Paclitaxel prodrug nano-micelle as well as preparation method and application thereof

Abstract

The invention relates to the field of medicines, and particularly discloses a paclitaxel prodrug nano-micelle as well as a preparation method and application thereof. According to the paclitaxel-retinoic acid prodrug nano-micelle, a paclitaxel-retinoic acid prodrug, sodium retinoate and sodium cholate are subjected to self-assembly in water to form the paclitaxel-retinoic acid prodrug nano-micelle. According to the invention, paclitaxel and retinoic acid are covalently bound to prepare a micromolecular prodrug, and the micromolecular prodrug is co-delivered to a tumor site to synergistically exert an anti-cancer effect. According to the invention, sodium cholate is introduced as a surfactant and is subjected to hydrophilic modification, so that the water solubility of the medicine is improved; by utilizing the characteristic that sodium cholate promotes dissolution of membrane protein, PTX is promoted to quickly enter tumor cells, and the clinical application prospect of PTX is improved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a paclitaxel prodrug nano-micelle and a preparation method and application thereof. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant digestive system tumor and the most common histological subtype of pancreatic cancer, accounting for over 90% of pancreatic cancer cases. The incidence of PDAC increases by 0.5% to 1.0% annually, and it is projected to become the second leading cause of cancer-related mortality by 2030. Among the various chemotherapeutic agents currently available, paclitaxel (PTX), whether used alone or in combination with other chemotherapeutic agents or immunotherapies, effectively prolongs the survival of PDAC patients. Currently, the combination of PTX albumin nanoformulation and gemcitabine (Gem) is the preferred treatment option for patients with advanced and late-stage PDAC. While this approach improves efficacy compared to monotherapy, drug resistance is one of the most common reasons for clinical treatment failure. Furthermore, acquired drug resistance resulting from long-term PTX treatment enhances the metastatic and invasive capabilities of tumor cells. Therefore, the development of novel PDAC-targeted drug delivery systems that can both enhance the anticancer effects of PTX and overcome drug resistance is crucial.

[0003] Retinoic acid (RA), a derivative of vitamin A, primarily regulates target gene transcription through binding to the nuclear receptors RAR and RXR, thereby influencing cellular physiological functions. RA can induce pancreatic stellate cells (PSCs) to return to a quiescent state by improving their accumulation of vitamin A lipid droplets, reducing interstitial fibrosis and enabling chemotherapeutic drug delivery to the tumor site. RA can also ameliorate the immunosuppressive and angiogenic effects of PSCs in the PDAC microenvironment by reversing PSC activation, enhancing drug availability and inhibiting tumor growth. RA also inhibits PSC proliferation by arresting the cell cycle at the G1 phase. Multiple clinical trials have demonstrated that RA is an effective and promising antitumor drug for various cancer types, including head and neck, breast, ovarian, pancreatic, and lung cancers. Furthermore, combination therapy with RA and PTX can inhibit multiple cell survival factors by downregulating NF-κB and BIRC, as well as proteins that promote Bcl-2 phosphorylation. RA can also induce pancreatic cancer cell death by reducing the Wnt / β-catenin signaling pathway. However, the strategy of covalent conjugation to prepare small molecule prodrugs has not been reported.

[0004] In recent years, the development of nanoparticles utilizing specific hydrophobic interactions has greatly enriched drug delivery strategies. Because assembly occurs in an aqueous environment, it exhibits unique advantages and holds enormous potential in the field of anticancer drug delivery. Cholic acid (CA), a natural surfactant secreted in mammalian bile, possesses a planar, rigid structure and surface amphiphilicity. Studies have shown that chemical modification of the CA sterol backbone and physical conjugation with various amphiphilic molecules can effectively enhance the solubility of micellar systems. Compared to traditional nanomedicines, CA self-assembled nanomicelles offer a simpler preparation process and a more stable structure, effectively extending drug circulation in the body and enhancing passive targeting of drugs to tumors. Furthermore, the unique dendritic structure of CA's hydrophobic core significantly increases the loading capacity of hydrophobic drugs, regulating their targeted release. It can also interact with phospholipids in cell membranes, enhancing drug penetration across various biological membranes. It can also inhibit the invasion and migration of pancreatic cancer cells by altering the epithelial-mesenchymal transition (EMT) process. Furthermore, high concentrations of CA, through the membrane potential of pancreatic cancer cells, increase the production of reactive oxygen species, thereby promoting cell death. Therefore, membrane disruption strategies hold great promise not only for achieving low-throughput delivery of molecules but also for overcoming cellular drug resistance.

[0005] Neoadjuvant chemotherapy strategies are gaining increasing attention in the treatment of PDAC, not only effectively improving the patient's survival index but also further enhancing the quality of life. However, the multidrug resistance of pancreatic cancer cells seriously affects the efficacy of chemotherapy for pancreatic cancer. Therefore, effectively matching drugs to the resistance mechanism of single drugs and synergizing them to exert efficient anti-cancer effects is a difficult problem that needs to be solved urgently. Based on the EPR effect, nano-drug delivery systems can accumulate in the tumor microenvironment of PDAC through systemic circulation, but the heterogeneity of the tumor microenvironment greatly limits the uptake of drugs by pancreatic cancer cells. Therefore, it is still necessary to develop a nano-carrier system that can achieve targeted transport of drug payloads and the PDAC tumor microenvironment, antagonize multidrug resistance, and effectively kill tumor cells. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to address the deficiencies of the existing technology and provide a paclitaxel prodrug nano-micelle and its preparation method and application.

[0007] Inventive Concept: Based on the pancreatic cancer microenvironment and cellular heterogeneity, this invention combines the advantages of nano-drug delivery systems and utilizes the synergistic effect of paclitaxel (PTX) and retinoic acid (RA). Using PTX as a drug model and RA as an adjuvant, the two are ester-linked to form a small molecule prodrug, PTX-RA. This is then self-assembled into nanomicelles in water with a certain ratio of sodium retinoate and sodium cholate, creating a novel paclitaxel prodrug nano-delivery system, namely, paclitaxel-retinoic acid prodrug nanomicelles (PTX-RA@RAS NPs). The constructed PTX-RA@RAS NPs can prolong circulation time in vivo. This prodrug nanosystem passively accumulates in solid tumors based on the EPR effect and is delivered to the lysosomes of tumor cells for degradation, releasing PTX and RA, exerting a synergistic anti-tumor effect and exhibiting significant anti-tumor activity.

[0008] In order to solve the above technical problems, the present invention discloses the following technical solutions:

[0009] In a first aspect, the present invention discloses a paclitaxel-retinoic acid prodrug.

[0010] Wherein, in the paclitaxel-retinoic acid prodrug, paclitaxel and retinoic acid are covalently linked via an ester bond.

[0011] Specifically, the paclitaxel-retinoic acid prodrug is prepared by the following method: in an organic solvent, paclitaxel and retinoic acid react under the action of a base and a catalyst.

[0012] Wherein, the molar ratio of paclitaxel to retinoic acid is 1:1-3, such as 1:2.

[0013] Wherein, the organic solvent is dichloromethane.

[0014] Wherein, the base is triethylamine.

[0015] Wherein, the catalyst is p-dimethylaminopyridine.

[0016] Wherein, the molar volume ratio of paclitaxel to the organic solvent is 0.008-0.016 mmol / mL, such as 0.012 mmol / mL.

[0017] Wherein, the molar ratio of paclitaxel to base is 1:7-11, such as 1:9-10

[0018] Wherein, the molar ratio of paclitaxel to catalyst is 1:0.4-1, such as 1:0.6-0.7.

[0019] Furthermore, the reaction system further comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide; preferably, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is added under stirring; and / or the molar ratio of paclitaxel to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:3-6, such as 1:4-5.

[0020] Furthermore, a base and a catalyst are added to a mixed solution of paclitaxel, retinoic acid and an organic solvent, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide is added to react to prepare a paclitaxel-retinoic acid prodrug.

[0021] Wherein, the reaction temperature is room temperature.

[0022] In a second aspect, the present invention discloses a method for preparing paclitaxel-retinoic acid prodrug.

[0023] The method comprises: reacting paclitaxel with retinoic acid in an organic solvent under the action of a base and a catalyst.

[0024] In the prepared paclitaxel-retinoic acid prodrug, paclitaxel and retinoic acid are covalently linked via an ester bond.

[0025] Wherein, the molar ratio of paclitaxel to retinoic acid is 1:1-3, such as 1:2.

[0026] Wherein, the organic solvent is dichloromethane.

[0027] Wherein, the base is triethylamine.

[0028] Wherein, the catalyst is p-dimethylaminopyridine.

[0029] Wherein, the molar volume ratio of paclitaxel to the organic solvent is 0.008-0.016 mmol / mL, such as 0.012 mmol / mL.

[0030] Wherein, the molar ratio of paclitaxel to base is 1:7-11, such as 1:9-10

[0031] Wherein, the molar ratio of paclitaxel to catalyst is 1:0.4-1, such as 1:0.6-0.7.

[0032] Furthermore, the reaction system further comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide; preferably, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is added under stirring; and / or the molar ratio of paclitaxel to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:3-6, such as 1:4-5.

[0033] Furthermore, a base and a catalyst are added to a mixed solution of paclitaxel, retinoic acid and an organic solvent, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide is added to react to prepare a paclitaxel-retinoic acid prodrug.

[0034] Wherein, the reaction temperature is room temperature.

[0035] In the third aspect, the present invention discloses a paclitaxel-retinoic acid prodrug nano-micelle with high stability and anticancer effect. powerful.

[0036] The paclitaxel-retinoic acid prodrug nanomicelles are self-assembled in water by the paclitaxel-retinoic acid prodrug described in the first aspect or the paclitaxel-retinoic acid prodrug prepared by the method described in the second aspect, sodium retinoate and sodium cholate to form the paclitaxel-retinoic acid prodrug nanomicelles.

[0037] The mass ratio of the paclitaxel-retinoic acid prodrug, sodium retinoate and sodium cholate is 1:0.4-0.8:0.8-1.0.

[0038] wherein a mixed solution of paclitaxel-retinoic acid prodrug, sodium retinoate, sodium cholate and an organic solvent is added to water, stirred to self-assemble to form nanoparticles, and the organic solvent in the system is removed to obtain paclitaxel-retinoic acid prodrug nanomicelles; preferably, the organic solvent is an alcohol, preferably methanol; preferably, in the mixed solution, the mass volume ratio of sodium retinoate to the organic solvent is 0.8-1.2 mg / mL; preferably, the mixed solution is obtained by dissolving paclitaxel-retinoic acid prodrug and sodium cholate in an organic solvent solution of sodium retinoate; and the stirring time is 1-5 min, such as 3 min.

[0039] In some embodiments, paclitaxel-retinoic acid prodrug and sodium cholate are mixed, a methanol solution of sodium retinoic acid is added to the mixture and completely dissolved. Subsequently, the mixture is slowly added dropwise to 5 mL of ultrapure water, and self-assembly is driven by solvent displacement in the methanol-water system to form nanoparticles. The reaction is stirred, and the methanol in the nanosystem is removed by reduced pressure distillation to obtain paclitaxel-retinoic acid prodrug nanomicelles.

[0040] In a fourth aspect, the present invention discloses the paclitaxel-retinoic acid prodrug described in the first aspect or the paclitaxel-retinoic acid prodrug described in the second aspect. The invention relates to a paclitaxel-retinoic acid prodrug prepared by the method or a use of the paclitaxel-retinoic acid prodrug nano-micelles described in the third aspect.

[0041] Wherein, the use is the use of the paclitaxel-retinoic acid prodrug or the paclitaxel-retinoic acid prodrug nano-micelle in the preparation of anti-pancreatic cancer drugs.

[0042] Wherein, the anti-pancreatic cancer treatment is targeted treatment of pancreatic cancer.

[0043] In a fifth aspect, the present invention discloses an anti-pancreatic cancer drug.

[0044] Wherein, the drug includes the paclitaxel-retinoic acid prodrug described in the first aspect, the paclitaxel-retinoic acid prodrug prepared by the method described in the second aspect, or the paclitaxel-retinoic acid prodrug nanomicelles described in the third aspect.

[0045] Wherein, the anti-pancreatic cancer treatment is targeted treatment of pancreatic cancer.

[0046] Beneficial effects:

[0047] (1) The present invention covalently binds paclitaxel (PTX) and retinoic acid (RA) to form a small molecule prodrug (PTX-RA), which is co-delivered to the tumor site to exert a synergistic anti-cancer effect. Simultaneously, the present invention utilizes RA as an anti-stromal agent to inhibit the proliferation of pancreatic stellate cells (PSCs), reduce interstitial fibrosis, and increase the tumor permeability of PTX.

[0048] (2) The present invention introduces sodium cholate as a surfactant to modify the hydrophilicity of the small molecule prodrug (PTX-RA), further improving the water solubility of PTX-RA. At the same time, the present invention utilizes the properties of sodium cholate to promote membrane protein solubility, promoting the rapid entry of PTX into tumor cells, and improving the clinical application prospects of PTX.

[0049] (3) Based on the amphiphilicity of sodium retinoic acid and sodium cholate, the present invention can spontaneously form prodrug nano-micelles, thereby improving the drug loading rate and enhancing the tumor accumulation of functional molecules.

[0050] (4) The present invention can be stably delivered to the tumor site through systemic circulation, and the drug can be rapidly released after being taken up by tumor cells, thereby exerting an efficient anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0052] Figure 1 This is a synthetic route for paclitaxel-retinoic acid prodrugs.

[0053] Figure 2 It is the ultraviolet absorption spectrum of PTX-RA prepared in Example 1.

[0054] Figure 3 TEM morphology diagrams of PTX-RA@RAS NPs prepared in Example 1 at 200 nm (A) and 100 nm (B).

[0055] Figure 4 The changes in particle size (A) and PDI (B) of PTX-RA@RAS NPs prepared in Example 1 in different solutions (n=3).

[0056] Figure 5 The critical micelle concentration of PTX-RA@RAS NPs prepared in Example 1 is detected. 373 / I 384 Ratio change trend chart.

[0057] Figure 6 Graph showing the in vitro drug release capacity test results of PTX (A) and PTX-RA (B) of the PTX-RA@RAS NPs prepared in Example 1 at pH 5.0, pH 7.4, and with / without tissue proteases.

[0058] Figure 7 These are graphs showing the apoptosis induction of PANC-1 cells (A, C) and HPSC cells (B, D) by different drug groups such as PTX-RA and PTX-RA@RAS NPs prepared in Example 1 at a concentration of 1 μM, as well as quantitative analysis of the cell apoptosis rate (n=3).

[0059] Figure 8 Migration diagrams of PANC-1 cells and (A, C) HPSC cells (B, D) treated with different drug groups such as PTX-RA and PTX-RA@RAS NPs prepared in Example 1 at different time points and quantitative analysis of cell migration rate (n=3) (scale: 100 μm).

[0060] Figure 9 Figure 1 is a graph showing the tumor growth curve (A) of nude mice in different drug groups including PTX-RA and PTX-RA@RAS NPs prepared in Example 1 and the weight change curve (B) of nude mice during the treatment period (n=5).

[0061] Figure 10 Graphs showing the anti-tumor activity test results of PTX-RA and PTX-RA@RAS NPs prepared in Example 1: (A) photos of tumor-bearing nude mice, (B) photos of solid tumors, and (C) weights of solid tumors (n=5). DETAILED DESCRIPTION

[0062] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0063] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0064] Example 1 Preparation of Paclitaxel-Retinoic Acid Prodrug Nanomicelles (PTX-RA@RAS NPs)

[0065] (1) Synthesis of Paclitaxel-Retinoic Acid Prodrug (PTX-RA)

[0066] 204.11 mg of paclitaxel (PTX, 0.24 mmol) and 139.52 mg of retinoic acid (RA, 0.46 mmol) were accurately weighed and dissolved in 20 mL of dichloromethane (DCM). After complete dissolution, 225.24 mg of triethylamine (TEA, 2.23 mmol) and 20 mg of p-dimethylaminopyridine (DMAP, 0.16 mmol) were added. 0.1672 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 1.1 mmol) was added under stirring. The reaction was stirred at room temperature for 4 hours and purified by column chromatography to obtain the prodrug paclitaxel-retinoic acid prodrug (PTX-RA). The structure of PTX-RA was verified by 1H NMR: 1H NMR(400MHz,Chloroform-d)δ8.16–8.09(m,2H),7.79–7.70(m,2H),7.65–7.57(m,1H),7.5 1(td,J=7.7,5.6Hz,4H),7.45–7.36(m,6H),7.37–7.29(m,1H),7.06(dd,J=15.0,11.4Hz,1 H),6.98(d,J=9.0Hz,1H),6.39–6.30(m,3H),6.30–6.22(m,2H),5.93(dd,J=9.0,3.4Hz,1H ),5.69(d,J=7.1Hz,1H),5.54(d,J=3.4Hz,1H),4.98(dd,J=9.6,2.3Hz,1H),4.46(s,1H),4. 32(d,J=8.4Hz,1H),4.20(d,J=8.4Hz,1H),3.82(d,J=7.0Hz,1H),2.63–2.43(m,5H),2.41– 2.31(m,4H),2.23(s,3H),2.21–2.14(m,2H),2.13–2.00(m,6H),1.96(d,J=1.4Hz,3H),1.8 8(ddd,J=17.7,9.9,4.4Hz,2H),1.74–1.57(m,14H),1.51–1.43(m,3H),1.29(d,J=7.0Hz,2H),1.25(d,J=7.1Hz,9H),1.13(s,4H),1.03(s,6H),0.98–0.83(m,11H),0.81(s,1H). Figure 1 shown.

[0067] (2) Synthesis of sodium retinoate (RA-Na)

[0068] 1.01 g of retinoic acid (3.33 mmol) was weighed and dissolved in 5 mL of tetrahydrofuran (THF, 43.91 mmol). 174.63 mg of sodium hydroxide (NaOH, 4.37 mmol) was weighed and dissolved in 500 μL of water. The sodium hydroxide aqueous solution was slowly added dropwise while stirring. After stirring at room temperature for 2 hours, 30 mL of ethyl acetate (EAC, 307.14 mmol) was added to the reaction solution and stirred at room temperature for 20 minutes. During stirring, a milky white precipitate was precipitated. After stirring, the precipitate was filtered and dried in an oven to obtain sodium retinoate (RA-Na).

[0069] (3) Preparation of Paclitaxel-Retinoic Acid Prodrug Nanomicelles (PTX-RA@RAS NPs)

[0070] Accurately weigh 5.03 mg of PTX-RA and 5.08 mg of sodium cholate (NaC) into 1.5 mL of EP for later use. Weigh 3.09 mg of sodium retinoic acid (RA-Na) and dissolve it in 3 mL of methanol to prepare a 1.04 mg / mL sodium retinoic acid methanol solution. Add 1 mL of this sodium retinoic acid methanol solution to the EP tube and allow it to completely dissolve. Under stirring, the mixed solution was slowly added dropwise to 5 mL of ultrapure water. Nanoparticles were formed through self-assembly driven by solvent displacement in the methanol-water system. After stirring for approximately 3 minutes, the methanol in the nanosystem was removed by vacuum distillation to obtain an aqueous solution containing paclitaxel-retinoic acid prodrug nanomicelles (PTX-RA@RAS NPs).

[0071] Detection:

[0072] (1) Full wavelength scanning was performed on the standard solutions of PTX, RA and PTX-RA. The results were as follows: Figure 2 As shown in the figure, within the wavelength range of 200-600 nm, the characteristic absorption peak of PTX is located at 227 nm, the characteristic absorption peak of RA is located at 352 nm, and PTX-RA has two characteristic absorption peaks, which are located near 229 nm and 364 nm respectively. Compared with PTX and RA, the position of the longitudinal absorption peak has hardly changed, indicating that RA has successfully modified PTX.

[0073] (2) The microscopic morphology of the PTX-RA@RAS NPs prepared in this example was observed using a transmission electron microscope. The process was as follows: PTX-RA@RAS NPs were diluted to an appropriate concentration with ultrapure water, and the diluted sample was dropped onto a copper mesh covered with a carbon film. After standing for 3 minutes, the excess solution was gently absorbed with filter paper. After natural drying at room temperature, the morphological characteristics of the PTX-RA@RAS NPs were observed and photographed using a transmission electron microscope (TEM). The results are shown in the figure. At the 200nm and 100nm scales, the PTX-RA@RAS NPs were all round particles with good dispersion, with an average diameter of approximately 82.54±6.79nm.

[0074] Experimental Example 2 The stability of PTX-RA@RAS NPs prepared in Example 1 was investigated

[0075] PTX-RA@RAS NPs were diluted with physiological saline, ultrapure water, pH 7.4 buffer containing 10% serum, and DMEM containing 10% serum, with the volume dilution multiples of 2, 5, 10, 30, 50, 80, and 100 times, respectively. The particle size and PDI of PTX-RA@RAS NPs in different dilution media were measured using a Malvern nanoparticle sizer, and their stability was evaluated based on the changes in particle size and PDI. The results are shown in Figure 2. Figure 4 .

[0076] Figure 4 The particle size changes of the PTX-RA@RAS NPs prepared in Example 1 were analyzed in different dilutions. The results showed that after dilution with various multiples of pure water, saline, and pH 7.4 buffer containing 10% serum, the particle size of the PTX-RA@RAS NPs ranged from 80 to 100 nm, showing no significant differences compared to the original solution. The PDI of the PTX-RA@RAS NPs remained below 0.2 in all dilution media, meeting the requirements for nanoformulation.

[0077] Experimental Example 3 Detection of the critical micelle concentration of PTX-RA@RAS NPs prepared in Example 1

[0078] Under light-proof conditions, 3.14 mg of pyrene was accurately weighed and placed in a 50 mL centrifuge tube. Acetone was added to the scale and the volume was adjusted to 3 × 10 -4 mol / L pyrene working solution, take 30 μL of pyrene working solution into a 2 mL EP tube, and completely evaporate the acetone solution at 50°C in the dark. Dilute PTX-RA@RAS NPs into nanoparticle solutions with a concentration range of 0.01-100 μg / mL, and take 1.5 mL of PTX-RA@RAS NPs of different concentrations and add them into the EP tube containing pyrene. The final concentration of pyrene is 6×10 -6mol / L, and allowed to stand at room temperature for 4 h. Subsequently, the samples were vortexed for 2 min and ultrasonicated for 30 min to achieve uniform dispersion. The samples were shaken at 100 rpm on a shaker for 24 h at a constant temperature of 25°C. Three parallel experiments were performed for each sample.

[0079] The fluorescence intensity of the above-mentioned different solution groups was measured using a fluorescence spectrophotometer with an excitation wavelength of 330 nm and an emission wavelength of 373 nm and 384 nm, and I was calculated. 373 / I 384 By establishing I 373 / I 384 The critical micelle concentration of PTX-RA@RAS NPs was determined by the intersection of the two linear intervals (the curve mutation point). Figure 5 .

[0080] Figure 5 The I of PTX-RA@RAS NPs prepared in Example 1 373 / I 384 The ratio change trend graph shows that the critical micelle concentration of PTX-RA@RAS NPs is 0.59 μg / mL.

[0081] Experimental Example 4: The in vitro drug release ability of PTX-RA@RAS NPs prepared in Example 1 was tested.

[0082] 1mL PTX-RA@RAS NPs were dissolved in 1ml of pH 5.0 acetate buffer (simulating lysosomal uptake) containing cathepsin B, enzyme-free, and pH 7.4 phosphate buffer (simulating systemic circulation), respectively, and transferred to a dialysis bag. 30mL of the corresponding pH buffer was used as the release medium and shaken in a 37°C constant temperature shaker. 1mL of the release solution was taken at different time points (0, 1, 2, 4, 6, 8, 12, and 24h), and the same volume of blank medium was added to supplement it and returned to the shaker. The obtained 1mL sample was centrifuged at 13000rpm for 15min, and 150μL of the supernatant was taken into a sample injection bottle. The cumulative release of PTX was detected by HPLC. The results are shown in Figure 2. Figure 6 .

[0083] Figure 6 The release curves of PTX and PTX-RA from the PTX-RA@RAS NPs prepared in Example 1 in different solvents show no significant difference in the cumulative release of PTX-RA in the presence or absence of cathepsins in a pH 5.0 buffer, both significantly exceeding the PTX-RA content in a pH 7.4 buffer. Furthermore, cathepsins accelerated the dissociation of PTX-RA, resulting in a cumulative PTX release of 92.18 ± 3.75% over 24 hours.

[0084] Experimental Example 5: PTX-RA and PTX-RA@RAS NPs prepared in Example 1 were used for cell culture to detect their cytotoxicity.

[0085] (1) Pancreatic cancer cells (PANC-1), pancreatic stellate cells (HPSC), and cell lines co-cultured with PANC-1 and HPSC at a ratio of 1:1 and 1:2 (P:H = 1:1 and P:H = 1:2) in the logarithmic growth phase were digested and centrifuged to disperse the cells into single cells and adjust the density to 1×10 4 cells / mL, and the above cell suspension was evenly spread in a 96-well cell culture plate, 200 μL per well, and 100 μL PBS buffer was added around the well plate. After spreading, the cells were shaken to disperse evenly at the bottom of the well, and cultured in a cell culture incubator (37°C, 5% CO2) for 24 h.

[0086] (2) The cell growth status was observed under a microscope, the old culture medium was discarded, and drug-containing culture medium with different concentrations of PTX, RA, a physical mixture of PTX and RA at a molar ratio of 1:1 (RTX+RA), PTX-RA, and PTX-RA@RAS NPs was added. The final concentration of PTX equivalent in each experimental group ranged from 0.01 to 500 μM, and 5 replicates were set for each concentration. The negative control group was: an equal volume of the corresponding cell suspension was added without drug administration; the blank control group was: an equal volume of DMEM / F12 complete culture medium (supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution) was added, and the cells were placed in an incubator and cultured for 72 h.

[0087] (3) Aspirate the drug-containing culture medium, rinse the cells with PBS buffer, add 200 μL of DMEM / F12 complete culture medium to each well, add 20 μL of MTT solution (5 mg / mL) in the dark, and continue culturing in an incubator for 4 hours. Aspirate the culture medium in the wells, rinse the cells with PBS buffer, add 150 μL of DMSO, and shake at 37°C for about 10 minutes to completely dissolve the formazan.

[0088] (4) The absorbance of each well sample at 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) and the cell viability of each experimental group was calculated using the following formula: cell viability = (AB) / (CB) × 100%, where A is the OD value of each experimental group; B is the OD value of the blank control group; and C is the OD value of the negative control group. The results are shown in Table 1.

[0089] Table 1 IC values ​​of PTX, RA, PTX+RA, PTX-RA, and PTX-RA@RAS NPs co-incubated with PANC-1, HPSC, P:H=1:1, and P:H=1:2 cells for 72 h 50 Value (n=5)

[0090]

[0091] * P<0.05vs PTX+RAgroup; # P<0.05 vs PTX-RA group

[0092] As shown in Table 1, the IC 50 The IC values ​​of PTX-RA@RAS NPs group were significantly lower than those of single drugs (PTX and RA) and physical mixture (PTX+RA). 50 The values ​​were the lowest, and their toxicities were 247.33 times, 28.33 times, 94.69 times, and 2239.33 times that of PTX, respectively, showing stronger cell killing ability.

[0093] Experimental Example 6: The PTX-RA and PTX-RA@RAS NPs prepared in Example 1 were used for cell culture to detect their cell apoptosis induction rates.

[0094] (1) PANC-1 and HPSC cells in the logarithmic growth phase were digested with trypsin (containing EDTA), centrifuged at 1000 rpm for 5 min, and the cell pellet was collected. The cells were resuspended in complete medium containing 10% fetal bovine serum and dispersed into single cells at a density of 1×10 5 cells / mL, and evenly spread the above cell suspension in a 6-well cell culture plate, 2 mL per well, ensuring that the cells are evenly distributed at the bottom of the well, and culture in an incubator for 24 h.

[0095] (2) After removing the original culture medium, culture medium containing PTX, PTX-RA, and PTX-RA@RAS NPs was added. The PTX equivalent concentration was 1 μM. Three replicate wells were set for each concentration, and a drug-free culture medium was set up as a negative control. The culture was continued for 72 h.

[0096] (3) Aspirate the drug-containing culture medium, gently wash the cells twice with PBS buffer, digest the cells with EDTA-free trypsin, centrifuge at 1000 rpm for 5 minutes, and collect the intact cells.

[0097] (4) Wash twice with pre-cooled PBS (1000 rpm, 5 min) to remove cell debris. After retaining the precipitate, add 500 μL Binding Buffer to resuspend the cells in a dark environment. Add 5 μL Annexin V-FITC, mix well, and then add 5 μL Propidium Iodide. Incubate at room temperature for 5-10 min. The samples of each group were detected using a flow cytometer. The results are as follows. Figure 7 .

[0098] Figure 7 Figure 1 shows the apoptosis induction of PANC-1 cells and HPSC cells by PTX-RA and PTX-RA@RAS NPs at a concentration of 1 μM, as well as quantitative analysis of the apoptosis rates. The PTX-RA group exhibited higher apoptosis induction rates in both PANC-1 and HPSC cells than the PTX group. The PTX-RA@RAS NPs group exhibited even higher antitumor activity, with apoptosis rates reaching 54.43% (PANC-1) and 39.91% (HPSC), respectively, significantly higher than those in the other experimental groups and consistent with the MTT results.

[0099] Experimental Example 7: The PTX-RA and PTX-RA@RAS NPs prepared in Example 1 were used for cell culture, and the cell migration and growth were detected by the “scratch” test.

[0100] (1) PANC-1 and HPSC cells in the logarithmic proliferation phase were digested and centrifuged, and the cells were dispersed into single cells and the density was adjusted to 1×10 5 cells / mL, and the above cell suspension was evenly spread in a 6-well cell culture plate, 2 mL per well. After spreading, the cells were shaken to disperse evenly at the bottom of the well, and cultured in a cell culture incubator (37°C, 5% CO2) for 24 h.

[0101] (2) Remove the old culture medium and use a 10 μL pipette tip to draw a “scar area” in the middle of the culture plate. After washing lightly with PBS once, add drug-containing culture medium containing PTX, PTX-RA, and PTX-RA@RAS NPs to make the PTX equivalent concentration 1 μM. Set up a negative control treated with basal culture medium and continue culturing in the incubator.

[0102] (3) Discard the supernatant, wash gently with PBS three times, observe and photograph under an inverted microscope at 24h and 48h, and then perform quantitative analysis of the migration results. Figure 8 .

[0103] Figure 8This is an analysis of the cell migration rate of PANC-1 cells and HPSC cells at different time points after treatment with PTX-RA and PTX-RA@RAS NPs prepared in Example 1. Compared with the control group, the migration speed of PANC-1 and HPSC cells to the scratch area slowed down after treatment with different drugs. Among them, the scratch area of ​​the PTX-RA and PTX-RA@RAS NPs groups was significantly larger than that of the PTX group. The results of quantitative analysis of migration rate showed that the migration rate of PANC-1 cells co-incubated with PTX-RA@RAS NPs for 48 hours was not significantly different from that of PTX-RA, but was significantly lower than that of the single drug group. The migration inhibition of HPSC cells was most obvious when they were incubated with PTX-RA@RAS NPs for 48 hours, with a migration rate of only 13.67±2.08%.

[0104] Experimental Example 8 The anti-tumor activity of PTX-RA and PTX-RA@RAS NPs prepared in Example 1 was detected

[0105] (1) Cultivate a co-cultured cell line that is in good growth condition and in the logarithmic growth phase (P:H=1:2), digest and centrifuge, discard the supernatant, collect the cells, resuspend the cells in PBS buffer, and place on ice for later use. Use a syringe to draw 100 μL (1×10 7 cells / mL) cell suspension, excluding air, was subcutaneously injected into the right shoulder of nude mice.

[0106] (2) Wait until the tumor volume of nude mice grows to 100-150mm 3 The mice were randomly divided into 5 groups, with 11 mice in each group. On the 1st, 7th, and 14th days, the nude mice in each experimental group were injected with normal saline, PTX, PTX-RA, and PTX-RA@RASNPs via tail vein technique, with the PTX equivalent being 5 mg / kg. A 21-day treatment cycle was set up, and the weight of the nude mice and the long diameter (L) and short diameter (S) of the tumor were measured every other day. The nude mice were treated according to the formula (V = 0.5 × L × S 2 ) to calculate the tumor volume, and construct a weight-time growth curve based on the time series to systematically analyze the weight fluctuation characteristics of tumor-bearing mice. Figure 9 .

[0107] (3) After the treatment, 5 nude mice were killed in each group, and the tumor tissue and major organs (heart, liver, spleen, lung, kidney, and pancreas) were completely removed. After washing with saline, they were weighed and photographed. The remaining mice were used for the survival rate study experiment and continued to be fed in a conventional manner. The double-blind method was used to observe the survival period of the mice in each experimental group at regular intervals every day, and the survival period of the mice in each experimental group was accurately recorded (based on the cessation of breathing and heartbeat as the judgment standard). The results are as follows: Figure 10 .

[0108] Figure 9The figure shows the growth inhibitory activity and body weight change curves of normal saline, PTX, and PTX-RA and PTX-RA@RAS NPs prepared in Example 1 on tumor growth. The results showed that the PTX group, PTX-RA group, and PTX-RA@RAS NPs group all had inhibitory effects on tumor growth. The three administrations of PTX-RA showed high inhibitory activity within 4 days after each administration, the tumor growth trend was significantly slowed, and the final tumor volume was significantly smaller than that of the PTX group. The tumor in the PTX-RA@RAS NPs group grew relatively slowly within 21 days, showing stronger tumor inhibitory activity, and the final tumor volume was significantly different from that of the PTX group and PTX-RA group. The body weight was maintained in a relatively stable range within 21 days of administration, without a significant decrease, and the in vivo safety indicators met the experimental expectations.

[0109] Figure 10 Figure 2 shows tumor analysis of nude mice in each experimental group after 21 days of treatment with PTX-RA and PTX-RA@RAS NPs prepared in Example 1. Results show that the tumor volume in the PTX-RA@RAS NPs group was significantly smaller than that in the other experimental groups, demonstrating a stronger anti-tumor effect. Tumor weight analysis revealed that the average tumor weights in both the PTX-RA and PTX-RA@RAS NPs groups were smaller than those in the PTX group, with the PTX-RA@RAS NPs group demonstrating more effective tumor growth inhibition.

[0110] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A paclitaxel-retinoic acid prodrug, characterized in that: In the paclitaxel-retinoic acid prodrug, paclitaxel and retinoic acid are covalently linked via an ester bond.

2. The paclitaxel-retinoic acid prodrug according to claim 1, characterized in that The preparation method is as follows: in an organic solvent, paclitaxel and retinoic acid react under the action of a base and a catalyst.

3. The method for preparing the paclitaxel-retinoic acid prodrug according to claim 1, characterized in that: include: In an organic solvent, paclitaxel reacts with retinoic acid in the presence of a base and a catalyst.

4. The paclitaxel-retinoic acid prodrug according to claim 1 or 2 or the preparation method according to claim 3, characterized in that: The molar ratio of paclitaxel to retinoic acid is 1:1-3.

5. The paclitaxel-retinoic acid prodrug according to claim 2 or the preparation method according to claim 3, characterized in that: The organic solvent is dichloromethane; and / or the base is triethylamine; and / or the catalyst is p-dimethylaminopyridine; and / or the molar volume ratio of paclitaxel to the organic solvent is 0.008-0.016 mmol / mL; and / or the molar ratio of paclitaxel to the base is 1:7-11; and / or the molar ratio of paclitaxel to the catalyst is 1:0.4-1; Preferably, the reaction system further comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide; preferably, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is added under stirring; and / or the molar ratio of paclitaxel to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:3-6; preferably, the reaction temperature is room temperature.

6. A paclitaxel-retinoic acid prodrug nanomicelle, characterized in that: The paclitaxel-retinoic acid prodrug according to claim 1, 2, 4 or 5, sodium retinoic acid and sodium cholate are self-assembled in water to form paclitaxel-retinoic acid prodrug nanomicelles.

7. The paclitaxel-retinoic acid prodrug nanomicelle according to claim 6, characterized in that: The mass ratio of the paclitaxel-retinoic acid prodrug, sodium retinoate and sodium cholate is 1:0.4-0.8:0.8-1.0; preferably, a mixed solution of paclitaxel-retinoic acid prodrug, sodium retinoate, sodium cholate and an organic solvent is added to water, stirred to self-assemble to form nanoparticles, and the organic solvent in the system is removed to obtain paclitaxel-retinoic acid prodrug nanomicelles; preferably, the organic solvent is an alcohol, preferably methanol; preferably, in the mixed solution, the mass volume ratio of sodium retinoate and the organic solvent is 0.8-1.2 mg / mL; preferably, the mixed solution is obtained by dissolving paclitaxel-retinoic acid prodrug and sodium cholate in an organic solvent solution of sodium retinoate; and the stirring time is 1-5 min.

8. Use of the paclitaxel-retinoic acid prodrug according to claim 1, 2, 4 or 5, or the paclitaxel-retinoic acid prodrug nanomicelle according to any one of claims 6 to 9 in the preparation of an anti-pancreatic cancer drug.

9. An anti-pancreatic cancer drug, characterized in that: The invention comprises the paclitaxel-retinoic acid prodrug according to claim 1, 2, 4 or 5, or the paclitaxel-retinoic acid prodrug nano-micelle according to any one of claims 6 to 9.

10. The use according to claim 8 or the medicine according to claim 9, characterized in that The anti-pancreatic cancer treatment is targeted treatment of pancreatic cancer.

Images