Disulfide-bridged SN38 dimer prodrug and self-assembled nanoparticles thereof, preparation method and application

Abstract

The disulfide-bridged SN38 dimer prodrug and its self-assembled nanoparticles, preparation method and application belong to the field of medical technology, and relate to the preparation of disulfide-bridged SN38 dimer prodrug and its self-assembled nanoparticles, and its application in drug delivery. The preparation method of the present invention is simple and easy, and the effect of the length of the connecting chain containing disulfide bonds on the chemical stability and self-assembly ability of the SN38 dimer prodrug, and the resulting effect on the drug release behavior, cytotoxicity, pharmacokinetics, tissue distribution and anti-tumor effect of the prodrug nanoassembly are investigated. It provides new ideas for the development of SN38 nanoformulations, solves the urgent demand for high-efficiency and low-toxic chemotherapy preparations in clinical practice, and the demand of enterprises for the development of SN38 nanoformulations.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and relates to a preparation method and application of a disulfide-bridged SN38 dimer prodrug and a self-assembled nanoparticle thereof, and specifically relates to the synthesis of a SN38 dimer prodrug with disulfide bonds located at adjacent carbonyl α, β and γ positions, the preparation of a SN38 dimer prodrug self-assembled nanoparticle with disulfide bonds located at adjacent carbonyl β and γ positions, and the application of the nanoparticle in drug delivery. Background Art

[0002] Cancer poses a serious threat to human health. Drug therapy is still one of the most commonly used strategies for cancer treatment, especially for advanced tumors, tumors that cannot be removed surgically, and tumors that have metastasized and spread. However, the existing formulation technology in clinical practice has low delivery efficiency, and anti-tumor drugs have poor efficacy and serious adverse reactions. For example, SN38 has super strong pharmacodynamic activity, but its water solubility is extremely low, and it is almost insoluble even in pharmaceutical excipients, which seriously limits its formulation development. Its clinically used formulation irinotecan (a water-soluble prodrug of SN38) cannot be efficiently converted into SN38 in vivo, has limited efficacy and serious toxic side effects. Although there are endless studies on the delivery system of SN38 drugs, SN38 has a poor affinity with most nanocarriers, resulting in the common defects of low drug loading and poor stability of nanoformulations encapsulating SN38. Therefore, it is of great significance to construct a new type of high-efficiency and low-toxic SN38 delivery system.

[0003] Small molecule prodrug self-assembled nanoparticles combine the advantages of prodrugs and nanotechnology. The obtained hydrophobic drugs can spontaneously assemble into nanoformulations in water, with high drug loading, and avoid adverse reactions caused by carrier materials. In addition, the preparation process is simple, reproducible, and has good application transformation prospects. In particular, homodimer prodrug self-assembled nanoparticles have ultra-high drug loading (greater than 50%) and unique structural characteristics, and are expected to efficiently release SN38. However, poor self-assembly ability has become a key factor restricting the application of homodimer prodrug self-assembled nanoparticles. Designing and developing dimeric prodrug self-assembled nanoparticles with good stability remains a major challenge, especially for drugs such as SN38, which have very strong structural rigidity.

[0004] The flexibility of the prodrug structure can significantly affect the self-assembly performance of the prodrug. The longer the carbon chain length, the more σ bonds that can rotate freely in the prodrug structure, the more flexible the prodrug structure, and the more conducive it is for the prodrug to adjust to the lowest energy state during the self-assembly process to form a stable nanostructure. Therefore, by increasing the length of the connecting chain, it is expected to improve the self-assembly ability of the prodrug, thereby improving the stability, in vivo fate and anti-tumor effect of the nanoparticles. In order to efficiently and specifically release active drugs in tumor tissues, disulfide bonds are used to construct tumor microenvironment-sensitive smart responsive prodrugs. Smart responsive prodrugs exist in the form of inactive prodrugs in the systemic circulation to avoid toxic side effects on normal tissues of the body; after reaching the tumor site, the disulfide bonds are quickly broken under the stimulation of the highly expressed glutathione in the tumor microenvironment to release the drug, exerting a highly effective anti-tumor effect. Summary of the invention

[0005] The purpose of the present invention is to design and synthesize SN38 dimer prodrugs with different lengths of disulfide bond-bridged connecting chains, prepare SN38 dimer prodrug self-assembled nano drug delivery systems, and investigate their application in drug delivery. The effects of the length of the connecting chain on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution and pharmacodynamics of prodrug self-assembled nanoparticles are explored to provide new ideas for the development of stable prodrug self-assembled nano drug delivery systems, meet the urgent clinical demand for highly efficient chemotherapy preparations and the needs of enterprises for the development of SN38 nano preparations.

[0006] In order to achieve the above object, the present invention provides a SN38 dimer prodrug bridged by disulfide bonds at different positions as shown in the general formula (I).

[0007]

[0008] Among them, n=1-3.

[0009] Preferably, the disulfide-bridged SN38 dimer prodrug is a SN38 dimer prodrug prepared by connecting SN38 via 2,2'-dithiodiacetic acid, 3,3'-dithiodipropionic acid or 4,4'-dithiodibutyric acid, wherein the disulfide bonds are located at the α, β or γ positions of adjacent carbonyl groups.

[0010] The structure of the SN38 dimer prodrug with the disulfide bond located at the α position of the adjacent carbonyl group is:

[0011]

[0012] The structure of the SN38 dimer prodrug with the disulfide bond located at the β position of the adjacent carbonyl group is:

[0013]

[0014] The structure of the SN38 dimer prodrug with the disulfide bond located at the γ position of the adjacent carbonyl group is:

[0015]

[0016] The present invention provides a method for preparing a disulfide-bridged SN38 dimer prodrug, comprising the following steps:

[0017] 2,2'-dithiodiacetic acid, 3,3'-dithiodipropionic acid or 4,4'-dithiodibutyric acid is dissolved in chloroform and stirred evenly; part of 4-dimethylaminopyridine (DMAP), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and SN38 are dissolved in chloroform and stirred evenly, and then mixed with the above 2,2'-dithiodiacetic acid, 3,3'-dithiodipropionic acid or 4,4'-dithiodibutyric acid solution respectively, and stirred at room temperature for 10-12 hours under nitrogen protection; the remaining EDCI and DMAP are added to the mixed solution, and stirring is continued at room temperature for 12-24 hours under nitrogen protection, and the obtained product is separated and purified by preparative liquid phase.

[0018] A pharmaceutical composition comprising the disulfide-bridged SN38 dimer prodrug, and a pharmaceutically acceptable carrier and excipient.

[0019] The present invention also provides the use of the disulfide-bridged SN38 dimer prodrug or a pharmaceutical composition comprising the prodrug in the preparation of anti-tumor drugs.

[0020] The present invention also provides the use of the disulfide-bridged SN38 dimer prodrug or a pharmaceutical composition comprising the prodrug in the preparation of a drug delivery system.

[0021] The present invention also provides the use of the disulfide-bridged SN38 dimer prodrug or a pharmaceutical composition comprising the prodrug in the preparation of an injection, oral administration or local administration system.

[0022] The present invention also provides self-assembled nanoparticles of the disulfide-bridged SN38 dimer prodrug, wherein the self-assembled nanoparticles may be non-PEGylated dimer prodrug self-assembled nanoparticles or PEG-modified dimer prodrug self-assembled nanoparticles. The preparation method is a nanoprecipitation method, including a high-speed stirring method and an ultrasonic method.

[0023] The preparation method of the disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles is as follows:

[0024] The disulfide-bridged SN38 dimer prodrug and the PEG modifier (the non-PEGylated dimer prodrug self-assembled nanoparticles do not contain the PEG modifier) ​​are dissolved in a solvent, and the solution is slowly added dropwise into water under stirring. The prodrug spontaneously forms uniform nanoparticles. The solvent is removed by reduced pressure distillation to obtain a nanocolloid solution free of organic solvent.

[0025] The PEG modifier is selected from TPGS, DSPE-PEG, PLGA-PEG and PE-PEG, and the preferred PEG modifier is DSPE-PEG. The molecular weight of the PEG is 1000-5000, preferably 1000, 2000 and 5000, and more preferably the molecular weight of the PEG is 2000.

[0026] The solvent is selected from ethanol, dimethyl sulfoxide, N,N'-dimethylformamide, tetrahydrofuran, and acetone.

[0027] The weight ratio of the disulfide-bridged SN38 dimer prodrug to the PEG modifier is 90:10 to 70:30. Under this range, the prodrug nanoparticles can exert a better anti-tumor effect.

[0028] The present invention also provides the use of the disulfide bond-bridged SN38 dimer prodrug self-assembly nanoparticles in the preparation of a drug delivery system.

[0029] The present invention also provides the use of the disulfide-bridged SN38 dimer prodrug self-assembly nanoparticles in the preparation of anti-tumor drugs.

[0030] The present invention also provides the use of the disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles in the preparation of injection, oral or local drug delivery systems.

[0031] The technical problem solved by the present invention is to introduce disulfide bonds into SN38 dimer prodrugs and self-assembled nanoparticles, design disulfide-bridged redox-sensitive dimer prodrugs, and use the dimer prodrugs for the construction of self-assembled nanoparticles, thereby achieving high drug loading, good stability, low toxicity and side effects, and tumor site-specific rapid drug release, thereby improving the therapeutic effect. At the same time, the differences in the self-assembly of SN38 prodrugs with different lengths of disulfide bond-connected chains are investigated, as well as the effects on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution, and pharmacodynamics of prodrug self-assembled nanoparticles.

[0032] The advantages of the present invention are:

[0033] (1) We designed and synthesized SN38 dimer prodrugs containing disulfide bonds of different lengths, and the synthesis method was simple and easy;

[0034] (2) Uniform dimer prodrug self-assembled nanoparticles were prepared. The preparation method is simple and easy to implement, and the drug can be efficiently loaded, with an ultra-high drug loading of more than 63%;

[0035] (3) The differences in self-assembly of disulfide-linked chains of different lengths were investigated, as well as the effects on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution and pharmacodynamics of prodrug self-assembled nanoparticles. Based on the experimental results, the SN38 dimer prodrug with disulfide bonds located at the β and γ positions of adjacent carbonyl groups has suitable chemical stability. At the same time, the SN38 dimer prodrug with disulfide bonds located at the γ positions of adjacent carbonyl groups has the best assembly ability. The present invention provides new strategies and more options for the development of intelligent responsive drug delivery systems for tumor microenvironment, meeting the urgent clinical demand for highly efficient chemotherapy preparations and the needs of enterprises for the development of SN38 nanoformulations. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the mass spectrum of the SN38 dimer prodrug (α-SN38-SS-SN38) in Example 1 of the present invention, in which the disulfide bond is located at the α position adjacent to the carbonyl group.

[0037] Figure 2 This is the structural confirmation of the SN38 dimer prodrug (β-SN38-SS-SN38) in Example 2 of the present invention, in which the disulfide bond is located at the β position of the adjacent carbonyl group.

[0038] A: Mass spectrum of β-SN38-SS-SN38.

[0039] B: β-SN38-SS-SN38 1 H-NMR spectrum.

[0040] Figure 3 This is the structural confirmation of the SN38 dimer prodrug (γ-SN38-SS-SN38) in Example 3 of the present invention, in which the disulfide bond is located at the γ position adjacent to the carbonyl group.

[0041] A: Mass spectrum of γ-SN38-SS-SN38.

[0042] B:γ-SN38-SS-SN38 1 H-NMR spectrum.

[0043] Figure 4 The chemical stability of the disulfide-bridged SN38 dimer prodrug in Example 4 of the present invention at room temperature.

[0044] Figure 5 This is the purity confirmation of the disulfide-bridged SN38 dimer prodrug prepared in Example 5 of the present invention.

[0045] A: Purity results of β-SN38-SS-SN38.

[0046] B: Purity results of γ-SN38-SS-SN38.

[0047] Figure 6 This is the stability result of the non-PEGylated disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles in Example 6 of the present invention.

[0048] A: Stability of non-PEGylated disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles at room temperature for 48 h.

[0049] B: Formulation properties of non-PEGylated prodrug nanoparticles at 0 h.

[0050] C: Formulation properties of non-PEGylated prodrug nanoparticles at 3 h.

[0051] Figure 7 This is the stability result of the PEG-modified disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles in Example 6 of the present invention.

[0052] A: Stability of PEG-modified disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles at room temperature for 48 h.

[0053] B: Formulation properties of PEG-modified prodrug nanoparticles at 0 h.

[0054] C: Formulation properties of PEG-modified prodrug nanoparticles at 4 h.

[0055] Figure 8 This is an in vitro release test of the PEG-modified disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles under reducing and oxidizing conditions in Example 7 of the present invention.

[0056] A: Blank media.

[0057] B: 0.1 mM DTT was added to the medium.

[0058] C: 1 mM DTT was added to the medium.

[0059] D: Add 0.1 mM H2O2 to the medium.

[0060] E: Add 1 mM H2O2 to the medium.

[0061] Fig. 9 This is a diagram showing the cytotoxicity of the PEG-modified disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles in Example 8 of the present invention.

[0062] A: The half inhibitory concentration of disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles on 4T1 cells.

[0063] B: The half-maximal inhibitory concentration of disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles on CT26 cells.

[0064] C: The half inhibitory concentration of disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles on L02 cells.

[0065] Fig.10 These are the in vivo anti-tumor experimental results of the disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles in Example 10 of the present invention.

[0066] A: Growth curve of Balb / C tumor-bearing mice after treatment with disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles.

[0067] B: Tumor bearing rate of Balb / C tumor-bearing mice after treatment with disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles.

[0068] C: Tumor photos of Balb / C tumor-bearing mice after treatment with disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles.

[0069] D: Body weight changes of Balb / C tumor-bearing mice after treatment with disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles. DETAILED DESCRIPTION

[0070] The present invention is further described below by way of examples, but the invention is not limited to the scope of the examples.

[0071] Example 1: Synthesis of SN38 dimer prodrug with disulfide bonds located at the α position of adjacent carbonyl groups (α-SN38-SS-SN38)

[0072] 2,2'-Dithiodiacetic acid (182.2 mg, 1 mmol), EDCI (287.6 mg, 1.5 mmol) and DMAP (122.2 mg, 1 mmol) were added to a 50 mL round-bottom flask and dissolved with 20 mL chloroform. Then a chloroform solution (5 mL) containing SN38 (784.8 mg, 2 mmol) was added dropwise. The reaction was allowed to react at room temperature for 10 h under nitrogen protection. EDCI (191.7 mg, 1 mmol) and DMAP (61.1 mg, 0.5 mmol) were then added to the reaction system, and the reaction was continued for 12 h under nitrogen protection. The reaction progress was monitored by thin layer chromatography. The chloroform in the system was removed by a rotary evaporator, and the product was dissolved in DMF and separated and purified by preparative liquid phase (acetonitrile: water = 70:30, 1‰ formic acid) (8.5 mg, yield 0.9%).

[0073] Mass spectrometry was used to determine the molecular weight of α-SN38-SS-SN38 prepared in Example 1. The results are as follows: Figure 1 As shown. [M+H] was detected + Peak 931.23 proved that α-SN38-SS-SN38 was successfully synthesized.

[0074] Example 2: Synthesis of SN38 dimer prodrug with disulfide bond located at the β position of adjacent carbonyl group (β-SN38-SS-SN38)

[0075] The preparation method of Example 1 was used to replace 2,2'-dithiodiacetic acid with 3,3'-dithiodipropionic acid to obtain a β-position disulfide-bridged SN38 dimer prodrug.

[0076] The structure of β-SN38-SS-SN38 prepared in Example 2 was determined by mass spectrometry and nuclear magnetic resonance hydrogen spectroscopy. Figure 2 shown.

[0077] The results of NMR spectroscopy analysis are as follows:

[0078] 1 H NMR(400MHz,CDCl3)ppm 7.84(d,2H,Ar-H,J=9.14Hz),7.58(d,2H,Ar-H,J=2.38Hz),7.42(dd,2H,Ar-H,J=9.14,2.40Hz),7.26(s,2H,Ar-H),5.65(d,2 H,22-CH2-αH,J=16.38Hz),5.19(dd,4H,5-CH2,J=17.55,8.12Hz),5.07(d,2H,22-CH2-βH,J=18.75Hz),3.29-3.00(m,12H,CH2 CH2 SS CH2 CH2, 23-CH2), 1.86 (td, 4H, 18-CH2, J = 14.28, 7.02Hz), 1.39 (t, 6H, 24-CH3, J = 7.66Hz), 0.99 (t, 6H, 29-CH3, J = 7.38Hz).

[0079] Mass spectrometry results are MS (ESI) m / z [M+H] + =959.26367.

[0080] Example 3: Synthesis of SN38 dimer prodrug with disulfide bond located at the γ position adjacent to the carbonyl group (γ-SN38-SS-SN38)

[0081] The preparation method of Example 1 was used to replace 2,2'-dithiodiacetic acid with 4,4'-dithiodibutyric acid to obtain the γ-position disulfide-bridged SN38 dimer prodrug.

[0082] The structure of γ-SN38-SS-SN38 prepared in Example 3 was determined by mass spectrometry and nuclear magnetic resonance hydrogen spectroscopy. Figure 3 shown.

[0083] The results of NMR spectroscopy analysis are as follows:

[0084] 1 H NMR (400MHz, CDCl3) δppm 7.74(d,2H,Ar-H,J=9.13Hz),7.65(d,2H,Ar-H,J=1.97Hz),7.35(s,2H,Ar-H),7.33(dd,2H,Ar-H,J=9.20,2.14Hz),5.66 (d,2H,22-CH2-αH,J=16.39Hz),5.21(t,4H,5-CH2,J=17.00Hz),5.10(d,2H,22-CH2-βH,J=18.70Hz),3.09(d,4H,CH2CH2 CH2 SS CH2 CH2CH2,J=7.72Hz),2.93(dt,4H,CH2 CH2 CH2SSCH2 CH2 CH2,J=6.63,1.73Hz),2.86(t,4H, CH2 CH2CH2SSCH2CH2 CH2, J=6.89Hz), 2.33-2.20(m,4H,23-CH2), 1.92-1.78(m,4H,18-CH2), 1.43(t,6H,J=7.59Hz), 0.99(t,6H,J=7.34Hz).

[0085] Mass spectrometry results are MS (ESI) m / z [M+H] + =987.29493.

[0086] Example 4: Chemical stability study of disulfide-bridged SN38 dimer prodrug

[0087] Weigh an appropriate amount of prodrug powder (α-SN38-SS-SN38, β-SN38-SS-SN38 or γ-SN38-SS-SN38) and dissolve it in DMF. Immediately record the peak area of ​​the prodrug by high performance liquid chromatography (0h). The sample was placed at room temperature for 9h. At different time points, the peak area of ​​the prodrug was detected by liquid chromatography, and the relative purity (the ratio of the prodrug peak area at different time points to the prodrug peak area at 0h) was calculated. The results are shown in Figure 4 As shown in the figure, after 9 hours at room temperature, the purity of β-SN38-SS-SN38 and γ-SN38-SS-SN38 remained basically unchanged, while the purity of α-SN38-SS-SN38 dropped rapidly to about 60%. This shows that the position of the disulfide bond in the prodrug can significantly affect the chemical stability of the SN38 dimer prodrug. The stability of α-SN38-SS-SN38 is extremely poor and does not have drugability, so we chose β-SN38-SS-SN38 and γ-SN38-SS-SN38 for subsequent experiments.

[0088] Example 5: Purity Confirmation of Disulfide-Bridged SN38 Dimer Prodrug

[0089] The purity of β-SN38-SS-SN38 and γ-SN38-SS-SN38 was determined by HPLC. Figure 5 As shown, the purity of the two prodrugs was above 99.5%, which met the requirements of subsequent experiments.

[0090] Example 6: Preparation and stability study of non-PEGylated / PEG-modified disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles

[0091] Accurately weigh 0.2 mg of β-SN38-SS-SN38 or γ-SN38-SS-SN38, dissolve it in 200 μL of N,N-dimethylformamide (DMF), and drop it into 2 mL of deionized water at a stirring speed of 1000 r / min. The prodrug will self-assemble into non-PEGylated nanoassemblies (non-PEGylated β-SN38-SS-SN38 NPs, non-PEGylated γ-SN38-SS-SN38 NPs). At room temperature, DMF was removed by dialysis. The results are shown in Table 1. The particle size of non-PEGylated β-SN38-SS-SN38 NPs is about 115 nm, while the particle size of non-PEGylated γ-SN38-SS-SN38 NPs is smaller, about 90 nm. The stability of the non-PEGylated prodrug nanoassemblies was investigated. The results are shown in Table 1. Figure 6As shown in the figure, after being placed at room temperature for 48 hours, the particle size of non-PEGylated γ-SN38-SS-SN38 NPs remained basically unchanged, and the properties of the preparation did not change significantly. However, non-PEGylated β-SN38-SS-SN38 NPs precipitated after being placed at room temperature for 3 hours. This shows that the position of the disulfide bond in the prodrug structure affects the self-assembly ability of the prodrug and the stability of the nanoassembly. γ-SN38-SS-SN38 has a stronger self-assembly ability and the formed nanoassembly has better stability.

[0092] Accurately weigh 1 mg of prodrug (β-SN38-SS-SN38 or γ-SN38-SS-SN38) and 0.25 mg of DSPE-PEG 2K , dissolved in 200 μL of DMF, and dripped into 2 mL of deionized water at a stirring speed of 1000 r / min. The prodrug self-assembled into PEGylated nanoassemblies (β-SN38-SS-SN38 NPs, γ-SN38-SS-SN38 NPs). At room temperature, DMF was removed by dialysis. As shown in Table 2, the particle size of the DSPE-PEG2K modified prodrug nanoassemblies is about 100 nm, the particle size distribution is uniform, and the surface charge is about -20 mV, which is beneficial to prevent the aggregation of nanoparticles. The change in surface charge also proves that PEG has been successfully modified on the surface of the nanoparticles. The stability of the PEGylated prodrug nanoassemblies at room temperature was investigated. The results are as follows Figure 7 As shown in the figure, after 4 hours, β-SN38-SS-SN38 NPs precipitated, while the particle size of γ-SN38-SS-SN38 NPs remained unchanged, further proving that the self-assembly performance of γ-SN38-SS-SN38 dimer prodrug is better than that of β-SN38-SS-SN38. PEG modification of the surface of prodrug nanoassemblies can improve their stability to a certain extent, but it will not change the difference in stability between different prodrug nanoassemblies.

[0093] Table 1 Particle size, particle size distribution, surface charge and drug loading of non-PEG-modified dimer prodrug self-assembled nanoparticles

[0094]

[0095] Table 2 Particle size, particle size distribution, surface charge and drug loading of PEG-modified dimer prodrug self-assembled nanoparticles

[0096]

[0097] Example 7: In vitro release test of PEG-modified disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles

[0098] The drug release of disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles was investigated using PBS (pH 7.4, containing 30% anhydrous ethanol) buffer containing different concentrations of hydrogen peroxide (H2O2, a commonly used ROS substitute) or dithiothreitol (DTT, a substitute for glutathione) as the release medium. The specific operation is as follows: 1 mL of PEG-modified disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles (SN38 equivalent mass is 200 μg) prepared in Example 5 is added to a 50 mL centrifuge tube, and 30 mL of release medium is added, and then the entire system is placed in a 37°C constant temperature shaker with an oscillation speed of 100 r / min. 200 μL of samples are taken at preset time points, and the SN38 content therein is determined using a high performance liquid chromatograph. The reduction release results are shown in FIG. Figure 8 As shown in A-8C, in the blank medium, the drug release rate of β-SN38-SS-SN38 NPs is much greater than that of γ; under the action of 0.1mM DTT and 1mM DTT, the drug release rate of γ-SN38-SS-SN38 NPs is greater than that of β-SN38-SS-SN38 NPs. This is because the stability of β-SN38-SS-SN38 NPs is poor and they are easy to disintegrate, resulting in easy hydrolysis of the prodrug, indicating that they will release drugs at low GSH levels in the blood and normal tissues. While γ-SN38-SS-SN38 NPs remain stable under normal conditions, they are more sensitive to GSH and can release the parent drug under the action of high concentrations of GSH in the tumor microenvironment, which ensures the anti-tumor effect and reduces the toxic side effects to normal tissues. The results of oxidative responsive drug release of prodrug nanoassemblies are shown in Figure 2. Figure 8 As shown in D-8E, the drug release rate of β-SN38-SS-SN38 NPs is faster than that of γ-SN38-SS-SN38 NPs under both low and high concentrations of hydrogen peroxide.

[0099] Example 8: Cytotoxicity of PEG-modified disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles

[0100] The MTT method was used to investigate the cytotoxicity of the PEG-modified SN38 dimer prodrug nanoassembly to two different tumor cells and one normal cell, in order to explore whether the prodrug nanoassembly can effectively inhibit tumor cell proliferation and whether it can distinguish tumor cells from normal cells. First, the cells with good morphology were digested, diluted to 5000 cells / mL with culture medium, and then 100 μL of cell suspension was added to each well of a 96-well plate, and incubated in an incubator for 24 hours to adhere to the wall. After the cells adhered to the wall, SN38 solution or PEG-modified disulfide-bridged SN38 dimer prodrug nanoparticles prepared in Example 5 were added. In this experiment, the preparation and dilution of the drug solution and the nanoparticle preparation were all made with the culture medium of the corresponding cells and aseptically filtered with a 0.22 μm filter membrane. 100 μL of the test solution was added to each well, and 3 parallel wells were added for each concentration. In the control group, no test solution was added, and 100 μL of culture medium was added alone, and the cells were incubated in an incubator. 48 hours after drug addition, the 96-well plate was taken out, 20 μL of 5 mg / mL MTT solution was added to each well, and the medium was discarded after incubation in an incubator for 4 hours. The 96-well plate was inverted on filter paper to fully absorb the residual liquid, and 200 μL DMSO was added to each well and oscillated on an oscillator for 10 minutes to dissolve the blue-purple crystals. A1 well (containing only 200 μL DMSO) was set as the zero well. The absorbance value of each well after zeroing was measured at 570 nm using an ELISA reader.

[0101] The results are as follows Fig. 9 As shown in the figure, the inhibitory effects of β-SN38-SS-SN38 NPs and γ-SN38-SS-SN38 NPs on tumor cells are stronger than that of the parent drug SN38, and the cytotoxicity of γ-SN38-SS-SN38 NPs is stronger. This is because both prodrugs are dimeric prodrugs, and one molecule of prodrug contains two molecules of SN38.

[0102] Example 9: Pharmacokinetic study of PEG-modified disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles

[0103] SD rats weighing between 200-250g were randomly divided into groups, fasted for 12h before administration, and allowed to drink water freely. SN38 solution and PEGylated SN38 dimer prodrug self-assembled nanoparticles prepared in Example 5 were intravenously injected respectively. The dose of SN38 was 2.5mg / kg. Blood was collected from the eye sockets at the specified time points, and plasma was separated. The drug concentration in plasma was determined by liquid chromatography-mass spectrometry.

[0104] Due to the large molecular weight of the SN38 dimer prodrug, the existing mass spectrometry conditions make it difficult to detect stable ion peaks and quantify the prodrug in plasma. Therefore, only the blood concentration of SN38 was detected, and the results are shown in Table 3. The area under the curve and peak concentration of SN38 released from the two dimer prodrug nanoassemblies are higher than those of the SN38 solution. Among them, the area under the curve and peak concentration of SN38 released from γ-SN38-SS-SN38 NPs are significantly higher than those from β-SN38-SS-SN38 NPs, indicating that γ-SN38-SS-SN38 NPs have better stability and can more effectively improve the pharmacokinetic behavior of SN38.

[0105] Table 3 Pharmacokinetic parameters of PEG-modified disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles

[0106]

[0107] Example 10: In vivo antitumor experiment of PEG-modified disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles

[0108] 100 μL of 5 × 10 7 Cells / mL of 4T1 cell suspension was inoculated into the right back of Balb / C mice by subcutaneous injection. 3 The tumor-bearing mice were randomly divided into 4 groups (n=5) and injected with normal saline (Saline), SN38 solution, β-SN38-SS-SN38 NPs or γ-SN38-SS-SN38 NPs (SN38 equivalent dose is 2.5 mg / kg) through the tail vein. The drug was administered once every other day, for a total of 5 times. The long diameter, short diameter and weight of the tumor and the weight of the mouse were measured and recorded every day, and the tumor volume was calculated. The calculation method of tumor volume is: long diameter × short diameter × short diameter × 0.5. One day after the last administration, the mice were killed, the tumor tissue was isolated, weighed and photographed, and the tumor-bearing rate was calculated (tumor weight / mouse weight × 100%).

[0109] like Fig.10 As shown, the volume of the saline group reached 600mm on the ninth day. 3 The SN38 solution group had a certain therapeutic effect, but the tumor volume still reached 300mm 3 The tumor volume of mice in the β-SN38-SS-SN38 NPs group reached 200 mm 3, and there was no significant difference with the SN38 solution group. γ-SN38-SS-SN38 NPs had the best antitumor effect, which could significantly inhibit tumor growth, and the tumor-bearing rate was significantly lower than that of other formulation groups. γ-SN38-SS-SN38 NPs showed stronger antitumor effect than SN38 solution, which was determined by the pharmacological advantages of prodrug nanoassemblies over solution: (1) long in vivo circulation time and higher AUC; (3) strong tumor accumulation ability; (2) high cellular uptake efficiency; (4) specific rapid drug release in tumor cells. Because β-SN38-SS-SN38 NPs have poor stability and will quickly disintegrate after intravenous injection into the systemic circulation, they lose the pharmacological advantages of nanoparticles, so their antitumor effect is not as good as γ-SN38-SS-SN38 NPs.

Claims

1. Use of a disulfide-bridged SN38 dimer prodrug self-assembled nanoparticle in the preparation of an anti-tumor drug or a drug delivery system, characterized in that: The disulfide-bridged SN38 dimer prodrug structural formula is: Preparation method: dissolve 4,4'-dithiodibutyric acid in chloroform and stir evenly; dissolve part of DMAP, EDCI and SN38 in chloroform and stir evenly, then mix with 4,4'-dithiodibutyric acid solution, stir at room temperature under N2 protection; add remaining EDCI and DMAP to the mixed solution, continue stirring at room temperature under nitrogen protection, and separate and purify the product by preparative liquid phase; The disulfide-bridged SN38 dimer prodrug self-assembled nanoparticles are specifically PEG-modified dimer prodrug self-assembled nanoparticles; the preparation method comprises: dissolving the disulfide-bridged SN38 dimer prodrug and the PEG modifier in a solvent, slowly dropping the solution into water under stirring, the prodrug spontaneously forms uniform nanoparticles, and removing the solvent by dialysis to obtain a nano-colloid solution free of organic solvent.

2. The use of the self-assembled nanoparticles of the disulfide-bridged SN38 dimer prodrug as claimed in claim 1 in the preparation of anti-tumor drugs or drug delivery systems, characterized in that: The PEG modifier is selected from TPGS, DSPE-PEG, PLGA-PEG and PE-PEG; the molecular weight of the PEG is 1000-5000; the solvent is selected from ethanol, dimethyl sulfoxide, N,N'-dimethylformamide, tetrahydrofuran, acetone; the weight ratio of the disulfide-bridged SN38 dimer prodrug to the PEG modifier is 90:10 to 70:30.

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