Preparation method of tanshinone IIA nanoparticles specifically released at inflammatory site

Abstract

The invention discloses a preparation method of tanshinone IIA nanoparticles specifically released at an inflammatory site, and belongs to the technical field of pharmaceutical preparations. The nano particle comprises a PEG (polyethylene glycol)-PLGA (poly (lactic-co-glycolic acid)) block copolymer nano carrier modified by tanshinone IIA and 1, 4-butanediamine. According to the invention, 1, 4-butanediamine is taken as a modification chain, a pH-sensitive PEG-PLGA block copolymer is constructed to prepare nanoparticles, targeted delivery of an inflammatory site of insoluble molecule tanshinone IIA is realized, drug enrichment of the tanshinone IIA at the inflammatory site is improved, and anti-inflammatory efficiency is increased.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to a kind of inflammation microenvironment-based specific release at inflammation site salvianate II A PEG-PLGA nanoparticle and preparation method thereof. BACKGROUND

[0002] Inflammation is the pathological basis of various diseases. Normal inflammatory response can resist external invasion and promote tissue repair. However, excessive inflammatory response can lead to pathological changes in tissues and cause serious damage. Therefore, controlling inflammation is a key measure for treating various diseases. Currently, various natural drug extracts have been proven to have excellent anti-inflammatory activity, such as salvianate II A, curcumin, astaxanthin, etc. The anti-inflammatory activity of some of these extracts is even better than that of the classic anti-inflammatory drug non-steroidal anti-inflammatory drugs, and the adverse reactions are fewer, such as salvianate II A. However, these drugs also have their own defects, such as poor solubility and poor pharmacokinetic properties, which make it difficult to achieve effective therapeutic concentration at the inflammation site, and their application is therefore greatly limited.

[0003] The pathological and physiological differences between inflammation sites and normal tissues are the basis for the development of targeted drug carriers. Inflammation microenvironment is a special microenvironment composed of pro-inflammatory enzymes, inflammatory cells, inflammatory mediators and other inflammation-related substances in inflammatory tissues. Targeted drug carriers based on inflammation microenvironment can deliver drugs to inflammation sites and achieve specific release, improving drug safety and efficacy, and therefore have attracted widespread attention. The targeting strategies of such carriers are diverse, including targeting inflammatory cells, inflammatory mediators, reactive oxygen species, and pH differences at inflammation sites. The increased metabolic activity of inflammatory tissues can lead to hypoxia, which in turn induces anaerobic glycolysis, producing lactic acid and hydrogen ions, making the inflammation microenvironment acidic. Developing acid-responsive materials to prepare inflammation microenvironment-targeted carriers is considered an effective strategy to improve the effectiveness of anti-inflammatory treatment.

[0004] Poly (lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) are excellent materials for preparing drug delivery carriers due to their good biocompatibility and biodegradability. PEG and PLGA can be synthesized into two-block amphiphilic copolymer PEG-PLGA with hydrophobic segment (PLGA) and hydrophilic segment (PEG). The copolymer can self-assemble into nanoparticles in aqueous medium, in which the hydrophobic drug is wrapped in the core of the nanoparticles, and the hydrophilic PEG on the surface of the nanoparticles can avoid being recognized and phagocytosed by macrophages. In addition, PEG-PLGA is easy to modify the structure during synthesis to endow special properties, for example, connecting a pH-sensitive chemical bond between PEG and PLGA can make PEG-PLGA pH-responsive. In addition, PEG-poly (caprolactone) (PEG-PCL), PLGA-PLGA, and PEG-PLA can also be used as carrier materials by structural modification. The present application uses 1,4-butanediamine as a linking molecule to modify and construct various block copolymers, so that the copolymers are broken at the inflammation site and release the drug. SUMMARY

[0005] The present application aims to provide a tanshinone ⅡA PEG-PLGA nanoparticle based on the inflammatory microenvironment, which targets the inflammation site and is specifically released at the inflammation site, and a preparation method thereof. The preparation method uses 1,4-butanediamine as a modification chain to construct a pH-sensitive PEG-PLGA block polymer to prepare nanoparticles, realizes the inflammation site targeted delivery of the poorly soluble molecule tanshinone ⅡA, improves the drug enrichment at the inflammation site, and increases the anti-inflammatory efficiency.

[0006] To achieve the above-mentioned purpose, the technical solutions adopted by the present application are as follows:

[0007] A tanshinone ⅡA nanoparticle based on the inflammatory microenvironment, which is specifically released at the inflammation site, comprises tanshinone ⅡA and a 1,4-butanediamine modified block copolymer nanocarrier. The nanocarrier is PEG-PLGA, PEG-PCL, PEG-PLA, or PLGA-PLGA, preferably PEG-PLGA.

[0008] A preparation method of a tanshinone ⅡA nanoparticle based on the inflammatory microenvironment, which is specifically released at the inflammation site, comprises the following steps:

[0009] Step one: methoxypolyethylene glycol (mPEG) (molecular weight: 2000-20000 Da) or PLGA 1-10 g was dissolved in 100 mL tetrahydrofuran, stirred at 50 °C for 10 min, N,N'-carbonyldiimidazole (CDI) (1-10 g) was dissolved in 80 mL tetrahydrofuran, then the CDI solution was slowly dropped into the mPEG solution under continuous stirring at 45 °C and the reaction was continued for 8-18 h, then the solution was concentrated by rotary evaporation, washed with excess cold tert-butyl methyl ether, and the obtained mPEG-CDI or PLGA-CDI product was dried at 0 °C under vacuum for 24 h.

[0010] Step two: 1,4-butanediamine bis-hydrochloride (0.5-1 g) was dissolved in 40 mL tetrahydrofuran, and the mPEG-CDI or PLGA-CDI (1-2 g) prepared in step one was dissolved in 50 mL tetrahydrofuran. The mPEG-CDI or PLGA-CDI solution was slowly dropped into the 1,4-diaminobutane solution under continuous stirring at 45 °C, and the reaction was continued for 8-18 h at 45 °C, then the solution was concentrated by rotary evaporation, washed with excess cold tert-butyl methyl ether, and the obtained mPEG-NH2 or PLGA-NH2 product was dried at 0 °C under vacuum for 24 h.

[0011] Step three: PLGA or PCL or PLA (0.5-1.5 g) was dissolved in 40 mL dichloromethane, N-hydroxysuccinimide (10-100 mg) and dicyclohexyl carbodiimide (50-150 mg) were added under stirring at 45 °C, and the reaction was continued for 8-18 h under N2 protection, then the solution was concentrated by rotary evaporation, washed with excess cold tert-butyl methyl ether, and the obtained PLGA-NHS or PCL-NHS or PLA-NHS product was dried at 0 °C under vacuum for 24 h.

[0012] Step four: the PLGA-NHS or PCL-NHS or PLA-NHS (50-150 mg) prepared in step three was dissolved in 10 mL dichloromethane, and the mPEG-NH2 or PLGA-NH2 (50-150 mg) prepared in step two was dissolved in 10 mL tetrahydrofuran and slowly dropped into the PLGA-NHS or PCL-NHS or PLA-NHS solution, and the reaction was continued for 8-18 h under stirring at 45 °C, then the solution was concentrated by rotary evaporation, washed with excess cold tert-butyl methyl ether, and the obtained 1,4-butanediamine-modified block copolymer PEG-PLGA or PEG-PCL or PEG-PLA or PLGA-PLGA product was dried at 25 °C under vacuum for 24 h.

[0013] Step 5: Dissolve the 1,4-butanediamine-modified block copolymer PEG-PLGA or PEG-PCL or PEG-PLA or PLGA-PLGA copolymer (10-100 mg) and tanshinone IIA (1-10 mg) obtained in Step 4 in 1 mL of dichloromethane, then slowly drop it into 4 mL of 1% PVA aqueous solution, stir continuously at 1000 rpm for 10 minutes, then sonicate for 4 minutes at an ultrasonic power of 100-800 W, and finally stir and evaporate at 30 °C for 24 h to remove the dichloromethane, allowing the dispersed nanodroplets to solidify in the aqueous solution to obtain pH-sensitive tanshinone IIA@PEG-PLGA nanoparticles.

[0014] Preferably, in the preparation method of PEG-PLGA, the molecular weight of mPEG in step one is 2000~20000 Da, and / or the ratio of mPEG to CDI in step one is 1:10~10:1, and / or the ratio of 1,4-butanediamine dihydrochloride to mPEG-CDI in step two is 1:4~1:1 g, and / or the ratio of PLGA to hydroxysuccinimide in step three is 150:1~5:1, and / or Alternatively, the ratio of PLGA to dicyclohexylcarbodiimide used in step three may be 30:1 to 1:3, and / or the ratio of PLGA-NHS to mPEG-NH2 used in step four may be 1:3 to 3:1, and / or the ratio of PEG-PLGA to tanshinone IIA used in step five may be 1:1 to 10:1, and / or the reaction time in steps one to four may be 8 to 18 hours, and / or the ultrasonic power in step five may be 100 to 800 W.

[0015] This invention provides a method for preparing tanshinone IIA nanoparticles specifically released at the inflammatory site based on the inflammatory microenvironment. Various block copolymers are constructed using 1,4-butanediamine as a modifying chain to prepare nanoparticle drug carriers, achieving targeted delivery of the poorly soluble tanshinone IIA molecule to the inflammatory site, improving drug accumulation at the inflammatory site, and increasing anti-inflammatory efficiency. This preparation process is simple, with mild reaction conditions and low equipment requirements. The drug carrier breaks down rapidly in the acidic pH environment of inflammation, achieving targeted drug delivery to the inflammatory site, significantly improving drug accumulation at the lesion site, reducing drug concentration at non-lesion sites, greatly improving drug bioavailability, and reducing side effects. Preferred carrier materials further enhance the high affinity of the nanocarrier for hydrophobic molecules, significantly improving the drug loading efficiency for the hydrophobic tanshinone IIA molecule. Attached Figure Description

[0016] Figure 1 This is the nuclear magnetic resonance spectrum of the PEG-PLGA block copolymer in Example 1 of the present invention.

[0017] Figure 2 This is a transmission electron microscope image of tanshinone IIA@PEG-PLGA nanoparticles in Example 1 of the present invention.

[0018] Figure 3 The figures show the drug release curves of tanshinone IIA nanoparticles in Examples 1, 4, 5, and 6 of this invention. The release curves for Examples 1, 4, 5, and 6 are as follows: Figure 3 (A), 3 (B), 3 (C), and 3 (D).

[0019] Figure 4 This is a comparison chart of the drug release capabilities of DiD@PEG-PLGA nanoparticles in inflammatory and normal cellular environments in Example 6 of the present invention.

[0020] Figure 5 This describes the inhibitory effect of tanshinone IIA nanoparticles on IL-1β in inflammatory cells in Examples 1, 4, 5, and 6 of this invention. Detailed Implementation

[0021] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments do not constitute a limitation of the present invention.

[0022] Example 1: Preparation of Tanshinone IIA@PEG-PLGA Nanoparticles Targeting Inflammatory Sites

[0023] (1) Weigh 6g of mPEG (molecular weight 5000Da), add 100mL of tetrahydrofuran, stir at 50℃ for 10 minutes, and dissolve for later use;

[0024] (2) Weigh 5.1g of CDI, dissolve it in 80 mL of tetrahydrofuran, slowly add the mPEG solution, stir and react at 45℃ for 12 h, then concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the mPEG-CDI product under vacuum at 0℃ for 24 h for later use.

[0025] (3) Weigh 0.75 g of 1,4-butanediamine dihydrochloride, add 40 mL of tetrahydrofuran, stir at 40 °C for 10 minutes, and dissolve for later use;

[0026] (4) Weigh 1.5g of mPEG-CDI prepared in step (2), dissolve it in 50 mL of tetrahydrofuran, and slowly add 1,4-butanediamine solution dropwise under stirring at 45°C. Stir the reaction at 45°C for 12 hours, then concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the obtained mPEG-NH2 product under vacuum at 0°C for 24 hours for later use.

[0027] (5) Weigh 0.9g of PLGA and add 40mL of dichloromethane. Stir at 40℃ for 10min to dissolve and set aside.

[0028] (6) Weigh 57 mg of N-hydroxysuccinimide and 102 mg of dicyclohexylcarbodiimide, add them to the PLGA solution, stir and react for 12 hours at 45 °C under N2 protection, then concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the obtained PLGA-NHS product under vacuum at 0 °C for 24 hours for later use.

[0029] (7) Weigh 90 mg of the prepared PLGA-NHS, add 10 mL of dichloromethane, stir at 40 °C for 10 min, and dissolve for later use;

[0030] (8) Weigh 80 mg of the prepared mPEG-NH2, dissolve it in 10 mL of tetrahydrofuran, slowly add PLGA-NHS solution, stir and react at 45 °C for 12 hours, then concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the obtained PEG-PLGA product under vacuum at 25 °C for 24 h for later use.

[0031] (9) Weigh 30 mg of the prepared PEG-PLGA and 2 mg of tanshinone IIA, dissolve them in 1 mL of dichloromethane, and then slowly drop them into 4 mL of 1% PVA aqueous solution. Stir continuously at 1000 rpm for 10 minutes, then sonicate for 4 minutes with an ultrasonic power of 240 W. Finally, stir and evaporate at 30 °C for 24 h to remove the dichloromethane, so that the dispersed nanodroplets can be solidified in the aqueous solution to obtain pH-sensitive tanshinone IIA@PEG-PLGA nanoparticles.

[0032] Example 2: Analysis of the molecular composition of PEG-PLGA block copolymer by nuclear magnetic resonance wave method

[0033] The PEG-PLGA block copolymer prepared in Example 1 was dissolved in 0.5 mL of deuterated chloroform and used for detection by proton nuclear magnetic resonance spectroscopy. Spectroscopic analysis is as follows: Figure 1 As shown.

[0034] Example 3: Microscopic morphology observation of tanshinone IIA@PEG-PLGA nanoparticles targeting inflammatory sites

[0035] The morphology of the tanshinone IIA@PEG-PLGA nanoparticles prepared in Example 1 was observed using a transmission electron microscope, as shown below. Figure 2 As shown, the tanshinone IIA@PEG-PLGA nanoparticles are uniformly distributed with a particle size of approximately 200 nm.

[0036] Example 4: Preparation of Tanshinone IIA@PEG-PCL Nanoparticles

[0037] Tanshinone IIA@PEG-PCL nanoparticles were prepared using the same method as in Example 1, with the specific steps as follows.

[0038] Step 1: Dissolve 6g of mPEG (molecular weight 5000Da) in 100mL of tetrahydrofuran, dissolve 5.1g of CDI in 80mL of tetrahydrofuran, and slowly add the mPEG solution dropwise. Stir and react at 45℃ for 12 h. Then, concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the mPEG-CDI product under vacuum at 0℃ for 24 h for later use.

[0039] Step 2: Weigh 0.75 g of 1,4-butanediamine dihydrochloride and dissolve it in 40 mL of tetrahydrofuran. Dissolve 1.5 g of the prepared mPEG-CDI in 50 mL of tetrahydrofuran and slowly add the 1,4-butanediamine solution dropwise. Stir and react at 45 °C for 12 hours. Then, concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the obtained mPEG-NH2 product under vacuum at 0 °C for 24 hours for later use.

[0040] Step 3: Weigh 0.9g of PCL and dissolve it in 40mL of dichloromethane. Weigh 57mg of N-hydroxysuccinimide and 102mg of dicyclohexylcarbodiimide and add them to the PCL solution. Stir and react at 45℃ under N2 protection for 12 hours. Then, concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the obtained PCL-NHS product under vacuum at 0℃ for 24 hours for later use.

[0041] Step 4: Weigh 90 mg of the prepared PCL-NHS and dissolve it in 10 mL of dichloromethane. Weigh 80 mg of the prepared mPEG-NH2 and dissolve it in 10 mL of tetrahydrofuran. Slowly add the PCL-NHS solution dropwise and stir the reaction at 45 °C for 12 hours. Then, concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the obtained PEG-PCL product under vacuum at 25 °C for 24 hours for later use.

[0042] Step 5: Weigh 30 mg of the prepared PEG-PCL and 2 mg of tanshinone IIA, dissolve them in 1 mL of dichloromethane, and then slowly drop them into 4 mL of 1% PVA aqueous solution. Stir continuously at 1000 rpm for 10 minutes, then sonicate for 4 minutes at an ultrasonic power of 240 W. Finally, stir and evaporate at 30 °C for 24 h to remove the dichloromethane, allowing the dispersed nanodroplets to solidify in the aqueous solution to obtain tanshinone IIA@PEG-PCL nanoparticles.

[0043] Example 5: Preparation of Tanshinone IIA@PEG-PLA Nanoparticles

[0044] Tanshinone IIA@PEG-PLA nanoparticles were prepared using the same method as in Example 1, with the specific steps as follows.

[0045] Step 1: Dissolve 6g of mPEG (molecular weight 5000Da) in 100mL of tetrahydrofuran, dissolve 5.1g of CDI in 80mL of tetrahydrofuran, and slowly add the mPEG solution dropwise. Stir and react at 45℃ for 12 h. Then, concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the mPEG-CDI product under vacuum at 0℃ for 24 h for later use.

[0046] Step 2: Weigh 0.75 g of 1,4-butanediamine dihydrochloride and dissolve it in 40 mL of tetrahydrofuran. Dissolve 1.5 g of the prepared mPEG-CDI in 50 mL of tetrahydrofuran and slowly add the 1,4-butanediamine solution dropwise. Stir and react at 45 °C for 12 hours. Then, concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the obtained mPEG-NH2 product under vacuum at 0 °C for 24 hours for later use.

[0047] Step 3: Weigh 0.9g of PLA and dissolve it in 40mL of dichloromethane. Weigh 57mg of N-hydroxysuccinimide and 102mg of dicyclohexylcarbodiimide and add them to the PLA solution. Stir and react at 45℃ under N2 protection for 12 hours. Then, concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the obtained PLA-NHS product under vacuum at 0℃ for 24 hours for later use.

[0048] Step 4: Weigh 90 mg of the prepared PLA-NHS and dissolve it in 10 mL of dichloromethane. Weigh 80 mg of the prepared mPEG-NH2 and dissolve it in 10 mL of tetrahydrofuran. Slowly add the PLA-NHS solution dropwise and stir the reaction at 45 °C for 12 hours. Then, concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the obtained PEG-PLA product under vacuum at 25 °C for 24 hours for later use.

[0049] Step 5: Weigh 30 mg of the prepared PEG-PLA and 2 mg of tanshinone IIA, dissolve them in 1 mL of dichloromethane, and then slowly drop them into 4 mL of 1% PVA aqueous solution. Stir continuously at 1000 rpm for 10 minutes, then sonicate for 4 minutes at an ultrasonic power of 240 W. Finally, stir and evaporate at 30 °C for 24 h to remove the dichloromethane, allowing the dispersed nanodroplets to solidify in the aqueous solution to obtain tanshinone IIA@PEG-PLA nanoparticles.

[0050] Example 6: Preparation of Tanshinone IIA@PLGA-PLGA Nanoparticles

[0051] Tanshinone IIA@PLGA-PLGA nanoparticles were prepared using the same method as in Example 1, with the specific steps as follows.

[0052] Step 1: Dissolve 6g PLGA in 100mL tetrahydrofuran, dissolve 5.1g CDI in 80mL tetrahydrofuran, and slowly add the PLGA solution dropwise. Stir and react at 45℃ for 12 h. Then, concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the PLGA-CDI product under vacuum at 0℃ for 24 h for later use.

[0053] Step 2: Weigh 0.75 g of 1,4-butanediamine dihydrochloride and dissolve it in 40 mL of tetrahydrofuran. Dissolve 1.5 g of the prepared PLGA-CDI in 50 mL of tetrahydrofuran and slowly add the 1,4-butanediamine solution dropwise. Stir the reaction at 45 °C for 12 hours. Then, concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the obtained PLGA-NH2 product under vacuum at 0 °C for 24 hours for later use.

[0054] Step 3: Weigh 0.9g of PLGA and dissolve it in 40mL of dichloromethane. Weigh 57mg of N-hydroxysuccinimide and 102mg of dicyclohexylcarbodiimide and add them to the PLGA solution. Stir and react at 45℃ under N2 protection for 12 hours. Then, concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the obtained PLGA-NHS product under vacuum at 0℃ for 24 hours for later use.

[0055] Step 4: Weigh 90 mg of the prepared PLGA-NHS and dissolve it in 10 mL of dichloromethane. Weigh 80 mg of the prepared PLGA-NH2 and dissolve it in 10 mL of tetrahydrofuran. Slowly add the PLGA-NHS solution dropwise and stir the reaction at 45 °C for 12 hours. Then, concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the obtained PLGA-PLGA product under vacuum at 25 °C for 24 hours for later use.

[0056] Step 5: Weigh 30 mg of the prepared PLGA-PLGA and 2 mg of tanshinone IIA, dissolve them in 1 mL of dichloromethane, and then slowly add them dropwise to 4 mL of 1% PVA aqueous solution. Stir continuously at 1000 rpm for 10 minutes, then sonicate for 4 minutes at an ultrasonic power of 240 W. Finally, stir and evaporate at 30 °C for 24 h to remove the dichloromethane, allowing the dispersed nanodroplets to solidify in the aqueous solution to obtain tanshinone IIA@PLGA-PLGA nanoparticles.

[0057] Example 7: Determination of drug loading and encapsulation efficiency of tanshinone IIA nanoparticles

[0058] The content of tanshinone IIA was determined by high performance liquid chromatography (HPLC). A SunFire C18 column (4.6 mm × 250 mm, 5 μm) was used; the mobile phase was methanol-water (90:10), and the flow rate was 1.0 mL / min. -1The column temperature was 30℃; the detection wavelength was 270nm; and the injection volume was 10μL.

[0059] Concentrations of 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, and 20.0 mg·L were taken respectively. -1 The tanshinone IIA reference solution was tested according to chromatographic conditions, and a regression equation was established by fitting the peak area to the tanshinone IIA concentration. 1.0 mg of tanshinone IIA nanoparticles prepared in Examples 1, 4, 5, and 6 were added to 10.0 mL of anhydrous ethanol, ultrasonically dispersed for 30 min, then centrifuged at 15000 rpm for 20 min. The supernatant was filtered through a 0.22 μm microporous membrane, and the peak area was determined by HPLC. The tanshinone IIA content in the solution was calculated based on the standard curve. The drug loading and encapsulation efficiency of the microspheres were calculated using the following formula:

[0060] Encapsulation efficiency % = (Drug loading on nanoparticles / Total drug loading) × 100%;

[0061] Drug loading % = (Drug loading on nanoparticles / Total mass of nanoparticles) × 100%.

[0062] The encapsulation efficiency and drug loading of various 1,4-butanediamine-modified block copolymer nanocarriers were obtained based on HPLC analysis and calculations of encapsulation efficiency and drug loading, and the results are listed in Table 1.

[0063] Table 1. Encapsulation efficiency and drug loading of 1,4-butanediamine-modified block copolymer nanocarriers

[0064]

[0065] Example 8: In vitro pH-sensitive drug release performance of tanshinone IIA nanoparticles

[0066] Tanshinone IIA nanoparticles containing 2 mg of tanshinone IIA as described in Examples 1, 4, 5, and 6 were dispersed in 5 mL of dissolution medium (pH 5.0 or 7.4 phosphate buffer), placed in a dialysis bag (molecular weight cutoff of 10,000), and added to 195 mL of phosphate buffer at the same pH. The mixture was then shaken in a constant temperature water bath at (37±0.5)℃ at 100 r·min. -1 Samples were taken in 5 mL at 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h, and the same volume of blank medium was added. The contents of the samples were determined by high performance liquid chromatography after filtration through a 0.22 μm microporous membrane, and the cumulative release rate was calculated according to the formula.

[0067]

[0068] In the formula: Q is the cumulative release rate; This refers to the cumulative release amount; The concentration of tanshinone IIA in the solution during the nth sampling is given; V is the solution volume. The concentration of tanshinone IIA in the sample solution during the i-th sampling in the first n-1 sampling operations; Let be the volume of the sample solution taken during the i-th sampling.

[0069] The release results of the tanshinone IIA@PEG-PLGA nanoparticles prepared in Example 1 are as follows: Figure 3 As shown in Figure A, it exhibits a distinct pH-sensitive drug release characteristic, rapidly releasing the drug in an acidic environment while releasing it slowly in a neutral environment.

[0070] For the release results of tanshinone IIA nanoparticles prepared in other embodiments, please refer to [link / reference]. Figure 3 As can be seen from BD, compared with Example 1, the pH-sensitive drug release characteristics are not obvious, and the release is slower in acidic and neutral environments.

[0071] Example 9: Study on inflammatory cell uptake of PEG-PLGA nanoparticles

[0072] By replacing tanshinone IIA with the fluorescent agent DiD, DiD@PEG-PLGA nanoparticles containing the fluorescent agent were prepared according to the method in Example 1.

[0073] Human lung epithelial cells BEAS-2B were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 0.05 mM β-mercaptoethanol in a humidified incubator at 37°C under a 5% CO2 atmosphere.

[0074] BEAS-2B cells were cultured in 6-well plates. LPS (10 mg / L) was added to induce inflammation, or no LPS was added as a normal cell control, according to the experimental design. After 24 hours of incubation, DiD@PEG-PLGA (equivalent to 2 μM DiD) was added, and the cells were further incubated for specified times (5, 15, 30, 60 min). Subsequently, the cells were washed three times with PBS, and the amount of DiD absorbed by the cells was observed under a fluorescence microscope.

[0075] The results are as follows Figure 4 As shown, the DiD@PEG-PLGA nanoparticles prepared by the method in Example 1 can significantly release drugs and enhance cell fluorescence under inflammatory conditions, while under normal cell conditions, the drugs are hardly released and the cell fluorescence signal is weak.

[0076] Example 10: In vitro anti-inflammatory performance of tanshinone IIA nanoparticles

[0077] Human lung epithelial cells BEAS-2B were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 0.05 mM β-mercaptoethanol in a humidified incubator at 37°C under a 5% CO2 atmosphere.

[0078] BEAS-2B cells were cultured in 6-well plates. Depending on the experimental design, LPS (10 mg / L) was added to induce inflammation, or no LPS was added as a normal cell control. Alternatively, LPS (10 mg / L) + tanshinone IIA nanoparticles (including nanoparticles prepared in Examples 1, 4, 5, and 6) were added. After 24 hours of incubation, the cell supernatant was collected, and the interleukin-1β (IL-1β) content in the cell culture medium was determined by ELISA. The procedure was followed according to the kit instructions, and the A450 nm value was measured using a microplate reader. The content was calculated based on the standard curve.

[0079] The results are as follows Figure 5 As shown, the tanshinone IIA@PEG-PLGA nanoparticles prepared by the method of Example 1 have a significant ability to inhibit the expression of inflammatory factors in inflammatory cells. The anti-inflammatory effects of the tanshinone IIA nanoparticles prepared in other examples are significantly lower than those prepared in Example 1.

[0080] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and modifications according to the present invention, but these corresponding changes and modifications should all fall within the protection scope of the appended claims of the present invention.

Claims

1. A tanshinone IIA nanoparticle specifically released at the site of inflammation based on an inflammatory microenvironment, comprising tanshinone IIA and a 1,4-butanediamine-modified block copolymer nanocarrier, wherein the 1,4-butanediamine-modified block copolymer nanocarrier is 1,4-butanediamine-modified PEG-PLGA, 1,4-butanediamine-modified PEG-PCL, 1,4-butanediamine-modified PEG-PLA, or 1,4-butanediamine-modified PLGA-PLGA.

2. A tanshinone IIA nanoparticle specifically released at the site of inflammation based on the inflammatory microenvironment as described in claim 1, characterized in that... The nanocarrier is PEG-PLGA modified with 1,4-butanediamine.

3. A method for preparing tanshinone IIA nanoparticles specifically released at the inflammatory site based on the inflammatory microenvironment as described in claim 1, characterized in that, Includes the following steps: Step 1: Polymer A with a molecular weight of 2000-20000 Da is dissolved in tetrahydrofuran and stirred at 50°C for 10 minutes. N,N'-carbonyldiimidazole (CDI) is dissolved in tetrahydrofuran. Subsequently, under continuous stirring at 45°C, the CDI solution is slowly added dropwise to the polymer A solution and the reaction continues. The solution is then concentrated by rotary evaporation, washed with excess cold tert-butyl methyl ether, and the obtained polymer A-CDI is vacuum dried at 0°C for 24 hours. The polymer A is methoxy polyethylene glycol (mPEG) or polylactic acid-glycolic acid copolymer (PLGA). Step 2: 1,4-Butanediamine dihydrochloride is dissolved in tetrahydrofuran. The polymer A-CDI prepared in Step 1 is dissolved in tetrahydrofuran. Under continuous stirring at 45°C, the polymer A-CDI solution is slowly added dropwise to the 1,4-butanediamine dihydrochloride solution. The reaction is stirred at 45°C. Then, the solution is concentrated by rotary evaporation. Excess cold tert-butyl methyl ether is added for washing. The resulting polymer A-NH2 product is dried under vacuum at 0°C for 24 hours. Step 3: Polymer B is dissolved in dichloromethane, and N-hydroxysuccinimide and dicyclohexylcarbodiimide are added under stirring at 45°C. The reaction is carried out under N2 protection. The solution is then concentrated by rotary evaporation, washed with excess cold tert-butyl methyl ether, and the resulting polymer B-NHS product is dried under vacuum at 0°C for 24 hours. The polymer B is PLGA, polycaprolactone (PCL), or PLA. Step 4: Dissolve the polymer B-NHS prepared in Step 3 in dichloromethane; dissolve the polymer A-NH2 prepared in Step 2 in tetrahydrofuran and slowly add it dropwise into the polymer B-NHS solution. Stir the reaction at 45°C, then concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the resulting 1,4-butanediamine-modified block copolymer under vacuum at 25°C for 24 h. Step 5: Dissolve the 1,4-butanediamine-modified block copolymer and tanshinone IIA obtained in Step 4 in dichloromethane, then slowly drop it into a 1% PVA aqueous solution while continuously stirring. Then, ultrasonically break it up with an ultrasonic power of 100-800W. Finally, stir and evaporate at 30°C for 24 hours to remove the dichloromethane, allowing the dispersed nanodroplets to solidify in the aqueous solution, thus obtaining tanshinone IIA nanoparticles that are specifically released at the inflammatory site.

4. A method for preparing tanshinone IIA nanoparticles specifically released at inflammatory sites based on an inflammatory microenvironment as described in claim 2, characterized in that... The preparation steps include the following: Step 1: Methoxy polyethylene glycol (mPEG), with a molecular weight of 2000-20000 Da, is dissolved in tetrahydrofuran and stirred at 50°C for 10 minutes. N,N'-carbonyl diimidazole (CDI) is dissolved in tetrahydrofuran. Then, under continuous stirring at 45°C, the CDI solution is slowly added dropwise to the mPEG solution and the reaction is continued for 8-18 hours. The solution is then concentrated by rotary evaporation, washed with excess cold tert-butyl methyl ether, and the obtained mPEG-CDI is dried under vacuum at 0°C for 24 hours. Step 2: Dissolve 1,4-butanediamine dihydrochloride in tetrahydrofuran. Dissolve the mPEG-CDI prepared in Step 1 in tetrahydrofuran. Under continuous stirring at 45°C, slowly add the mPEG-CDI solution dropwise to the 1,4-butanediamine dihydrochloride solution. Stir and react at 45°C for 8-18 hours. Then, concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether for washing, and dry the obtained mPEG-NH2 product under vacuum at 0°C for 24 hours. Step 3: PLGA is dissolved in dichloromethane, and N-hydroxysuccinimide and dicyclohexylcarbodiimide are added with stirring at 45°C. The reaction is carried out under N2 protection for 8-18 hours. The solution is then concentrated by rotary evaporation, washed with excess cold tert-butyl methyl ether, and the obtained PLGA-NHS product is dried under vacuum at 0°C for 24 hours. Step 4: Dissolve the PLGA-NHS prepared in Step 3 in dichloromethane, dissolve the mPEG-NH2 prepared in Step 2 in tetrahydrofuran, and slowly add it dropwise into the PLGA-NHS solution. Stir the reaction at 45°C for 8-18 hours, then concentrate the solution by rotary evaporation, add excess cold tert-butyl methyl ether to wash, and dry the obtained 1,4-butanediamine-modified PEG-PLGA product under vacuum at 25°C for 24 hours. Step 5: Dissolve the 1,4-butanediamine-modified PEG-PLGA block copolymer and tanshinone IIA obtained in Step 4 in dichloromethane, then slowly drop it into a 1% PVA aqueous solution while continuously stirring and reacting. Then, it is ultrasonically broken up with an ultrasonic power of 100-800W. Finally, it is stirred and evaporated at 30℃ for 24h to remove the dichloromethane, allowing the dispersed nanodroplets to solidify in the aqueous solution to obtain tanshinone IIA nanoparticles that are specifically released at the inflammatory site.

5. A preparation method as described in claim 4, characterized in that: The ratio of mPEG to CDI in step one is 1:10 to 10:1, preferably 6:5.

1.

6. A preparation method according to claim 4 or 5, wherein the ratio of 1,4-butanediamine to mPEG-CDI in step two is 1:4 to 1:

1.

7. A preparation method according to any one of claims 4-6, wherein the ratio of PLGA to hydroxysuccinimide in step three is 150:1 to 5:1, and the ratio of PLGA to dicyclohexylcarbodiimide is 30:1 to 1:

3.

8. A preparation method according to any one of claims 4-7, wherein the ratio of PLGA-NHS to mPEG-NH2 in step four is 1:3 to 3:

1.

9. A preparation method according to any one of claims 4-8, wherein the ratio of PEG-PLGA to tanshinone IIA in step five is 1:1 to 10:1; and the reaction time in step five is 8 to 18 hours.

10. Use of a nanoparticle as described in any one of claims 1-2 or a nanoparticle prepared by the preparation method of claims 3-9 in the preparation of an anti-inflammatory drug.

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