Preparation and application of responsive nano assembly capable of overcoming chemotherapy resistance and improving immunotherapy effect at same time
By designing nano-self-assembled carriers that respond to the tumor microenvironment, the problems of chemotherapy drug loss in blood circulation and insufficient tumor penetration were solved, and the synergistic therapeutic effects of chemotherapy sensitization and immune activation were achieved, overcoming chemotherapy resistance and TME inhibition, and enhancing the anti-tumor immune response.
Patent Information
- Application Number
- CN202410335771.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-22
- Publication Date
- 2025-09-23
AI Technical Summary
Existing chemotherapy drugs such as doxorubicin are lost during blood circulation, lack specific penetration into tumors, and have large side effects. In addition, the inhibitory nature of the tumor microenvironment limits the effectiveness of chemotherapy and immunotherapy, and chemotherapy resistance and TME inhibition have not been effectively overcome.
A nanodrug carrier composed of chitosan oligosaccharide, carboxylazobenzene, PEG-modified lipids, chemotherapy sensitizers and immune activators was designed. The nano-self-assembly was formed through amide bond coupling. The drug was released in response to tumor hypoxia and acidic environment, and synergistic treatment was achieved by combining chemotherapy sensitizers and immune activators.
It achieves efficient penetration and responsive release of chemotherapy drugs at the tumor site, reduces chemotherapy resistance, activates the immune response, and enhances the anti-tumor effect of chemotherapy.
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Figure CN120678729A_ABST
Abstract
Description
Technical field:
[0001] The present invention belongs to the field of drug carriers, and specifically relates to the preparation of a tumor microenvironment responsive nano self-assembly and its application in overcoming tumor drug resistance and enhancing chemotherapy-immunosynergistic therapy. Background technology:
[0002] Cancer, as a major human disease, has caused the deaths of tens of millions of people worldwide. Despite many improvements in improving treatment efficacy, surgery and chemotherapy remain the most commonly used treatment strategies in clinical practice. Among various chemotherapeutic drugs, doxorubicin (DOX) is the most widely used chemotherapeutic drug, which exerts its anti-tumor effect by preventing the replication of cancer cells. However, the loss of the drug during blood circulation, the lack of specific penetration into tumors, and numerous side effects limit its further application in anti-tumor therapy. In fact, tumor resistance to chemotherapy limits the successful treatment of cancer. For example, resistance to intracellular reactive oxygen species (ROS) produced by DOX has attracted great attention in cancer treatment. Glutathione (GSH) is an antioxidant molecule that acts as an intracellular ROS scavenger, helps to defend against intracellular oxidative stress, and has great potential in combating chemoresistance.
[0003] Furthermore, the suppressive tumor microenvironment (TME) limits the efficacy of chemotherapy. Currently, chemotherapy combined with immunotherapy has become a first-line approach for treating various cancers. Immunotherapy can directly kill tumor cells by activating immune cells to eliminate them. However, satisfactory efficacy has not been achieved to date because the suppressive nature of the TME limits the infiltration of effector T cells. Therefore, reversing the suppressive nature of the TME is a key factor for effective immunotherapy, such as inhibiting myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), promoting the maturation of APCs, and activating effector T cells. Therefore, treatments that can activate key immune response processes or enhance the entire tumor-immune cycle are expected to achieve satisfactory immunotherapy results. In recent years, many researchers have begun to focus on the anti-tumor effects of statins. Among various statins, simvastatin (SIM) inhibits protein preacylation through the mevalonate signaling pathway, thereby enhancing antigen presentation and adaptive immune responses. Although SIM's anti-tumor effects have been demonstrated, its poor water solubility and suboptimal dosing for cancer cells still limit its anti-tumor efficacy. Summary of the invention:
[0004] The purpose of the present invention is to overcome the drug resistance caused by current chemotherapy drugs and nanomedicines, and to provide a nano delivery carrier self-assembled from different types of copolymers. The slight negative charge on the surface of the carrier ensures long-term circulation in the blood. After being enriched in the tumor site through the EPR effect, the nano-composite drug is released in the low acid and hypoxic environment of the tumor to overcome tumor resistance. At the same time, the adjuvant or activator loaded in the core layer further activates immunity, thereby realizing combined treatment of chemotherapy and immunotherapy.
[0005] In one aspect, the present invention provides a nanomedicine self-assembled from a block copolymer that responds to the hypoxic environment of a tumor. The nanomedicine carrier is made of chitosan oligosaccharide, carboxyazobenzene, PEG-modified lipids, a chemotherapy sensitizer, a chemotherapy drug, and an immune activator. The preparation method of the nanomedicine is as follows:
[0006] S1) chitosan oligosaccharide and carboxyazobenzene are coupled via an amide bond to obtain carboxyazobenzene-modified chitosan oligosaccharide;
[0007] S2) coupling the carboxyazobenzene-modified chitosan oligosaccharide with a chemosensitizer to obtain a chemosensitizer-modified chitosan oligosaccharide;
[0008] S3) coupling the chemosensitizer-modified chitosan oligosaccharide with a PEG-modified lipid to obtain a chitosan oligosaccharide co-modified with a long-circulating lipid and a chemosensitizer;
[0009] S4) mixing the carboxyazobenzene-modified chitosan oligosaccharide with a chemotherapeutic drug in a solution to obtain the carboxyazobenzene-modified chitosan oligosaccharide that adsorbs the chemotherapeutic drug, and then coupling it with a PEG-modified lipid to obtain a long-circulating lipid-modified chitosan oligosaccharide that adsorbs the chemotherapeutic drug;
[0010] S5) mixing the chitosan oligosaccharide co-modified with the chemotherapy sensitizer obtained in step S3) with the chitosan oligosaccharide modified with the long-circulating lipid and adsorbed with the chemotherapy drug obtained in step S4), and adding an immune activator to form a nano drug carrier adsorbing the immune activator.
[0011] Furthermore, the molecular weight of the chitosan oligosaccharide is 1000-4000 Da, for example, 1000 Da, 2000 Da, 3000 Da, or 4000 Da.
[0012] Furthermore, the carboxyazobenzene is azobenzene-4,4-dicarboxylic acid.
[0013] Furthermore, the chemotherapy drug is doxorubicin, paclitaxel, cisplatin or their derivatives.
[0014] Furthermore, the chemotherapy sensitizer is one of 2-(3,4-dihydroxyphenyl)ethylamine, resveratrol, vitamin K2, shikonin and curcumin.
[0015] Furthermore, the PEG-modified lipid is DSPE-PEG, and the PEG terminal is modified with one of N-hydroxysuccinimide (NHS) and carboxyl (COOH).
[0016] Furthermore, the molar ratio of the chitosan oligosaccharide co-modified with the chemotherapy sensitizer in step S5) to the chitosan oligosaccharide modified with the long-circulating lipid and adsorbed chemotherapy drug obtained in step S4) is 1:4-4:1, and further 4:1, 3:1, 2:1, 1:1, 2:1, 3:1, 4:1.
[0017] Furthermore, the immune activator is one or more of nucleic acid drugs, nano drugs, small molecule drugs, macromolecular drugs, and antibody drugs, for example, one or more of simvastatin, aluminum phosphate, aluminum hydroxide, and potassium aluminum sulfate.
[0018] Furthermore, the amide bond coupling method in step S1) is to couple by adding a condensing agent, and the condensing agent is, for example, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.
[0019] Furthermore, the coupling method in step S2) is to perform coupling by adding a condensing agent, and the condensing agent is, for example, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.
[0020] Furthermore, the coupling method in step S3) is to perform coupling by adding a condensing agent, and the condensing agent is, for example, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.
[0021] Furthermore, the coupling method in step S4) is to perform coupling by adding a condensing agent, and the condensing agent is, for example, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.
[0022] Furthermore, in step S4), the adsorption is performed on the chitosan oligosaccharide modified with carboxyazobenzene and the chemotherapy drug at a molar ratio of 1:1-1.5, and the unadsorbed chemotherapy drug is removed by centrifugation or dialysis.
[0023] Another aspect of the present invention provides a method for preparing the above-mentioned nano drug carrier, the preparation method comprising the following steps:
[0024] S1) chitosan oligosaccharide and carboxyazobenzene are coupled via an amide bond to obtain carboxyazobenzene-modified chitosan oligosaccharide;
[0025] S2) coupling the carboxyazobenzene-modified chitosan oligosaccharide with a chemosensitizer to obtain a chemosensitizer-modified chitosan oligosaccharide;
[0026] S3) coupling the chemosensitizer-modified chitosan oligosaccharide with a PEG-modified lipid to obtain a chitosan oligosaccharide co-modified with a long-circulating lipid and a chemosensitizer;
[0027] S4) mixing the carboxyazobenzene-modified chitosan oligosaccharide with a chemotherapeutic drug in a solution to obtain the carboxyazobenzene-modified chitosan oligosaccharide that adsorbs the chemotherapeutic drug, and then coupling it with a PEG-modified lipid to obtain a long-circulating lipid-modified chitosan oligosaccharide that adsorbs the chemotherapeutic drug;
[0028] S5) mixing the chitosan oligosaccharide co-modified with the chemotherapy sensitizer obtained in step S3) with the chitosan oligosaccharide modified with the long-circulating lipid and adsorbed with the chemotherapy drug obtained in step S4), and adding an immune activator to form a nano drug carrier adsorbing the immune activator.
[0029] Another aspect of the present invention provides the use of the above-mentioned nanomedicine in preparing a drug for treating tumors.
[0030] Another aspect of the present invention provides use of drug-loaded nanomedicine in preparing a drug for improving the sensitivity of drug-resistant tumors.
[0031] Another aspect of the present invention provides the use of nanomedicine in preparing drugs for treating tumors and enhancing the sensitivity of drug-resistant tumors for synergistic treatment.
[0032] Another aspect of the present invention provides the use of drug-loaded nanomedicines in the preparation of drugs that simultaneously improve the sensitivity of drug-resistant tumors and enhance the immunotherapy effect.
[0033] Furthermore, the tumor is a drug-sensitive tumor or a drug-resistant tumor. Compared with the prior art, the present invention has the following beneficial effects:
[0034] Chemotherapy drugs, active molecules that reduce chemotherapy drug resistance, and immune-activating drugs are cleverly integrated into nanocarriers. The resulting nanocarriers, due to their slightly negative surface charge and small particle size (20nm), ensure stable blood circulation and better penetration into deep tumor layers. Tumor microenvironment-responsive release ensures the effective release of chemotherapy drugs and chemotherapy sensitizers in the tumor microenvironment, while achieving chemotherapy for the tumor while reducing the tumor's chemotherapy resistance through chemotherapy sensitizers. At the same time, the released immune-activating drugs and the in situ neoantigens produced by chemotherapy further activate dendritic cells and effector T cells, thereby further stimulating the immune system for tumor immunotherapy. Description of the drawings:
[0035] Figure 1 (a) The synthesis route of CS-Azo and (b) the H NMR spectrum.
[0036] Figure 2(a) The synthesis route of CS-SENSITIZER and (b) the H NMR spectrum.
[0037] Figure 3 Fourier transform infrared spectrum of the prepared DSPE-SENSITIZER.
[0038] Figure 4 This is the Zeta potential result of the nanomaterial prepared in Example 5.
[0039] Figure 5 Scanning electron microscope image and particle size distribution diagram of the prepared SIM@NPs-SENSITIZER / DOX.
[0040] Figure 6 This is the DOX release curve of the nanodrug in a simulated slightly acidic environment in vitro and its killing effect on tumor cells B16F10.
[0041] Figure 7 To detect the cytotoxicity and apoptosis of nanomedicines on tumor cells and DOX-resistant tumor cells.
[0042] Figure 8 To test the effect of nanomedicine in promoting antigen presentation, the expression of antigen peptides on the cell surface was detected by flow cytometry.
[0043] Figure 9 This is the tumor growth curve of the tumor-bearing mice constructed by subcutaneous injection of B16F10 cells after nanomedicine treatment in Example 9.
[0044] Figure 10 This is the tumor growth curve of Example 10 after nanomedicine treatment in the tumor-bearing mouse model constructed by subcutaneous injection of chemotherapy-resistant MCF / ADR cells and MCF.
[0045] Figure 11 This is the tumor growth curve of the tumor-bearing mouse model constructed by subcutaneous injection of chemotherapy-resistant B16F10ADR and B16F10 cells after nanodrug treatment in Example 11. Specific implementation method:
[0046] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.
[0047] The term "anti-tumor drug" refers to a drug that kills tumor cells or induces apoptosis of tumor cells by physical or chemical means, and the anti-tumor drug can be loaded onto a nanocarrier.
[0048] The term "immunomodulatory drugs" refers to a class of drugs that act on and through the immune system, activating the immune system and triggering an immune response, thereby suppressing tumors. They do not directly kill tumor cells through chemical or physical means, nor do they directly induce apoptosis of tumor cells.
[0049] The present invention will be further described below through specific embodiments in conjunction with the accompanying drawings.
[0050] Example 1: Preparation of carboxyazobenzene-modified chitosan oligosaccharide CS-Azo
[0051] 400 mg of chitosan oligosaccharide with a molecular weight of 2000 Da was added to 10 mL of dimethyl sulfoxide (DMSO) and fully dissolved in a 55 ° C water bath. 0.2 mM N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 54 mg of azobenzene-4,4-dicarboxylic acid were added to 10 mL of DMSO. After complete dissolution, the two solutions were mixed in a volume ratio of 1:1. The reaction was allowed to proceed overnight at room temperature and dialyzed for 24 hours with a dialysis bag with a molecular weight cutoff of 1 kDa to remove residual DMOS and some unreacted raw materials. The obtained product was named CS-Azo, and the reaction formula is as follows: Figure 1 As shown in a.
[0052] Experimental results: The H NMR spectrum of CS-Azo prepared in this example is as follows: Figure 1 As shown in b.
[0053] Example 2: Preparation of chemosensitizer-modified chitosan oligosaccharide CS-SENSITIZER
[0054] CS-Azo (70 mg), N-hydroxysuccinimide (23 mg), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (38 mg) were dissolved in 3 mL of DMSO and reacted for 6 hours. 38 mg of the hydrochloric chemosensitizer 2-(3,4-dihydroxyphenyl)ethylamine was added and the pH of the reaction system was adjusted to 8 with 0.1 M sodium hydroxide solution. The reaction was allowed to proceed overnight at room temperature in the dark. The solution was dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 1 kDa to remove residual DMSO and some unreacted raw materials. The obtained product was named CS-SENSITIZER, and the reaction formula is as follows: Figure 2 As shown in a.
[0055] Experimental results: The H NMR spectrum of the CS-SENSITIZER prepared in this example is as follows: Figure 2 As shown in b.
[0056] Example 3: Preparation of chitosan oligosaccharide DSPE-SENSITIZER modified with long-circulating lipids and chemosensitizers
[0057] 0.2 mM N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 55.45 mg of CS-SENSITIZER were dissolved in 3 mL of DMSO. After 6 hours, 9 mg of DSPE-PEG was added to the mixture. The mixture was allowed to react overnight in the dark and dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 1 kDa to remove residual DMSO and some unreacted raw materials.
[0058] The PEG terminal in the DSPE-PEG is carboxyl modified or NHS modified.
[0059] The obtained product was named DSPE-SENSITIZER.
[0060] Experimental results: The Fourier transform infrared spectrum of the DSPE-SENSITIZER prepared in this example is as follows: Figure 3 shown.
[0061] Example 4: Preparation of long-circulating lipid-modified chitosan oligosaccharide DSPE-DOX that adsorbs chemotherapeutic drugs
[0062] The block copolymer CS-Azo prepared in Example 1 was mixed with DOX in a molar ratio of 1:1, stirred and reacted for 24 hours, and then centrifuged to remove unadsorbed DOX.
[0063] 0.2 mM N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 55.45 mg of DOX-adsorbed CS-Azo were dissolved in 3 mL of DMSO. After 6 hours, 9 mg of DSPE-PEG was added to the mixture and the mixture was reacted overnight in the dark. The reaction was then dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 1 kDa to remove residual DMSO and some unreacted raw materials.
[0064] The PEG terminal in the DSPE-PEG is carboxyl modified or NHS modified.
[0065] The obtained product was named DSPE-DOX.
[0066] Example 5: Preparation and detection of nanomedicine
[0067] This embodiment relates to a multifunctional nanomedicine that can simultaneously release chemotherapy drugs in response to the micro-acidic environment of the tumor and release chemotherapy sensitizers in response to the hypoxic environment of the tumor, and has dual-response release with immune-activating drugs loaded inside the nanomaterial.
[0068] In this example, the two tumor microenvironment-responsive block copolymers (DSPE-SENSITIZER and DSPE-DOX) prepared in Examples 3 and 4 were mixed in a molar ratio of 1:1 and then prepared by self-assembly. The immune-activating drug simvastatin (SIM) was added during the self-assembly process to prepare SIM@NP-sensitizer / DOX.
[0069] The specific steps include:
[0070] DSPE-SENSITIZER and DSPE-DOX were both dispersed in water at a concentration of 0.5 mM. SIM was added to the reaction system at a concentration of 0.2 mg / mL. The reaction was accelerated at a stirring speed of 100 rpm / min. After 24 hours of reaction, the mixture was dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 1 kDa to remove impurities and unencapsulated SIM. The mixture was freeze-dried in a freeze dryer at a vacuum of 1 Pa and a temperature of -80°C for 24 hours. The resulting powder was stored at 4°C for later use.
[0071] SIM@NP-sensitizer: DSPE-SENSITIZER was dispersed in water at a concentration of 1.0 mM. SIM was added to the reaction system at a concentration of 0.2 mg / mL. The reaction was accelerated at a stirring speed of 100 rpm / min. After 24 hours of reaction, the product was dialyzed using a dialysis bag with a molecular weight cutoff of 1 kDa for 24 hours to remove impurities and unencapsulated SIM. The product was freeze-dried in a freeze dryer at a vacuum of 1 Pa and a temperature of -80°C for 24 hours. The resulting powder was stored at 4°C for later use.
[0072] SIM@NPs-DOX:DSPE-DOX were both dispersed in water at a concentration of 1.0 mM. SIM was added to the reaction system at a concentration of 0.2 mg / mL. The reaction process was accelerated at a stirring speed of 100 rpm / min. After 24 hours of reaction, the product was dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 1 kDa to remove impurities and unencapsulated SIM. The product was freeze-dried in a freeze dryer at a vacuum of 1 Pa and a temperature of -80°C for 24 hours. The obtained powder was stored at 4°C for later use.
[0073] Experimental results: Zeta potential of the above nanoparticles was measured, and the results are shown in Figure 4 It can be seen that the nanomedicine SIM@NP-sensitizer / DOX of the present invention has a slight electronegativity, which is conducive to its delivery in blood vessels and ensures the long-term circulation of the nanomedicine in the blood.
[0074] The scanning electron microscopy image and particle size distribution of SIM@NPs-sensitizer / DOX prepared in this example are shown in Figure 2. Figure 5As shown. Figure 5 It can be seen that nanoparticles are formed, and the particle size is uniform, concentrated between 10-30nm.
[0075] Example 6: In vitro simulated response release assay
[0076] This example is a response release test of the nanomedicine prepared in Example 5 under a simulated slightly acidic environment in vitro.
[0077] The specific steps include:
[0078] The nanodrug prepared in Example 5, loaded with 10 μg of SIM and 20 μg of DOX, was dispersed in 1 mL of PBS at pH 6.5 or pH 7.0 and placed in a dialysis bag with a molecular weight cutoff of 3500 Da. The dialysis bag was placed in 50 mL of PBS at pH 6.5 or pH 7.0. The liquid in the beaker was collected at fixed time points, 0, 1, 2, 4, 6, 8, 16, 24, 32, and 40 h, concentrated by ultrafiltration tube, resuspended in 2 mL of PBS, and its absorbance at 461 nm was measured using a UV spectrophotometer.
[0079] CCK-8 was used to detect the cytotoxicity of SIM@NPs-sensitizer / DOX on B16F10 cells. The steps are as follows:
[0080] B16F10 cells were seeded into 96-well plates at a density of 1x10 4 The cells were cultured at 37°C in 5% CO2 overnight, and the culture medium was replaced with culture medium containing different concentrations of SIM@NPs-PDA / DOX (0, 1.25, 2.5, 5, 10, 20, 40, and 80 μg / mL) and incubated at 37°C for 24 h.
[0081] At a ratio of 1:10, 10 μL of CCK-8 solution was added to each well, incubated for 30 min, and the absorbance at 450 nm was measured.
[0082] The cell activity was calculated according to the following formula: Cell activity = (A 450 Treatment Group-A 450 Blank group) / (A 450 Control group-A 450 blank group)*100%.
[0083] Experimental results: Figure 6 As shown, Figure 6 a shows that in the slightly acidic environment of the tumor simulated in vitro, the nanomedicine can release 77.26% of the drug, while at a neutral pH value, only 15.86% of the drug can be released, indicating that the drug of the present invention can release chemotherapy drugs in a targeted manner at the tumor site.
[0084] Figure 6 b shows (where NPs-sensitizer represents SIM@NPs-sensitizer, NPs-DOX represents SIM@NPs-DOX, and NPs-sensitizer / DOX represents SIM@NPs-sensitizer / DOX). The nanocarriers loaded with DOX simultaneously exhibited significant killing effects on melanoma cancer cells in a dose-dependent manner. Although SIM@NPs-sensitizer did not exhibit any anti-tumor activity even at a concentration of 80 μg / mL, when used in combination, a synergistic effect was demonstrated, and SIM@NPs-sensitizer / DOX exhibited superior anti-tumor activity compared to SIM@NPs-sensitizer and SIM@NPs-DOX.
[0085] Example 7: Cytotoxicity and cell apoptosis detection
[0086] This example is to detect the cytotoxicity and apoptosis of the nanomedicine prepared in Example 5 on tumor cells and DOX-resistant tumor cells. The specific steps include:
[0087] (1) During the cell culture process, by gradually increasing the concentration of DOX in the culture, DOX-resistant melanoma cells B16F10 / ADR and human breast cancer tumor cells MCF-7 / ADR were screened.
[0088] (2) B16F10, B16F10 / ADR, MCF-7, and MCF-7 / ADR cells were cultured at 10 5 The cells were seeded at a density of cells / well in a 6-well plate. After culturing for 24 hours, DOX or nanodrug SIM@NPs-SENSITIZER / DOX loaded with an equal amount of DOX was added to each cell type. After 24 hours of treatment, the cells were collected and apoptotic cells were detected using a cell apoptosis detection kit.
[0089] Experimental Results: As shown in Figure 7, DOX induced a 32.78% apoptosis rate in the MCF-7 cell model, but only a 5.88% apoptosis rate in the DOX-resistant MCF-7 / ADR cell model. Notably, the nanodrug SIM@NPs-SENSITIZER / DOX (represented in the figure as NPs-SENSITIZER / DOX) achieved high apoptosis rates of 67.13% and 67.50%, respectively, in both MCF-7 and MCF-7 / ADR cells.
[0090] In the B16F10 cell model, DOX induced a 14.34% apoptosis rate, but in the DOX-resistant B16F10 / ADR cell model, it only induced a 2.31% apoptosis rate. Notably, the nanodrug SIM@NPs-SENSITIZER / DOX (represented in the figure as NPs-PDA / DOX) achieved high apoptosis rates of 49.4% and 45.3%, respectively, in both B16F10-7 and B16F10 / ADR cells.
[0091] Example 8: Detection of the effect of promoting antigen presentation
[0092] This example is a test of the effect of the nanomedicine prepared in Example 5 on promoting antigen presentation. The specific steps include:
[0093] (1) DC2.4 cells were cultured at a rate of 1×10 5 The cells were seeded at a density of 1 μM in a 6-well plate and cultured for 24 h. Then, 1 μM SIM or nanodrug SIM@NPs-SENSITIZER / DOX loaded with an equal amount of SIM was added.
[0094] (2) Two hours later, fluorescein isothiocyanate-labeled ovalbumin (FITC-OVA) was added as a model antigen at a concentration of 10 μg / mL, and the fluorescence intensity at different time points was analyzed by fluorescence microscopy and flow cytometry.
[0095] (3) Antigen peptides presented on the cell surface are collected, stained with corresponding antibodies, and analyzed by flow cytometry.
[0096] Experimental results: Figure 8 As shown in the figure, compared with the FITC-OVA treatment group alone, the pre-addition of SIM@NPs-SENSITIZER / DOX can significantly promote the expression of antigen peptides, indicating that the SIM-loaded nanoparticles have a significant immune activation effect.
[0097] Example 9: Mouse tumor test
[0098] This example is a test of the effect of the nanomedicine prepared in Example 5 on treating mouse tumors. The specific steps include:
[0099] (1) Six-week-old female C57BL / 6J mice were injected subcutaneously on the flanks of the back with 10 B16F10 cells per flank. 5 tumor cells.
[0100] (2) The average tumor volume of mice reached 50 mm 3 The mice were randomly divided into 6 groups.
[0101] (3) SIM / DOX complex or nanodrug loaded with SIM and DOX was intravenously injected every three days, and the length (L) and width (W) of the mouse tumor were recorded every other day.
[0102] The dosing groups were as follows: saline injection group (control group), DOX solution group (DOX), solution group containing DOX / SIM at a molar ratio of 1:1 (DOX / SIM=1:1), solution group containing DOX / SIM at a molar ratio of 2:1 (DOX / SIM=2:1), DOX / SIM@NPs=1:1 group (product of Example 5), and DOX / SIM@NPs=2:1 group (prepared in the same manner as Example 5, except that DSPE-SENSITIZER and DSPE-DOX were mixed at a molar ratio of 1:2).
[0103] (4) Tumor volume is calculated as L×W 2 / 2, signs of death or tumor volume exceeds 2000mm 3 When the patient is found dead, he or she is deemed dead.
[0104] Experimental results: Figure 9 As shown in the figure, it can be calculated that in the DOX treatment group alone, the tumor inhibition rate can only reach 2.12%, while for the nanodrug treatment group loaded with DOX and SIM, the tumor inhibition rate reached 26.80% and 27.80%. This shows that the prepared nanodrug can achieve a significant effect of inhibiting tumor growth by enhancing penetration in the tumor microenvironment and responsive release. The formula for calculating the tumor growth inhibition rate is: relative tumor volume (RTV) = Vt / V0. Vt: that is, at the end of one experimental cycle (the tumor volume of the control group reaches 2000mm 3 V0: Tumor volume of mice in each group at the start of the experiment. Tumor growth inhibition rate = [1-RTV(experimental group) / RTV(control group)]*100%.
[0105] Example 10:
[0106] This example is a test of the effect of the nanomedicine prepared in Example 5 on treating drug-resistant tumors in mice. The specific steps include:
[0107] (1) Six-week-old female BALB / c nude mice were subcutaneously injected with MCF-7 or MCF-7 / ADR cells on their flanks. 5×10 cells were inoculated on each side. 6 tumor cells.
[0108] (2) The average tumor volume of mice reached 50 mm 3 The mice were randomly divided into 6 groups.
[0109] (3) DOX or nanodrugs loaded with SIM and DOX were intravenously injected every three days, and the length (L) and width (W) of the mouse tumor were recorded every other day.
[0110] The dosing groups were as follows: normal saline injection group (control group), drug-resistant mice injected with normal saline group (control group / ADR), DOX solution group (DOX group), drug-resistant mice injected with DOX solution group (DOX / ADR), product of Example 5 (SIM@NPs-sensitizer / DOX group), and drug-resistant mice injected with the product of Example 5 (SIM@NPs-sensitizer / DOX / ADR group).
[0111] (4) Tumor volume is calculated as L×W 2 / 2, signs of death or tumor volume exceeds 2000mm 3 When , it is determined to be dead. Figure 10 As shown, the DOX administration group alone achieved a tumor inhibition rate of 22.10% in the MCF-7 tumor model. In the MCF-7 / ADR tumor model, the tumor growth inhibition rate was only 9.23%. In the nanomedicine treatment group loaded with DOX and SIM, the tumor inhibition rates reached 58.30% and 60.59%. This shows that the prepared nanomedicine can significantly inhibit the growth of DOX-resistant tumors by enhancing penetration in the tumor microenvironment and responsive release. The formula for calculating the tumor growth inhibition rate is: relative tumor volume (RTV) = Vt / V0. Vt: that is, at the end of one experimental cycle (the tumor volume of the control group reaches 2000mm 3 V0: Tumor volume of mice in each group at the start of the experiment. Tumor growth inhibition rate = [1-RTV(experimental group) / RTV(control group)]*100%.
[0112] Example 11:
[0113] This example is a test of the effect of the nanomedicine prepared in Example 5 on treating drug-resistant tumors in mice. The specific steps include:
[0114] Six-week-old female C57BL / 6J nude mice were subcutaneously injected with B16F10 or B16F10 / ADR cells on their flanks, with 1×10 cells inoculated on each side. 5 tumor cells.
[0115] (1) The average tumor volume of mice reached 50 mm 3 The mice were randomly divided into 6 groups.
[0116] (2) DOX or nanodrugs loaded with SIM and DOX were intravenously injected every three days, and the length (L) and width (W) of the mouse tumors were recorded every other day.
[0117] The dosing groups were as follows: normal saline injection group (control group), normal saline injection group for drug-resistant mice (control group / ADR), DOX solution group (DOX), drug-resistant mice injected with DOX solution group (DOX / ADR), product of Example 5 (SIM@NPs-sensitizer / DOX group), and drug-resistant mice injected with the product of Example 5 (SIM@NPs-sensitizer / DOX / ADR group).
[0118] (3) Tumor volume is calculated as L×W 2 / 2, signs of death or tumor volume exceeds 2000mm 3 When the patient is found dead, he or she is deemed dead.
[0119] Experimental results: Figure 11 As shown, the tumor inhibition rates of the DOX- and SIM-loaded nanodrug treatment groups reached 69.35% and 70.67%, respectively, indicating that the prepared nanodrugs can significantly inhibit the growth of DOX-resistant tumors by enhancing their penetration into the tumor microenvironment and responsive release.
[0120] The formula for calculating tumor growth inhibition rate is: relative tumor volume (RTV) = Vt / V0. Vt is the time when the tumor volume of the control group reaches 2000 mm at the end of one experimental cycle. 3 V0: Tumor volume of mice in each group at the start of the experiment. Tumor growth inhibition rate = [1-RTV(experimental group) / RTV(control group)]*100%.
Claims
1. A nanomedicine self-assembled from a block copolymer that responds to the hypoxic environment of a tumor, characterized in that: The nano drug carrier is made of chitosan oligosaccharide, carboxylazobenzene, PEG-modified lipid, chemotherapy sensitizer, chemotherapy drug and immune activator. The preparation method of the nano drug is as follows: S1) chitosan oligosaccharide and carboxyazobenzene are coupled via an amide bond to obtain carboxyazobenzene-modified chitosan oligosaccharide; S2) coupling the carboxyazobenzene-modified chitosan oligosaccharide with a chemosensitizer to obtain a chemosensitizer-modified chitosan oligosaccharide; S3) coupling the chemosensitizer-modified chitosan oligosaccharide with a PEG-modified lipid to obtain a chitosan oligosaccharide co-modified with a long-circulating lipid and a chemosensitizer; S4) mixing the carboxyazobenzene-modified chitosan oligosaccharide with a chemotherapeutic drug in a solution to obtain the carboxyazobenzene-modified chitosan oligosaccharide that adsorbs the chemotherapeutic drug, and then coupling it with a PEG-modified lipid to obtain a long-circulating lipid-modified chitosan oligosaccharide that adsorbs the chemotherapeutic drug; S5) mixing the chitosan oligosaccharide co-modified with the chemotherapy sensitizer obtained in step S3) with the chitosan oligosaccharide modified with the long-circulating lipid and adsorbed with the chemotherapy drug obtained in step S4), and adding an immune activator to form a nano drug carrier adsorbing the immune activator.
2. The nanomedicine according to claim 1, characterized in that The molecular weight of the chitosan oligosaccharide is 1000-4000Da.
3. The nanomedicine according to claim 1, characterized in that The carboxyazobenzene is azobenzene-4,4-dicarboxylic acid.
4. The nanomedicine according to claim 1, characterized in that The chemotherapy drug is doxorubicin, paclitaxel, cisplatin or a derivative thereof.
5. The nanomedicine according to claim 1, characterized in that The chemotherapy sensitizer is one of 2-(3,4-dihydroxyphenyl)ethylamine, resveratrol, vitamin K2, shikonin and curcumin.
6. The nanomedicine according to claim 1, characterized in that The PEG-modified lipid is DSPE-PEG, and the PEG terminal is modified with one of N-hydroxysuccinimide (NHS) and carboxyl (COOH).
7. The nanomedicine according to claim 1, characterized in that The molar ratio of the chitosan oligosaccharide modified with the chemotherapy sensitizer in step S5) to the chitosan oligosaccharide modified with the long-circulating lipid and adsorbed chemotherapy drug obtained in step S4) is 1:4-4:1, preferably 4:1, 3:1, 2:1, 1:1, 2:1, 3:1, 4:
1.
8. The nanomedicine according to claim 1, characterized in that The immune activator is one or more of nucleic acid drugs, nano drugs, small molecule drugs, macromolecular drugs, and antibody drugs; preferably one or more of simvastatin, aluminum phosphate, aluminum hydroxide, and potassium aluminum sulfate.
9. A method for preparing the nanomedicine according to any one of claims 1 to 8, comprising the following steps: S1) chitosan oligosaccharide and carboxyazobenzene are coupled via an amide bond to obtain carboxyazobenzene-modified chitosan oligosaccharide; S2) coupling the carboxyazobenzene-modified chitosan oligosaccharide with a chemosensitizer to obtain a chemosensitizer-modified chitosan oligosaccharide; S3) coupling the chemosensitizer-modified chitosan oligosaccharide with a PEG-modified lipid to obtain a chitosan oligosaccharide co-modified with a long-circulating lipid and a chemosensitizer; S4) mixing the carboxyazobenzene-modified chitosan oligosaccharide with a chemotherapeutic drug in a solution to obtain the carboxyazobenzene-modified chitosan oligosaccharide that adsorbs the chemotherapeutic drug, and then coupling it with a PEG-modified lipid to obtain a long-circulating lipid-modified chitosan oligosaccharide that adsorbs the chemotherapeutic drug; S5) mixing the chitosan oligosaccharide co-modified with the chemotherapy sensitizer obtained in step S3) with the chitosan oligosaccharide modified with the long-circulating lipid and adsorbed with the chemotherapy drug obtained in step S4), and adding an immune activator to form a nano drug carrier adsorbing the immune activator.
10. Use of the nanomedicine according to any one of claims 1 to 8 in the preparation of a drug for treating tumors, increasing the sensitivity of drug-resistant tumors, synergistically treating tumors and increasing the sensitivity of drug-resistant tumors, or simultaneously increasing the sensitivity of drug-resistant tumors and enhancing the immunotherapy effect.