A kind of tripterygium wilfordii prodrug delivery gelatin micelle and preparation method thereof

Abstract

The present invention relates to a tripterygium wilfordii prodrug delivery gelatin micelle and a preparation method thereof. The tripterygium wilfordii prodrug delivery gelatin micelle comprises a gelatin micelle for delivering tripterygium wilfordii and tripterygium wilfordii encapsulated in the gelatin micelle; the gelatin micelle is gelatin first modified with lipoic acid and then aminated and reduced with ethylenediamine to obtain reduced aminated lipoic acid gelatin, which is an amphiphilic molecular micelle; through the synergistic effect of micellar thiol and o-formylphenylboronic acid, tripterygium wilfordii and gelatin micelle are reversibly covalently bonded to form tripterygium wilfordii prodrug delivery gelatin micelle. The obtained delivery micelle can reduce the toxicity of tripterygium wilfordii, improve the water solubility of tripterygium wilfordii, and can improve the micelle integrity through cross-linking and have tumor cell selectivity.

Description

Technical Field

[0001] The present invention belongs to the field of chemical drug preparations, and in particular relates to a tripterygium wilfordii prodrug delivery gelatin micelle and a method for in situ generating the tripterygium wilfordii prodrug delivery gelatin micelle. Background Art

[0002] Celastrol (CE), also known as celastrol, is a pentacyclic triterpenoid compound isolated from the root of the traditional Chinese medicine Tripterygium wilfordii. It has significant inhibitory effects on lung cancer, liver cancer, breast cancer, osteosarcoma, gastric cancer, leukemia, cervical cancer cells, etc., and has significant anti-tumor activity. However, the high toxicity and poor water solubility of celastrol limit its clinical application. One of the solutions is to use different polymer carriers (such as micelles) for delivery. For example, according to literature reports, Zhou et al. used polyethylene glycol-grafted polylactic acid-co-glycolic acid copolymer (PEG-PLGA) nanoparticles (NPs / CEL) to load celastrol, and used neutrophil membranes for coating to obtain (NNPs / CEL) for the treatment of acute pancreatitis. In order to improve the water solubility and toxic side effects of celastrol and optimize its anti-tumor efficacy, Chen et al. developed galactosylated liposomes. Galactose-modified 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-polyethylene glycol (C-GPL) was used to deliver triptolide.

[0003] Polymer micelles are composed of a unique core-shell structure. Amphiphilic polymers are high molecular weight polymers composed of at least two parts: hydrophilic and lipophilic. The hydrophobic core can encapsulate poorly soluble drugs, and the hydrophilic shell separates the drugs from the water-soluble environment to stabilize the micelles and prevent the micelles from being excreted from the body by the reticuloendothelial system. Polymer micelles can not only increase the solubility of anticancer drugs in water, but also have the potential for precise targeted release, thereby improving drug efficacy. However, polymer micelles have the risk of drug leakage before reaching the target site. Summary of the invention

[0004] The object of the present invention is to provide a tripterygium wilfordii prodrug delivery micelle and a preparation method thereof, which can reduce the toxicity of tripterygium wilfordii, improve the water solubility of tripterygium wilfordii and improve the integrity of the micelle by cross-linking.

[0005] The solution adopted by the present invention to solve the above technical problems is:

[0006] A tripterygium wilfordii prodrug delivery gelatin micelle, comprising a gelatin micelle for delivering tripterygium wilfordii and tripterygium wilfordii encapsulated in the gelatin micelle; the gelatin micelle is a reduced aminated lipoic acid gelatin obtained by modifying gelatin with lipoic acid, aminating with ethylenediamine, and reducing the amino group, and is an amphiphilic molecular micelle; the tripterygium wilfordii and the gelatin micelle are reversibly covalently bonded to form the tripterygium wilfordii prodrug delivery gelatin micelle.

[0007] Furthermore, the tripterygium wilfordii and gelatin micelles are reversibly covalently bonded through the synergistic effect of micelle thiol and o-formylphenylboronic acid to form tripterygium wilfordii prodrug delivery gelatin micelles.

[0008] Furthermore, the drug loading rate of tripterygium wilfordii in the tripterygium wilfordii prodrug delivery gelatin micelles can reach 4.5% to 6.5%.

[0009] Furthermore, the amino amidation reaction ratio of gelatin in the gelatin micelles for delivering tripterygium wilfordii is 90%-97%, and further 95%-96%.

[0010] The present invention also provides a method for preparing the tripterygium wilfordii prodrug delivery gelatin micelles, comprising the following steps:

[0011] (1) Activating lipoic acid with EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) at a solution pH of 5.0 to 6.8;

[0012] (2) Activated lipoic acid is covalently bonded to gelatin via an amide bond to obtain lipoic acid gelatin;

[0013] (3) subjecting the obtained lipoic acid gelatin to an amination reaction with ethylenediamine to obtain ethylenediamine lipoic acid gelatin nano-micelles;

[0014] (4) reducing the obtained ethylenediamine lipoic acid gelatin nano-micelles using tri(2-carboxyethyl)phosphine, dialyzing and freeze-drying the product to obtain reduced aminated lipoic acid gelatin;

[0015] (5) mixing tripterygium wilfordii with the obtained reduced aminated lipoic acid gelatin, adding o-formylphenylboronic acid to react, dialyzing and freeze-drying the reaction product to obtain tripterygium wilfordii prodrug delivery gelatin micelles.

[0016] Furthermore, the solvent used in the chemical reaction of step (1) and step (2) is selected from ethanol, methanol, acetonitrile, and dimethyl sulfoxide; more preferably, the ethanol solution has a concentration of 50% to 70%.

[0017] Furthermore, in step (1), sodium hydroxide is used to adjust the pH.

[0018] Furthermore, in step (1), the mass ratio of EDC, NHS and lipoic acid is (1-2):1:(1-2), and more preferably 1.55:1:1.3.

[0019] Furthermore, the reaction conditions of step (1) include: temperature 25-40° C., more preferably 37° C.; reaction time 15-60 min, more preferably 20-30 min; stirring reaction.

[0020] Furthermore, the reaction conditions of step (2) include: temperature 25-40° C., more preferably 37° C.; reaction time 12-24 h, more preferably 20 h; stirring the reaction.

[0021] Furthermore, after the reaction in step (2) is completed, the product is collected by adding a solvent to precipitate the solid. Before the product is subjected to the next step of reaction, it is washed to remove the solvent and the unreacted raw materials.

[0022] Furthermore, the solvent used in the amination reaction in step (3) is selected from deionized water, alcohol / water mixture, and PBS buffer.

[0023] Furthermore, the reaction conditions of step (3) include: temperature 25-40°C, more preferably 37°C; reaction time 12-36h, more preferably 24h; reaction system pH 4-6, more preferably pH 5; using concentrated hydrochloric acid to adjust the pH; stirring the reaction.

[0024] Furthermore, after the reaction in step (3) is completed, the reaction solution is transferred to a dialysis bag, preferably MWCO 3500, and dialyzed in deionized water for 1 to 3 days. The dialyzate is freeze-dried to obtain a white fluffy solid.

[0025] Furthermore, the mass ratio of lipoic acid gelatin to ethylenediamine is 1:(2.0-3.5), and more preferably 1:2.7.

[0026] Furthermore, the solvent used in step (4) is selected from PBS buffer, deionized water, and ethanol; more preferably, 0.001-0.01 M PBS buffer, and more preferably, 0.01 M, pH 5 PBS buffer.

[0027] Furthermore, in step (4), the mass ratio of ethylenediamine lipoic acid gelatin nano-micelles to TCEP is (2-4):1, and more preferably 3:1.

[0028] Furthermore, the reaction conditions of step (4) include: an inert gas atmosphere, such as nitrogen; a temperature of 5 to 40° C., more preferably room temperature; a reaction time of 12 to 24 h, more preferably 16 h; a reaction system pH of 4 to 6, more preferably pH 5.

[0029] Furthermore, after the reaction in step (4) is completed, the reaction solution is transferred to a dialysis bag and dialyzed for 1 to 3 days, the dialysis bag preferably has a MWCO of 3500, and the dialysate is preferably deionized water whose pH value is adjusted to 3.5 to 4.5 with hydrochloric acid. The dialysate is freeze-dried to obtain a white fluffy solid.

[0030] Furthermore, in step (5), the mass ratio of celastrol, reduced aminated lipoic acid gelatin and o-formylphenylboronic acid is 1:(8-15):(0.8-1.2).

[0031] Furthermore, the solvent used to dissolve the reduced lipoic acid gelatin is selected from PBS buffer, deionized water, and ethanol; more preferably, 0.001-0.01 M PBS buffer, and more preferably, 0.01 M, pH 5 PBS buffer.

[0032] Furthermore, the solvent for dissolving tripterygium wilfordii is selected from dimethyl sulfoxide and N,N-dimethylformamide.

[0033] Furthermore, the reaction conditions include: temperature 1-10°C, more preferably 8°C; reaction time 10-24h, more preferably 12h; reaction system pH 7.0-8.0, more preferably pH 7.4, and sodium hydroxide is used to adjust the pH.

[0034] Further, after the reaction is completed, the reaction solution is transferred to a dialysis bag for dialysis for 0.5 to 2 days, the dialysis bag is preferably MWCO3500, the dialysis solution is preferably deionized water, and the dialysis temperature is 0-15° C., preferably 8° C. The dialysis solution is freeze-dried to obtain a white fluffy solid.

[0035] The present invention also provides the use of the tripterygium wilfordii prodrug delivery gelatin micelles in the preparation of anticancer drugs.

[0036] The main technical route of the present invention is: the amino group provided by gelatin and the carboxyl group provided by lipoic acid are covalently bonded by amide bonds, and then the remaining carboxyl groups of gelatin are ethylenediamined to obtain the amphiphilic lipoic acid gelatin amphiphilic molecule. Tripterygium wilfordii and the amino group on the ethylenediamine gelatin, the thiol group on the reduced lipoic acid, and o-formylphenylboronic acid are generated in situ by click reaction. The synthetic route is as follows: Figure 1 shown.

[0037] Gelatin nanoparticles are widely used to deliver hydrophilic and hydrophobic anticancer drugs. Gelatin, as the hydrophilic part, can be modified with lipophilic lipoic acid. The disulfide bonds in the lipoic acid gelatin amphiphilic molecules can be rearranged to tightly connect the lipoic acid gelatin amphiphilic molecular chains to obtain cross-linked micelles, thereby improving the integrity of the micelles and reducing the chance of drug leakage in the drug-loaded micelles.

[0038] The C-6 position in the structure of tripterygium wilfordii can undergo Michael addition reaction to form Michael addition products. When the environment changes (such as pH decreases), tripterygium wilfordii can be regenerated, which has the chemical structure basis for prodrug design. The formed tripterygium wilfordii prodrug delivery gelatin micelles can release tripterygium wilfordii molecules with high cytotoxicity around tumor cells to kill tumor cells; around normal cells, the tripterygium wilfordii prodrug remains unchanged with low cytotoxicity; it is beneficial to improve the selective killing of tumor cells by tripterygium wilfordii, low toxicity to normal cells, and improve safety.

[0039] The present invention utilizes for the first time a click reaction involving thiol, aldehyde, amino and boric acid groups to generate a prodrug of tripterygium wilfordii, and connects it to the lipoic acid gelatin amphiphilic molecule through a reversible covalent bond, thereby preparing a novel gelatin micelle for delivering the prodrug of tripterygium wilfordii, which has the advantages of significant low toxicity and tumor cell selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The synthetic route for the gelatin micelles for the prodrug delivery of tripterygium wilfordii; wherein:

[0041] Gelatin;

[0042] LA: lipoic acid;

[0043] GLA: lipoic acid gelatin;

[0044] EDA: ethylenediamine;

[0045] EDA-GLA: ethylenediamine-modified lipoic acid gelatin;

[0046] TCEP: tris(2-carboxyethyl)phosphine;

[0047] Re-EDA-GLA: reduced lipoic acid gelatin;

[0048] CE: tripterygium wilfordii;

[0049] 2-FPBA: o-Formylphenylboronic acid;

[0050] EDA-GLA / CE / 2-FPBA: ethylenediamine-modified lipoic acid gelatin encapsulating tripterygium wilfordii and o-formylphenylboronic acid (tripterygium wilfordii prodrug delivery gelatin micelles).

[0051] Figure 2 The chemical structure characterization of gelatin micelles for the delivery of tripterygium wilfordii prodrug; wherein:

[0052] (A) Re-EDA-GLA and EDA-GLA / CE / 2-FPBA 1 H NMR spectrum;

[0053] (B) is the infrared spectra of Re-EDA-GLA and EDA-GLA / CE / 2-FPBA;

[0054] (C) is the UV absorption spectrum of CE, EDA-GLA / 2-FPBA, EDA-GLA / CE / 2-FPBA and EDA-GLA / CE / 2-FPBA-Destroy, in which the starting points of the curves from high to low are: EDA-GLA / 2-FPBA, EDA-GLA / CE / 2-FPBA, EDA-GLA / CE / 2-FPBA-Destroy, CE;

[0055] (D) is the differential scanning calorimetry graph of EDA-GLA, EDA-GLA@CE and EDA-GLA / CE / 2-FPBA;

[0056] (E) is the effect of different concentrations of CE on the fluorescence intensity of FI, with an excitation wavelength of 337 nm, where the highest points of the curve are 0, 0.04, 0.08, and 0.12 from top to bottom;

[0057] (F) is the fluorescence intensity diagram of Re-EDA-GLA and FI, with an excitation wavelength of 337 nm. The highest curves are Re-EDA-GLA and FI from top to bottom.

[0058] Figure 3 Schematic diagram of the self-assembly of gelatin micelles for the delivery of tripterygium wilfordii prodrug; wherein:

[0059] (A) TEM image of EDA-GLA / CE / 2-FPBA micelles and solution appearance;

[0060] (B) TEM image and solution appearance image of EDA-GLA / CE / 2-FPBA micelles after being placed in pH 5.0 PBS buffer solution for 5 days.

[0061] Figure 4 The morphology of EDA-GLA / CE / 2-FPBA micelles in different solutions;

[0062] (A) Schematic diagram of EDA-GLA / CE / 2-FPBA micelle self-assembly;

[0063] (B) is the scanning electron microscopy (TEM) image of EDA-GLA / CE / 2-FPBA micelles and the actual image of PBS (pH 7.4) solution;

[0064] (C) is a scanning electron microscope (TEM) image of EDA-GLA / CE / 2-FPBA micelles after being placed in PBS (pH 5.0) solution for 5 days and a physical image of the solution;

[0065] (D) is a physical picture of CE in aqueous solution.

[0066] Figure 5 The release rate and particle size changes of gelatin micelles for the prodrug delivery of tripterygium wilfordii under different pH conditions; wherein:

[0067] (A) The release amount of EDA-GLA / CE / 2-FPBA micelles in PBS 7.4 and PBS 5.0 aqueous solutions changes with time;

[0068] (B) The particle size variation trend of EDA-GLA / CE / 2-FPBA micelles in PBS 7.4 and PBS 5.0 aqueous solutions over time.

[0069] Figure 6 The release rate and particle size changes of tripterygium wilfordii prodrug delivery gelatin micelles at different GSH concentrations; wherein:

[0070] (A) The release trend of EDA-GLA / CE / 2-FPBA micelles at different GSH concentration levels over time;

[0071] (B) Particle size variation trend of EDA-GLA / CE / 2-FPBA micelles at different GSH concentration levels over time.

[0072] Figure 7 The effects of blank micelles of different concentrations on B16 and HaCat cells.

[0073] Figure 8 This is a comparison of the viability of B16 and HaCat cells cultured with gelatin micelles containing different concentrations of tripterygium wilfordii prodrug after 48 hours of culture, as well as a comparison of the viability of HaCat cells cultured with gelatin micelles containing different concentrations of tripterygium wilfordii prodrug after 48 hours of culture.

[0074] Fig. 9 This is a laser confocal image of B16 cells cultured with FITC-labeled EDA-GLA / CE / 2-FPBA for 4 hours; from left to right are FITC staining (green), DAPI nuclear staining (blue) and the overlay of the two colors. DETAILED DESCRIPTION

[0075] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples.

[0076] Example 1

[0077] The gelatin micelles for delivering tripterygium wilfordii prodrug provided in this example have a hydrated particle size of 200-400 nm. The preparation method is as follows:

[0078] Add 260mg lipoic acid to 20ml of 60% ethanol, stir to dissolve, and adjust the solution pH to 5.9-6.0 with 1M sodium hydroxide solution. Add 310mg EDC and 200mg NHS, stir and react at 37°C for 25min to obtain an activated lipoic acid solution. Add 250mg gelatin (type B) to 30ml 60% ethanol, stir until dissolved, and then add the above activated lipoic acid solution. The reaction solution is stirred and reacted at 37°C for 16 hours. After the reaction is completed, add anhydrous ethanol to precipitate solids, and collect the solids after centrifugation (speed 5000rpm) for 20min. Rinse three times with anhydrous ethanol and a small amount of deionized water to remove residual ethanol, unreacted lipoic acid and gelatin. Redissolve the solid in 10ml water, and freeze-dry the solution to obtain a white fluffy solid, which is lipoic acid gelatin. Store in a vacuum for standby use.

[0079] Dissolve the above 500 mg lipoic acid gelatin in 30 mL deionized water, add 1.5 mL ethylenediamine, adjust the pH to about 5 with concentrated hydrochloric acid, add 0.9 g EDC, stir and react at 37°C for 24 hours, transfer the solution to a dialysis bag (MWCO3500), dialyze in deionized water for 48 hours, and freeze-dry the dialyzate to obtain a white fluffy solid, which is ethylenediamine-modified lipoic acid gelatin. Store in vacuum for later use.

[0080] The above 300 mg of ethylenediamine-modified lipoic acid gelatin was dissolved in 60 mL of 0.01 M (pH 5.0) PBS buffer solution, stirred and dissolved at room temperature, 100 mg of tris (2-carboxyethyl) phosphine (TCEP) was added, and the mixture was reacted at room temperature for 16 hours under nitrogen protection, and the reaction solution was transferred to a dialysis bag (MWCO 3500), and dialyzed in deionized water (hydrochloric acid adjusted to pH 4.0) for 48 hours. The dialyzate was freeze-dried to obtain a white fluffy solid, which was the reduced lipoic acid gelatin. The mixture was stored in a vacuum for standby use.

[0081] The above 40 mg reduced lipoic acid gelatin was dissolved in 40 mL 0.01 M (pH 5.0) PBS buffer solution, and stirred at room temperature to dissolve. Another 4 mg of tripterygium wilfordii was dissolved in 4 ml of dimethyl sulfoxide solution, added to the reduced lipoic acid gelatin solution, stirred at room temperature, and the solution was adjusted to pH 7.4 with 1 M sodium hydroxide solution, and 3.9 mg of o-formylphenylboronic acid was added, and stirred at 8 ° C for 12 hours. The reaction solution was transferred to a dialysis bag (MWCO 3500), and dialyzed in deionized water at 8 ° C for 24 hours. After the dialyzate was freeze-dried, a white fluffy solid was obtained, which was the tripterygium wilfordii prodrug delivery gelatin micelle. It was stored in a vacuum for standby use.

[0082] The structure of the product obtained in this example is confirmed as follows Figure 2 As shown, it can be seen in the infrared spectrum ( Figure 2B), compared with EDA-GLA, characteristic absorption peaks can be observed in the infrared spectrum of EDA-GLA / CE / 2-FPBA at 1736cm-1, 1154cm-1, 1062cm-1, 547cm-1, etc. In the NMR spectrum of the prodrug polymer ( Figure 2 A), confirming that 2-FPBA and CE have been successfully grafted onto the main chain of EDA-GLA. In the DSC spectrum ( Figure 2 D), proving that CE has been successfully grafted onto the side chain of EDA-GLA. In the UV spectrum ( Figure 2 C), indicating that CE has been bound to the main chain of the EDA-GLA polymer and its long conjugated structure has been destroyed and the color has disappeared, which is consistent with the literature report. After acid destruction, the EDA-GLA / CE / 2-FPBA micelle solution showed an obvious absorption peak at 450nm, indicating that CE has been separated from the main chain of the EDA-GLA polymer and the long conjugated structure has recovered and re-displayed color.

[0083] Example 2

[0084] The tripterygium wilfordii prodrug delivery gelatin micelles were dissolved in an appropriate amount of 0.01M (pH 7.4) PBS buffer solution to prepare a sample solution with a concentration of 1 mg / ml. 1 ml of the sample solution was added to the sample cell, and the particle size and Zeta potential of the micelles were measured using a Malvern laser particle size analyzer at 25°C.

[0085] Table 1. Particle size, PDI and Zeta potential of EDA-GLA / CE / 2-FPBA micelles in aqueous and organic phases

[0086]

[0087]

[0088] As shown in Table 1, the particle size of EDA-GLA / CE / 2-FPBA micelles in 0.01M PBS (pH 7.4) buffer is 292.27±13.52nm, and the PDI is 0.268±0.041. This indicates that the micelles are in a single micellar particle state and are evenly distributed in the aqueous solution. After the micelles are dissolved in the organic solvent dimethyl sulfoxide, the particle size of the micelles increases to 709.00±426.11nm, and the PDI increases to 0.481±0.371, still retaining the characteristics of the micelles, because the disulfide bonds form cross-linked bonds inside the micelles, which improves the stability of the micelles.

[0089] Example 3

[0090] The celastrol prodrug delivery gelatin micelles were dissolved in an appropriate amount of 0.01M (pH 7.4) PBS buffer solution to prepare a sample solution with a concentration of 1 mg / ml. The sample solution was dripped onto a carbon-coated copper grid and stained with 2% (w / v) phosphotungstic acid for 2 minutes. After drying, the surface morphology of the micelles was observed by transmission electron microscopy (JEM-1230HC, Tokyo, Japan). A small amount of acid was added to the sample solution until the solution showed a light red color. The above operation was repeated to observe the surface morphology of the micelles. The results are shown in FIG. Figure 3 shown.

[0091] Through transmission electron microscopy observation, Figure 3 (A) shows that the micelles have a uniform spherical morphology. Figure 3 (B) shows that although the prodrug structure of tripterygium wilfordii was destroyed and tripterygium wilfordii was released after acid destruction, the spherical morphology remained intact because the cross-linked micelles were still basically completed and tripterygium wilfordii was encapsulated in the cross-linked micelles.

[0092] Example 4

[0093] The tripterygium wilfordii prodrug delivery gelatin micelles were dissolved in pH 7.4 and pH 5.0 PBS buffer solutions, respectively, and the change in particle size of the micelles within 96 hours and the change in absorbance at a wavelength of 450 nm (the micelle aqueous solution has a strong absorption at 450 nm) were monitored.

[0094] The results are as follows Figure 5 As shown in the figure, the micelles are relatively stable in a pH 7.4 environment, with basically no release of tripterygium wilfordii; in a pH 5.0 environment, due to the breakage of the boronate bond, tripterygium wilfordii gradually dissociates from the polymer main chain and is gradually released from the micelles, resulting in an increase in the absorbance of the solution over time, showing obvious pH responsiveness. The micelle size did not change significantly within 96 hours.

[0095] The tripterygium wilfordii prodrug delivery gelatin micelles were dissolved in PBS buffer solutions with different glutathione concentrations (0 μM, 20 μM and 10 mM) under the same pH 7.4 condition, and the changes in particle size of the micelles within 96 hours and the changes in absorbance at a wavelength of 450 nm were monitored.

[0096] The results are as follows Figure 6 As shown in the figure, the micelles have good stability in the environment of pH 7.4. As the concentration of GSH increases, the absorbance and particle size in the solution increase over time. Comparison was made with 20μM GSH (normal cells) and 10mM GSH (tumor cells). At a concentration of 10mM, the release of tripterygium wilfordii was accelerated, showing obvious concentration responsiveness. This also reflects the potential of tripterygium wilfordii prodrug delivery gelatin micelles to selectively kill tumor cells.

[0097] Example 5

[0098] Melanoma cells (B16F10) and normal epithelial cells (HaCat) were taken at 1×10 per well. 4 The cells were plated in a 96-well plate at a density of 100 μl of RPMI-1640 medium and MEM medium mixed with 10% fetal bovine serum and 1% penicillin plus streptomycin, respectively, and cultured at 37°C and 5% CO2 for 24 hours. After the culture was completed, the medium in each well was replaced with RPMI-1640 medium containing different concentrations (0, 0.39, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25 and 50 μg / mL) of tripterygium wilfordii prodrug delivery gelatin micelles, and cultured at 37°C and 5% CO2 for 24 hours. After the culture was completed, the medium in each well was removed and the MTT reagent was added respectively, and cultured at 37°C and 5% CO2 for 4 hours. After the culture was completed, the MTT reagent in each well was removed, and 150 μl of dimethyl sulfoxide was added to dissolve the formazan produced by the living cells. The absorbance values ​​of all samples at 570 nm were measured respectively, and the cell viability was calculated according to the following formula: Cell viability (%) = As / Ac×100%; As is the absorbance value of the cell sample, and Ac is the absorbance value of the cell control.

[0099] The results are as follows Figure 7 The blank delivery gelatin micelle EDA-GLA / 2-FPBA showed no significant cytotoxicity after being co-cultured with B16 and HaCat cells for 48 hours, proving its high safety. Figure 8 The IC50 value of the tripterygium wilfordii prodrug delivery gelatin micelles in B16 cells is 1.53 μg / ml, and the IC50 value of HaCat cells is 3.03 μg / ml (the IC50 value of tripterygium wilfordii for HaCat cells is 0.69 μg / ml); the toxicity to normal cells is significantly lower than that of tripterygium wilfordii.

[0100] Example 6

[0101] Mouse melanoma cells (B16F10) were cultured at 1×10 per well. 5The cells were plated in a 24-well plate at a density of 100 cells / mL, and 1 ml of RPMI-1640 medium mixed with 10% fetal bovine serum and 1% penicillin plus streptomycin was added to each well, and cultured at 37°C and 5% CO2 for 24 hours. After the culture was completed, the medium in each well was replaced with RPMI-1640 medium solution containing FITC-labeled tripterygium wilfordii prodrug delivery gelatin micelles (concentration of 300 μg / ml), and cultured at 37°C and 5% CO2 for 12 hours. After the culture was completed, the medium in each well was removed, and the cells were washed three times with PBS solution. 450 μl of 4% paraformaldehyde solution was added to each well, and the cells were solidified at 37°C for 15 minutes. The paraformaldehyde solution in each well was removed, and the cells were washed three times with PBS solution. 500 μl of DAPI staining reagent solution was added, and the cell nuclei were stained at 37°C for 20 minutes, and then washed three times with PBS solution. Fluorescence images were obtained using a live cell imager.

[0102] The results are as follows Fig. 9 After 4 hours of co-culture of EDA-GLA / CE / 2-FPBA micelles labeled with FITC fluorescent marker and cells, obvious green fluorescence appeared in B16 cells, indicating that EDA-GLA / CE / 2-FPBA micelles can be effectively phagocytosed by B16 cells.

[0103] The above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principle of the present invention, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims

1. A gelatin micelle for delivering tripterygium wilfordii prodrug, characterized in that: The tripterygium wilfordii prodrug delivery gelatin micelles include gelatin micelles for delivering tripterygium wilfordii and tripterygium wilfordii encapsulated in the gelatin micelles; the gelatin micelles are reduced aminated lipoic acid gelatin obtained by modifying gelatin with lipoic acid, aminated with ethylenediamine, and reducing the amino group, and are amphiphilic molecular micelles; the tripterygium wilfordii and the gelatin micelles are reversibly covalently bonded to form tripterygium wilfordii prodrug delivery gelatin micelles; the preparation method of the tripterygium wilfordii prodrug delivery gelatin micelles includes the following steps: (1) Using EDC and NHS to activate lipoic acid at a solution pH of 5.0-6.8; (2) The activated lipoic acid is covalently bonded to gelatin via an amide bond to obtain lipoic acid gelatin; (3) the obtained lipoic acid gelatin is subjected to an amination reaction with ethylenediamine to obtain ethylenediamine lipoic acid gelatin nano-micelles; (4) reducing the obtained ethylenediamine lipoic acid gelatin nano-micelles using tri(2-carboxyethyl)phosphine, dialyzing and freeze-drying the product to obtain reduced aminated lipoic acid gelatin; (5) mixing tripterygium wilfordii with the obtained reduced aminated lipoic acid gelatin, adding o-formylphenylboronic acid to react, dialyzing and freeze-drying the reaction product to obtain tripterygium wilfordii prodrug delivery gelatin micelles.

2. The tripterygium wilfordii prodrug delivery gelatin micelle according to claim 1, characterized in that: The amino amidation reaction ratio of gelatin in the gelatin micelles used for delivering tripterygium wilfordii is 90% to 97%.

3. A method for preparing gelatin micelles for delivering tripterygium wilfordii prodrug, characterized in that: The steps include: (1) Using EDC and NHS to activate lipoic acid at a solution pH of 5.0-6.8; (2) The activated lipoic acid is covalently bonded to gelatin via an amide bond to obtain lipoic acid gelatin; (3) the obtained lipoic acid gelatin is subjected to an amination reaction with ethylenediamine to obtain ethylenediamine lipoic acid gelatin nano-micelles; (4) reducing the obtained ethylenediamine lipoic acid gelatin nano-micelles using tri(2-carboxyethyl)phosphine, dialyzing and freeze-drying the product to obtain reduced aminated lipoic acid gelatin; (5) mixing tripterygium wilfordii with the obtained reduced aminated lipoic acid gelatin, adding o-formylphenylboronic acid to react, dialyzing and freeze-drying the reaction product to obtain tripterygium wilfordii prodrug delivery gelatin micelles.

4. The preparation method according to claim 3, characterized in that: The solvent used in the chemical reaction of step (1) and step (2) is selected from ethanol, methanol, acetonitrile, dimethyl sulfoxide, and alcohol / water mixture; the solvent used in the amination reaction of step (3) is selected from deionized water, alcohol / water mixture, and buffered salt solution.

5. The preparation method according to claim 3, characterized in that: The mass ratio of lipoic acid and gelatin is (1.2~0.8):

1.

6. The preparation method according to claim 3, characterized in that: In step (3), the mass ratio of lipoic acid gelatin to ethylenediamine is 1:(2.0-3.5).

7. The preparation method according to claim 3, characterized in that: In step (4), the mass ratio of ethylenediamine lipoic acid gelatin and tri(2-carboxyethyl)phosphine is (2-4):

1.

8. The preparation method according to claim 3, characterized in that: In step (5), the mass ratio of tripterygium wilfordii, reduced aminated lipoic acid gelatin and o-formylphenylboric acid is 1:(8-15):(0.8-1.2).

9. The preparation method according to claim 3, characterized in that: The dialysis temperature was 0-15°C.

10. Use of the tripterygium wilfordii prodrug delivery gelatin micelles according to any one of claims 1 to 2 or the tripterygium wilfordii prodrug delivery gelatin micelles obtained by the preparation method according to any one of claims 3 to 9 in the preparation of anti-melanoma drugs.

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