the efficacy and reducing systemic toxic side effects.

Description

Technical Field

[0001] The invention belongs to the field of new excipients and new dosage forms of pharmaceutical preparations, and specifically relates to the construction of dexamethasone-linolenic acid small molecule prodrug self-assembled nanoparticles and the application of the nanoparticles in the preparation of a drug delivery system. Background Art

[0002] Dexamethasone (Dex), as a long-acting glucocorticoid drug, has anti-inflammatory, antioxidant and immunosuppressive effects and has been widely used in clinical practice. Due to the poor water solubility of dexamethasone, the current market varieties are mainly tablets, but its shortcomings such as slow onset and poor patient adaptability limit its use. The commercially available dexamethasone sodium phosphate injection solves the problem of poor water solubility of dexamethasone, and it takes effect quickly after intravenous administration, which is suitable for rapid treatment of diseases. However, after the above two dosage forms of drugs enter the blood circulation system, due to the presence of glucocorticoid receptors throughout the body's tissues, the drug will accumulate in the body's tissues. Large doses or long-term use of the drug will cause serious side effects such as Cushing's syndrome, which greatly limits its clinical application. In recent years, new drug delivery systems based on nanotechnology have attracted much attention, and many nano drug delivery systems have been constructed for the delivery of dexamethasone. Among them, some have been used in clinical practice, including dexamethasone palmitate lipid injection. Although nanotechnology has greatly improved the drugability of dexamethasone, these new nanoformulations also have some defects, including: carrier-related toxicity, low drug loading, poor stability, easy leakage during drug storage, crystallization, etc. Therefore, how to design an efficient and low-toxic drug delivery system for the delivery of dexamethasone is still a hot topic of research.

[0003] Prodrugs have also been widely studied to overcome the serious toxic and side effects of dexamethasone. Prodrugs themselves have no biological activity or very low activity. After being metabolized in the body, they become active substances to exert their efficacy. Prodrug strategies can improve the adverse properties of chemotherapeutic drugs through clever structural modifications, including poor water solubility, poor stability, and poor toxic and side effects. In addition, the construction of a new nano drug delivery system based on nanotechnology can significantly improve the pharmacokinetic properties of drugs, prolong the retention time of drugs in the body, and can increase the accumulation of drugs in inflammatory sites through active or passive targeting, increase the cellular uptake of drugs, and control the release rate of drugs, thereby improving the anti-inflammatory effect and reducing toxic and side effects. On this basis, the self-assembled nano drug delivery system based on small molecule prodrugs combines the advantages of prodrugs and nanotechnology. With its advantages of high drug loading, good stability, and low toxic and side effects, it has become a hot topic in the study of chemotherapeutic drug delivery systems in recent years.

[0004] Whether it is a prodrug or a nanodrug delivery system, the selective release of drugs at the target site by intelligent triggering is very important for the effectiveness and safety of the preparation. Compared with normal cells, there are higher concentrations of reactive oxygen species (ROS) and glutathione (GSH) in inflammation-related cells. This special cellular redox microenvironment has been widely used to design stimulus-responsive drug delivery systems. By analyzing the chemical properties of disulfide bonds, we found that they have good redox responsiveness and can respond to the microenvironment of the inflammatory site, which is expected to specifically release drugs to exert therapeutic effects and reduce side effects. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a dexamethasone-linolenic acid prodrug, which uses a redox-sensitive disulfide bond as a connecting bond. The prodrug can self-assemble into nanoparticles, thereby achieving the effects of high drug loading, good stability and specific release of drugs at the inflammatory site, wherein the synergistic effect of dexamethasone and the connecting group linolenic acid will further improve the anti-inflammatory activity and reduce toxic side effects.

[0006] The invention designs and constructs dexamethasone-linolenic acid self-assembled prodrug nanoparticles, and modifies them with PEG to give them a long circulation property in the blood, and applies them to anti-inflammatory research.

[0007] The purpose of the present invention is to design and synthesize redox-sensitive dexamethasone prodrugs with different linking groups, prepare prodrug self-assembly nano drug delivery systems, and compare the effects of different linking groups on anti-inflammatory effects.

[0008] The present invention achieves the above object through the following technical solutions:

[0009] The dexamethasone prodrugs of the present invention include dexamethasone-disulfide bond-linolenic acid prodrugs, dexamethasone-carbon chain-linolenic acid prodrugs and dexamethasone-thioether bond-linolenic acid prodrugs, and their structures are as follows:

[0010]

[0011] The method for synthesizing a dexamethasone linolenic acid prodrug provided by the present invention comprises the following steps:

[0012] Synthesis of dexamethasone-disulfide bond-linolenic acid small molecule prodrug: 2,2'-dithiodiacetic acid is dissolved in tetrahydrofuran at room temperature, dexamethasone and DMAP are added, and then a dichloromethane solution containing EDCI is slowly dripped into the above reaction solution under ice-salt bath, argon protection, stirring and reacting, and intermediate product I is separated; ethylene glycol and DMAP are dissolved in dichloromethane at room temperature, and a dichloromethane solution of linolenic acid and EDCI is slowly dripped into the above reaction solution under ice-salt bath, argon protection, stirring and reacting, and intermediate product II is separated; intermediates I and II are dissolved in tetrahydrofuran at room temperature, and then a dichloromethane solution containing EDCI and DMAP is added to the above reaction solution; argon protection, stirring and reacting, and the final product is separated.

[0013] Synthesis of dexamethasone-carbon chain-linolenic acid small molecule prodrug: adipic acid is dissolved in tetrahydrofuran at room temperature, dexamethasone and DMAP are added, and then a dichloromethane solution containing EDCI is slowly dripped into the above reaction solution under ice-salt bath, argon protection, stirring and reacting, and intermediate product I is separated; ethylene glycol and DMAP are dissolved in dichloromethane at room temperature, and a dichloromethane solution of linolenic acid and EDCI is slowly dripped into the above reaction solution under ice-salt bath, argon protection, stirring and reacting, and intermediate product II is separated; intermediates I and II are dissolved in tetrahydrofuran at room temperature, and then a dichloromethane solution containing EDCI and DMAP is added to the above reaction solution; argon protection, stirring and reacting, and the final product is separated.

[0014] The preparation method of the dexamethasone linolenic acid small molecule prodrug self-assembled nanoparticles provided by the present invention is as follows:

[0015] The dexamethasone linolenic acid small molecule prodrug is dissolved in ethanol, and the ethanol solution is slowly added dropwise into water under stirring, so that the dexamethasone linolenic acid small molecule prodrug spontaneously forms nanoparticles with uniform particle size. Finally, the ethanol is removed to obtain a nanoparticle colloidal solution free of organic solvent.

[0016] The dexamethasone linolenic acid small molecule prodrug nanoparticles can be non-PEGylated small molecule prodrug nanoparticles, PEG-modified small molecule prodrug nanoparticles, hydrophobic drug-encapsulated small molecule prodrug nanoparticles, and actively targeted small molecule prodrug nanoparticles.

[0017] (1) Preparation method of non-PEGylated small molecule prodrug self-assembled nanoparticles: The dexamethasone linolenic acid small molecule prodrug is dissolved in ethanol, and the ethanol solution is slowly added dropwise into water under stirring. The prodrug spontaneously forms nanoparticles with uniform particle size. Finally, the ethanol is removed to obtain a nanoparticle colloidal solution without organic solvent.

[0018] (2) Preparation method of PEG-modified small molecule prodrug self-assembled nanoparticles: PEG material and dexamethasone linolenic acid small molecule prodrug are dissolved in ethanol, and the ethanol solution is slowly added dropwise into water under stirring. The dexamethasone linolenic acid small molecule prodrug and PEG material spontaneously form nanoparticles with uniform particle size. Finally, the ethanol is removed to obtain a nanoparticle colloidal solution free of organic solvent.

[0019] (3) Preparation method of small molecule prodrug nanoparticles encapsulating hydrophobic drugs: PEG material, hydrophobic drugs and dexamethasone linolenic acid small molecule prodrug are dissolved in ethanol. Under stirring, the ethanol solution is slowly added dropwise into water. The dexamethasone linolenic acid small molecule prodrug and PEG material spontaneously form nanoparticles with uniform particle size. Finally, the ethanol is removed to obtain a nanoparticle colloidal solution free of organic solvent.

[0020] (4) Preparation method of actively targeted small molecule prodrug nanoparticles: PEG material and materials modified with targeting groups such as DSPE-PEG-folic acid or DSPE-PEG-sialic acid and dexamethasone linolenic acid small molecule prodrug are dissolved in ethanol. The ethanol solution is slowly added dropwise into water under stirring. The dexamethasone linolenic acid small molecule prodrug, PEG material and materials modified with targeting groups spontaneously form nanoparticles with uniform particle size. Finally, the ethanol is removed to obtain a nanoparticle colloidal solution free of organic solvent.

[0021] The dexamethasone-disulfide bond-linolenic acid small molecule prodrug of the present invention can be self-assembled into a uniform nano drug delivery system. The advantages of the nano drug delivery system are: (1) the one-step nano precipitation method is adopted, the preparation process is simple, and it is easy to industrialize; (2) the particle size is small and uniform (about 150nm), which is easy to enrich in the inflammatory site through the inflammation ELVIS effect to exert the therapeutic effect; (3) the ultra-high drug loading is conducive to reducing the adverse reactions caused by excipients and biomaterials; (4) it is easy to modify the surface, and PEG and active targeting modification can be used to effectively avoid the uptake of the reticuloendothelial system and increase the uptake of the nano drug by inflammation-related cells; (5) with the sensitivity of the chemical linker to the redox microenvironment, dexamethasone and linolenic acid are specifically released at the inflammatory site, improving the efficacy and reducing the systemic toxic side effects.

[0022] The dexamethasone described in the present invention may also be other steroidal or non-steroidal anti-inflammatory drugs.

[0023] The inflammatory microenvironment sensitive bond described in the present invention refers to a redox environment sensitive bond, and the redox sensitive bond may also be a thioether bond.

[0024] The present invention has the following beneficial effects: (1) a dexamethasone-linolenic acid small molecule prodrug is designed and synthesized, and the synthesis method is simple and easy; (2) by designing and comparing different linking groups, the compound with the best anti-inflammatory effect can be effectively screened; (3) small molecule prodrug self-assembled nanoparticles with uniform particle size are prepared, and the preparation method is simple and easy, with good stability and high drug loading; (4) PEG-modified small molecule prodrug nanoparticles can effectively prolong the circulation time of the drug in the blood; (5) the prepared small molecule prodrug nanoparticles have good anti-inflammatory effect; (6) linolenic acid itself has anti-inflammatory effect, and dexamethasone synergistically with it will exert a better anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The MS (A) and 1 H-NMR spectrum (B).

[0026] Figure 2 The MS (A) and 1 H-NMR spectrum (B).

[0027] Figure 3 This is a transmission electron microscopy image of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 3 of the present invention.

[0028] Figure 4 This is a graph showing the colloidal stability experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 4 of the present invention.

[0029] Figure 5 This is a diagram of the in vitro release experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 5 of the present invention.

[0030] Figure 6 This is a diagram showing the cytotoxicity of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 6 of the present invention.

[0031] Figure 7 This is a diagram of the in vitro anti-inflammatory experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 7 of the present invention.

[0032] Figure 8 This is the tissue distribution result of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 8 of the present invention.

[0033] Fig. 9 This is a diagram showing the results of treating acute pneumonia in vivo using the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 9 of the present invention.

[0034] Fig.10This is the side effect evaluation result of the PEG-modified small molecule prodrug self-assembled nanoparticles of Invention Example 10.

[0035] Fig.11 The chemical structure, MS and 1 H-NMR results.

[0036] Fig.12 This is the in vitro drug release curve of the PEG-modified thioether-linked dexamethasone-linolenic acid prodrug nanoparticles of Inventive Example 12.

[0037] Fig.13 This is a graph showing the in vivo treatment results of acute pneumonia using PEG-modified thioether-linked dexamethasone-linolenic acid prodrug nanoparticles of Invention Example 13.

[0038] Fig.14 This is the distribution of sialic acid-modified dexamethasone-disulfide-linolenic acid nanoparticles and dexamethasone-disulfide-linolenic acid nanoparticles in the lungs of mice with acute pneumonia in Example 14 of the invention.

[0039] Fig.15 This is a comparison of the therapeutic effects of sialic acid-modified dexamethasone-disulfide-linolenic acid nanoparticles and dexamethasone-disulfide-linolenic acid nanoparticles in the lungs of mice with acute pneumonia in Example 14 of the invention.

[0040] Fig.16 These are the experimental results of the anti-tumor effect of dexamethasone-disulfide-linolenic acid nanoparticles loaded with docetaxel in Example 15 of the invention. DETAILED DESCRIPTION

[0041] The present invention is further described below by way of examples, but the invention is not limited to the scope of the examples.

[0042] Example 1: Synthesis of dexamethasone-disulfide bond-linolenic acid

[0043] 2,2'-dithiodiacetic acid (11.62g, 63.75mmol, 2.5eq) was added to a 500mL three-necked flask, and 30mL tetrahydrofuran (THF) was added to dissolve at room temperature. Then, under the protection of argon, 100mL dexamethasone (10.0g, 25.50mmol, 1.0eq) and DMAP (1.56g, 12.75mmol, 0.5eq) THF solution and 120mL EDCI (12.22g, 63.75mmol, 2.5eq) anhydrous dichloromethane (DCM) solution were slowly added dropwise to the above reaction solution, and stirred in an ice-salt bath for 24 hours. After the reaction was completed, the solution was evaporated to dryness to obtain 35.28g of brown-red oil. The oil was then separated and purified by column chromatography to obtain 6.98g of white solid (intermediate I), with a yield of 47.5%. ESI-MS analysis revealed that the + and 579.1[M+Na] + . At room temperature, ethylene glycol (4.68g, 75.43mmol, 3.0eq) and DMAP (1.56g, 12.57mmol, 0.5eq) were added to a 250mL three-necked flask, 20mL of anhydrous DCM was added to dissolve, and then 100mL of THF solution containing dexamethasone (10.0g, 25.50mmol, 1.0eq) and EDCI (12.05g, 62.85mmol, 2.5eq) was added to react, and the solution was evaporated to dryness. Subsequently, 100mL of dichloromethane was added, and then washed three times with 100mL of 1M dilute hydrochloric acid and once with saturated brine. The organic layer was then dried over anhydrous sodium sulfate. After suction filtration, the filtrate was evaporated to dryness to obtain 6.22g of yellow oil. The substance was further separated and purified by column chromatography to obtain 4.01g of yellow oil in a yield of 59.23%. The compound (Intermediate II) was confirmed by ESI-MS and found to be 323.3 [M+H] + and 345.2[M+Na] + . At room temperature, intermediate I (2.68 g, 4.82 mmol, 1.0 eq) and intermediate II (1.86 g, 5.78 mmol, 1.2 eq) were dissolved in 30 mL of tetrahydrofuran, and then 30 mL of anhydrous DCM solution containing EDCI (1.85 g, 9.64 mmol, 2.0 eq) and DMAP (0.29 g, 2.41 mmol, 0.5 eq) was added to the above reaction solution, and the reaction was carried out under argon protection for 8 h. Subsequently, the crude product was purified by column chromatography to obtain a yellow oil, which was the final product. The yield was 43.6%. ESI-MS and 1 These compounds were identified by H-NMR. Figure 1 shown.

[0044] Example 2: Synthesis of dexamethasone-carbon chain-linolenic acid

[0045] At room temperature, 30 mL of anhydrous DCM was added to adipic acid (4.66 g, 31.88 mmol, 2.5 eq) to dissolve, and then dexamethasone (5.0 g, 12.75 mmol, 1.0 eq) was added. 60 mL of anhydrous DCM solution containing EDCI (6.11 g, 31.88 mmol, 2.5 eq) and DMAP (0.31 g, 2.55 mmol, 0.2 eq) was slowly added to the reaction solution, and the reaction was carried out under argon protection and ice-salt bath for 24 h. After the reaction was completed, the solution was evaporated to dryness to obtain 17.08 g of yellow oil. The oil was then separated and purified by column chromatography to obtain 2.72 g of white solid (intermediate I) with a yield of 41.01%. ESI-MS identification found 521.3 [M+H] + and 543.2[M+Na] + . At room temperature, ethylene glycol (4.68 g, 75.43 mmol, 3.0 eq) and DMAP (1.54 g, 12.57 mmol, 0.5 eq) were added to a 250 mL three-necked flask, 20 mL of anhydrous DCM was added to dissolve, and then 100 mL of THF solution containing dexamethasone (10.0 g, 25.50 mmol, 1.0 eq) and EDCI (12.05 g, 62.85 mmol, 2.5 eq) was added to react, and the solution was evaporated to dryness. Subsequently, 100 mL of dichloromethane (DCM) was added, and then washed three times with 100 mL of 1M dilute hydrochloric acid and once with saturated brine. The organic layer was then dried over anhydrous sodium sulfate. After suction filtration, the filtrate was evaporated to dryness to obtain 6.22 g of yellow oil. The substance was further separated and purified by column chromatography to obtain 4.01 g of yellow oil with a yield of 59.23%. These compounds (Intermediate II) were confirmed by ESI-MS and found to be 323.3 [M+H] + and 345.2[M+Na] + . At room temperature, intermediate I (2.73 g, 5.25 mmol, 1.0 eq) and intermediate II (1.86 g, 5.78 mmol, 1.2 eq) were dissolved in 30 mL of tetrahydrofuran, and then 30 mL of anhydrous DCM solution containing EDCI (1.85 g, 9.64 mmol, 2.0 eq) and DMAP (0.29 g, 2.41 mmol, 0.5 eq) was added to the above reaction solution, and the reaction was carried out under argon protection for 8 h. Subsequently, the crude product was purified by column chromatography to obtain a yellow oil, which was the final product. The yield was 97.8%. ESI-MS and 1 These compounds were identified by H-NMR. Figure 2 shown.

[0046] Example 3: Preparation of PEG-modified small molecule prodrug self-assembled nanoparticles

[0047] Accurately weigh DSPE-PEG 2k 1 mg and 4 mg of dexamethasone linolenic acid prodrug were dissolved in 0.5 mL of ethanol, and the ethanol solution was slowly added dropwise to 4 mL of deionized water under stirring to self-assemble into nanoparticles with uniform particle size (dexamethasone-disulfide bond-linolenic acid nanoparticles and dexamethasone-carbon chain-linolenic acid nanoparticles). The organic solvent in the nanoformulation was removed by dialysis with deionized water at 25°C or rotary evaporation at 50°C. As shown in Table 1, the particle size of the nanoparticles was about 150 nm, the dexamethasone drug loading was greater than 38%, and the linolenic acid drug loading was greater than 26%.

[0048] Table 1. Particle size, size distribution, surface potential and drug loading of PEG-modified small molecule prodrug self-assembled nanoparticles

[0049]

[0050] The particle size and morphology of the small molecule prodrug self-assembled nanoparticles prepared in Example 3 were measured by transmission electron microscopy. Figure 3 As shown, the transmission electron microscopy image shows that the drug-loaded nanoparticles are spherical with uniform particle size, and the particle size is about 150nm.

[0051] Example 4: Colloidal stability test of PEG-modified small molecule prodrug self-assembled nanoparticles

[0052] 1 mL of the PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 3 was added to a PBS solution containing 10% FBS, incubated at 37°C for 24 h, and the particle size change was measured by dynamic light scattering at predetermined time points. Figure 4 As shown, the particle size of the PEG-modified small molecule prodrug self-assembled nanoparticles did not change significantly within 24 hours, showing good colloidal stability.

[0053] Example 5: In vitro release test of PEG-modified small molecule prodrug self-assembled nanoparticles

[0054] Phosphate buffer (PBS) containing 30% ethanol at pH 7.4 was used as the release medium to investigate the in vitro release of small molecule self-assembled nanoparticles bridged with different connecting bonds. 1 mL of the PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 3 was added to 10 mL of the release medium. Samples were taken at set time points at 37°C, and the concentration of released dexamethasone was determined by high performance liquid chromatography. A certain concentration of hydrogen peroxide (H2O2, 1 mM) or dithiothreitol (DTT, 1 mM) was added to the release medium to investigate the release of the nanoparticles under redox conditions. The cumulative release of dexamethasone was determined at a wavelength of 240 nm using high performance liquid chromatography (HPLC). The results are shown in Figure 2. Figure 5 As shown, the results indicate that disulfide bond-linked small molecule prodrug nanoparticles have dual redox hypersensitivity. Under the action of ROS or GSH, the prodrug can quickly break and release dexamethasone, while the insensitive carbon chain-linked prodrug nanoparticles hardly release the drug.

[0055] Example 6: Cytotoxicity of PEG-modified small molecule prodrug self-assembled nanoparticles

[0056] The MTT method was used to investigate the cytotoxicity of PEG-modified small molecule prodrug self-assembled nanoparticles to mouse mononuclear macrophages. The cells in good condition were digested and diluted with culture fluid to a cell concentration of 10000 cells / ml. After blowing, 100 μL of cell suspension was added to each well of the 96-well plate, and the cells were placed in an incubator and incubated for 24 hours to adhere to the wall. After the cells adhered to the wall, 100 μL of dexamethasone solution, linolenic acid solution or two nanoparticles prepared in Example 3 were added, with 3-5 parallel wells for each concentration. The control group, that is, no test solution was added, a single 100 μL culture fluid was added, and the 96-well plate was taken out after incubation in the incubator for 24 hours, and 10 μL of 5 mg / ml MTT solution was added to each well, and then the plate was thrown after incubation in the incubator for 4 hours. After the 96-well plate was turned upside down on the filter paper and the residual liquid was fully absorbed, 200 μL DMSO was added to each well and shaken on the oscillator for 10 minutes to dissolve the blue-purple crystals. The absorbance of each well was measured at 490 nm using an ELISA reader. Figure 6 As shown, the cell viability of the two nanoparticles and dexamethasone solution and linolenic acid solution at different concentrations was similar, and the cell viability was greater than 80%, indicating that the nanoparticles were non-cytotoxic and had good safety.

[0057] Example 7: In vitro cell anti-inflammatory experiment of PEG-modified small molecule prodrug self-assembled nanoparticles

[0058] The ELISA method was used to investigate the anti-inflammatory effect of PEG-modified small molecule prodrug self-assembled nanoparticles on the inflammatory mouse mononuclear macrophage model. The cells in good condition were digested and diluted with culture medium to a cell concentration of 10,000 cells / ml. After blowing evenly, 1 mL of cell suspension was added to each well of a 12-well plate and incubated in an incubator for 24 hours to allow them to adhere to the wall. After the cells adhered to the wall, 100 ng / ml of lipopolysaccharide (LPS) solution was added. After incubation for 4 hours, they were divided into 6 groups and incubated with 1 mL of normal saline, dexamethasone, linolenic acid solution, a mixed solution of dexamethasone and linolenic acid, or the dexamethasone-disulfide bond-linolenic acid nanoparticles and dexamethasone-carbon chain-linolenic acid nanoparticles prepared in Example 3, with 3-5 parallel wells in each group. After incubation for 24 hours, the incubation solution was taken out, the supernatant was taken by centrifugation, and the levels of related inflammatory factors TNF-α, IL-6 and IL-1β in the supernatant were determined using a kit. The experimental results are as follows. Figure 7 As shown in the figure, the experimental results of inflammatory factors TNF-α, IL-6 and IL-1β were consistent. Among them, the dexamethasone group and the linolenic acid group showed certain inflammation inhibition activity compared with the saline group. The mixed solution group had stronger anti-inflammatory activity, which showed that the two had synergistic effects. In addition, the disulfide-linked small molecule prodrug self-assembled nanoparticles showed better activity than the carbon chain-linked dexamethasone-linolenic acid, which may be attributed to the redox sensitivity of the disulfide bond. Therefore, the synergistic anti-inflammatory disulfide-linked dexamethasone-linolenic acid prodrug self-assembled nanodrug delivery system will have great potential in the treatment of inflammation-related diseases.

[0059] Example 8: Distribution of PEG-modified small molecule prodrug self-assembled nanoparticles in mice with acute pneumonia

[0060] Mice with acute pneumonia were divided into three groups and intravenously injected with dexamethasone-disulfide-linolenic acid nanoparticles, dexamethasone-carbon chain-linolenic acid nanoparticles and dexamethasone solution (the dosage of each preparation was calculated as dexamethasone: 1 mg / kg). After 0.5h, 2h, 6h, 12h and 24h of administration, the mice were euthanized, and their hearts, livers, spleens, lungs and kidneys were removed. The tissues were prepared into 10% tissue homogenates, and the proteins were precipitated with acetonitrile. The concentrations of dexamethasone and prodrug in the supernatant were directly determined by ultra-performance liquid chromatography / tandem mass spectrometry (UPLC-MS / MS, Waters, USA). The experimental results are shown in the figure. Figure 8As shown, the distribution of dexamethasone and both nanoparticles in the heart and kidney was less, and no significant difference was observed between the nanoparticles and the dexamethasone solution. In contrast, the distribution of the nanoparticles in the liver and spleen was significantly higher than that of the dexamethasone free solution in these organs, which may be related to the phagocytosis of the nanoparticles by the reticuloendothelial system. In addition, the nanoparticles also significantly improved the distribution of the drug in the lungs, reaching a peak at 2h, which may be because the passive accumulation of nanoparticles in damaged lungs is a relatively slow process. In addition, it is worth noting that the prodrug nanoparticles are present in these organs in the form of prodrugs and part of the free dexamethasone, which is mainly related to the slow release of the prodrug nanoparticles in vivo. Limited dexamethasone exposure in non-target organs will help reduce the side effects caused by dexamethasone. In summary, dexamethasone-disulfide-linolenic acid nanoparticles have the characteristics of selectively releasing active drugs in a high redox environment, which can not only prolong the circulation time of the drug in the bloodstream and the targeted accumulation in the lungs, but also selectively release the drug in the pathological microenvironment, and have the potential for precise anti-inflammatory.

[0061] Example 9: Anti-inflammatory effect of PEG-modified small molecule prodrug self-assembled nanoparticles in mice with acute pneumonia

[0062] In this study, an acute pneumonia model in mice was induced by intratracheal instillation of lipopolysaccharide (LPS, 5 mg / kg; 50 μL / mouse). Sham surgery was performed by intratracheal injection of the same amount of normal saline. After 4 hours of LPS treatment, an acute pneumonia mouse model was successfully established. The mice were divided into 6 groups and given normal saline, dexamethasone solution, linolenic acid solution, a mixed solution of dexamethasone and linolenic acid, dexamethasone-disulfide-linolenic acid nanoparticles, and dexamethasone-carbon chain-linolenic acid nanoparticles (the dosage was equivalent to 2.5 mmol / kg dexamethasone or linolenic acid) by intravenous injection. The mice were killed 24 hours after administration, and the bronchoalveolar lavage fluid (BALF) was collected for the determination of white blood cell counts, protein concentrations, and inflammatory cytokines. The experimental results are shown in the following table. Fig. 9As shown in the figure, tracheal instillation of lipopolysaccharide led to a significant increase in immune cell infiltration and protein exudation in bronchoalveolar lavage fluid. In contrast, different drug treatment groups significantly alleviated these pathological manifestations, among which dexamethasone-disulfide-linolenic acid nanoparticles showed the best therapeutic effect. In addition, we measured the levels of key proinflammatory cytokines (IL-6, TNF-α, IL-1β) in bronchoalveolar lavage fluid by ELISA kit to evaluate the anti-inflammatory effect. The results showed that the cytokine levels in the drug treatment groups were reduced to varying degrees, and both free dexamethasone and linolenic acid had certain anti-inflammatory effects, and the effect of dexamethasone was better than that of linolenic acid. The mixed solution of the two was more effective, reflecting the synergistic effect of the two. In addition, the two prodrug nanoparticles also showed anti-inflammatory effects, among which the effect of dexamethasone-carbon chain-linolenic acid nanoparticles was weaker than that of the mixed solution group, which may be related to its incomplete release of drugs in vivo. Dexamethasone-disulfide-linolenic acid nanoparticles had the best effect, superior to the mixed solution group, mainly due to the nanoparticles' long circulation in the blood, lung targeting, and responsive rapid drug release under pathological conditions.

[0063] Example 10: Side effect evaluation of PEG-modified small molecule prodrug self-assembled nanoparticles

[0064] After 21 days of continuous administration of the drug to mice with acute pneumonia, glucocorticoid-related side effects, including weight loss, increased blood sugar, and immunosuppression, were evaluated. Whole blood samples were collected from mice and hematological parameters were analyzed using a Mindray veterinary fully automatic hematology analyzer (BC-2800vet). In addition, blood glucose levels were measured using a blood glucose meter. The experimental results are shown in Figure 2. Fig.10As shown, except for mice in the model group (normal saline group), mice treated with dexamethasone and a mixed solution of dexamethasone and linolenic acid showed the slowest weight recovery, while the linolenic acid and dexamethasone-disulfide-linolenic acid nanoparticles treated groups maintained a stable weight gain. This suggests that the nanoparticles can reduce the weight loss caused by dexamethasone. Hematological analysis showed that in mice treated with free dexamethasone, lymphocytes were significantly reduced and monocytes and granulocytes were increased. However, mice treated with dexamethasone-disulfide-linolenic acid nanoparticles did not show any obvious abnormal changes. In addition, the results of blood glucose analysis showed that the group containing free dexamethasone significantly increased the blood glucose levels of mice compared with the sham operation group. This increase in blood glucose may be related to glucocorticoid-induced insulin resistance and glucose synthesis. In contrast, the nanoparticles did not cause a significant increase in blood glucose in mice, which is mainly related to the exposure of the active ingredient dexamethasone in the body. The prodrug nanoparticles have the property of selectively releasing active drugs in a high redox environment, which can reduce the leakage of active drugs in non-target organs. This result has been confirmed in tissue distribution experiments. It is worth mentioning that only disulfide-linked dexamethasone-linolenic acid nanoparticles were selected for research in this study because they performed best in efficacy studies. Dexamethasone-carbon chain-linolenic acid nanoparticles have a slower in vitro and in vivo drug release rate and have the same tissue distribution behavior as dexamethasone-disulfide-linolenic acid nanoparticles, so they will not cause dexamethasone-related side effects.

[0065] Example 11: Synthesis of Dexamethasone-Thioether-Linolenic Acid Prodrug

[0066] The synthesis method of dexamethasone-thioether bond-linolenic acid prodrug is basically the same as that in Example 1, except that 2,2'-dithiodiacetic acid (11.62 g, 63.75 mmol, 2.5 eq) is replaced with 2,2'-thiodiacetic acid (9.57 g, 63.75 mmol, 2.5 eq). The yield of the compound is 71.3%. Its chemical structure and ESI-MS and 1 The H-NMR results are as follows Fig.11 shown.

[0067] Example 12: Preparation of PEG-modified dexamethasone-thioether-linolenic acid prodrug nanoparticles and evaluation of their oxidation-sensitive drug release behavior

[0068] The preparation method of PEG-modified nanoparticles can refer to Example 3, except that the dexamethasone-disulfide bond-linolenic acid prodrug is replaced by dexamethasone-thioether bond-linolenic acid prodrug. The average particle size of the final nanoparticles is 153.36 nm and the PDI is 0.121. In addition, the in vitro release experiment of the nanoparticles refers to Example 5. The final in vitro drug release results are shown in Fig.12As shown in the figure, the nanoparticles basically do not release drugs under reducing conditions (DTT), indicating that the nanoparticles do not have reduction-responsive drug release behavior. However, under oxidative conditions, the nanoparticles can slowly release active drugs, and the release rate is dependent on the concentration of hydrogen peroxide.

[0069] Example 13: Anti-inflammatory effect of PEG-modified dexamethasone-thioether bond-linolenic acid prodrug nanoparticles in mice with acute pneumonia

[0070] The experimental method in this study is consistent with that in Example 9, and the carbon chain-linked dexamethasone-linolenic acid prodrug nanoparticles that respond to non-inflammatory microenvironment are used as the control group. Fig.13 As shown in the figure, tracheal instillation of lipopolysaccharide led to a significant increase in immune cell infiltration and protein exudation in bronchoalveolar lavage fluid. In contrast, different drug treatment groups significantly alleviated these pathological manifestations, among which dexamethasone-thioether bond-linolenic acid nanoparticles showed the best therapeutic effect. In addition, we measured the levels of key proinflammatory cytokines (IL-6, TNF-α, IL-1β) in bronchoalveolar lavage fluid by ELISA kit to evaluate the anti-inflammatory effect. The results showed that the cytokine levels in the drug treatment groups were reduced to varying degrees, and both free dexamethasone and linolenic acid had certain anti-inflammatory effects, and the effect of dexamethasone was better than that of linolenic acid. The mixed solution of the two had a better effect, reflecting the synergistic effect of the two. In addition, the two prodrug nanoparticles also showed anti-inflammatory effects, among which the effect of dexamethasone-carbon chain-linolenic acid nanoparticles was weaker than that of the mixed solution group, which may be related to its incomplete drug release in vivo. Dexamethasone-thioether-linolenic acid nanoparticles had the best effect, which was better than the mixed solution group. This was mainly due to the nanoparticles' long circulation in the blood, lung targeting, and responsive rapid release of drugs under pathological conditions. The results showed that dexamethasone-thioether-linolenic acid prodrug nanoparticles and dexamethasone-disulfide-linolenic acid prodrug nanoparticles had similar effects and were better than the mixed solution group and non-sensitive carbon chain-linked prodrug nanoparticles.

[0071] Example 14: Preparation, tissue distribution and pharmacodynamics study of DSPE-PEG-sialic acid modified dexamethasone-disulfide bond-linolenic acid prodrug nanoparticles

[0072] As a targeting group, sialic acid can specifically bind to the E-selectin receptor overexpressed on vascular endothelial cells at the site of inflammation. By utilizing this property, we modified the surface of dexamethasone-linolenic acid prodrug nanoparticles with DSPE-PEG-sialic acid to enable them to actively target the disease site. 2Kand sialic acid and dexamethasone-linolenic acid small molecule prodrugs are dissolved in ethanol, and the ethanol solution is slowly added dropwise to water under stirring. These components are co-assembled to form nanoparticles. Finally, the ethanol is removed to obtain DSPE-PEG-sialic acid-modified dexamethasone-linolenic acid prodrug nanoparticles without organic solvents. The average particle size is about 150 nm, and the PDI is 0.116, which is basically consistent with the particle size of the nanoparticles without sialic acid modification. Next, we studied the effects of sialic acid modification on the targeting and anti-inflammatory activity of the nanoparticles. First, according to the experimental method of tissue distribution in Example 8, the lung targeting of dexamethasone-linolenic acid prodrug nanoparticles and DSPE-PEG-sialic acid-modified dexamethasone-linolenic acid prodrug nanoparticles in mice with acute pneumonia was investigated. In addition, referring to the method in Example 9, the effects of the two nanoparticles in treating pneumonia in mice were investigated. The experimental results are as follows Fig.14 and Fig.15 As shown, sialic acid-modified nanoparticles significantly improved the distribution of nanoparticles in diseased lungs and also improved their effectiveness in treating pneumonia. The protein concentration, cell number and pro-inflammatory factor levels in bronchoalveolar lavage fluid were lower, indicating that nanoparticles modified with targeting groups can enhance the targeted therapeutic effect of the nanoparticles themselves.

[0073] Example 15: Preparation of dexamethasone-linolenic acid prodrug nanoparticles loaded with docetaxel and evaluation of antitumor activity

[0074] DSPE-PEG 2K 2 mg, 4 mg of anti-tumor drug docetaxel and 4 mg of dexamethasone-disulfide-linolenic acid small molecule prodrug were dissolved in ethanol, and the ethanol solution was slowly added to water under stirring. These components were assembled into nanoparticles with uniform particle size. Finally, ethanol was removed to obtain dexamethasone-linolenic acid prodrug nanoparticles containing docetaxel without organic solvent. The average particle size of the nanoparticles was 150-200 nm, and the docetaxel encapsulation rate was greater than 80%. In addition, the nanoparticles were used for combined tumor treatment research. 4T1 cell suspension (5x 10 6 cells / 100 μL) were inoculated subcutaneously on the right back side of female nude mice. 3 The tumor-bearing mice were randomly divided into 5 groups, with five mice in each group: blank control group (PBS), dexamethasone-linolenic acid prodrug nanoparticle group, docetaxel group, dexamethasone-linolenic acid prodrug nanoparticle and docetaxel mixed solution group, and dexamethasone-linolenic acid prodrug nanoparticle group loaded with docetaxel. The drug was administered once every other day for 5 consecutive times. The dosage was 5 mg / kg based on docetaxel and 2 mg / kg based on dexamethasone. After administration, the survival status of the mice was observed every day, their body weight was weighed, and the tumor volume was measured. The experimental results are as follows: Fig.16As shown in the figure, compared with the control group, dexamethasone-linolenic acid nanoparticles showed certain tumor inhibitory activity. In contrast, the anti-tumor drug docetaxel showed stronger anti-tumor activity than dexamethasone-linolenic acid nanoparticles. It is worth noting that the anti-tumor effect was better after the two were administered together. The dexamethasone-linolenic acid prodrug nanoparticle group encapsulating docetaxel showed the best therapeutic effect, with basically no tumor growth. This shows the advantage of nanoparticles in synchronous drug delivery, which is an effective anti-tumor drug delivery system.

Claims

1. A dexamethasone-linolenic acid small molecule prodrug, characterized in that: Dexamethasone and linolenic acid are linked by disulfide bonds or thioether bonds that are sensitive to the inflammatory microenvironment. Their structures are as follows:

2. The method for preparing the self-assembled nanoparticles of dexamethasone-linolenic acid small molecule prodrug according to claim 1, characterized in that: The following steps are involved: The self-assembled nanoparticles of dexamethasone-linolenic acid small molecule prodrug are small molecule prodrug nanoparticles modified with PEG, and the PEG is DSPE-PEG; First, dexamethasone is reacted with a dibasic acid to form an intermediate I, and linolenic acid is reacted with a diol to form an intermediate II. Finally, intermediates I and II are subjected to an esterification reaction to form a dexamethasone-linolenic acid small molecule prodrug; Then, DSPE-PEG and dexamethasone-linolenic acid small molecule prodrug are dissolved in ethanol, and the ethanol solution is slowly added dropwise into water under stirring. The dexamethasone-linolenic acid small molecule prodrug and DSPE-PEG spontaneously form nanoparticles with uniform particle size. Finally, the ethanol is removed to obtain a nanoparticle colloidal solution free of organic solvent.

3. Use of the dexamethasone-linolenic acid small molecule prodrug according to claim 1 or the self-assembled nanoparticles of the dexamethasone-linolenic acid small molecule prodrug prepared by the preparation method according to claim 2 in preparing a drug delivery system.

4. Use of the dexamethasone-linolenic acid small molecule prodrug according to claim 1 in the preparation of anti-inflammatory or anti-tumor drugs.

5. Use of the dexamethasone-linolenic acid small molecule prodrug according to claim 1 or the self-assembled nanoparticles of the dexamethasone-linolenic acid small molecule prodrug according to claim 2 in the preparation of drugs for injection, oral administration or topical administration.

6. Use of the self-assembled nanoparticles of the dexamethasone-linolenic acid small molecule prodrug according to claim 2 in the preparation of anti-inflammatory or anti-tumor drugs.

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