Preparation method of active oxygen response intestinal cancer targeting nanoparticles and radiotherapy combined application of active oxygen response intestinal cancer targeting nanoparticles

Abstract

The invention discloses a preparation method of active oxygen response intestinal cancer nanoparticles and radiotherapy combined application of the active oxygen response intestinal cancer nanoparticles. The invention relates to the field of cancers, in particular to a method for treating colorectal cancer by using an active oxygen responsive nanoparticle targeted delivery system. According to the invention, a therapeutic drug is loaded by nano-particles, and an external radiotherapy technology is combined, so that efficient targeted therapy of colorectal cancer is realized. The preparation method comprises the following steps: firstly, carrying out connection reaction on PLGA-PEG-NHS and QRH polypeptide to synthesize PLGA-PEG-QRH, carrying out self-assembly on the PLGA-PEG-QRH and mPEG-TK-PLGA to form nanoparticles QRH-TK (at) NP, then loading regorafenib (RG) into the QRH-TK (at) NP, and carrying out self-assembly to form the nanoparticles QRH-TK (at) RG. The nanoparticles prepared by the preparation method disclosed by the invention have low toxicity and good biocompatibility, are directionally enriched in colorectal cancer tissues and have ROS responsiveness.

Description

Technical Field

[0001] The present invention relates to the field of cancer treatment, in particular to a method for treating colorectal cancer by combining radiotherapy with a functional nanoparticle targeted delivery system. The present invention loads therapeutic drugs on nanoparticles and combines external radiotherapy technology to achieve efficient targeted treatment of colorectal cancer. Background Art

[0002] Colorectal cancer is one of the common malignant tumors worldwide. Traditional treatment methods include surgery, chemotherapy, and radiotherapy. However, these methods have limitations, such as the systemic toxicity of chemotherapy drugs, the damage of radiotherapy to normal tissues, and tumor drug resistance. In recent years, nanoparticles have shown great potential as drug delivery systems in cancer treatment. They can improve the targeting of drugs, reduce side effects, and enhance the therapeutic effect. However, the advantages of nanoparticles and radiotherapy have not been fully combined in the prior art, especially in the treatment of colorectal cancer. In current clinical practice, the treatment of early colorectal cancer usually mainly relies on surgical resection, while for mid- and late-stage colorectal cancer, a comprehensive treatment plan of surgery combined with drug treatment is mostly adopted. Although these treatment means can control the growth of tumors to a certain extent, there may be incomplete resection during surgery. At the same time, due to the non-specific action of chemical drugs, they will affect all body cells, resulting in strong toxic side effects. In addition, these treatment methods also face the problem of a relatively high risk of cancer cell metastasis and recurrence. Therefore, the treatment of colorectal cancer still faces many severe challenges.

[0003] To address these challenges, there is provided a method including developing a radiotherapy combined with ROS-responsive nanoparticle drug delivery system to reduce systemic toxicity and increase the enrichment amount in the tumor region. ROS-responsive nanoparticles can release drugs in a tumor microenvironment with a high concentration of ROS, thus promising to achieve precise therapeutic effects, that is, precisely release drugs at the tumor site without affecting surrounding healthy cells.

[0004] It should be noted that the above content belongs to the technical cognition scope of the inventor and does not necessarily constitute the prior art. Summary of the Invention

[0005] To solve the above problems, the object of the present invention is to provide a preparation method and application of a radiotherapy combined with ROS-responsive nanoparticle drug delivery. The prepared ROS nanoparticles have an organ-targeted amphiphilic polymer nanovesicle system. Its vesicular structure can attach chemical drugs to the inner layer, and the surface-modified polypeptide QRH can improve the biocompatibility and targeting of the nanoparticles. The chemical bond thioacetal bond has an obvious response effect to high-concentration ROS in tumors and can release drugs at a fixed point in the tumor microenvironment.

[0006] To achieve the above object, the present invention provides a radiotherapy combined with a ROS-responsive nanoparticle drug delivery system. To achieve the above object of the invention, the technical solution of the present invention is as follows:

[0007] An organ-targeting polymeric nanovesicle system is prepared by co-assembling an amphiphilic triblock polymer and a small molecule drug. The amphiphilic triblock polymer has the following chemical structural formula:

[0008]

[0009] In the present invention, in the amphiphilic triblock polymer, the molecular weight of polyethylene glycol is 5000 Da; the molecular weight of the hydrophobic segment is about 3 times that of the PEG molecular weight, and the hydrophobic segment is a poly(lactic-co-glycolic acid) copolymer (75:25); the polypeptide is QRH. The amphiphilic triblock polymer is denoted as PLGA-PEG-QRH. For example, the preparation method of PLGA-PEG-QRH includes the following steps: QRH and PLGA-PEG-NHS are prepared into PLGA-PEG-QRH through an amidation reaction.

[0010] PLGA-PEG-NHS has the following chemical structural formula:

[0011]

[0012] In the present invention, for PLGA-PEG-NHS, the molecular weight of polyethylene glycol is 5000 Da; the molecular weight of the hydrophobic segment is about 3 times that of the PEG molecular weight, and the hydrophobic segment is a poly(lactic-co-glycolic acid) copolymer (75:25);

[0013] In the polymeric nanovesicles of the present invention, an amphiphilic triblock polymer and a small molecule drug are co-assembled by a solvent displacement method, and the hydrophilic inner shell is an amino acid polypeptide, which can achieve efficient loading of the small molecule drug.

[0014] In the present invention, the small molecule drug is the multi-kinase inhibitor regorafenib (RG).

[0015] The present invention discloses a treatment method for targeted treatment of colorectal cancer by polymeric nanovesicles combined with radiotherapy.

[0016] The preparation method of the colorectal cancer-targeting polymeric nanovesicles in the present invention can be as follows:

[0017] (1) Activate PLGA-PEG with EDC to form PLGA-PEG-NHS, and then react with QRH polypeptide to obtain PLGA-PEG-QRH;

[0018] (2) The mixed solution of the synthesized PLGA-PEG-QRH and mPEG-TK-PLGA polymers was dropped into the stirred PBS buffer solution and stirred, followed by dialysis to obtain QRH-TK@NP;

[0019] (3) Under stirring conditions, the mixed solution of PLGA-PEG-QRH and mPEG-TK-PLGA polymers (DMSO, 20 mg / mL, mass ratio 30:70) was dropped into PBS (pH 7.4) containing 5% regorafenib (RG) and stirred for 30 minutes, followed by dialysis with PBS to obtain QRH-TK@RG;

[0020] The present invention relates to a targeted polymer nanovesicle system for the treatment of colorectal cancer, its preparation method and medical application. The nanovesicles adopt a three-layer composite structure design: an inner core structure composed of amphiphilic amino acid polypeptides, a middle hydrophobic membrane layer formed by poly(lactic-co-glycolic acid) (PLGA), and an outer hydrophilic surface composed of polyethylene glycol (PEG) segments, and specific charged functional groups are embedded in the PEG layer by molecular engineering methods. This nanocarrier has the following innovative features: (1) The unique multi-layer vesicle structure can achieve co-loading of amphiphilic drugs, where the inner core cavity can efficiently encapsulate hydrophilic therapeutic agents, and the hydrophobic membrane layer can stably load lipophilic drug molecules; (2) The surface-modified PEG chains significantly enhance the blood circulation time and biocompatibility of the nanoparticles through steric stabilization effects; (3) By regulating the type and density of the charged groups on the outer layer, active targeting delivery based on the characteristics of the tumor microenvironment (such as abnormal vascular permeability, specific charged targets, etc.) can be achieved. Among them, the positively charged groups can mediate the selective accumulation in tumor tissues through electrostatic interactions, and the negatively charged groups are beneficial for targeting tumor-associated macrophages. Experimental data show that this intelligent nanosystem exhibits excellent drug delivery efficiency and targeting specificity both in vitro and in vivo in a colorectal cancer model, and has important clinical transformation value. Description of the Drawings

[0021] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0022] Figure 1 It is a schematic diagram of the process for synthesizing the PLGA-PEG-QRH polymer material in Example 1 of the present invention, the schematic diagram of the nanoparticle QRH-TK@RG, and the treatment mechanism schematic diagram of the colorectal cancer model;

[0023] Figure 2 It is the dynamic light scattering particle size diagram, transmission electron microscope diagram and stability evaluation of the polymer nanovesicles prepared in Example 2 of the present invention;

[0024] Figure 3 Results of flow cytometry and confocal laser scanning microscopy in Example 3 of the present invention

[0025] Figure 4 Cytotoxicity experiment of the polymer in Example 4 of the present invention

[0026] Figure 5 In vivo efficacy evaluation of biodistribution and radiotherapy combined nanoparticle targeted delivery system in Example 5 of the present invention Detailed implementation manners

[0027] In order to more clearly illustrate the overall concept of the present invention, the following will be described in detail by way of examples in conjunction with the accompanying drawings of the specification.

[0028] Example 1

[0029] A preparation method and efficacy evaluation of a radiotherapy combined ROS-responsive nanoparticle drug delivery system, including the following steps:

[0030] Appendix Figure 1 The research adopted a stepwise synthesis method to prepare a nanovesicle system co-loading QRH and TK (QRH-TK@RG). The specific steps are as follows: First, the terminal carboxyl group of PLGA was activated by N-hydroxysuccinimide to obtain the PLGA-PEG-NHS active intermediate, and then an amidation reaction was carried out with the QRH polypeptide containing an amino terminal to successfully construct the amphiphilic diblock copolymer PLGA-PEG-QRH. During the nano-assembly process, PLGA-PEG-QRH and mPEG-TK-PLGA were dissolved in dimethyl sulfoxide (DMSO) at a mass ratio of 30:70 to form a mixed polymer solution (the total polymer concentration was 20 mg / mL), and this solution was slowly dropped into the continuously stirred phosphate buffer solution (PBS, pH 7.4) by a micro-injection pump. After magnetic stirring for 30 minutes to complete the nano self-assembly, the mixed system was transferred to a dialysis bag with a molecular weight cut-off of 5 kDa and dynamically dialyzed in PBS buffer solution for 48 hours to remove organic solvents, and finally the QRH-TK@NP with a vesicle structure was obtained.

[0031] Using the same method, the mixed solution of PLGA-PEG-QRH and mPEG-TK-PLGA polymers was synchronously dropped into the PBS buffer solution containing 5% regorafenib (RG) under stirring conditions for co-assembly, and the QRH-TK@RG nano-delivery system with a vesicle structure was successfully constructed. The drug-loaded nanoparticles showed significant therapeutic effects in a subcutaneous xenograft model of colorectal cancer, and their responsive drug release characteristics and radiosensitization effects were verified by radiotherapy. The experimental results showed that the nano-system had a synergistic effect in tumor targeting and radiotherapy.

[0032] Example 2

[0033] Characterization data of QRH-TK and QRH-TK@RG nanoparticles:

[0034] Appendix Figure 2 A, B Particle size analyzer (Malvern Zetasizer) and appendix Figure 2 C Transmission electron microscope (TEM) characterization shows that the hydrodynamic diameters of QRH-TK and QRH-TK@RG nanoparticles are (240.5 ± 3.2) nm and (259.8 ± 4.1) nm, respectively. Appendix Figure 2 D Under the low-temperature storage condition of -4°C, the nano-dispersion system exhibits excellent colloidal stability within 7 days, with stable particle size changes and a polydispersity index (PDI) maintained within the range of 0.12 ± 0.03. Appendix Figure 2 E In vitro simulated physiological environment experiments show that in a 37°C culture medium containing 10% fetal bovine serum (FBS), the nanoparticles have a low amplitude of particle size fluctuation within 48 hours, and the PDI value is stable below 0.2, confirming their good serum stability.

[0035] Appendix Figure 2 F To verify the ROS-responsive release characteristics, the dynamic dialysis method was used to investigate the drug release behavior of the nanoparticles in phosphate buffer (PBS, pH 7.4) containing 20 mM H2O2. Under the condition of constant temperature oscillation (100 rpm) at 37°C, the cumulative drug release rate of the H2O2 treatment group was significantly higher than that of the PBS control group within 48 hours, indicating that the nanoparticles can achieve controllable drug release through ROS triggering.

[0036] Example 3

[0037] Flow cytometry for quantitative analysis of nanoparticle endocytosis efficiency and laser confocal microscopy for visualizing nanoparticle endocytosis behavior:

[0038] Appendix Figure 3 A, B An in vitro model was established by selecting CT26 mouse colon cancer cells. 1×10 5 cells / well were seeded in a 12-well plate and cultured in RPMI 1640 medium containing 10% fetal bovine serum for 24 hours until 70% confluence. The nanoparticle working solution was prepared according to the following protocol:

[0039] Experimental group (Group A): The QRH-TK system was vortex-emulsified by mixing 30 μL of PLGA-PEG-QRH (20 mg / mL), 70 μL of mPEG-TK-PLGA (20 mg / mL), and 5 μL of DiD fluorescent probe (1 mg / mL) in 1.9 mL of PBS (pH 7.4), and then sterilized through a 0.22 μm filter membrane.

[0040] Control group (Group B): The TK system was prepared with the same process using 100 μL of mPEG-TK-PLGA (20 mg / mL) and 5 μL of DiD (1 mg / mL).

[0041] For the cell intervention experiment, the time gradient method was used: 50 μL of the corresponding nano-suspension (final concentration 200 μg / mL) was added to each well at 6, 4, 2, and 1 h before treatment, and the operation was carried out in the dark throughout the process. After the treatment, the cells were digested with 0.25% trypsin-EDTA, washed 3 times with PBS, resuspended in 500 μL of pre-cooled PBS, and immediately detected using a flow cytometer (BD FACSCelesta). The excitation / emission wavelength was 640 / 670 nm. Data analysis showed that under the conditions of 4 h and 6 h of treatment, the endocytosis efficiency in Group A (experimental group) was significantly higher than that in Group B, confirming that QRH modification could significantly enhance the active targeting uptake ability of nanoparticles.

[0042] Appendix Figure 3 C An in vitro model was established using CT26 human colon cancer cells. 1×10 5 cells / dish were seeded in a special culture dish for laser confocal microscopy (MatTek, P35G-1.5-14-C) and cultured in RPMI 1640 medium (containing 10% FBS) at 37 °C and 5% CO2 for 24 h until 60% confluence.

[0043] Nanoparticle intervention: TK and QRH-TK (final concentration 100 μg / mL) were added to the cells respectively, and co-incubated at 37 °C for 6 h;

[0044] Cell membrane staining: 1×CellMask was used TM Stained in the dark for 15 min (washed 3 times with PBS);

[0045] Cell fixation: Fixed with 4% paraformaldehyde (PFA) at room temperature for 15 min, and rinsed with PBS to remove the residual fixative;

[0046] Nuclear staining and mounting: A quenching-resistant mounting medium containing DAPI (1 μg / mL) was added dropwise and cured in the dark for 24 h.

[0047] In the QRH-TK group, a significantly enhanced red fluorescence signal of DiD was presented in the cytoplasm, which was consistent with the flow cytometry data, confirming that the targeted modification could improve the cellular uptake efficiency of nanoparticles.

[0048] Example 4

[0049] Biocompatibility evaluation of a nano-drug delivery system based on normal and immune cell models and in vitro anti-tumor effects of radiotherapy dose gradient synergistic nano-formulations:

[0050] Appendix Figure 4To evaluate the safety advantages of RG nanoparticles in the system, NIH / 3T3 mouse embryonic fibroblasts (normal cell model) and RAW264.7 mouse mononuclear macrophages (immune cell model) were selected in this study for comparative analysis. 3×10 3 cells / well were seeded in 96-well plates respectively. After 24 h of culture, the following were added respectively: free RG (concentration 30 μg / mL), drug-loaded nanoparticles QRH-TK@RG (equivalent RG concentration). After 48 h of intervention in each group, cell viability was detected by the CCK-8 method. The experimental data showed that: at a high concentration of 30 μg / mL, the survival rate of NIH / 3T3 cells in the QRH-TK@RG group was significantly higher than that in the free RG group; the survival rate in the RAW264.7 cells in the QRH-TK@RG group was also significantly better than that in the free group. The results indicated that the nanocarrier could reduce the non-specific toxicity of RG to normal cells and immune cells through its slow-release characteristics, and the mechanism might be related to reducing the mitochondrial membrane potential damage caused by drug exposure.

[0051] Appendix Figure 4 To explore the regulatory effect of radiotherapy radiation dose on the drug release of ROS-responsive nanoparticles, a gradient radiotherapy dose (0, 4, 6 Gy) and a combined QRH-TK@RG nanoparticle intervention system were designed in this study to systematically evaluate its synergistic killing effect on CT26 colon cancer cells. The results showed that high-dose radiotherapy could significantly enhance the therapeutic gain ratio of the nanoparticles.

[0052] Example 5

[0053] Evaluation of the combined radiotherapy-nanodrug treatment effect in tumor-bearing mice:

[0054] Appendix Figure 5 A In this study, in vivo treatment experiments were carried out through a subcutaneous transplantation tumor model of CT26 colon cancer in BALB / c mice (female, 6-8 weeks old, average body weight 20±2 g): 25 tumor-bearing mice (tumor volume ≈ 100 mm 3 ) were randomly divided into 5 groups (n = 5). PBS control group: PBS (pH 7.4); free RG group: 6 mg / kg RG solution (dissolved in 5% DMSO-PBS); X-ray monotherapy group: 4 Gy local radiotherapy (dose rate 3 Gy / min); nanoparticle group: QRH-TK@RG (200 mg / kg nanoparticles, equivalent to 6 mg / kg RG); combined treatment group: QRH-TK@RG (same as the nanoparticle group) + X-ray (4 Gy, implemented 24 h after drug administration). Tail vein injection was used, once every 4 days, for a total of 2 treatments. The tumor volume in the combined treatment group was significantly smaller than that in the nanoparticle group and the free RG group ( Figure 5 B), and there was no significant weight loss in the mice in all treatment groups ( Figure 5C). Through the mouse survival experiment, the results showed that the survival rate of mice in the combination treatment group was also much higher than that of other groups. Figure 5 D).

[0055] The results of HE staining and immunofluorescence showed that Figure 5 E), radiotherapy can significantly improve the in vivo anti-tumor efficacy of nanoparticles through the dual mechanisms of expanding tumor vascular permeability (CD34 + microvessel density decreased) and activating ROS-responsive drug release.

Claims

1. A colorectal cancer treatment system based on radiotherapy combined with targeted delivery of nanoparticles, characterized in that Functional nanoparticles are loaded with chemotherapy drugs; targeted modification units modify the surface of nanoparticles with colorectal cancer-specific ligands to achieve specific targeting of colorectal cancer sites; selective killing of tumor cells is achieved through the combined effects of targeted delivery of nanoparticles and radiotherapy.

2. The system according to claim 1, characterized in that The nanoparticles have an average particle size of 268.5 nm and are made of biodegradable materials.

3. The system according to claim 1, characterized in that The chemotherapy drug is selected from multi-kinase inhibitors, including but not limited to other drugs combined with radiotherapy, such as platinum drug SN-38, etc.

4. The system according to claim 1, characterized in that The colorectal cancer specific ligands include anti-EGFR antibodies, folic acid, and ligands targeting colorectal cancer stem cell surface markers (such as CD44 and LGR5).

5. A method for treating colorectal cancer by combining radiotherapy with targeted delivery of nanoparticles, characterized in that The following steps are involved: Nanoparticles loaded with chemotherapeutic drugs are prepared; colorectal cancer-specific ligands are bound to the surface of the nanoparticles through surface modification technology; the nanoparticles are delivered to the colorectal cancer lesion area by intravenous injection; after the nanoparticles reach the tumor site, external radiotherapy is applied with a dose of 4Gy and the irradiation time is 24 hours after the nanoparticle administration.

6. The method according to claim 5, characterized in that The release mechanism of the nanoparticles is RO S responsive release.

7. The method according to claim 5, characterized in that The combination of radiotherapy and nanoparticles enhances the efficacy by: -Radiotherapy promotes drug release from nanoparticles; - Chemotherapy drugs in nanoparticles enhance the killing effect of radiotherapy on tumor cells; - Targeted delivery reduces damage to normal tissues.

8. A nanoparticle preparation prepared according to the system or method according to any one of claims 1 to 8, characterized in that: The preparation comprises a pharmaceutically acceptable carrier and is suitable for intravenous administration.

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