Colon cancer multi-mode collaborative treatment auxiliary system

Abstract

The invention relates to the field of cancers, in particular to a colon cancer multi-mode collaborative treatment auxiliary system which comprises an LPDAM nano-platform, the LPDAM nano-platform comprises a medicine prepared from Lapacone, polydopamine and manganese ions, the medicine is applied to cancer cells, a photo-thermal module is used for acting on the cancer cells to which the LPDAM nano-platform is applied by utilizing a photo-thermal effect, and the LPDAM nano-platform is used for treating the cancer cells to which the LPDAM nano-platform is applied. A magnetic resonance module for magnetic resonance imaging of a patient whose cancer cells have been administered an LPDAM nanoplatform. By adopting the technical scheme of the invention, the Lapacone micelle can be modified by polydopamine to maintain the stability of the micelle, and manganese ions are chelated on the polydopamine to form the LPDAM nano platform with small and stable particle size, so that the primary tumor can be ablated, the distant metastasis and pulmonary metastasis of the tumor can be obviously reduced, and the toxic reaction of the whole body is not caused.

Description

Technical Field

[0001] The present invention relates to the field of cancer, and in particular to a multi-mode collaborative treatment auxiliary system for colon cancer. Background Art

[0002] Colon cancer has become one of the most common malignant tumors in the world today. According to the statistics of the World Health Organization in 2022, the number of new cases of colorectal cancer in 2022 reached 1,926,118, and the incidence rate ranked third, second only to lung cancer and breast cancer. The mortality rate ranked second, with 903,859 deaths, accounting for 9.3% of the total cancer deaths.

[0003] Lapachone is a chemotherapy drug with anti-tumor properties. Normally, the NQO1 enzyme is highly expressed in tumor cells, and Lapachone takes advantage of the high NQO1 enzyme environment in tumor cells to catalyze the production of H2O2, thereby causing oxidative stress damage to tumor cells. Currently, the anti-cancer activity of Lapachone has been verified in multiple studies, including in vitro cell experiments and in vivo animal experiments. Lapachone can also be used in combination with other anti-cancer drugs to enhance the anti-cancer effect and reduce side effects, and has broad application prospects.

[0004] Manganese is a very important trace element for the human body, and manganese ions have excellent chemodynamic therapeutic effects on tumors. Manganese ions catalyze Fenton-like reactions, catalyzing H2O2 to generate highly oxidizing hydroxyl radicals (·OH). These active oxygen species have a good killing effect on tumors.

[0005] Polydopamine is a common photosensitizer that can generate heat under near-infrared light irradiation. In view of the heat-intolerant characteristics of tumor cells, photothermal therapy can effectively inhibit tumors. Photothermal therapy can also promote cell immunogenic death, secrete ATP and HMBG1, and express CRT, thereby promoting the maturation of DC cells and activating the anti-tumor immune system.

[0006] Immunotherapy is a promising anti-tumor therapy. cGAS-STING signaling is a DNA sensing mechanism that plays a key role in connecting tumor cells and immune cells. When cGAS senses tumor-derived dsDNA that should not be present, it activates its enzymatic activity to generate cyclic guanosine monophosphate (cGAMP). Then cGAMP binds to the interferon-stimulated protein (STING) dimer to induce STING oligomerization activation. The STING protein is transported along the endoplasmic reticulum to the Golgi apparatus, gradually recruiting the key kinase TBK1, phosphorylating and activating the interferon regulatory factor (IRF3). Activated IRF3 enters the nucleus, promoting the synthesis of type 1 interferon (IFN) and the secretion of pro-inflammatory cytokines. These factors trigger the maturation of dendritic cells (DC) for antigen presentation, thereby promoting the activation of T cells to produce anti-tumor immunity. However, single immunotherapy still has certain limitations. Therefore, a multimodal synergistic treatment adjuvant system for colon cancer is needed to obtain better therapeutic effects. Summary of the invention

[0007] To solve the above problems, the present invention provides a multimodal synergistic treatment auxiliary system for colon cancer. Lapachone micelles are modified with polydopamine to maintain the stability of the micelles, and then manganese ions are chelated on the polydopamine to form a small and stable LPDAM nanoplatform, which can ablate the primary tumor and significantly reduce the distant metastasis and lung metastasis of the tumor without causing systemic toxic reactions.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows: a multi-mode collaborative treatment auxiliary system for colon cancer, comprising:

[0009] LPDAM nanoplatform: includes drugs made of lapachone, polydopamine and manganese ions, which are used to administer to cancer cells.

[0010] Further, the preparation method of the LPDAM nanoplatform includes:

[0011] Step 1: Prepare PEG-PLA and Lapachone, and prepare Lapachone micelles using thin film hydration method;

[0012] Step 2: Prepare dopamine hydrochloride, dissolve dopamine hydrochloride and Lapachone micelles in Tris buffer and stir, put into ultrafiltration tube for centrifugation, after centrifugation, take the supernatant and wash with deionized water to obtain LPDA modified with dopamine Lapachone micelles;

[0013] Step 3: Prepare soluble manganese metal salt, dissolve the manganese metal salt and LPDA in deionized water and stir, put into an ultrafiltration tube for centrifugation, and after centrifugation, take the supernatant and wash it with deionized water to obtain LPDAM with LPDA chelated by metal manganese ions.

[0014] Furthermore, in step 1, the thin film hydration method is to dissolve PEG-PLA and Lapachone in acetonitrile to obtain a solvent, evaporate the solvent by a rotary evaporator to form a thin film, add physiological saline after the film is formed, and perform ultrasonic treatment to obtain a drug-loaded micelle aqueous solution, and filter the drug-loaded micelle aqueous solution through a filter to remove unencapsulated drug aggregates to obtain Lapachone micelles.

[0015] Furthermore, the filter in step one is a 0.45 μm nylon filter, and the ultrafiltration tubes in steps two and three are both 100,000 kDa ultrafiltration tubes.

[0016] Furthermore, in step 2 and step 3, the centrifugation time is 20-30 min, the centrifugation speed is 2000 rpm, and the deionized water washing is performed three times.

[0017] Furthermore, in step 1, the mass ratio of PEG-PLA to Lapachone is 9:1.

[0018] Furthermore, in step 1, the evaporation temperature of the rotary evaporator is 60°C.

[0019] Furthermore, the manganese metal salt is MnCl2.

[0020] Furthermore, in step 2, the mass ratio of Lapachone micelles to dopamine hydrochloride is 1:1, and in step 3, the mass ratio of LPDA to MnCl2 is 1:1.

[0021] Furthermore, it also includes:

[0022] Photothermal module: used to use the photothermal effect to act on cancer cells to which the LPDAM nanoplatform is applied;

[0023] Magnetic resonance module: used for magnetic resonance imaging of patients in which the LPDAM nanoplatform has been administered to cancer cells.

[0024] The technical principles of the above scheme are as follows:

[0025] The surface of Lapachone micelles was further modified with dopamine to maintain the stability of the micelles. In an alkaline environment, dopamine molecules formed a black polydopamine shell on the surface of Lapachone micelles by self-polymerization. Finally, metal manganese ions were chelated on the surface of polydopamine nanospheres.

[0026] After the LPDAM nanoplatform acts on cancer cells. Lapachone uses the high NQO1 enzyme environment in tumor cells to catalyze the production of H2O2, and manganese ions catalyze the Fenton-like reaction, catalyzing H2O2 to generate highly oxidizing hydroxyl radicals (·OH), thereby enhancing the effect on tumor damage. The combination of Lapachone and manganese ions promotes the amplified production of reactive oxygen species, and the photothermal effect of polydopamine further enhances the reaction effect of the Fenton-like reaction. Reactive oxygen species damage mitochondria, and the dsDNA released by mitochondria activates the cGAS-STING pathway together with manganese ions, thereby inducing the maturation of DC cells. At the same time, the ICD mediated by photothermal therapy further promotes the maturation of DC cells. This ultimately leads to the activation of the anti-tumor immune system and the initiation of T cell infiltration. The LPDAM nanoplatform combines chemotherapy / chemodynamic therapy, photothermal therapy, and immunotherapy in a synergistic manner.

[0027] In terms of photothermal effect, dopamine itself is an excellent photothermal agent. The heating effect of LPDAM is slightly lower than that of LPDA, which may be due to the fact that at the same mass concentration, the PDA content in the LPDAM nanoplatform is slightly lower than that in the LPDA nanoplatform. However, the chelation of manganese ions does not affect the photothermal effect of PDA, and the photothermal effect it produces is sufficient to cause damage to cancer cells.

[0028] The LPDAM nanoplatform chelated with manganese ions also has the function of magnetic resonance imaging. Its nanoparticle solution detection shows a significant concentration-dependent signal enhancement effect, which makes it have a certain MRI contrast agent effect, making MRI imaging clearer and making it easier to monitor tumor changes through MRI imaging during the treatment cycle.

[0029] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the working principle of an embodiment of the multi-modal collaborative treatment auxiliary system for colon cancer of the present invention.

[0031] Figure 2 Schematic diagram of the photothermal, chemical kinetic and MRI performances of the LPDAM nanoplatform in an embodiment of the multi-modal collaborative treatment auxiliary system for colon cancer of the present invention.

[0032] Figure 3 Schematic diagram of the stability of the LPDA and LPDAM nanoplatforms in an embodiment of the multi-modal collaborative treatment auxiliary system for colon cancer of the present invention.

[0033] Figure 4Schematic diagram of the photothermal, chemical kinetic and MRI performances of the LPDAM nanoplatform in an embodiment of the multi-modal collaborative treatment auxiliary system for colon cancer of the present invention.

[0034] Figure 5 Schematic diagram of the photothermal performance of the LPDA and LPDAM nanoplatforms in an embodiment of the multi-modal collaborative treatment auxiliary system for colon cancer of the present invention.

[0035] Figure 6 Schematic diagram of the absorption spectrum of MB degradation rate at different LPDAM concentrations in an embodiment of the multi-modal synergistic treatment auxiliary system for colon cancer of the present invention.

[0036] Figure 7 Schematic diagram of LPDAM-mediated Lapachone / chemodynamic / photothermal synergistic therapy inducing tumor cell damage in an embodiment of the multi-modal synergistic treatment auxiliary system for colon cancer of the present invention.

[0037] Figure 8 Schematic diagram of the effect of the Lapachone / chemodynamic / photothermal synergistic LPDAM nanoplatform on MC38 cells in an embodiment of the multi-modal synergistic treatment auxiliary system for colon cancer of the present invention.

[0038] Fig. 9 This is a schematic diagram of the morphology of CT26 cells after different treatments observed under a light microscope in an embodiment of the multi-modal collaborative treatment auxiliary system for colon cancer of the present invention.

[0039] Fig.10 This is a schematic diagram of the cell apoptosis rate statistics using flow cytometry in an embodiment of the multi-modal collaborative treatment auxiliary system for colon cancer of the present invention.

[0040] Fig.11 This is a schematic diagram of detecting cell apoptosis by Annexin V-FITC / PI staining according to an embodiment of the multi-modal synergistic treatment auxiliary system for colon cancer of the present invention.

[0041] Fig.12 Schematic diagram of the LPDAM nanoplatform promoting immune activation through immunogenic cell death and cGAS-STING signaling pathway in an embodiment of the multimodal synergistic treatment auxiliary system for colon cancer of the present invention.

[0042] Fig.13 This is a schematic diagram of the body weight curve after different treatments of the in vivo synergistic lapachone / chemodynamic / photothermal therapy a of a tumor in an embodiment of the multi-modal synergistic treatment auxiliary system for colon cancer of the present invention.

[0043] Fig.14 Schematic diagram of the LPDAM nanoplatform activating systemic anti-tumor immune response in vivo according to an embodiment of the multi-modal synergistic treatment auxiliary system for colon cancer of the present invention.

[0044] Fig.15 Schematic diagram of in vitro lung images of different groups after treatment with the anti-tumor efficacy of the LPDAM nanoplatform in lung metastases according to an embodiment of the multi-modal synergistic treatment auxiliary system for colon cancer of the present invention.

[0045] Fig.16 Schematic diagram of the body weight curve of mice treated with intravenous LPDAM nanoplatform for acute toxicity evaluation of intravenous LPDAM nanoplatform in an embodiment of the multi-modal synergistic treatment auxiliary system for colon cancer of the present invention.

[0046] Fig.17 This is a schematic diagram of the toxicity test of PDA empty micelles at different concentrations in an embodiment of the multi-modal synergistic treatment auxiliary system for colon cancer of the present invention. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0049] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0050] The following is further described in detail through specific implementation methods:

[0051] As attached Figure 1-Figure 17 Shown: A multi-modal collaborative treatment auxiliary system for colon cancer, comprising:

[0052] LPDAM nanoplatform: includes drugs made of lapachone, polydopamine and manganese ions, which are used to administer to cancer cells.

[0053] Photothermal module: used to use the photothermal effect to act on cancer cells to which the LPDAM nanoplatform is applied.

[0054] Magnetic resonance module: used to perform magnetic resonance imaging of patients in which the LPDAM nanoplatform has been administered to cancer cells.

[0055] The preparation method of the LPDAM nanoplatform includes, first, preparing Lapachone-loaded micelles by thin film hydration method. 9 mg of PEG-PLA and 1 mg of Lapachone are dissolved in acetonitrile to obtain a solvent, and the solvent is evaporated at 60 degrees Celsius by a rotary evaporator to form a thin film. Then, physiological saline is added and ultrasonically treated for five minutes to obtain a drug-loaded micelle aqueous solution. The drug-loaded micelle aqueous solution is filtered through a 0.45 μm nylon filter to remove unencapsulated drug aggregates in the solution.

[0056] Take 10 mg of Lapachone micelles and 10 mg of dopamine hydrochloride, dissolve in Tris buffer (10 mM, pH 8.5), and stir at room temperature for 6 hours. Place the product in a 100000 kDa ultrafiltration tube, centrifuge at 2000 rpm for 20 minutes, take the supernatant, wash with deionized water and repeat the centrifugation washing three times to obtain LPDA.

[0057] Take 15 mg LPDA and 15 mg MnCl2, dissolve in 8 ml deionized water, and stir at room temperature for 24 hours. Place the product in a 100000 kDa ultrafiltration tube, centrifuge at 2000 rpm for 20 minutes, take the supernatant, wash with deionized water and repeat the centrifugation washing three times to obtain LPDAM.

[0058] The verification experiment is as follows:

[0059] When preparing Lapachone micelles, empty micelles were also prepared as a control group.

[0060] Characterization of LPDAM Nanoplatform:

[0061] The morphology of LPDAM was characterized by transmission electron microscopy. The dynamic light scattering and zeta potential of the LPDAM nanoplatform were detected using a laser particle sizer. The absorption spectrum and Fourier infrared spectrum of the nanoplatform were detected by UV-visible spectrometer and infrared spectrometer, respectively. The LPDAM nanoplatform was dispersed in deionized water, saline and culture medium containing 10% serum. After standing for 0, 1, 3, 5, and 7 days, the absorption spectrum was detected to confirm the stability of the LPDAM nanoplatform. The content of Lapachone was confirmed by high performance liquid chromatography. The content of manganese ions was confirmed by inductively coupled plasma emission spectrometry.

[0062] Fenton reaction performance evaluation:

[0063] LPDAM with different concentrations was mixed with 20 mM hydrogen peroxide and 10 mg / mL methylene blue solution, and incubated at 37° C. or 60° C. for 15 or 30 minutes. The absorption spectrum of the mixture was detected and the degradation rate of methylene blue was calculated.

[0064] Photothermal performance evaluation:

[0065] LPDA and LPDAM were prepared into a dispersion with a concentration of 1 mg / mL or 2 mg / mL. 1 mL of water and LPDA or LPDAM dispersion were placed in a quartz cuvette. 808 nm near-infrared lasers with different powers (0.5, 1.0, 1.5, 2.0, 2.5 W / cm2) were used for 300 s. The thermal imager read the temperature data and collected images every 10 s. Then, 1 mg / mL of LPDA or LPDAM dispersion was taken and 2.5 W / cm 2 The laser was turned on / off for 5 mins, and five heating / cooling cycles were performed to evaluate the photothermal stability of the nanoplatform. Finally, the LPDA or LPDAM dispersion was irradiated with laser until the dispersion reached thermal equilibrium and then naturally cooled to room temperature. The dispersion temperature data was collected and the photothermal conversion efficiency (η) was calculated.

[0066] MRI performance evaluation:

[0067] MesoMR23-060H-I magnetic resonance apparatus (0.5T) was used to scan LPDAM dispersions of different concentrations (15.625, 31.25, 62.5, 125, 250 and 500 μg / mL), record T1 and T2 relaxation times and obtain T1-weighted MR images. In order to further demonstrate the magnetic resonance imaging effect of LPDAM in vivo, tumor-bearing mice were prepared and LPDAM dispersions were injected into the tumor tissues of tumor-bearing mice. The mice were scanned by MRI before and after the injection.

[0068] Cell culture:

[0069] Two cell lines were used in the experiment, namely Mouse Colorectal Carcinoma Cells (CT26, mouse colorectal cancer cells) and Mouse Colon Cancer Cells (MC38, mouse colon cancer cells). The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and the culture environment was 37°C and 5% CO2. The control group, NIR (near infrared light) group, LPDA group, LPDAM group, PDA (dopamine) + NIR group, and LPDM + NIR group were set up respectively.

[0070] Cellular Uptake:

[0071] After LPDAM was loaded with FITC fluorescent probe, it was co-cultured with CT26. After 4 hours, the unphagocytosed LPDAM was washed away with PBS, and the cell nuclei were stained with Hoechst, and the cell fluorescence images were taken.

[0072] PDA cytotoxicity assessment:

[0073] The cells in exponential growth phase were 4 The cells were evenly seeded into a 96-well plate at a density of 10 cells per well and incubated overnight in a cell culture incubator. Next, fresh culture medium containing different concentrations of empty micelle PDA was added to the wells and incubated for 24 hours. The culture medium containing nanoparticles was then removed and washed with PBS. Finally, CCK-8 solution was added and incubated for 4 hours, and the absorbance of the solution was measured at 450nm using an enzyme reader to calculate the cell survival rate.

[0074] Evaluation of the synergistic therapeutic effect of Lap / CDT / PTT in vitro:

[0075] The cells in exponential growth phase were 4 The cells were evenly inoculated into a 96-well plate at a density of 10 cells per well and incubated overnight in a cell culture incubator. Fresh culture medium containing different concentrations of nanodispersions was added to each group of wells and incubated for 24 hours (the near-infrared light group was irradiated with a near-infrared laser for five minutes 4 hours after administration, and then incubated for 20 hours). The culture medium containing nanoparticles was aspirated and washed with PBS. Finally, CCK-8 solution was added and incubated for 4 hours, and the absorbance of the solution was measured at 450nm with an enzyme marker and the cell survival rate was calculated to evaluate the combined efficacy of Lap / CDT / PTT in cells.

[0076] Bio-TEM mitochondrial morphology analysis:

[0077] After the cells were treated with fresh medium containing 300 μg / mL LPDAM for 4 hours (the control group was only replaced with fresh medium without LPDAM), the medium was removed and 2.5% glutaraldehyde was added for fixation. The cells were gently scraped and centrifuged at 2000 rpm for five minutes and the cells were collected. Finally, the cells were embedded and ultrathinly sectioned (Leica UC7), and the mitochondrial morphology in the cells was observed using Bio-TEM.

[0078] Apoptosis assay:

[0079] The Annexin V-FITC / PI kit was used to evaluate cell apoptosis and necrosis. First, the cells were inoculated in a 12-well plate and incubated overnight, and then fresh culture medium was replaced (including control group, NIR group, LPDA group, LPDAM group, PDA+NIR group, LPDM+NIR group), and the equivalent concentration of the nanodispersion in each group of culture medium was controlled to be 300μg / mL, and incubated for 24 hours. (The NIR-related group was treated with laser irradiation of 0.625W / cm2, 5mins four hours after administration, and then continued to incubate for 20 hours.) After 24 hours, Annexin V-FITC / PI fluorescent staining cell imaging was performed or cells were collected and stained with Annexin V-FITC / PI for flow cytometry analysis.

[0080] Evaluation of Tumor Cell Immunogenic Death:

[0081] The cells were inoculated in 24-well plates overnight, and then fresh culture medium was replaced (including control group, NIR group, LPDA group, LPDAM group, PDA+NIR group, LPDM+NIR group). The equivalent concentration of nanodispersed liquid in each group of culture medium was controlled to be 300μg / mL, and incubated for 8 hours. (The NIR group was treated with laser irradiation of 0.625W / cm2, 5mins 4 hours after administration, and then incubated for 4 hours). Then the supernatant of each group was collected, and the ATP level in the cell supernatant was detected using an ATP detection kit. Alternatively, the cells were immunofluorescently stained with CRT and HMGB1 antibodies, and cell fluorescence imaging was collected.

[0082] Western blot detection of cGAS-STING signaling pathway activation:

[0083] First, the cells were inoculated in a 12-well plate and incubated overnight, and then fresh culture medium was replaced (including control group, NIR group, LPDA group, LPDAM group, PDA+NIR group, LPDM+NIR group), and the equivalent concentration of the nanodispersion in each group of culture medium was controlled to be 300μg / mL, and incubated for 8 hours. (The NIR group was treated with laser irradiation of 0.625W / cm2, 5mins 4 hours after administration, and then continued to incubate for 4 hours.) After 24 hours, the cells were collected and the total protein concentration was determined by BCA protein assay kit. Subsequently, cGAS-STING pathway-related proteins were determined by Western blot with GAPDH as a reference.

[0084] ELISA to determine the biochemical changes of immune cells stimulated by LPDAM:

[0085] The cell supernatant and tumor tissue homogenate supernatant were collected, and the expression levels of TNF-α and IFN-β were detected using ELISA detection kits.

[0086] ELISA detects the biochemical effects of immune cells:

[0087] The cell culture supernatant and tumor tissue homogenate supernatant were collected, and the expression levels of TNF-α and IFN-β were detected by ELISA kits.

[0088] Flow cytometric analysis of dendritic cell maturation:

[0089] Prepare mice, separate the bone marrow of the mouse tibia and fibula, extract and separate bone marrow cells, incubate the cells with IL-4 (10ng / mL) and GM-CSF (20ng / mL), and after seven days of induction, the cells will differentiate into BMDC (bone marrow-derived dendritic cells). At the same time, CT26 cells were inoculated in a 12-well plate, and CT26 was incubated for 24 hours in groups (control group, NIR group, LPDA group, LPDAM group, PDA+NIR group, LPDM+NIR group). Subsequently, the supernatant of each group of CT26 was collected as a culture medium for BMDC for 24 hours. Finally, BMDC was collected and stained with fluorescent antibodies, and flow cytometry was performed to analyze the maturity of BMDC.

[0090] Construction of animal model and efficacy evaluation:

[0091] CT26 cells in exponential growth phase were inoculated subcutaneously into Balb / c mice (4-5 weeks old). 6The cells were suspended in 50ul serum-free medium and injected subcutaneously in mice. Tumor size was measured using a vernier caliper during the experiment. Tumor volume was calculated as V=W×L / 2, where W and L are the width and length of the tumor, respectively. When the tumor volume reached 50-60mm3, the mice were randomly divided into six groups: (control group, NIR group, LPDA group, LPDAM group, PDA+NIR group, LPDM+NIR group), and the corresponding nanodispersions were injected into the tumor according to the group. The equivalent concentration of the nanodispersions in each group was controlled to be 1mg / mL, and the volume was 50ul. After the drug diffused for 2h, the tumors of mice in groups II, V, and VI were irradiated with near-infrared laser at 2W / cm2 for 5mins. The temperature changes of mice were recorded with a thermal imager. The weight and tumor volume of the mice were measured every other day. After 20 days, the mice were euthanized, and the tumors of the mice were obtained for photography.

[0092] Analysis of different immune cells in vivo:

[0093] Construct a subcutaneous tumor-bearing mouse model. When the tumor volume reaches 50-60mm 3 The mice were randomly divided into six groups: control group, NIR group, LPDA group, LPDAM group, PDA+NIR group, LPDM+NIR group. The corresponding nanodispersions were injected into the tumor according to the group. The equivalent concentration of the nanodispersions in each group was controlled at 1 mg / mL and the volume was 50 ul. After the drug diffused for 2 hours, the tumors of mice in groups II, V, and VI were exposed to 1 W / cm 2 , 5mins of near-infrared laser irradiation. After 20 days, the mice were divided into groups for treatment and euthanized, and the inguinal lymph nodes of each group of mice were obtained. After grinding, single cell suspension was obtained through a 70μm filter. The cells were incubated with CD11c, CD80, and CD86 antibodies, and the maturity of DC cells in mouse lymph node swelling was examined by flow cytometry.

[0094] Immunohistochemical analysis of cGAS-STING pathway activation in tumor tissues:

[0095] Tumor tissues were fixed with paraformaldehyde and then embedded in paraffin for sectioning. HE staining and immunohistochemical staining of the cGAS-STING pathway were performed on the tumor sections.

[0096] Evaluation of distal tumor suppression effect:

[0097] 10 6 CT26 cells were suspended in 50ul serum-free medium and injected subcutaneously on the right side of the mouse. 3The mice were randomly divided into groups: (control group, NIR group, LPDA group, LPDAM group, PDA+NIR group, LPDM+NIR group), and injected with the corresponding nanodispersions according to the grouping. The equivalent concentration of the nanodispersions in each group was controlled at 1 mg / mL and the volume was 50 ul. The tumors of mice in the photothermal group received 1 W / cm 2 , 5mins of near-infrared laser irradiation. Intratumoral injection and laser irradiation were once a day. After two treatments, 5*10 5 CT26 cells were added to the mice, and the weight of the mice and the volume of the contralateral tumor were measured every other day. After 14 days, the mice were euthanized and the bilateral tumors were removed for subsequent experiments.

[0098] Evaluation of lung metastases:

[0099] When the tumor volume reaches 50-60 mm 3 At the same time, the mice were randomly divided into groups and treated twice, and 5×10 CT26 cells were injected into the tail vein. 6 Each mouse was euthanized 20 days later, and the lung tissues were removed and fixed with paraformaldehyde. The lung tissues were photographed, and the number of lung metastatic nodules was counted, followed by paraffin-embedded sections and HE staining.

[0100] In vivo toxicity assessment:

[0101] Balb / c mice were randomly divided into four groups and injected with LPDAM at concentrations of 0, 20, 40 and 60 mg / kg through the tail vein. The weight of mice in each group was collected every other day. After 28 days, the mice were euthanized and the blood and internal organs of mice in each group were collected. The organs were then photographed and weighed to calculate the organ coefficient. Blood routine and blood biochemistry analysis were performed on the mouse blood.

[0102] Experimental conclusion:

[0103] Preparation and characterization of LPDAM nanoplatform:

[0104] like Figure 2 As shown in part a of FIG, PEG-PLA micelles loaded with Lapchone were synthesized and characterized by TEM. The Lapchone micelles were spherical and had a particle size of about 20 nm. Figure 1 As shown in the ef part, the hydrodynamic diameter of the micelle is 50.7nm and the zeta potential is -1.34mv. UV-visible spectrometer detection shows that the characteristic absorption peak of Lapchone is located at 252nm. The characteristic absorption peak in the spectrum of PEG-PLA-Lapchone confirms the effective loading of Lapchone. HPLC confirmed that the actual loading amount of Lapchone is 1.74%.

[0105] The surface of Lapachone micelles was further modified with dopamine to maintain the stability of the micelles. In an alkaline environment, dopamine molecules formed a black polydopamine shell on the surface of Lapachone micelles by self-polymerization. Finally, metal manganese ions were chelated on the surface of polydopamine nanospheres. The nanoparticles were characterized by TEM, such as Figure 2 As shown in parts b and c of the figure, the obtained PDA and PDAM both present spherical structures with an average diameter of about 20 nm. Dynamic laser scattering (DLS) studies the synthesis process of the material by measuring the changes in particle size and zeta potential during the synthesis process. DLS shows that with the formation of the PDA coating and the chelation of manganese ions, Figure 2 As shown in part e, the hydrodynamic diameter of the nanoparticles changed from 58.8 nm to 91.3 nm, and the polydispersity index (PDI) changed from 0.152 to 0.197. Due to the chelation of positively charged Mn ions, the Zeta potential of the nanoparticles changed from -20.55 mv to -12.95 mv.

[0106] The concentration quantitative curve of LPDAM nanoplatform in the 550nm band was confirmed by UV-Vis spectrometer. The stability of the drug was evaluated by detecting the spectrum. The LPDAM nanoplatform was dispersed in physiological saline, PBS and culture medium containing 10% serum and allowed to stand for seven days. Figure 2 The i part, Figure 3 As shown, the spectrum and physical photos show that the PDA coating gives the LPDAM nanoplatform good stability.

[0107] The above characterization results show that the LPDAM nanoplatform successfully loaded Lapachone and encapsulated PDA to chelate manganese ions. At the same time, the LPDAM nanoplatform also has a good particle size, good dispersibility and stability.

[0108] Photothermal, chemical kinetics, and MR performance of the LPDAM nanoplatform:

[0109] PDA itself is an excellent photothermal agent. In order to detect the photothermal effect of the LPDAM nanoplatform, LPDA and LPDAM were irradiated with 808nm near-infrared light to obtain the temperature rise curve, temperature rise and fall curve and repeatability curve. Figure 4 Part a Figure 5 , Figure 6 Compared with the deionized water group, the difference is that the temperature of the nanodispersion liquid is positively correlated with the nanoparticle concentration, laser irradiation power density and irradiation time, and Figure 4The temperature changes of the nanoparticles shown in part e are consistent. After five minutes of laser irradiation of 1mg / ml nanoparticles at a power density of 1.5W per square centimeter, the temperature of LPDA can reach 13.5 degrees Celsius, and the temperature of LPDAM can reach 13.26 degrees Celsius. The heating effect of LPDAM is slightly lower than that of LPDA, which may be because the PDA content in the LPDAM nanoplatform is slightly lower than that in the LPDA nanoplatform at the same mass concentration. The chelation of manganese ions does not affect the photothermal effect of PDA, which is sufficient to cause damage to tumor cells.

[0110] To evaluate the photothermal repeatability, Figure 4 As shown in part c, LPDA and LPDAM still maintain good heating and cooling conditions after five heating-cooling cycles, and the highest temperature of the nanoparticles in the five cycles does not change significantly. Therefore, it is inferred that LPDA and LPDAM have excellent photothermal stability.

[0111] Manganese ions chelated on PDA give the LPDAM nanoplatform the ability of tumor chemodynamic therapy. 2+ It can convert H2O2 into OH by catalyzing a Fenton-like reaction, thus causing the fading of methylene blue. Figure 4 The f and g parts, Figure 6 As shown in the figure, the absorbance of methylene blue decreased with the increase of LPDAM concentration and incubation time. In addition, at 60 degrees Celsius, LPDAM showed a stronger degradation effect of methylene blue than at 37 degrees Celsius ( Figure 4 The h and i parts, Figure 6 ). This proves that high temperature not only does not affect the catalytic activity of manganese ions in Fenton-like reactions, but increases the catalytic effect of this reaction.

[0112] The LPDAM nanoplatform that chelates manganese ions also has the function of magnetic resonance imaging. Figure 4 As shown in part j, the longitudinal relaxation rate (r1) and transverse relaxation rate (r2) of LPDAM are 0.0226 and 0.0549 mM-1s-1, respectively, and r2 / r1 is 2.4335. The nanoparticle solution shows a significant concentration-dependent signal enhancement effect.

[0113] Figure 4 As shown in part k, T1-weighted signal enhancement was detected in the tumor site. The above experiments prove that LPDAM is a suitable MRI contrast agent.

[0114] In vitro cytotoxicity and synergistic therapeutic effects of LPDAM nanoplatform:

[0115] To verify the effect of LPDAM nanoparticles on tumor cells, LPDAM labeled with the fluorescent dye FITC was first synthesized. Figure 7 As shown in part a, the nanoparticles can be successfully internalized into the cytoplasm by tumor cells.

[0116] Next, we first ruled out the possibility that the PDA nanoplatform with empty micelles had no toxic effect on cells ( Fig.17 ). CCK8 was used to evaluate the killing effect of LPDAM nanoplatform on tumor cells. Figure 7 As shown in part b, with the increase of nanoparticle concentration, the survival rate of tumor cells gradually decreased. When the nanoparticle concentration reached 800 micrograms per milliliter, the survival rate of tumor cells in the LPDAM group was 0, and the survival rate of tumor cells in the LPDA group was 39.8%. This shows that Lapchone has a damaging effect on tumor cells, and the Fenton-like reaction catalysis of manganese ions increases the killing effect of Lapchone on tumor cells. Secondly, the killing effect of different laser power densities on tumor cells was explored, such as Figure 7 As shown in part c, the equivalent concentration of LPDA and LPDAM was controlled to be 300ug / ml. As the laser power density increased, the cell survival rate continued to decrease. At the same time, the killing effect of the LPDAM group was significantly better than that of the LPDA group. Figure 7 Part e confirms that when the laser power is controlled to 0.625 W / cm 2 The combined therapeutic effect of LPDAM nanoplatform and photothermal therapy.

[0117] MC38 cells once again verified the tumor killing effect of LPDAM combined with photothermal therapy ( Figure 8 ). This demonstrates that the LPDAM nanoplatform successfully integrates the enhanced combined efficacy of Lapchone / chemodynamic therapy / photothermal therapy in tumor cells.

[0118] The synergistic relationship between photothermal and LPDAM concentration is beneficial for tumor cells ( Figure 7 d), it was determined that the subsequent cell experiments would use a laser power density of 0.625 W / cm2 and a dosing concentration of 300 mg / ml.

[0119] To further explore the mechanism of the LPDAM nanoplatform killing tumor cells, first, we took biological electron microscopy images of cells treated with LPDAM ( Figure 7 e and f), it was found that compared with the control group, the number of mitochondria in the LPDAM group was reduced, the mitochondria were obviously swollen and deformed, and the mitochondrial cristae were reduced or disappeared. At the same time, the morphology of tumor cells also changed significantly ( Fig. 9). Compared with the control group, the tumor cells became loosely attached to the wall after drug administration and photothermal treatment, and the cells were spherical and aggregated, which was the result of damage to CT26 cells and a precursor to death. The above phenomena may be caused by the photothermal heating after LPDAM enters the cells, and the generation of reactive oxygen species through the Fenton-like reaction at high temperature, which damages mitochondria and cells. Therefore, the level of reactive oxygen species in cells after drug administration was further explored. Figure 7 As shown in part g, the tumor cells produced obvious DCFH-DA green fluorescence after drug administration and photothermal treatment, which means that the tumor cells did produce a large amount of reactive oxygen species after treatment.

[0120] Subsequently, the cell apoptosis and necrosis were further investigated. Figure 7 The h part, Fig.10 As shown, the apoptosis and necrosis rates of the LPDAM and PDA+NIR groups reached 34.4% and 25.2%, respectively, while the apoptosis and necrosis rate of the LPDAM+NIR group reached 54.5%.

[0121] Secondly, after drug treatment and photothermal treatment, flow cytometry results showed that tumor cells aggregated in the FITC / PI double positive area, and fluorescent staining also found that the cells showed FITC / PI double staining ( Fig.11 ), which may be because LPDAM and photothermal treatment directly caused cell necrosis.

[0122] Based on the above experimental results, the LPDAM nanoplatform has good synergistic therapeutic effects of chemotherapy, chemokinetics and photothermal therapy. The LPDAM nanoplatform stimulates cells to produce a large amount of reactive oxygen species, causing cell and mitochondrial damage and inducing tumor cell necrosis.

[0123] LPDAM nanoplatform induces immunogenic apoptosis of tumor cells:

[0124] Tumor immunotherapy is a promising treatment method. Mild photothermal therapy can promote the maturation of DC cells through tumor immunogenic death and induce the body's own anti-tumor immune response. Therefore, the three key indicators of immunogenic death (CRT, HMGB1, ATP) were further tested for verification. The exposure of cellular calreticulin (CRT) on the cell membrane and the release of high mobility group protein-1 (HMGB1) were detected by immunofluorescence. Compared with the control group, the LPDAM group had a small amount of CRT green fluorescence expression, while the two photothermal groups showed a large amount of green fluorescence ( Fig.12 This indicates that the stimulation of LPDAM and photothermal heating can cause intracellular CRT to translocate to the cell membrane surface, releasing the "eat me" signal. At the same time, the expression of HMGB1 protein in the photothermal group was also significantly reduced ( Fig.12This indicates that HMGB1 was secreted from cells under drug induction and photothermal stimulation. Then, the ATP level in the cell supernatant was detected, and it was found that the ATP level in the photothermal treatment group was significantly increased ( Fig.12 The above results indicate that the immunogenic death effect in the LPDAM nanoplatform is mainly triggered by PTT. The induction of ICD is expected to promote the maturation of DC cells.

[0125] Studies have shown that reactive oxygen species damage mitochondria, and mitochondrial DNA release activates the cGAS-STING pathway, thereby promoting the maturation of DC cells. It has been confirmed above that the LPDAM nanoplatform induces cells to produce reactive oxygen species and damages cell mitochondria. Therefore, we further studied the activation of the cGAS-STING pathway by the LPDAM nanoplatform. Western blotting showed ( Fig.12 (d), cGAS expression increased after LPDA and photothermal treatment, and cGAS protein expression increased significantly in the LPDAM and LPDAM plus photothermal groups. Similarly, the expression of phosphorylated STING reached the highest level in the LPDAM plus photothermal group. At the same time, the expression of downstream proteins related to the cGAS-STING pathway in cells treated with LPDAM and photothermal treatment also increased significantly. In addition, the expression of IFN-β and TNF-α also showed the same trend ( Fig.12 e and f parts), which further indicates that the cGAS-STING pathway is activated. To further verify the anti-tumor immunity mediated by the cGAS-STING pathway, the supernatant of tumor cells in each group was used to culture bone marrow-derived DC cells. The results showed that compared with the DC maturity of the control group (7.89%), the DC maturity of the LPDA and photothermal groups was 12.67% and 18.17%, respectively, the LPDAM group was 35.7%, and the LPDAM plus photothermal group reached 46.6% ( Fig.12 g and h). This indicates that the LPDAM nanoplatform effectively promotes the maturation of DCs. At the same time, Lapachone / PTT / CDT plays a synergistic role. The DC maturation of the LPDAM group and the LPDAM+NIR group is relatively higher. This is not only due to the synergistic treatment of Lapachone and CDT, but may also be the result of PTT warming up to amplify the efficacy of reactive oxygen species, and may also be caused by Mn ion sensitization of the cGAS-STING pathway.

[0126] Based on the above experimental results, it is concluded that PTT can activate DC cells through ICD, and manganese ions can synergize Lapachone and PTT through CDT to produce incremental reactive oxygen species to damage mitochondria and then release dsDNA, sensitize and activate the cGAS-STING pathway, and promote the maturation of DC cells.

[0127] In vivo synergistic therapeutic effects of LPDAM nanoplatform:

[0128] In order to study the therapeutic effect of the LPDAM nanoplatform in vivo, a mouse subcutaneous tumor-bearing model was constructed to observe the tumor progression after intratumoral injection and laser irradiation treatment. Fig.13 Part a shows that LPDAM and photothermal treatment had no significant effect on the weight of mice, proving that the administration and photothermal treatment methods had no toxic effects on mice. During the photothermal treatment, thermal imaging showed that the tumor temperature of the PDA+NIR LPDAM+NIR group rose rapidly to about 51°C under laser irradiation, while the normal saline+near infrared group only rose by about 2°C. Fig.13 Part b shows that compared with the saline group, the tumor volume in the LPDA group was reduced, but the tumor growth curve still showed an upward trend, which may be because the therapeutic effect of a single dose of Lapachone is limited. At the same time, the tumor inhibition rate of LPDAM reached, and the tumor inhibition rate of the PDA+NIR group and the LPDAM+NIR group reached about 100%. This may be because the relatively high laser power was used, which made the treatment effect of the PDA+NIR group and the LPDAM+NIR group significantly better than that of the LPDAM group. After 20 days of treatment, the actual pictures of the tumors in each group of mice were consistent with the above results. In summary, the LPDAM nanoplatform can achieve the treatment of tumors in vivo by coordinating Lapachone / CDT / PTT.

[0129] The LPDAM nanoplatform elicits systemic immunity and inhibits distant tumors and lung metastasis;

[0130] Furthermore, in order to evaluate the effect of the LPDAM nanoplatform on tumor immunity in mice, a bilateral tumor-bearing model was constructed. The primary side (right side) tumors were divided into groups for intratumoral injection and laser irradiation treatment. The laser power density was reduced and the number of treatments was increased. The anti-tumor immune response in mice was evaluated by monitoring the occurrence and development of distant tumors ( Fig.14 As expected, the distant tumors in the control group showed uncontrolled growth, while the distant tumor inhibition rate in the LPDA-treated group was 42.78%, and the distant tumor inhibition rates in the LPDAM and PDA+photothermal treatment groups were 73.29% and 57.7%, respectively. The tumors in the LPDAM+photothermal group showed a significant tumor inhibition effect in terms of tumor volume, with a tumor inhibition rate of 91.87% ( Fig.14 The tumor mass and tumor images of mice showed the same trend as the above experiment ( Fig.14c and d). Furthermore, the activation of the cGAS-STING pathway was determined by immunohistochemical staining. As expected, the expression of cGAS and p-STING proteins in the nanoplatform treatment and photothermal groups increased to varying degrees, with the highest expression in the LPDAM+NIR group. Subsequently, we further explored whether the LPDAM nanoplatform could promote the maturation of DC cells. Fig.14 As shown in parts f and g, the DC maturity of the LPDAM+NIR group (32.5%) was higher than that of the LPDAM group (26.83%) and the NIR group, and was approximately 3.25 times that of the control group (9.99%), indicating that chemotherapy / PTT / CDT combined therapy achieved a stronger anti-tumor immune response.

[0131] At the same time, the LPDAM nanoplatform triggered the secretion of TNF-α in mice. Compared with the control group, the secretion of LPDA increased by 3.8 times, the LPDAM and PDA+photothermal treatment groups increased by 21 times and 10.6 times, and the LPDAM+photothermal group was as high as 33.4 times.

[0132] Finally, the inhibition of tumor lung metastasis by the LPDAM nanoplatform was evaluated. Fig.15 As shown in parts a and b of the figure, compared with the control group, the number of lung tumor nodules in the LPDAM+NIR group was the least among all treatment groups, and the number of lung tumor nodules in the other treatment groups was reduced to varying degrees. At the same time, H&E staining was performed on the lungs for pathological analysis ( Fig.15 In the c part of the figure, the lung tissues of the control group and the NIR group were heavily infiltrated by tumors. In contrast, the tumor infiltration of the lungs in the LPDA, LPDAM, and PDA+NIR groups was alleviated to varying degrees, while only a small number of lung nodules were observed in the LPDAM+NIR group.

[0133] Based on the above experiments, it is concluded that the LPDAM nanoplatform combined with Lapachone / PTT / CDT treatment can activate the cGAS-STING pathway in mice, promote DC cell maturation, and thus activate the immune response in mice, thereby achieving the purpose of inhibiting tumor recurrence and distant metastasis.

[0134] In vivo evaluation of acute toxic effects of LPDAM nanoplatform:

[0135] To confirm the safety of LPDAM, healthy Barb / c mice were randomly divided into four groups and injected intravenously with LPDAM at concentrations of 0, 20, 40, and 60 mg / kg, and the body weight of mice in each group was collected every other day. During the toxicity test, the body weight of mice in each group increased steadily without statistical difference, and no death occurred ( Fig.16 The mice were euthanized 28 days later, and the blood and internal organs of each group of mice were collected. There was no statistical difference in the organ coefficients of each group of mice ( Fig.16 The color and morphology of the organ did not change, and there was no visible lesion ( Fig.16 C), H&E staining also did not reveal any pathological changes in the organs ( Fig.16 Then, blood routine and blood biochemistry tests were performed on the mice ( Fig.16 Similarly, all indicators in each group were within the normal range.

[0136] So far, LPDAM has been shown to have good biocompatibility and can be used safely in mice.

[0137] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A multi-modal collaborative treatment auxiliary system for colon cancer, characterized in that: include: LPDAM nanoplatform: includes drugs made of lapachone, polydopamine and manganese ions, which are used to administer to cancer cells.

2. The multi-modal collaborative treatment auxiliary system for colon cancer according to claim 1, characterized in that: The preparation method of the LPDAM nanoplatform includes: Step 1: Prepare PEG-PLA and Lapachone, and prepare Lapachone micelles using thin film hydration method; Step 2: Prepare dopamine hydrochloride, dissolve dopamine hydrochloride and Lapachone micelles in Tris buffer and stir, put into ultrafiltration tube for centrifugation, after centrifugation, take the supernatant and wash with deionized water to obtain LPDA modified with dopamine Lapachone micelles; Step 3: Prepare soluble manganese metal salt, dissolve the manganese metal salt and LPDA in deionized water and stir, put into an ultrafiltration tube for centrifugation, and after centrifugation, take the supernatant and wash it with deionized water to obtain LPDAM with LPDA chelated by metal manganese ions.

3. The multi-modal collaborative treatment auxiliary system for colon cancer according to claim 2, characterized in that: In step 1, the thin film hydration method is to dissolve PEG-PLA and Lapachone in acetonitrile to obtain a solvent, evaporate the solvent by a rotary evaporator to form a thin film, add physiological saline after the film is formed, and perform ultrasonic treatment to obtain a drug-loaded micelle aqueous solution, and filter the drug-loaded micelle aqueous solution through a filter to remove unencapsulated drug aggregates to obtain Lapachone micelles.

4. The multi-modal collaborative treatment auxiliary system for colon cancer according to claim 3, characterized in that: The filter in step 1 is a 0.45 μm nylon filter, and the ultrafiltration tubes in steps 2 and 3 are both 100,000 kDa ultrafiltration tubes.

5. The multi-modal collaborative treatment auxiliary system for colon cancer according to claim 4, characterized in that: In step 2 and step 3, the centrifugation time is 20-30 min, the centrifugation speed is 2000 rpm, and the deionized water washing is performed three times.

6. The multi-modal collaborative treatment auxiliary system for colon cancer according to claim 5, characterized in that: The mass ratio of PEG-PLA to Lapachone in step 1 is 9:

1.

7. The multi-modal collaborative treatment auxiliary system for colon cancer according to claim 6, characterized in that: The evaporation temperature of the rotary evaporator in step 1 is 60°C.

8. The multi-modal collaborative treatment auxiliary system for colon cancer according to claim 7, characterized in that: The manganese metal salt is MnCl2.

9. The multi-modal collaborative treatment auxiliary system for colon cancer according to claim 8, characterized in that: In step 2, the mass ratio of Lapachone micelles to dopamine hydrochloride is 1:1, and in step 3, the mass ratio of LPDA to MnCl2 is 1:

1.

10. The colon cancer multi-modal collaborative treatment auxiliary system according to claim 9, characterized in that: Also includes: Photothermal module: used to use the photothermal effect to act on cancer cells to which the LPDAM nanoplatform is applied; Magnetic resonance module: used for magnetic resonance imaging of patients in which the LPDAM nanoplatform has been administered to cancer cells.

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