ATRA and ATO composite nano sustained-release microsphere as well as preparation method and application thereof

Abstract

The invention provides a preparation method of all-transretinoic acid and arsenic trioxide composite nano sustained-release microspheres, which comprises the following steps: a, adding albumin and L-reduced glutathione into deionized water, and carrying out reduction reaction on disulfide bonds in albumin molecules to obtain a reduced albumin aqueous solution; b, taking a sodium hydroxide solution of ATO, and then adding the sodium hydroxide solution of ATO into the reductive albumin aqueous solution obtained in the step a to obtain an ATO-albumin solution; c, ATRA is added into absolute ethyl alcohol and stirred, and an ethyl alcohol solution of ATRA is obtained; d, dropwise adding the ATRA ethanol solution obtained in the step c into the ATO-albumin solution obtained in the step b by using a micro-injection pump to obtain an ATRA and ATO composite nano sustained-release microsphere crude product; and e, centrifuging the crude product, cleaning with deionized water, and freeze-drying to obtain the microsphere product. The preparation method of the product is rapid, simple and convenient, the targeting property is strong, the effect of resisting tumor growth and metastasis of the medicine can be obviously enhanced, and the biological safety is good.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an ATRA and ATO composite nano slow-release microsphere and a preparation method and application thereof. BACKGROUND

[0002] Molecular targeted drugs are an important method for treating diseases such as cancer in clinical treatment, but the treatment effect on solid tumors is poor at present. The main reason is that most of the existing molecular targeted drugs are used for tumor treatment by targeting a single or a few cancer signal pathways, while in fact the malignant growth and metastasis of cancer involve the activation of multiple signal pathways. Therefore, to improve the treatment effect on cancer, it is necessary to develop molecular targeted drugs that can simultaneously block multiple cancer signal pathways. In the cell proliferation and transformation signal pathway, the main common mechanism is the protein phosphorylation guided by proline, which is regulated by many kinases and phosphatases, and the structure and function of the phosphorylated protein are further regulated by Pin1. Two specific inhibitors of Pin1, all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), ATRA can specifically bind to PIN1 and degrade PIN1, thereby blocking multiple Pin1-regulated oncogenic signaling pathways; ATO binds to PIN1 through non-covalent bonds, and ATO can synergistically degrade PIN1 with ATRA, thereby synergistically blocking multiple Pin1-regulated oncogenic signaling pathways.

[0003] In clinical practice, ATRA and ATO have been used in combination to treat acute promyelocytic leukemia. However, ATRA and ATO have poor treatment effect on solid tumors. The reason is that ATRA and ATO each have a deficiency in drug development. ATRA has the disadvantages of poor water solubility, short half-life, easy inactivation by light and heat, etc. ATO has the disadvantages of poor uptake by solid tumor cells, large side effects at high doses, and easy clearance by the body at low doses, etc. Therefore, it is necessary to develop new dosage forms for loading ATRA and ATO single drugs and double drugs to overcome the respective defects of ATRA and ATO, and to enable ATRA and ATO to reach the tumor site at the optimal ratio and concentration. SUMMARY

[0004] In order to overcome the defects of the prior art, the present application provides the following technical solutions: The first aspect of the present application provides a preparation method of an all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite nano slow-release microsphere, comprising the following steps: a. adding albumin and L-reduced glutathione to deionized water, then stirring at a certain temperature to perform a reduction reaction on the disulfide bond in the albumin molecule, to obtain a reduced albumin aqueous solution; b. Take the sodium hydroxide solution of ATO, and then add to the aqueous solution of reduced albumin obtained in step a to obtain an ATO-albumin solution; c. Add ATRA to anhydrous ethanol, stir to obtain an ethanol solution of ATRA; d. Use a micro-injection pump to drop the ATRA ethanol solution obtained in step c into the ATO-albumin solution obtained in step b to obtain a crude product of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite nano-release microspheres (i.e. ATRA-ATO-NPs); e. Centrifuge the above-mentioned crude product, then wash with deionized water, and freeze-dry to obtain the ATRA-ATO-NPs product; Further, in step a, the albumin is selected from any one of bovine serum albumin, human serum albumin; Further, in step a, the concentration of the aqueous albumin solution is 2-6 m / v%, and the mass ratio of L-reduced glutathione to albumin is 30-45 wt%; Further, in step a, the reduction reaction temperature is 38-42°C; Further, in step a, the reduction reaction time is 40 min-2 h; Further, in step a, the stirring speed is 60-200 rpm; Further, in step b, the concentration of the ATO sodium hydroxide aqueous solution is 0.005-0.04 kg / L; preferably, the concentration of the ATO sodium hydroxide aqueous solution is 0.02 kg / L; Further, in step b, the volume ratio of the ATO solution to the aqueous albumin solution is 0.1-2 v / v%; Further, in step c, the concentration of the ATRA anhydrous ethanol solution is 0.00008-0.0008 kg / L; Further, in step d, the mass ratio of ATRA-ATO-NPs ATO:ATRA is 4%-13%; Further, in step d, the speed of dropping the ATRA ethanol solution obtained in step c into the ATO-albumin solution obtained in step b is 0.5-4 mL / min; Further, in step d, the volume ratio of the ATRA ethanol solution to the ATO-albumin solution is 3:1-6:1; Further, in step e, the centrifugation temperature is 0-8°C, the centrifugation speed is 10000-20000 rpm, and the centrifugation time is 20-50 min; preferably, the centrifugation temperature is 4°C, the centrifugation speed is 15000 rpm, and the centrifugation time is 30 min; The second aspect of the present application provides a composite nano-sustained release microsphere of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), which is prepared by any of the above methods; Further, the particle size of the composite nano-sustained release microsphere is 90 nm to 180 nm; Further, in the sustained release nano-microsphere, the molar ratio of ATRA:ATO is 5 to 15:1; further preferably, in the sustained release nano-microsphere, the molar ratio of ATRA:ATO is 10:1; The third aspect of the present application provides the use of any of the above composite nano-sustained release microspheres of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) in the preparation of a drug for treating Pin1 overexpression diseases; Further, the Pin1 overexpression disease is a tumor, inflammation, lupus erythematosus, or asthma; Further, the anti-tumor effect is inhibition of tumor growth or inhibition of tumor metastasis; Further, the tumor is selected from any one or two of liver cancer and lung cancer; Further, the inflammation is arthritis; Beneficial effects

[0005] The present application uses albumin as a drug-loaded component, which provides a new biodegradable sustained release preparation as an endogenous protein, and further provides a preparation method and application. The technical scheme of the present application avoids the problems of traditional preparation methods, such as complicated operation, time-consuming, and severe preparation conditions, and quickly and simply prepares drug-loaded nanoparticles, and illustrates that the drug-loaded nanoparticles have the effects of enhancing drug resistance to tumor growth and metastasis, and illustrates the mechanism of action. The nano-microsphere prepared by the present application has good targeting ability, can accurately reach the lesion, has good anti-liver cancer and anti-lung metastatic nodule effects, and has high biological safety. BRIEF DESCRIPTION OF DRAWINGS

[0006] Figure 1 SEM image of ATRA-ATO-NPs nanoparticles prepared in Example 1; Figure 2 SEM images of (a) ATRA-NPs and (b) ATO-NPs nanoparticles prepared in Example 1; Figure 3 In vitro release profile of ATRA-ATO-NPs nano-microspheres in three different medium solutions; Figure 4 Cell uptake diagram of FITC-loaded albumin nano-microspheres; Figure 5Figure for the effect of blank albumin nanoparticle (Blank NPs), ATRA, ATO, ATRA / ATO combination drug, ATRA-ATO-NPs nanoparticle treatment on the viability of hepatoma cells HuH7, PLC, Hepa1-6, normal liver cells L-02; Figure 6 Figure for the effect of ATRA, ATO, ATRA / ATO combination drug, ATRA-ATO-NPs nanoparticle treatment on the wound healing, migration and invasion of hepatoma cells HuH7; Figure 7 Figure for the effect of blank albumin nanoparticle (Blank NPs), ATRA, ATO, ATRA / ATO combination drug, ATRA-ATO-NPs nanoparticle treatment on the expression of Pin1, Pin1-regulated oncogene, cancer metastasis marker protein in hepatoma cells HuH7, PLC; Figure 8 Figure for the in vivo anti-tumor growth effect of blank albumin nanoparticle (Blank NPs), ATRA, ATO, ATRA / ATO combination drug, ATRA-NPs, ATO-NPs, ATRA-ATO-NPs nanoparticle; Figure 9 Figure for the in vivo anti-tumor metastasis effect of ATRA, ATO, ATRA / ATO combination drug, ATRA-NPs, ATO-NPs, ATRA-ATO-NPs nanoparticle; Figure 10 Figure for the inhibitory effect of ATRA, ATO, ATRA / ATO combination drug treatment on the viability of hepatoma cells HuH7, PLC and synergistic index analysis; Figure 11 Figure for the in vivo pharmacokinetics and tissue distribution of ATRA, ATO, ATRA-NPs, ATO-NPs, ATRA-ATO-NPs nanoparticle; Figure 12 Figure for the in vivo biological safety of ATRA-ATO-NPs nanoparticle. DETAILED DESCRIPTION

[0007] The present application will be described in detail below with reference to the accompanying drawings and examples. The ATRA, ATO, L-reduced glutathione used in the following examples of the present application are purchased from Sigma Company of the United States; human serum albumin is purchased from Solabio Technology Co., Ltd. of Beijing, China. Other conventional reagents are commercially available. (I) Examples

[0008] Example 1

[0009] The albumin and L-reduced glutathione are added to deionized water, and placed in a constant temperature shaker, incubated at 40°C and 100 rpm for 1 h, to open the intramolecular disulfide bond of albumin, expose free thiol, and obtain a reduced albumin aqueous solution, wherein the concentration of albumin is 4% (% is mass volume percentage, the embodiment of the present application refers to kg / L), the mass ratio of glutathione and albumin is 38.5%; an ATO solution with a concentration of 0.1 M / L is prepared by using sodium hydroxide, and the ATO solution is added to the reduced albumin aqueous solution to obtain an ATO-albumin solution, wherein the volume ratio of the ATO solution and the albumin aqueous solution is 0.8%; ATRA is added to anhydrous ethanol, and an ATRA ethanol solution is obtained by stirring, and the ATRA ethanol solution is added to the ATO-albumin solution at a speed of 1.5 mL / min by using a micro-injection pump, wherein the mass ratio of ATO and ATRA is 12.7%, and the volume ratio of the ATRA ethanol solution to the ATO-albumin solution is 5:1, to produce co-loaded ATRA and ATO albumin nanoparticles (ATRA-ATO-NPs) by desolvation and self-assembly method, and at the same time, the ethanol inactivates the reduction of glutathione, and then the drug-loaded nanoparticles are solidified by cross-linking between the free thiol groups of albumin molecules, the ATRA-ATO-NPs are collected by centrifugation, the centrifugation conditions are 4°C, 15000 rpm, and 30 min, the ATRA-ATO-NPs are washed and centrifuged by using deionized water, and then the ATRA-ATO-NPs are obtained by freeze-drying.

[0010] Example 2

[0011] Albumin and L-reduced glutathione were added to deionized water, and placed in a constant temperature shaker, incubated at 38°C, 150 rpm, for 50 min, to open the intramolecular disulfide bond of albumin, expose free thiol, and obtain a reduced albumin aqueous solution, wherein the concentration of albumin was 3% (% is mass volume percentage, the embodiment of the present application refers to kg / L), the mass ratio of glutathione and albumin was 30%; an ATO solution with a concentration of 0.05 M / L was prepared by using sodium hydroxide, and the ATO solution was added to the reduced albumin aqueous solution to obtain an ATO-albumin solution, wherein the volume ratio of the ATO solution and the albumin aqueous solution was 0.2%; ATRA was added to anhydrous ethanol, and ATRA ethanol solution was obtained by stirring, and the ATRA ethanol solution was added to the ATO-albumin solution at a speed of 1 mL / min by using a micro-injection pump, wherein the mass ratio of ATO and ATRA was 8%, and the volume ratio of ATRA ethanol solution:ATO-albumin solution was 3:1, co-loaded ATRA and ATO albumin nanoparticles (ATRA-ATO-NPs) were produced by desolvation and self-assembly method, at the same time, ethanol deactivated the reduction of glutathione, and then the drug-loaded nanoparticles were solidified by cross-linking between albumin molecules through free thiol, ATRA-ATO-NPs were collected by centrifugation, the centrifugation conditions were 2°C, 12000 rpm, 40 min, ATRA-ATO-NPs were washed and centrifuged with deionized water, and then ATRA-ATO-NPs were obtained by freeze-drying.

[0012] Example 3 Albumin and L-reduced glutathione were added to deionized water, and incubated in a constant temperature shaker at 42°C and 100 rpm for 1 h to open the intramolecular disulfide bond of albumin, expose free thiol groups, and obtain a reduced albumin aqueous solution, wherein the concentration of albumin was 4% (% is mass volume percentage, and the embodiment of the present application refers to kg / L), and the mass ratio of glutathione to albumin was 42%; an ATO solution with a concentration of 0.2 M / L was prepared by using sodium hydroxide, and the ATO solution was added to the reduced albumin aqueous solution to obtain an ATO-albumin solution, wherein the volume ratio of the ATO solution to the albumin aqueous solution was 2%; ATRA was added to anhydrous ethanol, and an ATRA ethanol solution was obtained by stirring; the ATRA ethanol solution was added to the ATO-albumin solution at a speed of 3 mL / min by using a micro-injection pump, wherein the mass ratio of ATO to ATRA was 18%, and the volume ratio of ethanol to albumin aqueous solution was 6:1; co-loaded ATRA and ATO albumin nanoparticles (ATRA-ATO-NPs) were generated by desolvation and self-assembly, and at the same time, ethanol deactivated the reduction of glutathione, and the drug-loaded nanoparticles were solidified by cross-linking between albumin molecules through free thiol groups; the ATRA-ATO-NPs were collected by centrifugation under the conditions of 4°C, 15000 rpm, and 30 min; the ATRA-ATO-NPs were washed and centrifuged by using deionized water, and then obtained by freeze-drying. (B) Comparative Example

[0013] Comparative Examples 1-4

[0014] ATRA-NPs, ATO-NPs, Blank NPs (empty nanomicrospheres), and FITC-NPs (nanomicrospheres loaded with isothiocyanate fluorescein) were prepared under the same experimental conditions as in Example 1. Figure 1 , Figure 2 As shown in Table 1, the results of scanning electron microscopy (SEM) showed that the ATRA-ATO-NPs, ATRA-NPs, and ATO-NPs had good sphericity and uniform particle size. Figure 1 SEM image of the ATRA-ATO-NPs nanoparticles prepared in Example 1; Figure 2 SEM images of (a) ATRA-NPs and (b) ATO-NPs nanoparticles prepared in Example 1.

[0015] Comparative Example 5 Peng et al. used folic acid grafted human serum albumin as an encapsulating material, reduced the disulfide bond of human serum albumin to free thiol group through glutathione (GSH), and prepared ATO-loaded nanoparticles by forming a sulfur-arsenic bond spontaneously under low pH conditions (Peng Y, Zhao Z, Liu T et al. Smart human-serum-albumin-As2O3 nanodrug with self-amplified folate receptor-targeting ability for chronic myeloid leukemia treatment. Angewandte Chemie International Edition 2017; 56: 10845-10849.). However, using this method, ATRA-loaded albumin nanoparticles (ATRA-NPs) cannot be prepared, and albumin nanoparticles co-loaded with ATRA and ATO (ATRA-ATO-NPs) cannot be prepared. The reasons are: 1) ATRA is almost insoluble in water, while albumin is easily soluble in water, and ATRA cannot be uniformly mixed with the albumin aqueous solution; 2) albumin cannot directly encapsulate ATRA. In the preparation method described in the present application, ATRA is dissolved in ethanol, and then the ATRA ethanol solution is added to the ATO-albumin aqueous solution at a certain flow rate under stirring. On the one hand, ATRA and ATO-albumin can be fully mixed, and on the other hand, as the ethanol content increases, the solubility of albumin decreases, so that albumin nanoparticles can be generated by desolvation-self-assembly method, and ATRA can be encapsulated in the process of crystal nucleus formation. At the same time, ethanol will inactivate the reducing effect of glutathione, which will cause the pre-reduced albumin to cross-link between molecules through thiol groups, form disulfide bonds, and thus solidify the drug-loaded albumin nanoparticles, thereby preparing ATRA-loaded and co-loaded ATRA and ATO albumin nanoparticles. Therefore, the preparation method reported by Peng et al. is only suitable for preparing albumin nanoparticles loaded with water-soluble drugs, and the preparation method described in the present application is not only suitable for preparing albumin nanoparticles loaded with water-soluble drugs, but also suitable for preparing albumin nanoparticles loaded with fat-soluble and co-loaded water-soluble and fat-soluble drugs. (Three) Experimental Examples

[0016] The ATRA-ATO-NPs nanospheres used in the experimental examples are all products prepared in Example 1.

[0017] Experimental Example 1 Take 4 mg ATRA-ATO-NPs nanospheres, add to 4 mL dimethyl sulfoxide, magnetically stir to dissolve completely, use a microplate reader to measure the absorbance of the solution at 360 nm. Use ATRA standard to establish a standard curve of ATRA, calculate the concentration of ATRA in dimethyl sulfoxide solution. In addition, take 5 mg ATRA-ATO-NPs nanospheres prepared in Example 1, add to 1 mL deionized water to mix, then add 2 mL 1 M / L concentration sodium hydroxide to the mixture to digest, then add deionized water to the digesting solution to a total volume of 5 mL, adjust the pH of the digesting solution to 6.8, then filter with a 0.22 μm filter membrane, finally use inductively coupled plasma mass spectrometry (ICP-MS) to determine the content of ATO in the digesting solution. Calculate the drug loading and encapsulation efficiency according to the formula. Drug loading (%) = (weight of ATRA or ATO in drug-loaded nanospheres / weight of drug-loaded nanospheres) x 100. Encapsulation efficiency (%) = (total weight of ATRA or ATO in drug-loaded nanospheres / initial weight of ATRA or ATO input) x 100. The results show that the drug loading of ATRA in ATRA-ATO-NPs nanospheres is 4.1 ± 0.3%, and the encapsulation efficiency is 72 ± 5%.

[0018] Experimental Example 2 Take 3 mg ATRA-ATO-NPs nanospheres, respectively disperse in 1.5 mL PBS (pH 7.4), DMEM medium, or DMEM medium containing 5 v / v% serum, place in a 37 °C, 100 rpm shaker, at different time points (2 h, 6 h, 12 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d, 9 d, 10 d, 11 d, 12 d, 13 d, 14 d, 15 d, 16 d, 17 d, 18 d, 19 d, 20 d), take out the experimental samples, centrifuge at 15000 rpm for 30 min, collect the supernatant, then resuspend the nanospheres in fresh release medium and continue the experiment. Determine the content of ATRA and ATO in each supernatant, and calculate the cumulative release rate of ATRA and ATO. Results, ATRA-ATO-NPs nanospheres can still release ATRA for 20 days even in DMEM medium containing serum; ATO can be released from ATRA-ATO-NPs nanospheres, but the release rate of ATO is faster than that of ATRA. That is, ATRA-ATO-NPs nanospheres can effectively release ATRA and ATO in vitro. Figure 3 The in vitro release profile of ATRA-ATO-NPs nanospheres in three different medium solutions, Figure 3 (a) is the release profile of ATRA; Figure 3 (b) is the release profile of ATO.

[0019] Experimental Example 3 Effects of ATRA, ATO, ATRA / ATO combination drug, and ATRA-ATO-NPs on liver cancer

[0020] (1) Internalization of FITC-NPs nanomaterials by liver cancer cells. Human liver cancer cells HuH7 were cultured and treated with FITC-NPs nanomaterials (0.13 mg / mL) for 1 h and 24 h. The cells were fixed with paraformaldehyde and stained with DAPI for cell nuclei, and then observed under a fluorescence microscope. The results showed that FITC could be released from the FITC-NPs nanomaterials and be taken up by the cells; the FITC-NPs nanomaterials themselves could be taken up by the cells. That is, the FITC-NPs nanomaterials could release drugs, the released drugs could be taken up by the cells, and the drug-loaded albumin nanomaterials themselves could be taken up by the cells. Figure 4 Cellular uptake of FITC-loaded albumin nanomaterials.

[0021] (2) The effects of ATRA, ATO, ATRA / ATO combination drugs, ATRA-NPs, ATO-NPs, ATRA-ATO-NPs on tumor cell proliferation, migration and invasion and related gene expression. Hepatoma cells HuH7 (3000 cells / well), PLC (6000 cells / well), Hepa1-6 (3500 cells / well) and normal liver cells L-02 (5000 cells / well) were inoculated in 96-well plates, and different concentrations of Blank NPs nanospheres (concentration 0-0.6 mg / mL), ATRA group, ATO group, ATRA / ATO combination drug group (ATRA content 10 µM, ATO content 1 µM), ATRA-ATO-NPs group (ATRA content 10 µM, ATO content 1 µM) were used to treat hepatoma cells and liver cells for 72 h, and untreated cells were used as controls. The effect of drug treatment on hepatoma cell proliferation was determined by MTT method. Hepatoma cells HuH7 (5×105 cells / well) were inoculated in 6-well plates and cultured for 24 h, then cultured in serum-free medium for 12 h, then the cells were scratched, and then treated with ATRA, ATO, ATRA / ATO combination drugs, ATRA-ATO-NPs (ATRA content 10 µM, ATO content 1 µM) for 72 h. The wound healing was photographed under a microscope, and the scratch area was measured using Image-Pro Plus 6.0 software. Hepatoma cells HuH7 (5×104 cells / well, 24-well plate) were inoculated in the upper chamber of the migration chamber, and the upper chamber medium did not contain serum but contained drugs (ATRA, ATO, ATRA / ATO combination drugs, ATRA-ATO-NPs), and the medium volume was 200 µL. The lower chamber medium contained 10% serum, and the medium volume was 500 µL. After 72 h of culture, the non-migrated cells on the surface of the upper membrane of the migration chamber were removed, the membrane was fixed and crystal violet stained, and the migrated cells were photographed and counted under a microscope. The effect of drug treatment on hepatoma cell invasion was determined using a migration chamber containing Matrigel, and the test method was the same as the above cell migration test method, but the inoculation amount of hepatoma cells HuH7 was 3×105 cells / well. Hepatoma cells HuH7 and PLC were cultured, and different concentrations of Blank NPs nanospheres (concentration 0-0.6 mg / mL), ATRA, ATO, ATRA / ATO combination drugs, ATRA-ATO-NPs (ATRA content 10 µM, ATO content 1 µM) were used to treat hepatoma cells for 72 h, and the total protein of the cells was extracted with RIPA lysis buffer, the protein was quantified with BCA kit, and the expression of Pin1, Pin1-regulated oncogenes and cancer metastasis-related proteins was detected by Western blot.

[0022] Cell proliferation results show that blank NPs treatment has no effect on the proliferation of hepatoma cells and liver cells, ATRA-ATO-NPs nanospheres can significantly improve the inhibitory effect of ATRA and ATO on the proliferation of hepatoma cells, that is, ATRA-ATO-NPs nanospheres can significantly enhance the synergistic inhibitory effect of ATRA and ATO on the proliferation of hepatoma cells, and albumin nanospheres themselves have good biocompatibility. Wound healing results show that ATRA-ATO-NPs can significantly enhance the inhibitory effect of ATRA, ATO, ATRA / ATO combined drugs on the migration of hepatoma cells. The results of the migration chamber test show that ATRA-ATO-NPs can significantly enhance the inhibitory effect of ATRA, ATO, ATRA / ATO combined drugs on the migration and invasion of hepatoma cells.

[0023] That is, ATRA-ATO-NPs can significantly enhance the synergistic inhibitory effect of ATRA and ATO on cancer cell migration and invasion. Western blot results show that blank NPs itself does not affect the expression of Pin1 in hepatoma cells, and ATRA-ATO-NPs can significantly enhance the synergistic inhibitory effect of ATRA and ATO on the expression of Pin1, Pin1-regulated oncogenes, and cancer metastasis marker proteins.

[0024] Figure 5 The figure of the effect of blank albumin nanospheres (Blank NPs), ATRA, ATO, ATRA+ATO, ATRA-ATO-NPs nanospheres on the viability of hepatoma cells HuH7, PLC, Hepa1-6, normal liver cells L-02. Figure 5 (a) is the effect of Blank NPs nanospheres on the viability of HuH7, PLC, L-02 cells treated for 72 h; Figure 5 (b) is the effect of ATRA, ATO, ATRA / ATO combined drugs, ATRA-ATO-NPs on the viability of HuH7 cells treated for 72 h; Figure 5 (c) is the effect of ATRA, ATO, ATRA / ATO combined drugs, ATRA-ATO-NPs on the viability of Hepa1-6 cells treated for 72 h.

[0025] Figure 6 The figure of the effect of ATRA, ATO, ATRA / ATO combined drugs, ATRA-ATO-NPs nanospheres on the wound healing, migration and invasion of hepatoma cells HuH7. Figure 6 (a) and (c) are the effects of ATRA, ATO, ATRA / ATO combined drugs, ATRA-ATO-NPs nanospheres on the wound healing of hepatoma cells HuH7; Figure 6Fig. 6. Effects of ATRA, ATO, ATRA / ATO combination drug, ATRA-ATO-NPs on the migration and invasion of hepatocarcinoma cells HuH7 in (b), (d) and (e).

[0026] Figure 7 Fig. 7. Effects of blank NPs, ATRA, ATO, ATRA / ATO combination drug, ATRA-ATO-NPs on the expression of Pinl, Pinl-regulated oncogenes and cancer metastasis marker proteins in hepatocarcinoma cells HuH7 and PLC. Figure 7 Fig. 8. Effects of blank NPs on the expression of Pinl in hepatocarcinoma cells HuH7 and PLC in (a-c). Figure 7 Fig. 9. Effects of ATRA, ATO, ATRA / ATO combination drug, ATRA-ATO-NPs on the expression of Pinl and Pinl-regulated oncogenes in hepatocarcinoma cells HuH7 and PLC in (d-e). Figure 7 Fig. 10. Effects of ATRA, ATO, ATRA / ATO combination drug, ATRA-ATO-NPs on the expression of Pinl and cancer metastasis marker proteins in hepatocarcinoma cells HuH7 in (f-g).

[0027] (3) Inhibition of tumor growth and Pinl and its regulated oncogene expression in vivo by ATRA, ATO, ATRA / ATO combination drug, ATRA-NPs, ATO-NPs, and ATRA-ATO-NPs. Hepatocarcinoma cell line HuH7 was cultured in vitro, and 3x106 HuH7 cells were injected subcutaneously into the root of the hind leg of a nude mouse to establish a tumor model. When the tumor grew to 50 cubic millimeters, the nude mice were randomly divided into 8 groups, namely, a normal saline group, a blank albumin nanoparticle (Blank NPs) group, an ATRA group, an ATO group, an ATRA / ATO combination drug group, an ATRA-NPs group, an ATO-NPs group, and an ATRA-ATO-NPs nanoparticle group. ATRA was dissolved in ethanol and diluted with corn oil; ATO was dissolved in sodium hydroxide and diluted with normal saline; and Blank NPs, ATRA-NPs, ATO-NPs, and ATRA-ATO-NPs nanoparticles were dispersed in normal saline. The drugs were administered intraperitoneally, in which ATRA in the ATRA group, the ATRA / ATO combination drug group, the ATRA-NPs group, and the ATRA-ATO-NPs group was injected at a dose of 15 mg / kg, ATO in the ATO group, the ATRA / ATO combination drug group, the ATO-NPs group, and the ATRA-ATO-NPs group was injected at a dose of 1 mg / kg, and the injection amount of Blank NPs was the same as that of ATRA-ATO-NPs nanoparticles, and the injection was performed twice a week for 3 consecutive weeks. The tumor size was measured every week with a vernier caliper, the tumor volume was calculated, and the formula was = length x width x width x 0.52, and the body weight of the nude mice was measured every week. After 3 weeks of administration, the nude mice were sacrificed, the tumor mass was collected, photographed, and weighed. The total protein in the tumor mass was extracted, and the expression of Pinl and its regulated oncogenes in the tumor mass was detected by Western blot. The results showed that Blank NPs had no inhibitory effect on tumor growth, ATRA and ATO had only a slight inhibitory effect on tumor growth, ATRA / ATO combination drug, ATRA-NPs, ATO-NPs, and ATRA-ATO-NPs nanoparticles had a significant inhibitory effect on tumor growth, the inhibitory effect of the ATRA / ATO combination drug group on tumor growth was basically the same as that of the ATRA-NPs group and the ATO-NPs group, and the inhibitory effect of ATRA-ATO-NPs nanoparticles on tumor growth was significantly higher than that of the ATRA / ATO combination drug group, the ATRA-NPs group, and the ATO-NPs group. That is, blank albumin nanoparticles themselves had no inhibitory effect on tumor growth, while ATRA-ATO-NPs nanoparticles could significantly enhance the synergistic inhibitory effect of ATRA and ATO on tumor growth. Western blot results showed that ATRA-ATO-NPs nanoparticles could significantly enhance the synergistic inhibitory effect of ATRA and ATO on the expression of Pinl and its regulated oncogenes in the tumor mass.

[0028] Figure 8 Figure 13 shows the in vivo anti-tumor growth effect of blank NPs, ATRA, ATO, ATRA / ATO combination drug, ATRA-NPs, ATO-NPs, and ATRA-ATO-NPs in nude mice. Figure 8 (a) shows the schematic diagram of nude mouse subcutaneous tumor model construction, drug administration, and tumor growth analysis; Figure 8 (b-d) respectively show the inhibitory effect of blank NPs, ATRA, ATO, ATRA / ATO combination drug, ATRA-NPs, ATO-NPs, and ATRA-ATO-NPs on the size, volume, and weight of tumors in nude mice; Figure 8 (e) shows the effect of each group of treatments on the body weight of nude mice; Figure 8 (f-g) respectively show the inhibitory effect of each group of treatments on Pin1 and its regulated oncogene expression in tumor mass (Western blot detection).

[0029] Figure 14 shows the inhibitory effect of ATRA, ATO, ATRA / ATO combination drug, ATRA-NPs, ATO-NPs, and ATRA-ATO-NPs on tumor lung metastasis and Pin1 and tumor metastasis marker gene expression in animals.

[0030] Hepa 1-6 cells were cultured in vitro, and 2.5 x 106Hepa 1-6 cells were injected into the tail vein of nude mice to establish a lung metastasis model of nude mice. One day after injection of cancer cells, the nude mice were randomly divided into 7 groups, namely, the normal saline group, the ATRA group, the ATO group, the ATRA / ATO combination drug group, the ATRA-NPs group, the ATO-NPs group, and the ATRA-ATO-NPs nanomicrosphere group. ATRA was dissolved with ethanol and diluted with corn oil; ATO was dissolved with sodium hydroxide and diluted with normal saline; ATRA-NPs, ATO-NPs, and ATRA-ATO-NPs nanomicrospheres were dispersed with normal saline. The drugs were administered by intraperitoneal injection, in which ATRA in the ATRA, ATRA / ATO combination drug, ATRA-NPs, and ATRA-ATO-NPs groups was injected at a dose of 15 mg / kg, and ATO in the ATO, ATRA / ATO combination drug, ATO-NPs, and ATRA-ATO-NPs groups was injected at a dose of 1 mg / kg, twice a week, for 3 weeks of continuous administration. The body weight of the nude mice was measured every week. After 3 weeks of administration, the nude mice were sacrificed, the lung tissues were collected and photographed, and the number of nodules on the surface of the lung tissues was counted. The lung tissues were paraffin-embedded, sectioned, HE-stained, and observed under a microscope. The total protein in the lung tissues was extracted, and the expression of Pin1 and tumor metastasis marker proteins in the lung tissues was detected by Western blot. The results showed that ATRA and ATO had only a slight inhibitory effect on tumor metastasis, and ATRA / ATO combination drug, ATRA-NPs, ATO-NPs, and ATRA-ATO-NPs nanomicrospheres had a significant inhibitory effect on the size and number of tumor metastasis nodules. The inhibitory effect of the ATRA / ATO combination drug group on tumor metastasis nodules was basically equivalent to that of the ATRA-NPs group and the ATO-NPs group, while ATRA-ATO-NPs nanomicrospheres could significantly enhance the inhibitory effect of ATRA and ATO on the number of tumor metastasis nodules. That is, ATRA-ATO-NPs nanomicrospheres could significantly enhance the synergistic inhibitory effect of ATRA and ATO on tumor metastasis. Western blot results showed that ATRA-ATO-NPs nanomicrospheres could significantly enhance the synergistic inhibitory effect of ATRA and ATO on the expression of Pin1 in lung metastasis tissues and the reverse effect of ATRA and ATO on the expression of tumor metastasis marker proteins in lung tissues.

[0031] Figure 9 The in vivo anti-tumor metastasis effect of ATRA, ATO, ATRA / ATO combination drug, ATRA-NPs, ATO-NPs, and ATRA-ATO-NPs nanomicrospheres. Figure 9 (a) shows a schematic diagram of the construction of a nude mouse tumor cell lung metastasis model, drug treatment, and tumor metastasis analysis; Figure 9 (b) shows the lung metastasis photos of each treatment group; Figure 9(c) HE staining micrographs of lung tissues of each treatment group; Figure 9 (d) The number of metastatic nodules on the lung surface of each treatment group; Figure 9 (e-f) The effect of each group of treatments on the expression of Pinl and tumor metastasis marker proteins in lung tissue (Western blot detection).

[0032] Experimental Example 5 Synergistic inhibitory effect of different proportions and concentrations of ATRA and ATO on tumor cell growth

[0033] Hepatoma cells HuH7 (3000 cells / well), PLC (6000 cells / well) were inoculated in 96-well plates, and treated with different concentrations of ATRA group, ATO group, ATRA / ATO combined drug group (the concentration of ATRA was 5 µM~30 µM, the concentration of ATO was 0.5 µM~2 µM, and the molar ratio of the drug concentration of ATRA and ATO was 5:1~15:1). After 72 h of treatment, the untreated cells were used as controls, and the MTT or CCK8 method was used to determine the effect of drug treatment on cancer cell proliferation. The synergistic index (CI value) of ATRA / ATO combined drug in inhibiting cancer cell proliferation was calculated by CalcuSyn software. When the CI value is <0.1, it indicates a very strong synergistic effect. When the CI value is in the range of 0.1-0.3, 0.3-0.7, 0.7-0.85, 0.85-0.9 or 0.9-1.1, it indicates strong synergistic effect, synergistic effect, moderate synergistic effect, slight synergistic effect, and approximately additive effect, respectively.

[0034] The results of cell proliferation showed that with the increase of drug concentration, the inhibitory effect of ATRA, ATO, and ATRA / ATO combination on cancer cell activity gradually increased. The synergistic index analysis results showed that when the molar ratio of ATRA and ATO combined drug concentration was 10:1, the synergistic effect of the two on cancer cell activity was the strongest, with ATRA concentration of 5~20 µM and ATO concentration of 0.5~2 µM. When the molar ratio of ATRA and ATO combined drug concentration was 5:1 or 15:1, the inhibitory effect of the two on cancer cell activity had moderate synergistic effect, with ATRA concentration of 5~30 µM and ATO concentration of 1~2 µM.

[0035] Figure 10 The inhibitory effect of ATRA, ATO, and ATRA / ATO combined drug treatment on the activity of hepatoma cells HuH7 and PLC and the synergistic index analysis chart. Figure 10 (a) and (b) are the effects of ATRA, ATO, and ATRA / ATO combined drug treatment on the activity of HuH7 cells for 72 h. Figure 1Figure 6A shows the effect of ATRA, ATO, ATRA / ATO combination drug treatment on cell viability of PLC cells for 72 h; Figure 10 Figure 6D shows the synergistic index analysis of ATRA / ATO combination drug treatment on cell viability inhibition of HuH7 cells. Figure 10 Figure 6E shows the synergistic index analysis of ATRA / ATO combination drug treatment on cell viability inhibition of PLC cells.

[0036] Experimental Example 6 In vivo pharmacokinetics and tissue distribution of ATRA, ATO, ATRA-NPs, ATO-NPs, ATRA-ATO-NPs nanospheres 8-week-old male BALB / c nude mice (purchased from Shanghai Slac Laboratory Animal Co. Ltd.) were randomly divided into 3 groups, namely ATRA group, ATO group, ATRA-NPs group, ATO-NPs group, and ATRA-ATO-NPs nanomicelle group. A single intraperitoneal injection was performed, and the ATRA dose in ATRA, ATO, ATRA-NPs, ATO-NPs, and ATRA-ATO-NPs was 15 mg / kg, and the ATO dose was 1 mg / kg. Each experimental group contained 3-4 nude mice at each experimental time point. At 0.5 h, 1 h, 3 h, 4 h, 6 h, 12 h, and 24 h after administration, about 0.2 mL of blood was taken from the retro-orbital plexus of the nude mice. The blood samples were centrifuged at 3000 rpm for 10 min at 4 ℃ to separate the plasma samples. 0.1 mL of plasma was taken, 1 mL of ethyl acetate was added, vortex mixed for 5 min, centrifuged at 10000 rpm for 10 min, and the residue was dissolved in 100 µL of mobile phase solution. The ATRA content in the sample was determined by liquid chromatography tandem mass spectrometry (LC-MS / MS). Another 100 µL of plasma was taken, 125 µL of nitric acid, 125 µL of hydrogen peroxide, and 100 µL of deionized water were added, vortex mixed, then digested at 65 ℃ and 95 ℃ water bath for 1 h, then diluted with deionized water and filtered with a 0.22 µm filter. The ATO content in the sample was determined by inductively coupled plasma mass spectrometry (ICP-MS). The pharmacokinetic curves of ATRA, ATO, ATRA-NPs, ATO-NPs, and ATRA-ATO-NPs were plotted. Hepatocarcinoma cells HuH7 were cultured in vitro, and 4×106 HuH7 cells were injected subcutaneously into the hind limb root of nude mice to establish a nude mouse tumor model. When the tumor grew to 50 cubic millimeters, the nude mice were given intraperitoneal injection of ATRA-NPs, ATO-NPs, and ATRA-ATO-NPs nanomicelles. After 4 h and 24 h of administration, the nude mice were sacrificed, and the tumor tissue and heart, liver, spleen, lung, and kidney tissues were collected. A certain weight of tissue was added to the tissue, and physiological saline (3 µL of physiological saline per 1 mg of tissue) was added and homogenized. Then 4 times the volume of methanol was added to the tissue homogenate and vortexed for 5 min, followed by centrifugation at 10000 rpm for 10 min at 4 ℃. The ATRA content in the tissue sample was determined by the above method. Another certain weight of tissue was digested with nitric acid and hydrogen peroxide according to the above method, and then the ATO content in the sample was determined by ICP-MS. The pharmacokinetic results showed that ATRA-NPs, ATO-NPs, and ATRA-ATO-NPs nanomicelles could effectively release ATRA and ATO in vivo.The biodistribution results show that ATRA-NPs, ATO-NPs, ATRA-ATO-NPs nanospheres can effectively deliver ATRA and ATO into tumor tissues, and can maintain the relative stability of drug content in tissues within 24 h.

[0037] Figure 11 The figure for the in vivo pharmacokinetics and tissue distribution of ATRA, ATO, ATRA-NPs, ATO-NPs, ATRA-ATO-NPs nanospheres. Figure 11 (a-d) represent the kinetic curves of ATRA and ATO in blood of each treatment group; Figure 11 (c-d) represent the ATRA and ATO contents in tumor and main organ tissues of ATRA-NPs, ATO-NPs, ATRA-ATO-NPs nanospheres treatment groups.

[0038] Experimental Example 7 Biological safety of ATRA-ATO-NPs nanospheres

[0039] Male BALB / c nude mice (purchased from Shanghai Slac Laboratory Animal Co., Ltd.) were randomly divided into 3 groups, namely, normal saline group, ATRA-ATO-NPs nanospheres low-dose group, and ATRA-ATO-NPs nanospheres high-dose group. Different doses (150 mg / kg, 600 mg / kg) of ATRA-ATO-NPs were injected into the tail vein of the nude mice once through intraperitoneal injection, and the nude mice were sacrificed after 48 hours. Blood samples and heart, liver, spleen, lung, kidney, and stomach were collected. Flow cytometry was used to determine the blood cell components and biochemical indicators in the blood samples, and paraffin embedding and HE staining were performed on the heart, liver, spleen, lung, kidney, and stomach tissues to evaluate the safety of ATRA-ATO-NPs. The results of blood biochemical indicator and blood cell component analysis showed that ATRA-ATO-NPs nanospheres had no significant effect on the biochemical indicators of heart, liver, and kidney and the blood cell components. The results of tissue HE staining showed that ATRA-ATO-NPs nanospheres had no significant damage to the heart, liver, spleen, lung, kidney, and stomach tissues. That is, ATRA-ATO-NPs nanospheres have good in vivo biological safety.

[0040] Figure 12 The figure for the in vivo biological safety of ATRA-ATO-NPs nanospheres. Figure 12 (a-b) represent the blood biochemical indicators and blood cell component contents of ATRA-ATO-NPs nanospheres treatment groups; Figure 12 (c) represents the microscope photos of HE staining of heart, liver, spleen, lung, kidney, and stomach tissues of ATRA-ATO-NPs nanospheres treatment groups.

Claims

1. A method for preparing all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite sustained-release nanospheres, characterized in that, Includes the following steps: a. Add albumin and L-reduced glutathione to deionized water, and then stir at a certain temperature to reduce the disulfide bonds in the albumin molecule to obtain a reduced albumin aqueous solution. b. Take the sodium hydroxide solution of ATO and add it to the reduced albumin aqueous solution obtained in step a to obtain the ATO-albumin solution; c. Add ATRA to anhydrous ethanol, stir, and obtain an ethanol solution of ATRA; d. Using a micro-injection pump, the ATRA ethanol solution obtained in step c is added dropwise to the ATO-albumin solution obtained in step b to obtain crude all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite nano-slow-release microspheres (i.e., ATRA-ATO-NPs); e. Centrifuge the above crude product, then wash it with deionized water and freeze-dry it to obtain the ATRA-ATO-NPs product.

2. The preparation method of the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite sustained-release nanospheres according to claim 1, characterized in that, In step a, the albumin is selected from either bovine serum albumin or human serum albumin.

3. The preparation method of the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite nanospheres according to claim 1, characterized in that, In step a, the concentration of the albumin aqueous solution is 2~6m / v%, and the mass ratio of L-reduced glutathione to albumin is 30~45wt%.

4. The preparation method of the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite sustained-release nanospheres according to claim 1, characterized in that, In step a, the reduction reaction temperature is 38~42℃.

5. The preparation method of the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite nanospheres according to claim 1, characterized in that, In step a, the reduction reaction time is 40 min to 2 h.

6. The method for preparing the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite sustained-release nanospheres according to claim 1, characterized in that, In step a, the stirring speed is 60~200 rpm.

7. The preparation method of the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite sustained-release nanospheres according to claim 1, characterized in that, In step b, the concentration of ATO in the sodium hydroxide aqueous solution is 0.005~0.04 kg / L; preferably, the concentration of ATO in the sodium hydroxide aqueous solution is 0.02 kg / L.

8. The method for preparing the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite nanospheres according to claim 1, characterized in that, In step b, the volume ratio of the ATO solution to the albumin aqueous solution is 0.1~2 v / v.

9. The method for preparing the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite sustained-release nanospheres according to claim 1, characterized in that, In step c, the concentration of the anhydrous ethanol solution of ATRA is 0.00008~0.0008 kg / L.

10. The method for preparing the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite sustained-release nanospheres according to claim 1, characterized in that, In step d, the mass ratio of ATO to ATRA in the ATRA-ATO-NPs is 4% to 13%.

11. The method for preparing all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite sustained-release nanospheres according to claim 1, characterized in that, In step d, the ATRA ethanol solution obtained in step c is added dropwise to the ATO-albumin solution obtained in step b at a rate of 0.5~4 mL / min.

12. The method for preparing all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite sustained-release nanospheres according to claim 1, characterized in that, In step d, the volume ratio of the ATRA ethanol solution to the ATO-albumin solution is 3:1 to 6:

1.

13. The method for preparing all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite sustained-release nanospheres according to claim 1, characterized in that, In step e, the centrifugation temperature is 0~8℃, the centrifugation speed is 10000~20000 rpm, and the centrifugation time is 20~50 min; preferably, the centrifugation temperature is 4℃, the centrifugation speed is 15000 rpm, and the centrifugation time is 30 min.

14. A composite sustained-release nanosphere of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), characterized in that, Prepared by the method of any one of claims 1 to 13; Furthermore, the particle size of the composite nano-sustaining microspheres is 90 nm to 180 nm; Furthermore, in the sustained-release nanospheres, the molar ratio of ATRA to ATO is 5 to 15:1; more preferably, the molar ratio of ATRA to ATO in the sustained-release nanospheres is 10:

1.

15. Use of the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) composite nano-release microspheres as described in claim 14 in the preparation of a medicament for treating Pin1 overexpression diseases; Furthermore, the diseases in which Pin1 is overexpressed include tumors, inflammation, lupus erythematosus, or asthma; Furthermore, the antitumor effect is the inhibition of tumor growth or the inhibition of tumor metastasis; Furthermore, the tumor is selected from one or both of liver cancer and lung cancer; Furthermore, the inflammation is arthritis.

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