Reduction-sensitive tetravalent oxaliplatin prodrug as well as preparation method and application thereof

Abstract

The invention relates to a reduction-sensitive tetravalent oxaliplatin prodrug as well as a preparation method and application thereof, and belongs to the technical field of medicines. The preparation method comprises the following steps: firstly, preparing a reduction-sensitive tetravalent oxaliplatin prodrug which has a structural formula shown in the specification and can be used for reducing the systematic toxicity of oxaliplatin to achieve the effects of enhancing the effect and reducing the toxicity; and then the oxaliplatin is combined with a ferroptosis inducer RSL3 to form a co-assembled nano preparation, so that the problems of drug resistance of oxaliplatin and the like are solved. The oxaliplatin prodrug disclosed by the invention can be reduced in a high-GSH environment of a tumor site, so that the oxaliplatin parent drug and RSL3 are released after the drug reaches the tumor site, tumor cells are killed, and the profound significance for enhancing the anti-tumor curative effect is achieved. The synthesis method of the oxaliplatin prodrug and the preparation method of the nano preparation provided by the invention are simple and easy to implement, and are beneficial to expanding the industrial production of the nano drug and solving the problem of clinical transformation. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a reduction-sensitive tetravalent oxaliplatin prodrug, a preparation method thereof and an application thereof, and belongs to the technical field of medicine. Background Art

[0002] The discovery and development of platinum drugs is one of the typical cases in the field of anticancer chemotherapy drugs. In the existing clinical chemotherapy combination drug regimens, 70% - 80% are mainly based on platinum drugs or have them participate in the compatibility, which is an important part of clinical chemotherapy administration. Among them, oxaliplatin, as the third-generation platinum drug, has a unique activity spectrum and cell damage recognition mechanism. Compared with cisplatin, at the same dose, oxaliplatin can generate fewer DNA adducts, but can produce stronger cytotoxicity. Although platinum drugs are widely used, they still have the following limitations: 1) Severe toxic side effects. Platinum drugs are rapidly absorbed, but their distribution is non-specific, resulting in toxicity to normal tissues, such as gastrointestinal toxicity, hematotoxicity, neurotoxicity, etc.; 2) Drug resistance problem. Platinum drugs will be "detoxified" by binding to high concentrations of glutathione (GSH) in tumor cells, resulting in drug resistance. In addition, the epithelial-mesenchymal transition (EMT) of tumor cells is also one of the important reasons for drug resistance.

[0003] The emergence of tetravalent platinum (Pt(IV)) provides a new option for improving the defects of platinum drugs. Traditional platinum drugs all exist in the form of divalent platinum (Pt(II)), and through a simple oxidation step, Pt(IV) can be obtained. Due to the structural inertness of the octahedral six-coordination mode of tetravalent platinum, tetravalent platinum needs to be reduced to active divalent platinum to exert its anti-tumor efficacy. This process not only ensures its safety to normal tissues, but also can release Pt(II) under the action of high concentrations of reducing substances (-GSH) in tumor cells, thereby damaging the DNA of tumor cells and alleviating the drug resistance problem to a certain extent. In addition, the two hydroxyl groups of Pt(IV) can be further modified to develop functional Pt(IV) prodrugs. However, the complex drug resistance problem of platinum drugs still needs to be solved.

[0004] Ferroptosis is a form of iron-dependent regulated cell death driven by excessive lipid peroxidation and has become a new target for cancer treatment. As a typical antioxidant enzyme, GPX4 is one of the main inhibitors of ferroptosis and can catalyze the degradation of intracellular lipid peroxides into non-toxic products. Ferroptosis is related to the drug resistance of various cancers, and those drug-resistant cancer cells with a high mesenchymal state rely on GPX4. GPX4 plays an important role in the drug resistance of cancer cells to various therapies, and the combined treatment of chemotherapeutic drugs and GPX4 inhibitors can effectively reduce the number of drug-resistant cells. Therefore, targeting GPX4 and inducing ferroptosis combined with platinum chemotherapy can effectively kill a large number of mesenchymal tumor cells generated during the EMT transformation, which may provide a new effective solution to overcome platinum drug resistance. RSL3 is one of the classical ferroptosis inducers, which can covalently bind to GPX4 and inactivate it, resulting in the accumulation of intracellular peroxides and triggering ferroptosis. Therefore, combining RSL3 with tetravalent platinum prodrugs can achieve the synergistic anti-cancer effect of ferroptosis combined with chemotherapy. On the one hand, ferroptosis improves the platinum resistance problem related to GSH by interfering with the GSH pathway; on the other hand, EMT-resistant tumor cells are highly sensitive to ferroptosis, further improving the drug resistance problem. However, RSL3 has problems such as poor solubility, poor delivery and uptake efficiency, non-specific distribution of targets, serious toxic and side effects, and the risk of toxicity superposition usually brought by multi-drug combination. Therefore, to achieve the effective combination of platinum drugs and ferroptosis inducers, more advanced drug delivery platforms need to be developed to improve the oxaliplatin resistance problem and enhance its anti-tumor efficacy.

[0005] The emergence of nanotechnology has enriched the application of anti-cancer drug delivery strategies, with various advantages such as improving the physicochemical properties of drugs, prolonging blood circulation time, enhancing the targeting of drugs, and enhancing anti-tumor effects. Among them, nano-drugs prepared by self-assembly of prodrug molecules have great application potential, showing ultra-high drug loading capacity and avoiding adverse reactions caused by excipients. In addition, most small molecule prodrug nanoparticles are simple to prepare, which is beneficial to expand industrial production and solve the problem of clinical transformation. Small molecule prodrug nanoparticles are widely used in combination therapy by co-delivering multiple drugs, such as chemotherapeutic drugs, photosensitizers, and immunomodulatory molecules. The multi-modal combined small molecule prodrug nano-delivery system is expected to solve the problems of tumor heterogeneity and drug resistance. The present invention achieves a better combined drug delivery effect by combining small molecule prodrug nanoparticles with ferroptosis inducers, effectively solving the problems of tumor heterogeneity and drug resistance.

[0006] In summary, designing and synthesizing tetravalent oxaliplatin prodrugs through structural modification and then forming co-assembled nanoparticles with the ferroptosis inducer RSL3 is of great significance for improving oxaliplatin resistance and enhancing anti-tumor efficacy. Summary of the Invention

[0007] To solve the problems of drug resistance of oxaliplatin in the existing technology and the poor solubility, delivery and uptake efficiency, non-specific distribution of the target, and serious toxic and side effects of the ferroptosis inducer RSL3, the present invention provides a reduction-sensitive tetravalent oxaliplatin prodrug, its preparation method and application. The present invention designs and synthesizes a novel reduction-sensitive tetravalent oxaliplatin prodrug, and then forms a co-assembled nanoformulation with the ferroptosis inducer RSL3. The co-assembled nanoformulation provided by the present invention enables oxaliplatin to stably exist in a normal physiological environment and achieve specific release in an abnormally highly reducing environment within tumor cells, thereby synchronously killing sensitive cells and drug-resistant cells, and having excellent prospects for clinical transformation.

[0008] To achieve the above object, the technical solution of the present invention is as follows:

[0009] A reduction-sensitive tetravalent oxaliplatin prodrug, the structural formula of the prodrug is shown as follows:

[0010]

[0011] Another object of the present invention is to provide a preparation method of a reduction-sensitive tetravalent oxaliplatin prodrug, comprising the following steps:

[0012] S1: React an aqueous solution of oxaliplatin and an aqueous solution of oxidant hydrogen peroxide under nitrogen conditions with stirring in the dark to obtain tetravalent oxaliplatin;

[0013] S2: Add a linoleic acid solution to a catalyst N,N'-dicyclohexylcarbodiimide solution, and react with stirring under nitrogen conditions to obtain an intermediate linoleic anhydride;

[0014] S3: Dissolve the tetravalent oxaliplatin obtained in S1 and the linoleic anhydride obtained in S2 in N,N-dimethylformamide, and carry out a heating and stirring reaction under nitrogen conditions. After completion, separate and purify to obtain a reduction-sensitive tetravalent oxaliplatin prodrug.

[0015] In the method of the present invention, in S1, the volume ratio of the aqueous solution of oxaliplatin to the aqueous solution of hydrogen peroxide is 10:5 to 7.

[0016] Furthermore, the mass-volume ratio of oxaliplatin to deionized water in the aqueous solution of oxaliplatin is 198.6 mg:8 to 12 mL.

[0017] Furthermore, the mass fraction of the aqueous solution of hydrogen peroxide is 20% to 40%.

[0018] In the method of the present invention, in S2, the volume ratio of the linoleic acid solution to the N,N'-dicyclohexylcarbodiimide solution is 10:1 to 3.

[0019] Further, the mass-volume ratio of linoleic acid to dichloromethane in the linoleic acid solution is 280 mg: 4-6 mL.

[0020] Further, the mass-volume ratio of N,N'-dicyclohexylcarbodiimide to dichloromethane in the N,N'-dicyclohexylcarbodiimide solution is 103 mg: 0.5-1.5 mL.

[0021] In the method of the present invention, in S3, the mass-volume ratio of tetravalent oxaliplatin to N,N-dimethylformamide is 50 mg: 8-12 mL; the mass-volume ratio of linoleic anhydride to N,N-dimethylformamide is 163 mg: 8-12 mL.

[0022] A preferred technical solution of the present invention:

[0023] A preparation method of a reduction-sensitive tetravalent oxaliplatin prodrug, comprising the following steps:

[0024]

[0025] S1: Oxaliplatin is dissolved in deionized water under stirring at a rotation speed of 45 °C and 100 rpm. After the solution becomes clear, an oxaliplatin aqueous solution is obtained. Then, an aqueous hydrogen peroxide solution is added, and stirring reaction is continued under nitrogen protection in the dark for 24 h and then stopped. Recrystallization is carried out overnight at 4 °C, centrifuged, the precipitate is collected, washed 3 times each with pre-cooled ethanol and ether in sequence, and dried to obtain tetravalent oxaliplatin;

[0026]

[0027] S2: Linoleic acid is dissolved in dichloromethane and stirred at 100-200 rpm in an ice-water bath for 1.5-2.5 h to obtain a linoleic acid solution; N,N'-dicyclohexylcarbodiimide is dissolved in dichloromethane and then added to the above linoleic acid solution. Stirring reaction is carried out at 100-200 rpm in an ice bath under nitrogen protection for 2 h. After completion, filtration is carried out, and the organic solvent is evaporated under vacuum to obtain the intermediate linoleic anhydride;

[0028]

[0029] S3: The tetravalent oxaliplatin obtained in S1 and the linoleic anhydride obtained in S2 are dissolved in N,N-dimethylformamide, stirred at 100-200 rpm at 75 °C for 10 h, the organic solvent is rotary evaporated, and after liquid-phase purification, a reduction-sensitive tetravalent oxaliplatin prodrug is obtained.

[0030] The tetravalent oxaliplatin prodrug provided by the present invention and the tetravalent oxaliplatin prodrug prepared by the above method can release the parent drug oxaliplatin after being activated by reduction substances.

[0031] Another object of the present invention is to provide a co-assembled nanoparticle based on the above-mentioned reduction-sensitive tetravalent oxaliplatin prodrug, and the co-assembled nanoparticle is a co-assembled nanoparticle of a reduction-sensitive oxaliplatin prodrug modified by a PEG modifier and a ferroptosis inducer RSL3.

[0032] Furthermore, the function of the ferroptosis inducer RSL3 is to be able to inhibit the level of GPX4 in tumor cells.

[0033] In the above technical solution, the molar ratio of the reduction-sensitive tetravalent oxaliplatin prodrug to the ferroptosis inducer RSL3 is 4:1.

[0034] In the above technical solution, the mass ratio of the sum of the masses of the reduction-sensitive tetravalent oxaliplatin prodrug and the ferroptosis inducer RSL3 to the mass of the PEG modifier is 5:1.

[0035] In the above technical solution, the particle size of the co-assembled nanoparticle is 120-140 nm.

[0036] In the above technical solution, the reduction-sensitive tetravalent oxaliplatin prodrug and the ferroptosis inducer RSL3 are co-assembled through intermolecular forces, and the intermolecular forces are electrostatic forces, hydrophobic forces and hydrogen bonds. Among them, the electrostatic force is the dominant force.

[0037] In the above technical solution, the PEG modifier is DSPE-PEG 2K 。

[0038] Another object of the present invention is to provide a preparation method of the co-assembled nanoparticle based on the above-mentioned reduction-sensitive tetravalent oxaliplatin prodrug, including the following steps: dissolving the reduction-sensitive tetravalent oxaliplatin prodrug, the ferroptosis inducer RSL3 and the PEG modifier into an organic solvent respectively, slowly dropping the mixed solution into water, stirring, and spontaneously forming a uniform co-assembled nanoparticle, and removing the organic solvent to obtain the product.

[0039] Furthermore, the organic solvent is methanol.

[0040] Furthermore, the stirring condition is stirring at 1000-1600 rpm for 20-40 min.

[0041] The tetravalent oxaliplatin prodrug of the present invention is a novel reduction-sensitive tetravalent oxaliplatin prodrug, which can be reduced in a high-GSH environment at the tumor site to release the parent drug oxaliplatin; at the same time, the co-assembled nanoparticle formed by co-assembling the above-mentioned oxaliplatin prodrug and the ferroptosis inducer RSL3 can release the oxaliplatin parent drug and RSL3 after reaching the tumor site, kill tumor cells, improve the problem of oxaliplatin resistance, synergistically induce tumor death, and achieve a high anti-tumor effect.

[0042] Another object of the present invention is to provide the use of the above-mentioned reduction-sensitive tetravalent oxaliplatin prodrug or co-assembled nanoparticles based on the above-mentioned reduction-sensitive tetravalent oxaliplatin prodrug in the preparation of a drug delivery system.

[0043] Another object of the present invention is to provide the use of the above-mentioned reduction-sensitive tetravalent oxaliplatin prodrug or co-assembled nanoparticles based on the above-mentioned reduction-sensitive tetravalent oxaliplatin prodrug in the preparation of drugs for cancer targeted therapy or the research and development of drugs to overcome drug resistance.

[0044] Advantages of the present invention:

[0045] 1. The present invention prepares oxaliplatin into a reduction-sensitive prodrug, reducing the systemic toxicity of oxaliplatin; and the reduction-sensitive tetravalent oxaliplatin prodrug provided by the present invention can achieve specific release of the parent drug oxaliplatin at the tumor site, achieving the effect of enhancing efficacy and reducing toxicity.

[0046] 2. The present invention combines the ferroptosis inducer RSL3 with the tetravalent oxaliplatin prodrug provided by the present invention to overcome the problem of oxaliplatin drug resistance.

[0047] 3. The synthesis method of the oxaliplatin prodrug and the preparation method of the nanoformulation provided by the present invention are simple and easy to implement. Description of the drawings

[0048] Figure 1 1H NMR spectrum and mass spectrum of the tetravalent oxaliplatin prodrug obtained in Example 1 of the present invention. 1 H NMR spectrum and mass spectrum.

[0049] Figure 2 Characterization diagram of OL@RNPs obtained in Example 2 of the present invention.

[0050] Figure 3 In vitro release experiment diagram and cytotoxicity diagram of OXA solution (OXA Sol), OXA+RSL3 solution (OR Sol), OL NPs and OL@RNPs in Example 2 of the present invention.

[0051] Figure 4 In vivo anti-tumor efficacy diagram of OXA solution (OXA Sol), OXA+RSL3 solution (OR Sol), OL NPs and OL@RNPs in Example 2 of the present invention.

[0052] Figure 5 Safety evaluation diagram of OXA solution (OXA Sol), OXA+RSL3 solution (OR Sol), OL NPs and OL@RNPs in Example 2 of the present invention. Detailed implementation manners

[0053] The following non-limiting examples can enable those of ordinary skill in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.

[0054] In the following examples, the test methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0055] Example 1

[0056] A preparation method of a reduction-sensitive tetravalent oxaliplatin prodrug, comprising the following steps:

[0057] S1: Under stirring at 45 °C and 100 rpm, dissolve oxaliplatin (0.5 mmol, 198.6 mg) in 10 mL of deionized water. After the solution becomes clear, add 6 mL of 30% H 2 O 2 , protect from light, continue stirring and reacting under nitrogen protection for 24 h, then stop stirring. Place the reaction solution at 4 °C for overnight recrystallization, centrifuge, collect the precipitate, wash it 3 times each with pre-cooled ethanol and ether, and dry it to obtain 156.3 mg of tetravalent oxaliplatin (OXA(IV)-2OH), which is a white powder with a yield of 72.5%;

[0058] S2: Dissolve linoleic acid (280 mg, 10 mmol) in 5 mL of dichloromethane (DCM), cool it in an ice-water bath, stir at a rate of 200 rpm, dissolve N,N'-dicyclohexylcarbodiimide (DCC, 103 mg, 5 mmol) in 1 mL of dichloromethane, and then add it to the above-obtained linoleic acid solution. Stir and react at 200 rpm in an ice bath under nitrogen protection for 2 h. After the reaction is completed, filter, and evaporate the solvent under vacuum to obtain the product linoleic anhydride;

[0059] S3: Add the OXA(IV) (50 mg, 0.15 mmol) obtained in S1 and the linoleic anhydride (163 mg, 0.3 mmol) obtained in S2 to 10 mL of N,N-dimethylformamide (DMF), stir at 200 rpm at 75 °C for 10 h. After the reaction is completed, use an oil pump and a rotary evaporator to evaporate the solvent to dryness, and purify it using a preparative liquid chromatograph (detection wavelength: 220 nm, mobile phase: 100% methanol) to obtain 176.7 mg of the tetravalent oxaliplatin prodrug, which is a pale yellow solid (OXA-LA) with a yield of 67%.

[0060] Use high-resolution mass spectrometry and proton nuclear magnetic resonance spectroscopy to confirm the structure of the above-obtained OXA-LA; use high-performance liquid chromatography to determine its purity.

[0061] The mass spectrum of OXA-LA, as Figure 1As shown in A, the mass spectrometry analysis results are as follows:

[0062] ESI-MS(m / z): [C 26 H 46 N 2 O 7 PtN][M-H] - =692.25 (calculated [M-H] - : 692.28).

[0063] The nuclear magnetic resonance spectrum of OXA-LA is as shown in Figure 1 B, and the nuclear magnetic resonance spectrum analysis results are as follows:

[0064] 1 H-NMR(400MHz, DMSO): 7.73(s, 4H, -NH 2 -), 5.33(m, 4H, -CH=CH-), 2.51(d, DMSO-d6), 2.18(t, 2H, J=7.5Hz, -CH 2 CO-), 1.98(m, 4H, -CH 2 CH=CHCH 2 -), 1.83(d, 10H, hexamethylene), 1.49(d, 2H, -CH 2 CH 2 CO-), 1.22(m, 20H, -CH 2 CH 2 CH 2 -), 0.86(t, 3H, -CH 2 CH 3 ).

[0065] Example 2

[0066] A preparation method of reduction-sensitive tetravalent oxaliplatin prodrug co-assembled nanoparticles (OL@RNPs) includes the following steps:

[0067] Weigh 5 mg of OXA-LA and ferroptosis inducer RSL3 obtained in Example 1 respectively, dissolve them in 1 mL of methanol solution to obtain 5 mg / mL OXA-LA solution and RSL3 solution; take 1 mg of OXA-LA as the mass, mix OXA-LA and RSL3 according to a molar ratio of 4:1, and then add 0.25 mg of DSPE-PEG 2K , to obtain a mixed solution; slowly drop the above mixed solution into 2 mL of deionized water, stir at a speed of 1300 rpm for 30 min to prepare co-assembled nanoparticles OL@RNPs wrapped with PEG.

[0068] With the mass of OXA-LA being 1 mg and the molar ratio of OXA-LA to RSL3 being 1:0, OLNPs were prepared by the same operation process. After diluting the OL NPs and the above-obtained OL@RNPs 10-fold with deionized water, the particle size, PDI, and Zeta potential were measured using a Malvern particle size analyzer. In addition, the prepared OL NPs and OL@R NPs were diluted 20-fold with PBS (pH = 7.4). 10 μL was taken and dropped onto a copper mesh covered with a carbon film. After standing for 30 s, it was suctioned with filter paper until only one layer of liquid remained. After naturally evaporating the remaining layer of liquid, negative staining was performed with 2% phosphotungstic acid, and the appearance morphologies of OL NPs and OL@RNPs were observed using a transmission electron microscope (TEM). The results are as Figure 2 shown. It can be seen that the average particle size of OL NPs is about 150 nm, and the average particle size of OL@R NPs is about 130 nm. The Zeta potential of both is in the range of -15 to -30 mV. In addition, the TEM images show that the appearances of OL NPs and OL@R NPs both present uniform spherical shapes.

[0069] For the stability evaluation of the obtained OL@RNPs in this example, with the change in its particle size as the index: 1 mL of OL@RNPs (concentration 0.5 mg / mL) was added to 9 mL of PBS (pH 7.4) and PBS (pH 7.4) containing 10% fetal bovine serum respectively, and incubated in a constant temperature shaker at 37 °C (rotation speed 100 rpm). The particle size changes were measured at preset time points (0, 1, 2, 4, 6, 8, 10, and 12 h). The results are as Figure 3 shown. It can be seen that the OL@RNPs modified with DSPE-PEG 2K can stably exist in PBS (pH 7.4), which is attributed to the PEG layer being able to effectively prevent salting-out. In addition, OL@RNPs can also stably exist in PBS (pH 7.4) containing 10% FBS, which provides conditions for the in vivo delivery of the nanoparticles.

[0070] In vitro release experiment of the obtained OL@RNPs in this example:

[0071] Using vitamin C (VC) as a reducing agent, PBS (pH 7.4) containing 5% dimethyl sulfoxide as the release medium, the in vitro release pattern was studied by dialysis method. Specific operation: Add 1 mL of OL@RNPs into the dialysis bag, then place the dialysis bag into 9 mL of release medium containing 0 or 10 mM VC, and incubate in a constant temperature shaking incubator at 37 °C (rotation speed is 100 rpm) to determine the release of oxaliplatin (OXA) and RSL3 in the release medium VC. At preset time points (1, 2, 4, 8, 12, and 24 h), take out 200 μL of the release medium and supplement the same volume of release medium. High performance liquid chromatography was used to determine the drug concentrations of OXA and RSL3 in the samples, and calculate the cumulative release amount and cumulative release percentage of the drugs. The results are as Figure 3 shown. It can be seen that the OL@RNPs provided by the present invention have the characteristics of reduction-sensitive drug release, can respond to the specific oxidative environment of tumor tissues, and achieve site-specific drug release at the tumor site.

[0072] Cytotoxicity experiment of the OL@RNPs obtained in this example:

[0073] The MTT method was used to evaluate the cytotoxicity of OL@RNPs. Specific operation: Inoculate A549 and LLC cells into 96-well plates at a density of 2×10 3 cells / well, and culture in an incubator containing 5% CO 2 at 37 °C for 12 h; then treat the cells with different concentrations of OXA solution (OXA Sol), OXA+RSL3 solution (OR Sol, that is, simply mix OXA and RSL3), OL NPs and OL@RNPs for 72 h, where the OXA concentration in the above solutions is the same; then, co-incubate with 5 mg / mL MTT (25 μL / well) at 37 °C for 4 h; discard the culture medium, and use DMSO (200 μL / well) to replace the culture medium to dissolve the generated formazan; finally, use a multifunctional microplate reader to measure the ultraviolet absorbance at 490 nm. The MTT results are as Figure 3 shown. It can be seen that the cytotoxicity of the combined treatment groups (i.e., the OXA+RSL3 solution group and the OL@R NPs group) is significantly greater than that of the OXA sol treatment group, which confirms the synergistic effect between OXA and RSL3; in addition, due to its efficient cell uptake and rapid intracellular drug release, OL@RNPs exhibit higher cytotoxicity than the OXA+RSL3 solution group.

[0074] It can be seen from the above in vitro release experiment and cytotoxicity experiment that the OL@RNPs provided by the present invention can respond to the specific redox environment of the tumor site, achieve site-specific drug release at the tumor site, and the combined use of OXA and RSL3 can effectively kill tumor cells and improve the anti-tumor effect of oxaliplatin.

[0075] In vivo pharmacodynamic experiment of the OL@RNPs obtained in this example:

[0076] The LLC cells were subcultured. When the cells grew to 80%, the LLC cells were digested with trypsin and the digestion was terminated with DMEM medium; the cells were collected by centrifugation and redispersed in PBS (pH 7.4) to a concentration of 5×10 7 cells / mL; 100 μL of the above cell suspension was subcutaneously injected into the right back of the mice to establish an LLC tumor-bearing mouse model. When the average tumor volume of the mice grew to about 150 mm 3 , the mice were randomly divided into 5 groups: control group (PBS), OXA Sol, OR Sol, OL NPs, and OL@R NPs. The drugs were administered every other day for a total of 5 times at an equal concentration of OXA (4 mg / kg). At the same time, the tumor volume and body weight of the mice were measured and recorded every day. Two days after the last treatment, the mice were weighed and sacrificed, the tumor tissues were separated, weighed and photographed, and the tumor-bearing rate was calculated according to the following formula:

[0077]

[0078] The results are as Figure 4 shown. It can be seen that the combined use of RSL3 and OXA is more effective than the use of OXA alone; OL@RNPs almost completely inhibited tumor growth throughout the treatment process. The two drugs in the OL@RNPs provided by the present invention can efficiently and synchronously reach the tumor site, have a synergistic anti-tumor effect, and improve the problem of oxaliplatin resistance.

[0079] Safety experiment of the OL@RNPs obtained in this example:

[0080] After administration, the body weight of the mice was measured every day, and the changes in the body weight of the mice in different administration groups were plotted. The safety of the drugs was preliminarily evaluated based on the changes in the body weight of the mice. Two days after the last treatment of the pharmacodynamics, the eyeballs were removed, blood was collected, the serum was obtained after centrifugation of the samples, and liver and kidney function analyses were performed. Among them, ALT (alanine aminotransferase), AST (aspartate aminotransferase), UREA (urea), and CREA (creatinine) were used as indicators to evaluate the liver and kidney function toxicity of the drugs. The results are as Figure 5 shown. It can be seen that OXA Sol and OXA+RSL3 Sol reduced the body weight of the tumor-bearing mice, but OL NPs and OL@RNPs had little effect on the body weight of the mice; in addition, no significant abnormalities were observed in the blood parameters of the liver and kidneys after treatment, indicating that OL@RNPs have good safety.

Claims

1. A reduction-sensitive tetravalent oxaliplatin prodrug, characterized in that: The structural formula of the prodrug is shown below:

2. A method for preparing a reduction-sensitive tetravalent oxaliplatin prodrug, characterized in that: The following steps are involved: S1: reacting an oxaliplatin aqueous solution and an oxidizing agent, hydrogen peroxide aqueous solution, with stirring under nitrogen and in the dark to obtain tetravalent oxaliplatin; S2: adding the linoleic acid solution to the catalyst N,N'-dicyclohexylcarbodiimide solution, stirring and reacting under nitrogen conditions to obtain the intermediate linoleic anhydride; S3: The tetravalent oxaliplatin obtained in S1 and the linoleic anhydride obtained in S2 are dissolved in N,N-dimethylformamide, and heated and stirred to react under nitrogen conditions, and then separated and purified to obtain a reduction-sensitive tetravalent oxaliplatin prodrug.

3. The preparation method according to claim 2, characterized in that: In S1, the volume ratio of the oxaliplatin aqueous solution to the hydrogen peroxide solution is 10:5-7, wherein the mass volume ratio of oxaliplatin to deionized water in the oxaliplatin aqueous solution is 198.6 mg:8-12 mL; the mass fraction of the hydrogen peroxide aqueous solution is 20%-40%; In S2, the volume ratio of the linoleic acid solution to the N,N'-dicyclohexylcarbodiimide solution is 10:1-3, wherein the mass volume ratio of linoleic acid to dichloromethane in the linoleic acid solution is 280 mg:4-6 mL; the mass volume ratio of N,N'-dicyclohexylcarbodiimide to dichloromethane in the N,N'-dicyclohexylcarbodiimide solution is 103 mg:0.5-1.5 mL; In S3, the mass volume ratio of tetravalent oxaliplatin to N,N-dimethylformamide is 50 mg:8-12 mL; the mass volume ratio of linoleic anhydride to N,N-dimethylformamide is 163 mg:8-12 mL.

4. The preparation method according to claim 2 or 3, characterized in that: The following steps are involved: S1: dissolving oxaliplatin in deionized water at 45°C and 100 rpm with stirring, obtaining an oxaliplatin aqueous solution after the solution is clarified, then adding an aqueous hydrogen peroxide solution, stirring and reacting for 24 hours under nitrogen protection and in the dark, then stopping, recrystallizing at 4°C overnight, centrifuging, collecting the precipitate, washing with pre-cooled ethanol and ether three times each, and drying to obtain tetravalent oxaliplatin; S2: dissolving linoleic acid in dichloromethane, stirring at 100-200 rpm in an ice-water bath for 1.5-2.5 hours to obtain a linoleic acid solution; dissolving N,N'-dicyclohexylcarbodiimide in dichloromethane and adding the mixture to the linoleic acid solution, stirring at 100-200 rpm in an ice-water bath under nitrogen protection for 2 hours, filtering after completion, and evaporating the organic solvent in vacuo to obtain an intermediate linoleic anhydride; S3: The tetravalent oxaliplatin obtained in S1 and the linoleic anhydride obtained in S2 are dissolved in N,N-dimethylformamide, stirred at 75°C at 100-200 rpm for 10 hours, and the organic solvent is rotary evaporated to obtain a reduction-sensitive tetravalent oxaliplatin prodrug after liquid phase purification.

5. A co-assembled nanoparticle based on the reduction-sensitive tetravalent oxaliplatin prodrug according to claim 1, characterized in that: The co-assembled nanoparticles are reduction-sensitive tetravalent oxaliplatin prodrugs modified with a PEG modifier and co-assembled nanoparticles with a ferroptosis inducer RSL3.

6. The co-assembled nanoparticles according to claim 5, characterized in that: The molar ratio of the reduction-sensitive tetravalent oxaliplatin prodrug to the ferroptosis inducer RSL3 is 4:1; the mass ratio of the sum of the masses of the reduction-sensitive tetravalent oxaliplatin prodrug and the ferroptosis inducer RSL3 to the mass of the PEG modifier is 5:

1.

7. The co-assembled nanoparticles according to claim 5, characterized in that: The particle size of the co-assembled nanoparticles is 120 to 140 nm; The reduction-sensitive tetravalent oxaliplatin prodrug and the ferroptosis inducer RSL3 are co-assembled through intermolecular forces, wherein the intermolecular forces are electrostatic forces, hydrophobic forces and hydrogen bonds, among which the electrostatic forces play a dominant role.

8. The method for preparing co-assembled nanoparticles according to any one of claims 5 to 7, characterized in that: The method comprises the following steps: dissolving a reduction-sensitive tetravalent oxaliplatin prodrug, a ferroptosis inducer RSL3 and a PEG modifier in organic solvents respectively, slowly dropping the mixed solution into water, stirring, spontaneously forming uniform co-assembled nanoparticles, and removing the organic solvent to obtain the nanoparticles.

9. Use of the reduction-sensitive tetravalent oxaliplatin prodrug according to claim 1 or the co-assembled nanoparticles based on the reduction-sensitive tetravalent oxaliplatin prodrug according to any one of claims 5 to 7 in the preparation of a drug delivery system.

10. Use of the reduction-sensitive tetravalent oxaliplatin prodrug of claim 1 or the co-assembled nanoparticles based on the reduction-sensitive tetravalent oxaliplatin prodrug of any one of claims 5 to 7 in the preparation of cancer targeted therapy or drug development to overcome drug resistance.

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