A conjugate with triple photo-immune function, its preparation method and application

Abstract

The present invention provides a conjugate with triple photo-immune function, its preparation method and application, belonging to the technical fields of biomedicine and molecular biology. The present invention innovatively uses amphiphilic cholesterol polyethylene glycol (Chol-PEG) to synthesize a polymer precursor and further modifies functional small molecules, including photosensitizer chlorin e6 (Ce6) and new indocyanine green (IR820), and TLR7 / 8 small molecule agonist IMDQ. Phototherapy synergistically enhances the anti-tumor immune response of the body through the TLR7 / 8 signaling pathway, thereby developing a functional amphiphilic Chol-PEG conjugate (FCC) system for photo-immune synergistic therapy, which has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to a conjugate with triple photo-immunological functions, a preparation method thereof, and applications thereof. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to enhance the overall understanding of the present invention, and does not necessarily constitute an admission or imply in any form that such information forms the prior art already known to those of ordinary skill in the art.

[0003] Tumor immunotherapy can effectively stimulate and activate the body's immune response, control and eliminate tumors, and inhibit their metastasis. This is the fourth-generation tumor treatment strategy after surgical operation, radiotherapy, and chemotherapy. Immune checkpoint inhibitors, such as PD-1 / PD-L1 and CTLA-4, are the most widely studied T cell-based anti-tumor immunity, but they currently mainly act on some specific tumor types, such as melanoma, lung cancer, and microsatellite instability (MSI) cancers with high mutation burden. However, even in this type of tumor, they do not fully respond to current checkpoint inhibitors. Clinical studies have shown that typical immune "cold" tumors exhibit an immunosuppressive state: (1) The lymphocyte infiltration rate in the tumor microenvironment is low, the number of immune cells is insufficient, the immunogenicity is low, and the immune system cannot recognize the tumor; (2) The immune cells in the tumor microenvironment are in an immunosuppressive state, which means they can recognize but not kill tumor cells, severely limiting the effectiveness of current tumor immunotherapy. Colorectal cancer (CRC) is a highly heterogeneous malignant tumor originating from the colorectal mucosa. Approximately 95% of advanced colorectal cancers belong to typical immune "cold" tumors. Therefore, it is necessary to adopt a multi-dimensional strategy to enhance the tumor immunogenicity of CRC and improve the tumor immunosuppressive microenvironment, thereby activating the anti-tumor immune response and effectively inhibiting tumor growth and recurrence.

[0004] Dendritic cells (DCs) are the most important antigen-presenting cells (APCs), responsible for collecting tumor-associated antigens, stimulating, and initiating immune responses. DCs play an important role in mediating innate immune responses and inducing adaptive immune responses, capable of activating both immune responses and immune memory. When DCs accumulate at the tumor site, DCs guide T cells to eliminate cancer cells. However, in most immune "cold" tumors, dendritic cells are most susceptible to an immunosuppressed state. Therefore, agonists that can reverse this state and activate DCs to reach the tumor site are the best choice for stimulating anti-tumor responses. Toll-like receptors (TLRs) are important pattern recognition receptors (PRRs) in innate immunity. By upregulating the expression of MHC molecules and co-stimulatory molecules CD80 / 86 on the surface of DCs, TLRs can initiate innate immune responses, promote DC maturation, the generation of antigen-specific immune responses, and build a bridge between innate and acquired immune responses. Toll-like receptor agonists (TLRa) as immune adjuvants have become important targets for cancer immunotherapy. Among them, small molecule TLR7 agonists have achieved some success in the treatment of skin malignancies including basal cell carcinoma, but further optimization is needed for other types of cancers. Imidazoquinoline derivatives (IMDQ) are TLR7 / 8 small molecule agonists. Like most small molecule immunomodulators, systemic administration is accompanied by uncontrollable pathogen-associated molecular patterns (PAMPs) that trigger systemic inflammatory responses, which severely limits their clinical translation. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide a conjugate with triple photo-immune function, its preparation method, and application. The present invention innovatively uses amphiphilic cholesterol polyethylene glycol (Chol-PEG) to synthesize polymer precursors and further modifies functional small molecules, including photosensitizers chlorin e6 (Ce6) and indocyanine green (IR820), and TLR7 / 8 small molecule agonist IMDQ. Phototherapy synergizes with the TLR7 / 8 signaling pathway to enhance the body's anti-tumor immune response, thus developing a functional amphiphilic Chol-PEG conjugate (FCC) system for photo-immune synergistic therapy, which has good practical application value.

[0006] In the first aspect of the present invention, there is provided a conjugate with triple photo-immune function, wherein the conjugate comprises amphiphilic cholesterol polyethylene glycol, and the amphiphilic cholesterol polyethylene glycol is modified with functional small molecules;

[0007] The functional small molecules include photosensitizers and TLR7 / 8 agonists.

[0008] Among them, the photosensitizer includes chlorin e6 (Ce6) and indocyanine green (IR820). Compared with normal tissues, due to local ischemia and hypoxia in tumors, energy is difficult to diffuse with the systemic circulation at the tumor site. By NIR, more light energy can be locally absorbed by the tumor to eliminate tumor cells and induce immunogenic cell death (ICD). The inventors surprisingly found that due to the overlap of the maximum absorption wavelengths of modified Ce6-PEG-Chol and IR820-PEG-Chol, the effects mediated by the conjugate system such as photodynamic therapy (PDT) and photothermal therapy (PTT) can be activated by simultaneous irradiation with a single-wavelength 660 nm near-infrared laser. Thereby effectively inducing immunogenic cell death (ICD) at the tumor site, affecting the HMGB1-(Tim-3) signaling pathway, downregulating the expression of T cell immunoglobulin-3 (Tim-3), increasing T cell infiltration at the tumor site, and synergistically enhancing the anti-tumor immune response of the body with the TLR7 / 8 signaling pathway, effectively inhibiting the growth of primary and distal ectopic tumors.

[0009] The TLR7 / 8 agonist is an imidazoquinoline derivative, namely 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ).

[0010] The conjugate with triple photo-immune function is a nanoparticle. It can be observed by TEM imaging and fluorescence imaging that it has a spherical morphology and has fluorescence characteristics; in a specific embodiment of the present invention, the conjugate with triple photo-immune function has a particle size of 270.33 ± 5.51 nm, and its corresponding zeta potential value is -9 mV.

[0011] In the second aspect of the present invention, a preparation method of the above-mentioned conjugate with triple photo-immune function is provided, and the preparation method includes:

[0012] Amphiphilic cholesterol polyethylene glycol modified with a photosensitizer and a TLR7 / 8 agonist are synthesized respectively. The above raw materials are dissolved in DMSO, and then added to water and stirred to obtain micelles by self-assembly.

[0013] In the third aspect of the present invention, an application of the above conjugate in the preparation of an anti-tumor drug delivery system is provided.

[0014] In the fourth aspect of the present invention, an anti-tumor delivery system is provided, and the active ingredient of the anti-tumor drug delivery system includes the above conjugate.

[0015] According to the present invention, the anti-tumor drug delivery system further includes at least one pharmaceutically inactive ingredient.

[0016] In the fifth aspect of the present invention, an anti-tumor system is provided, and the anti-tumor system includes:

[0017] a) The above conjugate or the above anti-tumor drug delivery system; and,

[0018] b) A laser irradiation device.

[0019] The sixth aspect of the present invention provides a method for treating tumors, the method comprising administering a therapeutically effective dose of the above conjugate, anti-tumor delivery system or anti-tumor system to a subject.

[0020] Advantageous technical effects of the above one or more technical solutions:

[0021] The above technical solution provides a conjugate system with triple photo-immune functions. Specifically, the above technical solution designs an amphiphilic cholesterol-polyethylene glycol conjugate (Chol-PEG) as a polymer precursor based on the inherent trend of local high-fat consumption at the tumor site and lipid-based element accumulation in lymphoid tissues, modifies the synthesized TLR7 / 8 receptor agonist imidazoquinoline derivative (IMDQ) and other functional small molecule drugs (Ce6 and IR820), and forms a functionalized amphiphilic Chol-PEG conjugate (FCC) system (i.e., the above conjugate) for photo-immune synergistic treatment effects.

[0022] The retention time of the modified FCC system at the local site is prolonged, effectively avoiding its systemic toxicity while maintaining the functional activity of small molecule drugs. The above technical solution simultaneously verified the polarization regulation effect of IMDQ-PEG-Chol as a TLR7 / 8 agonist on tumor-associated macrophages (TAM) from M2 type to M1 type through in vitro and in vivo experiments.

[0023] At the same time, since the maximum absorption wavelengths of Ce6-PEG-Chol and IR820-PEG-Chol overlap, the FCC system designed in the present invention can simultaneously excite local photothermal (PDT) and photodynamic (PTT) effects through 660 nm single-wavelength near-infrared laser irradiation, effectively induce immunogenic cell death (ICD) at the tumor site, affect the HMGB1-(Tim-3) signaling pathway, down-regulate the expression of T cell immunoglobulin-3 (Tim-3), increase T cell infiltration at the tumor site, and synergistically enhance the anti-tumor immune response of the body through the TLR7 / 8 signaling pathway, effectively inhibiting the growth of primary and distal ectopic tumors.

[0024] The above technical solution provides a new local single-wavelength laser-excited triple photo-immune therapy strategy, which can effectively regulate the anti-tumor immune microenvironment and enhance the anti-tumor immune response, and has great potential in the current treatment of colorectal cancer and the treatment of other immunologically "cold" tumors that are ineffective against existing immune checkpoint inhibitors. Therefore, it has good practical application value. Description of the Drawings

[0025] The accompanying drawings of the specification, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0026] Figure 1 It is a diagram related to the synthesis and properties of conjugates in the embodiments of the present invention; wherein, A) Schematic diagrams of the synthesis and modification routes of amphiphilic cholesterol-PEG modified functional drug conjugates: Ce6-PEG-Chol, IR820-PEG-Chol, and IMDQ-PEG-Chol. B) Ultraviolet-visible spectra of free Ce6, free IR820, Ce6-PEG-Chol, and IR820-PEG-Chol in DMSO. C) and E) Hydrodynamic diameters and surface zeta potentials of (Chol-PEG) NPs, (Ce6-PEG-Chol) NPs, (IR820-PEG-Chol) NPs, (IMDQ-PEG-Chol) NPs, (Ce6 / IR820-PEG-Chol) NPs, and (Ce6 / IR820 / IMDQ-PEG-Chol) NPs. D) TEM images; fluorescence microscope images of (Ce6 / IR820 / IMDQ-PEG-Chol) NPs; the scale bars are 2μm and 500nm respectively. F) Evaluation of the PDT effect of (Ce6 / IR820-PEG-Chol) NPs by the decrease in the ultraviolet-visible absorption at 420nm of the DPBF probe from 0s to 180s under 660nm laser irradiation. G) Infrared thermograms of H2O, free IR820, (IR820-Chol) NPs, and (Ce6 / IR820-PEG-Chol) NPs (IR820 equivalent concentration is 18.3μg / mL) in the range of 0 - 300s under 660nm laser irradiation;

[0027] Figure 2 It is the SEC analysis and data comparative analysis of single BOC-protected PEG, BOC-protected PEG-Chol, and NH2-PEG-Chol in the embodiments of the present invention;

[0028] Figure 3 It is the SEC analysis and data comparative analysis of IR820-PEG-Chol and NH2-PEG-Chol in the embodiments of the present invention;

[0029] Figure 4 It is the analysis of the content of IR820 in IR820-PEG-Chol by ultraviolet-visible spectrophotometry in the embodiments of the present invention;

[0030] Figure 5SEC analysis and data comparison analysis of Ce6-PEG-Chol and NH2-PEG-Chol in the embodiments of the present invention;

[0031] Figure 6 Analysis of the content of Ce6 in Ce6-PEG-Chol by ultraviolet-visible spectrophotometry in the embodiments of the present invention;

[0032] Figure 7 Uptake behavior of CT26 cells for IR820, IR820-PEG-Chol, Ce6 and Ce6-PEG-Chol for 24 h (low: 1 μg / mL; medium: 3 μg / mL; high: 6 μg / mL Ce6 / IR820). The data was processed using the Flow-Jo software package;

[0033] Figure 8 In vitro detection effect diagram of the conjugate; wherein, A1) and A2) Cellular uptake behavior of (Ce6-PEG-Chol) NPs, (IR820-PEG-Chol) NPs and (Ce6 / IR820-PEG-Chol) NPs at 4 °C and 37 °C (equal amount of Ce6-PEG-Chol or IR820-PEG-Chol at 4 μg / mL). The data was processed using the Flow-Jo software package. B1) Evaluation of the phototherapeutic effect of (Ce6-PEG-Chol) NPs and (IR820-PEG-Chol) NPs by CCK-8 kit (0.5 - 6 μg / mL). B2) Fluorescence microscope images of the cellular uptake behavior of free Ce6, (Ce6-PEG-Chol) NPs, free IR820, (IR820-PEG-Chol) NPs and (Ce6 / IR820-PEG-Chol) NPs. C1) and C2) Reactive oxygen species generation of free Ce6, (Ce6-PEG-Chol) NPs, free IR820, (IR820-PEG-Chol) NPs and (Ce6 / IR820-PEG-Chol) NPs under single-wavelength 660 nm near-infrared laser irradiation. D1), D2) and D3) Flow cytometry analysis of HMGB1 and CRT released by CT26 cells. The data was processed using the Flow-Jo software package. D4) ELISA analysis of ATP secreted by CT26 cells. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0034] Figure 9 Flow cytometry analysis results of macrophage repolarization experiment of RAW264.7 cells (IL-4 pre-stimulated cells polarized to M2 to simulate the in vivo tumor immunosuppressive microenvironment) in the embodiments of the present invention A) M1 macrophages (F4 / 80+ INOS + ,F4 / 80 + IL-12 + ,F4 / 80 + CD86 + and F4 / 80 + MHC II + ), and the percentage of (F4 / 80 + CD206 + and F4 / 80 + CD200R + ). C1) The ratio of M1 macrophages to M2 macrophages; C2) The M1 / M2 ratio, calculated from the analysis of M1 macrophages (F4 / 80 + MHC II + ) and M2 macrophages (F4 / 80 + CD200R + ). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001);

[0035] Figure 10 For the maturation, stimulation and activation of BMDC and the inhibition of Tim-3 in the examples of the present invention. The maturation and activation of BMDC were evaluated by stimulating with different sample groups A1) to A4), and detected by labeling with CD11c, CD80, MHC II, MHC I and PD-L1 respectively; B1) and B2) were labeled with CD103 and MHC II; C1) and C2) were labeled with CD8a and MHC II. D1) to D3) The expression of Tim-3 in different DC cell subsets under different concentrations of IMDQ and Chol-PEG treatment; E1) to E3) The effects of the culture supernatants of CT26 cells (equal concentrations of Chol-PEG and IMDQ-PEG-Chol, Ce6-PEG-Chol and IR820-PEG-Chol) treated with different sample groups on Tim-3 in different DC cell subsets of activated BMDC. The data were processed using the Flow-Jo software package. (*P < 0.05, **P < 0.01);

[0036] Figure 11Distribution and targeting localization of free Ce6, (Ce6-PEG-Chol) NPs, free IR820 and (IR820-PEG-Chol) NPs in vivo in the embodiments of the present invention. A1) and B1) IVIS images of CT26 tumor-bearing BALB / c mice treated with different concentrations of Ce6 (36 μg / mL) and IR820 (18.3 μg / mL). A2), B2) In vitro imaging of major organs (T: tumor, H&LN: heart and lymph nodes, S: spleen, LV: liver, B: brain, LU: lung, K: kidney);

[0037] Figure 12 Antitumor efficacy in vivo of different treatment groups in the embodiments of the present invention. A) Near-infrared photothermal conversion images of CT26 tumor-bearing BALB / c mice under irradiation with a single-wavelength 660 nm near-infrared laser at 1.0 W / cm 2 for 2 min; B) and C1) Photographs of tumor-bearing mice and tumors in different treatment groups of BALB / c mice. C2) and C3) Growth curves of primary tumors and distant tumors in different treatment groups. C4) Tumor volume [mm 3 of each mouse in different treatment groups. D1) and D2) Tumor weight and inhibition rate. E1) and E2) Body weight changes and survival curves of tumor-bearing mice in each group. F) H&E staining images (40X), Ki67 cell proliferation intensity staining images (400X), and TUNEL apoptosis staining images (400X) of tumors in different treatment groups: (1) PBS, (2) PBS + laser, (3) (IMDQ-PEG-Chol) NPs, (4) (Ce6-PEG-Chol) NPs + laser, (5) (IR820-PEG-Chol) NPs + laser, (6) (Ce6 / IR820-PEG-Chol) NPs + laser, (7) (Ce6 / IMDQ-PEG-Chol) NPs + laser, (8) (IR820 / IMDQ-PEG-Chol) NPs + laser, and (0) (Ce6 / IR820 / IMDQ-PEG-Chol) NPs + laser. Data are expressed as Mean ± SD (n = 7). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001);

[0038] Figure 13 Results of macrophage repolarization in the in vivo antitumor experiment of different treatment groups in the embodiments of the present invention. A) and C) The percentage of macrophages increases both locally and systemically; B) and D) The expression of M1 macrophage-specific factors (F4 / 80 + INOS + ) is upregulated, and the expression of M2 macrophage-specific factors (F4 / 80 + CD206 +) There was no significant change in expression; B3) and D3) Calculation of the proportions of M1 and M2 macrophages. The different treatment groups were (B3&D3). (1) PBS, (2) PBS + laser, (3) (IMDQ-PEG-Chol) NPs, (4) (Ce6-PEG-Chol) NPs + laser, (5) (IR820-PEG-Chol) NPs + laser, (6) (Ce6 / IR820-PEG-Chol) NPs + laser, (7) (Ce6 / IMDQ-PEG-Chol) NPs + laser, (8) (IR820 / IMDQ-PEG-Chol) NPs + laser, and (0) (Ce6 / IR820 / IMDQ-PEG-Chol) NPs + laser. Data are shown as mean ± SD (n = 7). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001);

[0039] Figure 14 These are the results of local immune analysis and systemic peripheral immune analysis in the examples of the present invention. A) Percentages and activation status of dendritic cells in local and systemic immune analyses; B) and C) CD8 + T cells and CD4 + T cell percentages and activation status; D) NK cell percentages and activation status. Data are presented as Mean SD (n = 7). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001);

[0040] Figure 15 These are H&E staining images (40X) of different organs in different treatment groups in the examples of the present invention. Detailed implementation manners

[0041] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0042] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0043] The present invention will be further described below in conjunction with specific examples. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by reagent companies; the reagents, consumables, etc. used in the following examples can be obtained from commercial channels without special instructions.

[0044] As shown above, dendritic cells (DCs) are the most important antigen-presenting cells (APCs), responsible for collecting tumor-associated antigens, stimulating and initiating immune responses. DCs play an important role in mediating innate immune responses and inducing adaptive immune responses, and can activate both immune responses and immune memory simultaneously. When DCs are enriched at the tumor site, DCs guide T cells to eliminate cancer cells. However, in most immune "cold" tumors, dendritic cells are most susceptible to an immunosuppressive state. Therefore, agonists that can reverse this state and activate DCs to reach the tumor site are the best choice for stimulating anti-tumor responses. Toll-like receptors (TLRs) are important pattern recognition receptors (PRRs) in innate immunity. By upregulating the expression of MHC molecules and co-stimulatory molecules CD80 / 86 on the surface of DCs, TLRs can initiate innate immune responses, promote DC maturation, the generation of antigen-specific immune responses, and build a bridge between innate and acquired immune responses. Toll-like receptor agonists (TLRa) as immune adjuvants have become important targets for cancer immunotherapy. Among them, small molecule TLR7 agonists have achieved some success in the treatment of skin malignancies including basal cell carcinoma, but further optimization is needed for other types of cancers. Imidazoquinoline derivatives (IMDQ) are TLR7 / 8 small molecule agonists. Like most small molecule immunomodulators, systemic administration is accompanied by uncontrollable pathogen-associated molecular patterns (PAMPs) that trigger systemic inflammatory responses, which severely limits their clinical translation.

[0045] In view of this, in a typical specific embodiment of the present invention, a conjugate with triple photo-immune function is provided, and the conjugate includes amphiphilic cholesterol polyethylene glycol, and the amphiphilic cholesterol polyethylene glycol is modified with a functional small molecule;

[0046] The functional small molecule includes a photosensitizer and a TLR7 / 8 agonist.

[0047] Among them, the photosensitizer includes chlorin e6 (Ce6) and indocyanine green (IR820). Compared with normal tissues, due to local ischemia and hypoxia in tumors, it is difficult for energy to diffuse with the systemic circulation at the tumor site. By NIR, more light energy can be locally absorbed by the tumor to eliminate tumor cells and induce immunogenic cell death (ICD). The inventors surprisingly found that due to the overlap of the maximum absorption wavelengths of modified Ce6-PEG-Chol and IR820-PEG-Chol, the effects of conjugate systems mediated by photodynamic therapy (PDT) and photothermal therapy (PTT) can be activated by simultaneous irradiation with a single-wavelength 660 nm near-infrared laser. Thus, it can effectively induce immunogenic cell death (ICD) at the tumor site, affect the HMGB1-(Tim-3) signaling pathway, down-regulate the expression of T cell immunoglobulin-3 (Tim-3), increase T cell infiltration at the tumor site, and synergistically enhance the anti-tumor immune response of the body with the TLR7 / 8 signaling pathway, effectively inhibiting the growth of primary and distal ectopic tumors.

[0048] The TLR7 / 8 agonist can be an imidazoquinoline derivative, namely 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ).

[0049] The conjugate with triple photo-immune function is a nanoparticle. It can be observed by TEM and fluorescence imaging that it has a spherical morphology and fluorescence characteristics; in a specific embodiment of the present invention, the particle size of the conjugate with triple photo-immune function is 270.33 ± 5.51 nm, and its corresponding zeta potential value is -9 mV.

[0050] In another specific embodiment of the present invention, a preparation method of the above conjugate with triple photo-immune function is provided, and the preparation method includes:

[0051] Amphiphilic cholesterol polyethylene glycol modified with a photosensitizer and a TLR7 / 8 agonist is synthesized respectively. The above raw materials are dissolved in DMSO, and then added to water and stirred to obtain micelles by self-assembly.

[0052] In another specific embodiment of the present invention, the specific method of "respectively synthesizing amphiphilic cholesterol polyethylene glycol modified with a photosensitizer and a TLR7 / 8 agonist" includes:

[0053] IR820-PEG-Chol, Ce6-PEG-Chol and IMDQ-PEG-Chol are synthesized respectively;

[0054] Among them, the synthesis method of the IR820-PEG-Chol specifically includes:

[0055] Dissolve NH2-PEG-cholesterol in anhydrous DMF under an inert atmosphere;

[0056] Dissolve IR820 in the above anhydrous DMF containing NH2-PEG-cholesterol; then mix the resulting solution with anhydrous N,N-diisopropylethylamine and stir overnight at a high temperature; after removing DMF, dissolve the resulting polymer in a water / methanol mixture, dialyze, and lyophilize to obtain.

[0057] The mass ratio of the NH2-PEG-cholesterol to IR820 is 3-6:1-3; in a specific embodiment of the present invention, the mass ratio is 4.57:1.62;

[0058] The specific temperature for the overnight stirring is controlled at 70-90 °C, preferably 80 °C;

[0059] The specific method of dialysis is as follows: Dialyze in 0.1% (v / v) ammonium hydroxide solution for one day and in methanol for 2-3 days. The dialysis membrane has a cut-off molecular weight (MWCO) of 0.5-5 kDa, preferably 1 kDa;

[0060] In another specific embodiment of the present invention, the specific preparation method of the above NH2-PEG-cholesterol includes:

[0061] Dissolve monoprotected PEG diamine (BOC-NH-PEG-NH2) with tert-butoxycarbonyl (BOC) in anhydrous CHCl3 under an inert gas, and then add it to anhydrous CHCl3 containing anhydrous triethylamine and cholesteryl chloroformate. After reacting and stirring overnight, evaporate the solvent CHCl3, redissolve the resulting polymer in CH2Cl2 and purify to obtain a BOC-protected PEG-cholesterol conjugate. Then dissolve it in dichloromethane, add trifluoroacetic acid thereto, stir and remove trifluoroacetic acid. Dissolve the resulting polymer in water, dialyze, and lyophilize to obtain.

[0062] Among them, the molar ratio of the monoprotected PEG diamine with tert-butoxycarbonyl, anhydrous triethylamine, and cholesteryl chloroformate is 1:1-2:1-2, preferably 1:1.5:1.1.

[0063] The specific method of dialysis is as follows: Dialyze in 0.1% (v / v) ammonium hydroxide solution for 3 days and in deionized water for 1 day; the dialysis membrane has a cut-off molecular weight (MWCO) of 0.5-5 kDa, preferably 1 kDa;

[0064] The specific synthesis method of the Ce6-PEG-Chol specifically includes:

[0065] Dissolve NH2-PEG-cholesterol in anhydrous DMF under an inert atmosphere;

[0066] Dissolve 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate in the above anhydrous DMF containing NH2-PEG-cholesterol, then mix it with anhydrous N,N-diisopropylethylamine and add Ce6. After stirring overnight, remove the solvent DMF. Dissolve the obtained polymer in a water / methanol mixture, dialyze it, and then lyophilize to obtain the product.

[0067] The specific method of the dialysis is as follows: Dialyze in a water / methanol (50 / 50, v / v) mixture for one day, and dialyze in methanol or acetonitrile for 2 - 3 days.

[0068] The preparation of the IMDQ-PEG-Chol can be completed referring to documents such as Vrieze, J.D., Louage, B., Swarte, K.D., Zhong, Z. & Geest, B. Potent Lymphatic Translocation and Spatial Control Over Innate Immune Activation by Polymer–Lipid Amphiphile Conjugates of Small-Molecule TLR7 / 8 Agonists. Angewandte Chemie International Edition 58, (2019) and Jangra, S., Vrieze, J.D. et al. Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Lipid Amphiphile Imidazoquinoline TLR7 / 8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine. Angewandte Chemie International Edition 60, 9467 - 9473 (2021), etc.

[0069] In another specific embodiment of the present invention, the application of the above conjugate in the preparation of an anti-tumor drug delivery system is provided.

[0070] Meanwhile, it should be noted that tumors are used in the present invention as known to those skilled in the art, including benign tumors and / or malignant tumors. Benign tumors are defined as the excessive proliferation of cells that cannot form invasive and metastatic tumors in the body. Conversely, malignant tumors are defined as cells with various cellular and biochemical abnormalities that can form a systemic disease (such as the formation of tumor metastases in distant organs).

[0071] In another specific embodiment of the present invention, the anti-tumor drug delivery system (drug) of the present invention can be used to treat malignant tumors. Malignant tumors include primary tumors in the said organs and corresponding secondary tumors (tumor metastasis) in distal organs. Examples of malignant tumors that can be treated with the drug of the present invention include solid tumors and hematological tumors. Preferably, they are solid tumors, so as to more favorably achieve intratumoral injection and / or peritumoral injection of the drug. Solid tumors can be tumors of the breast, bladder, bone, brain, central and peripheral nervous systems, endocrine glands (such as the thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, colon, rectum, kidney, small intestine, soft tissue, testis, stomach, skin (such as melanoma), ureter, vagina and vulva, etc.

[0072] In particular, the drug in the present invention is based on a triple photo-immunotherapy strategy excited by local single-wavelength laser, which can effectively regulate the anti-tumor immune microenvironment and enhance the anti-tumor immune response. It is particularly suitable for immunologically cold tumors that are ineffective against existing immune checkpoint inhibitors, such as colorectal cancer.

[0073] In another specific embodiment of the present invention, an anti-tumor drug delivery system is provided, and the active ingredient of the anti-tumor drug delivery system contains the above-mentioned conjugate.

[0074] According to the present invention, the anti-tumor drug delivery system further includes at least one pharmaceutically inactive ingredient.

[0075] The pharmaceutically inactive ingredient can be a carrier, excipient, diluent, etc. commonly used in pharmacy. Moreover, according to the usual methods, it can be made into dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral administration, external use, suppositories, and sterile injection solutions.

[0076] The carrier, excipient, diluent, etc. that can be included are well-known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.

[0077] In another specific embodiment of the present invention, the carrier, excipient, and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, arabic gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, etc.

[0078] In yet another specific embodiment of the present invention, the drug of the present invention can be administered into the body by known methods. For example, it can be delivered systemically via intravenous injection or locally injected into the tissue of interest. Optionally, it can be administered via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors, such as the target cells, the biological type or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, and so on.

[0079] In yet another specific embodiment of the present invention, the subjects to which the drug is administered can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, etc.

[0080] In yet another specific embodiment of the present invention, an anti-tumor system is provided, and the anti-tumor system includes:

[0081] a) the above conjugate or the above anti-tumor drug delivery system; and,

[0082] b) a laser irradiation device.

[0083] Wherein, the light source emitted by the laser irradiation device is a near-infrared light source. Specifically, the wavelength of the light source can be 660 nm. Since the maximum absorption wavelengths of the modified Ce6-PEG-Chol and IR820-PEG-Chol overlap, the PDT and PTT effects mediated by the drug can be activated by simultaneous irradiation with a single-wavelength 660 nm near-infrared laser. At the same time, in combination with IMDQ-PEG-Chol, the drug can effectively activate the TLR7 / 8 pathway, while inhibiting the HMGB1-(Tim-3) pathway, enhancing M1-type TAM polarization, promoting DCs maturation, activating and recruiting TIL, and upregulating local and systemic immune-related activation factors (such as TNF-α, IFN-γ, IL-12 and ICOS). At the same time, the present application also first verified the effective polarization regulation effect of IMDQ-PEG-Chol as a TLR7 / 8 agonist on M1-type tumor-associated macrophages (TAM). Therefore, the conjugates, drugs and systems of the present application are expected to achieve the therapeutic effect of "local stimulation activation - systemic effect - immune restoration - memory dormancy (reactivation potential)" (LSRM).

[0084] In yet another specific embodiment of the present invention, a method for treating tumors is provided, and the method includes administering a therapeutically effective dose of the above conjugate, anti-tumor delivery system or anti-tumor system to a subject.

[0085] The present invention will be further explained and illustrated by the following examples, which do not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The test methods without specific conditions in the following examples are usually carried out under conventional conditions.

[0086] Example

[0087] Method:

[0088] Synthesis of cholesterol-terminated polyethylene glycol:

[0089] Weigh monoprotected PEG diamine (BOC-NH-PEG-NH2, 3000 g / mol, 300 mg, 0.1 mmol) in a dry round-bottom flask, stir, and dissolve it in 5 mL of anhydrous CHCl3 under an inert gas. Then, add anhydrous triethylamine (20.91 μL, 0.15 mmol) and cholesteryl chloroformate (49.40 mg, 0.11 mmol) dissolved in 3 mL of anhydrous CHCl3 to the polymer mixture. The reaction is stirred overnight. Then, evaporate CHCl3, and the resulting polymer is redissolved in 1 mL of CH2Cl2. The polymer is purified by precipitation in a cold mixture of ether / methanol (95 / 5, v / v). This purification process is repeated twice to achieve higher purity. Vacuum drying is used to remove the remaining solvent in the polymer.

[0090] Next, the BOC protecting group is removed. Weigh the BOC-protected PEG-cholesterol conjugate (200 mg, 0.058 mmol) in a round-bottom flask, stir and dissolve it in 2 mL of dichloromethane. Add trifluoroacetic acid (TFA, 0.5 mL) to the solution and stir at room temperature in an open environment for 3 hours. To remove TFA, it can be evaporated three times by mixing with toluene. Finally, dissolve the resulting polymer in water, transfer it to a dialysis membrane (MWCO 1 kDa), and dialyze it in 0.1% v / v ammonium hydroxide solution for 3 days and in deionized water for 1 day. After lyophilization, SEC( Figure 2 ) is used to characterize the white fluffy powder.

[0091] Synthesis of IR820-PEG-Chol:

[0092] Weigh NH2-PEG-cholesterol (45.7 mg, 13.5 μmol) in a dry Schlenk tube and dissolve it in 1 mL of anhydrous DMF under inert gas protection with stirring. Then, weigh IR820 (16.2 mg, 19 mmol) in a glass bottle and dissolve it in 0.5 mL of anhydrous DMF. Add the IR820 mixture and anhydrous N,N-diisopropylethylamine (DIPEA, 5.3 μL, 31 mmol) to the Schlenk tube and stir overnight at 80 °C. The next day, remove the DMF under reduced pressure. The resulting polymer is dissolved in a water / methanol mixture, transferred to a dialysis membrane (MWCO 1 kDa), dialyzed in 0.1% v / v ammonium hydroxide solution for one day, and dialyzed in methanol for 2 - 3 days. After lyophilization, the blue powder is characterized by SEC( Figure 3 ) and UV-VIS( Figure 4 ).

[0093] Synthesis of Ce6-PEG-Chol:

[0094] Weigh NH2-PEG-cholesterol (45.7 mg, 13.5 μmol) in a dry round-bottom flask and dissolve it in 2 mL of anhydrous DMF under inert gas protection with stirring. Then, weigh 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU (8.7 mg, 23 mmol) in a glass bottle and dissolve it in 0.5 mL of anhydrous DMF. Add the HATU mixture and anhydrous N,N-diisopropylethylamine (DIPEA, 4 μL, 23 mmol) to the glass bottle containing Ce6 (10 mg, 17 mmol). This mixture is added to the flask containing the polymer and stirred overnight. The next day, the DMF is removed under reduced pressure. The resulting polymer is dissolved in a water / methanol mixture, transferred to a dialysis membrane (MWCO 1 kDa), and then dialyzed in a water / methanol (50 / 50 v / v) mixture for one day, and dialyzed in methanol or acetonitrile for 2 - 3 days. After lyophilization, the green powder is characterized by SEC( Figure 5 ) and UV-VIS( Figure 6 ).

[0095] Preparation and characterization of functional amphiphilic cholesterol-polyethylene glycol conjugate (FCC) system:

[0096] The grafting rates of Ce6-PEG-Chol and IR820-PEG-Chol were further determined by UV-visible spectroscopy. A functional amphiphilic cholesterol-polyethylene glycol conjugate (FCC) system was prepared by self-assembly technology. Ce6-PEG-Chol, IR820-PEG-Chol, and IMDQ-PEG-Chol were dissolved in DMSO at a concentration of 200 μg / mL, respectively. Then the above solutions were separately dropped into 1.5 mL of MilliQ water and stirred. Each suspension was stirred in the dark at room temperature for 30 min to obtain micelles. These functional nanoparticles were characterized by dynamic light scattering (DLS), and their particle sizes and ζ potentials were measured. The optical properties of (Ce6-PEG-Chol) NPs and (IR820-PEG-Chol) NPs were observed by fluorescence microscopy.

[0097] In vitro photodynamic effect (PDT):

[0098] UV-visible spectroscopy analysis of singlet oxygen generation was performed using 1,3-diphenylisobenzofuran (DPBF) as a probe. When the DPBF solution reacts with singlet oxygen, its absorbance at 426 nm decreases; the singlet oxygen generation ability of the samples under irradiation was measured using this method. In summary, DPBF (1.0 mg / mL in DMF, 6 μL) was mixed with 1 mL of water, free Ce6 (36 μg / mL), free IR820 (18.3 μg / mL), (Ce6-PEG-Chol) NPs (36 μg / mL of Ce6), (IR820-PEG-Chol) NPs (18.3 μg / mL IR820), and (Ce6 / IR820-PEG-Chol) NPs (36 μg / mL Ce6 and 18.3 μg / mL IR820). The samples were irradiated at a 660 nm laser wavelength of 1000 mW / cm 2 for 30 s, 60 s, 90 s, 120 s, 150 s, 180 s, and the characteristic absorbance was measured by UV-VIS spectroscopy at approximately 426 nm.

[0099] In vitro photothermal effect (PTT):

[0100] The (IR820-PEG-Chol) NPs and (Ce6 / IR820-PEG-Chol) NPs were measured at a 660 nm laser (1000 mW / cm 2)Irradiate from below for 5 min to compare the photothermal effects of the sample with water and IR820 solution. The study was carried out by monitoring the temperature changes of (IR820-PEG-Chol) NPs at different concentrations. Sample solutions (1 mL), including water, free Ce6 (36 μg / mL), free IR820 (9.15 μg / mL, 18.3 μg / mL, 36.6 μg / mL), (IR820-PEG-Chol) NPs (18.3 μg / mL IR820), and (Ce6 / IR820-PEG-Chol) NPs (36 μg / mL Ce6 and 18.3 μg / mL IR820), were placed in 1.5 mL EP tubes and then exposed to laser irradiation at a wavelength of 660 nm for 5 minutes (1000 mW / cm 2 ). During irradiation, a real-time thermometer and an infrared thermal imaging camera (Testo-ComSoft-Basic-5) were used to detect the temperature changes of the sample solution and capture thermal infrared images.

[0101] Results:

[0102] Based on the existing literature, imidazoquinoline 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ) and IMDQ-PEG-Chol were prepared. To synthesize Ce6-PEG-Chol and IR820-PEG-Chol, mono-BOC protected PEO diamine (3 kDa) was first treated with cholesteryl chloroformate and triethylamine. Then, the BOC protecting group was removed, and the released amine was coupled with Ce6 or IR820 ( Figure 1 A). The resulting conjugate compounds were analyzed by NMR, SEC, and UV-VIS. UV-VIS analysis of Ce6-PEG-Chol and IR820-PEG-Chol solutions in DMSO ( Figure 1B) shows that the maximum absorbance peak of the former is enhanced, while that of the latter is weakened. Interestingly, due to the overlap of the maximum absorption wavelengths of Ce6-PEG-Chol and IR820-PEG-Chol after modification, a dual phototherapy effect of the photodynamic effect (PDT) of Ce6-PEG-Chol and the photothermal therapy effect (PTT) can be achieved by laser irradiation stimulation at a single wavelength of 660 nm. According to the amphiphilic Ce6-PEG-Chol, IR820-PEG-Chol, and IMDQ-PEG-Chol self-assembled into nanoparticles together, their structural and kinetic diameters DLS and the corresponding zeta-potential values are: (Ce6-PEG-Chol) NPs, 355 nm and -13 mV, (IR820-PEG-Chol) NPs, 252 nm and -9 mV, (Ce6 / IR820-PEG-Chol) NPs, 245 nm and -11 mV, and (Ce6 / IR820 / IMDQ-PEG-Chol) NPs, 270.33 ± 5.51 nm and -9 mV( Figure 1 C and 1E). The fluorescence characteristics of (Ce6 / IR820 / IMDQ-PEG-Chol) NPs can be directly observed by fluorescence microscopy. At the same time, TEM imaging was used to observe its spherical morphology( Figure 1 D).

[0103] When free Ce6, (Ce6-PEG-Chol) NPs, and (Ce6 / IR820-PEG-Chol) NPs in aqueous solution were irradiated with a 660 nm laser, the PDT effect was confirmed( Figure 1 F), by measuring the absorbance of 1,3-diphenylisobenzofuran (DPBF), the absorbance of DPBF decreases in the presence of singlet oxygen (ROS) generation. The PTT effect of 660 nm laser irradiation was evaluated by an infrared thermal imager( Figure 1 G), showing the increase in solution temperature under laser irradiation. Interestingly, the temperature increase of IR820-PEG-Chol and Ce6 / IR820-PEG-Chol solutions is higher than that of the IR820 solution. This may be due to the left shift of the maximum absorbance wavelength of IR820-PEG-Chol compared to IR820, resulting in a better match with the wavelength of the laser source. In summary, these data indicate that Ce6 / IR820-PEG-Chol can be activated by a 660 nm laser for PDT and PTT treatments.

[0104] Methods:

[0105] Cell culture:

[0106] Both CT26.WT (mouse colon cancer cells) and RAW 264.7 (mouse monocyte-macrophage leukemia cells) are from the Chinese Academy of Sciences. Bone marrow-derived dendritic cells (BMDCs) were extracted from the hind limb bone marrow of C57BL / c mice. CT26 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (fetal bovine serum, pre-treated by inactivating in a 56°C water bath) and 1% antibiotics (streptomycin and penicillin); RAW 264.7 cells were cultured in DMEM (H) medium supplemented with 10% inactivated fetal bovine serum; BMDCs (mouse bone marrow-derived dendritic cells) were cultured in RPMI-1640 medium containing interleukin-4 (IL-4, 5 ng / ml) and granulocyte-macrophage colony-stimulating factor (GM-CSF, 10 ng / ml). The cells were cultured in a sterile environment controlled by a 37°C constant temperature incubator, with a relative humidity of 95% and a CO2 concentration of 5%.

[0107] In vitro cell uptake behavior:

[0108] Flow cytometry was used to qualitatively and quantitatively evaluate the cell uptake behavior of (Ce6-PEG-Chol) NPs, (IR820-PEG-Chol) NPs and (Ce6 / IR820-PEG-Chol) NPs in CT26 cells in vitro, and fluorescence microscopy was observed. CT26.WT (mouse colon cancer cells) were seeded in 24-well plates at a density of 250,000 cells / mL. One day later, the cells were pulsed with different concentrations of (Ce6-PEG-Chol) NPs, (IR820-PEG-Chol) NPs and (Ce6 / IR820-PEG-Chol) NPs. After incubation for 6 h, 12 h and 24 h, CT26 cells were separated with cell dissociation buffer, centrifuged at 100G for 5 min, and flow cytometry analysis was performed on a BD Accuri C6 Plus. The data was processed using the FlowJo software package. Fluorescence imaging: After 24 h of culture, the cells were fixed, permeabilized and stained with DAPI. Then the samples were imaged with a fluorescence microscope, and the images were processed using the BioTek CYTATION 1 imaging reading software package.

[0109] Generation of intracellular ROS in vitro:

[0110] The 2′,7′-dichlorofluorescein diacetate (DCFH DA) probe was used to evaluate the production of intracellular ROS, which is considered a key factor in killing cancer cells by causing irreversible damage. DCFH-DA can be hydrolyzed to DCFH in cancer cells, and then DCFH is oxidized to DCF by the generated ROS, and DCF can emit bright green fluorescence. Flow cytometry verified the experimental results. CT26.WT (mouse colon cancer cells) were seeded in 24-well plates at a density of 250,000 cells / mL. After one day, the cells were pulsed with different concentrations of (Ce6-PEG-Chol) NPs, (IR820-PEG-Chol) NPs, and (Ce6 / IR820-PEG-Chol) NPs. After incubation for 12 h, the medium in each well was replaced with fresh medium, and 2.0×10 -6 mol DCFH-DA was injected. After 20 min, the cells were irradiated with a 660 nm laser (1000 mW / cm 2 ) for 1 min or not irradiated. Then, the CT26 cells were separated with cell dissociation buffer, centrifuged at 100G for 5 min, and flow cytometry analysis was performed on a BD Accuri C6 Plus. The data were processed using the FlowJo software package.

[0111] In vitro photocytotoxicity assay:

[0112] The cell counting kit-8 (CCK-8) was used to evaluate the photocytotoxicity of (Ce6-PEG-Chol) NPs and (IR820-PEG-Chol) NPs. A new type of rapid and highly sensitive detection kit for cell proliferation and cytotoxicity based on the water-soluble tetrazolium salt (WST, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt). CT26.WT (mouse colon cancer cells) were seeded in 96-well plates at a density of 5,000 cells / mL. After one day, the cells were pulsed with different concentrations (0.5 - 6 μg / mL) of free Ce6, free IR820, (Ce6-PEG-Chol) NPs, and (IR820-PEG-Chol) NPs. After incubation for 24 h, the medium in each well was replaced with fresh medium, and the cells were irradiated with a 660 nm laser (1000 mW / cm 2 ) for 2 min or not irradiated for 2 min. After laser irradiation, 10 μL of the CCK-8 kit was added to each well, and then incubated for another 4 h. The absorbance at 450 nm was read using a microplate reader (LightCycler96).

[0113] Cell viability (%) = [A(sample) - A(blank)] / [A(medium) - A(blank)] × 100%

[0114] Among them, A (sample), A (blank), and A (culture medium) represent the absorbance of cells treated with different samples, cell culture medium without cells, and cells treated with CCK-8 solution, cell culture fluid without cells, and cells treated with CCK-8 solution, respectively.

[0115] In vitro induction of immunogenic cell death:

[0116] CT26.WT (mouse colon cancer cells) were seeded in 24-well plates at a density of 200,000 cells / mL. After one day, the cells were pulsed with free Ce6, free IR820, (Ce6-PEG-Chol) NPs, (IR820-PEG-Chol) NPs, and (Ce6 / IR820-PEG-Chol) NPs at a concentration of 4 μg / mL. After 12 h of incubation in the dark, the fresh medium in each well was replaced. Then, the cells were irradiated with 660 nm laser (1000 mW / cm 2 ) for 2 min and incubated in the dark for another 4 h. Then, the cells were blocked with 3% BSA solution (in PBS buffer at pH 7.4) for 30 min. The cells were labeled with anti-calreticulin antibody (CRT) and goat anti-rabbit IgG H&L / Alexa Fluor 488 antibody respectively for flow cytometry analysis and fluorescence microscopy imaging. To detect the released HMGB1, 0.1% Triton X-100 was used as a nuclear translocation reagent for 30 min after the blocking step. Then, the cells were labeled with anti-HMGB1 and goat anti-rabbit IgG H&L / Alexa Fluor 488 antibody respectively for flow cytometry analysis and fluorescence microscopy imaging. The secretion of adenosine triphosphate (ATP) was detected using an ATP detection kit.

[0117] Result:

[0118] The uptake behaviors of Ce6-PEG-Chol, IR820-PEG-Chol, and Ce6 / IR820-PEG-Chol on CT26 mouse colon cancer cells were detected in vitro. Flow cytometry analysis showed that both Ce6-PEG-Chol and IR820-PEG-Chol were cell-associated in a dose-dependent manner ( Figure 8 A1, 8A2 and Figure 7 ). Due to the different solubilities of Ce6 and IR820, the complexes showed energy-dependent cell uptake behaviors. Fluorescence microscopy images ( Figure 8B2) It was confirmed that the cellular uptake of the Ce6 / IR820-PEG-Chol conjugate was increased relative to Ce6-PEG-Chol, IR820-PEG-Chol, and their original counterparts. The cytotoxicity and photocytotoxicity of Ce6-PEG-Chol and IR820-PEG-Chol were determined by an in vitro Cell Counting Kit-8 (0.5 - 6 μg / mL) in CT26 cells ( Figure 8 B1). Both Ce6-PEG-Chol and IR820-PEG-Chol exhibited dose-dependent cytotoxicity and photocytotoxicity. In the absence of laser irradiation, the amphiphiles showed low to moderate dose-dependent cytotoxic effects on CT26 cells within the tested concentration range. After 660 nm laser irradiation, the cell viability of cells treated with Ce6-PEG-Chol and IR820-PEG-Chol decreased significantly.

[0119] Previous studies have shown that phototherapy can induce local strong immunogenic cell death (ICD), leading to heat stress responses and apoptosis in tumor cells. DCFH-DA was used as a probe to evaluate the generation of intracellular reactive oxygen species (ROS), which is necessary for the effective killing of cancer cells by causing irreversible damage to nucleic acids, proteins, and lipids. DCFH-DA can be hydrolyzed into DCFH inside cancer cells, and DCFH is then oxidized by ROS to DCF, which has bright green fluorescence (FLA-1) and can be detected by flow cytometry. Flow cytometry data showed that (Ce6-PEG-Chol) NPs and (Ce6 / IR820-PEG-Chol) NPs had higher fluorescence intensities, which was considered due to the higher internalization degree of the amphiphilic conjugate and the synergistic effect of Ce6 and IR820 in ROS generation ( Figure 8 C1, 8C2).

[0120] After treatment with different conjugates, damage-associated molecular patterns (DAMPs) were highly expressed on the surface of CT26 cells, such as the expression of calreticulin (CRT), the release of high-mobility group box 1 (HMGB1) from the nucleus, and the secretion of adenosine triphosphate (ATP) ( Figure 8 C and 8D). These signaling molecules release endogenous tumor-associated antigens, enhancing the immunogenicity of tumor cells.

[0121] Method:

[0122] In vitro repolarization study of macrophages by (IMDQ-PEG-Chol) NPs:

[0123] RAW 264.7 (mouse monocyte-macrophage leukemia cells) were seeded in 24-well plates at a density of 1,000,000 cells / mL for 12 hours, and then the cells were pulsed with 5 μL of IL-4 (15 ng / mL) to stimulate the differentiation of RAW 264.7 into M2 macrophages and further incubated for 12 hours. Then the medium was replaced with fresh medium containing different concentrations of IMDQ, cholesterol-PEG, and IMDQ-PEG-Chol. After further incubation for 24 h, all macrophages (F4 / 80 + )、M2 (F4 / 80 + CD206 + 、F4 / 80 + CD200R + ) and M1 (F4 / 80 + CD86 + 、F4 / 80 + INOS + 、F4 / 80 + IL-12 + 、F4 / 80 + MHC-II + ) were labeled. Flow cytometry was used to analyze the subtypes of RAW264.7 cells. The data were processed using the FlowJo software package.

[0124] Study on the inhibition of the HMGB1-(Tim-3) pathway in vitro:

[0125] CT26.WT (mouse colon cancer cells) were seeded in 24-well plates at a density of 200,000 cells / mL and pulsed with Chol-PEG, (IMDQ-PEG-Chol) NPs, (Ce6-PEG-Chol) NPs, (IR820-PEG-Chol) NPs, (Ce6 / IR820-PEG-Chol) NPs, and (Ce6 / IR820 / IMDQ-PEG-Chol) NPs at a concentration of 4 μg / mL. After dark incubation for 12 h, the fresh medium was replaced in each well, and the cells were irradiated with a 660 nm laser (1000 mW / cm 2 ) for 2 min and then incubated in the dark for 4 h. The cell culture medium was collected separately and added to BMDCs.

[0126] BMDCs were seeded in 48-well plates at a density of 100,000 cells / mL and replaced with the above CT26 cell medium in each well after one day. After incubating in the dark for 24 hours, the cells were blocked with 1% rat serum (PBS buffer, pH 7.4) for 30 min. Then we labeled BMDCs with different immunofluorescently labeled antibodies to evaluate their maturation and inhibition / activation of the HMGB1-(Tim-3) pathway (anti-CD45, fixable viability dye, anti-CD11c, anti-CD8a, anti-CD103, anti-CD80, anti-MHC-I, anti-MHC-II, anti-PD-L1 and anti-Tim-3).

[0127] BMDCs were seeded in 48-well plates at a density of 100,000 cells / mL and pulsed with IMDQ, Chol-PEG, and (IMDQ-PEG-Chol) NPs after one day. After incubating in the dark for 24 hours, the cells were blocked with 1% rat serum (PBS buffer, pH 7.4) for 30 min. Then we labeled BMDCs with different immunofluorescently labeled antibodies to evaluate their maturation and inhibition / activation of the HMGB1-(Tim-3) pathway (anti-CD45, fixable viability dye, anti-CD11c, anti-CD8a, anti-CD103, anti-CD80, anti-MHC-I, anti-MHC-II, anti-PD-L1 and anti-Tim-3).

[0128] Results:

[0129] The M2 polarization of RAW264.7 mouse macrophages pre-stimulated with IL-4 was evaluated using amphiphilic IMDQ-PEG-Chol. Flow cytometry analysis showed that the proportions of M1 macrophages (F4 / 80 + INOS + , F4 / 80 + IL-12 + , F4 / 80 + CD86 + and F4 / 80 + MHC II + ) increased significantly. Meanwhile, the proportions of M2 macrophages (F4 / 80 + CD206 + and F4 / 80 + CD200R + ) also increased slightly, which might be caused by an immune compensatory response ( Figure 9 A and 9B). Meanwhile, we observed that in the (IMDQ-PEG-Chol) NPs group ( Figure 9C1) There is a huge difference in the ratio of M1 macrophages to M2 macrophages. The M1 / M2 ratio is 5 times higher than that of IL-4. Compared with the IMDQ and Chol-PEG groups, the proportion of M1 macrophages (F4 / 80 + MHCII + ) and M2 macrophages (F4 / 80 + CD200R + ) ( Figure 9 C2). In summary, under the condition of the same low concentration of IMDQ (10 nM), the (IMDQ-PEG-Chol) NPs group can significantly induce the inflammatory response mediated by M1 polarization and relieve the immunosuppressive tumor microenvironment.

[0130] To further verify the immune activation effect of the FCC system, tumor cells CT26 were first incubated with different FCC conjugates and irradiated with 660 nm NIR. Then, bone marrow-derived dendritic cells (BMDCs) were incubated with the culture medium of CT26 cells in different treatment groups. Flow cytometry was used to detect the activation status of BMDCs and the expression of Tim-3 on BMDCs. The results showed that the CT26 culture medium treated with FCCs + laser could effectively induce the expression of costimulatory molecules CD80 and MHC-II on BMDCs, but did not affect the expression of MHC-I and PD-L1 ( Figure 10 A, 10B and 10C). At the same time, (IMDQ-PEG-Chol) NPs could significantly increase the proportion of cDC1 cells, such as CD8α + DCs and CD103 + DCs ( Figure 10 ). In addition, the (IMDQ-PEG-Chol) NPs and (Ce6 / IR820 / IMDQ-PEG-Chol) NPs + laser group-derived culture medium down-regulated the expression of Tim-3 on cDC1 ( Figure 10 ).

[0131] Method:

[0132] In vivo IVIS images and tissue biodistribution analysis:

[0133] Male BALB / c mice (3 - 5 weeks old) were subcutaneously injected with 1*10 6 CT26.WT (mouse colon cancer cells) in the left area of each mouse. When the tumor volume reached 300 mm 3When the time came, the mice were randomly divided into 8 groups. Equal amounts of free Ce6, free IR820, (Ce6-PEG-Chol) NPs and (IR820-PEG-Chol) NPs were injected into the tumor or the tail vein, respectively. Fluorescence real-time images were obtained at 2, 4, 8, 12, 24 and 48 h using a living imaging system (Xenogen IVIS Kinetic system, ex: 640 nm; em: ICG). After 48 h of drug administration, the tumors and major organs were excised and analyzed using the same method.

[0134] In vivo photothermal conversion performance:

[0135] Male BALB / c mice (3 - 5 weeks old) were subcutaneously injected with 1 * 10^6 CT26.WT (mouse colon cancer cells) in the left area of each mouse. When the tumor volume reached 100 mm 3 When the time came, the mice were randomly divided into 9 groups, and intratumoral injections of different formulations were given every 4 days for 5 consecutive times for 28 days. The doses of (IMDQ-PEG-Chol) NPs, (Ce6-PEG-Chol) NPs and (IR820-PEG-Chol) NPs were 0.1 mg / kg, 0.4 mg / kg and 0.4 mg / kg, respectively.

[0136] The different treatment groups included: (1) PBS, (2) PBS + laser, (3) (IMDQ-PEG-Chol) NPs, (4) (Ce6-PEG-Chol) NPs + laser, (5) (IR820-PEG-Chol) NPs + laser, (6) (Ce6 / IR820-PEG-Chol) NPs + laser, (7) (Ce6 / IMDQ-PEG-Chol) NPs + laser, (8) (IR820 / IMDQ-PEG-Chol) NPs + laser and (0) (Ce6 / IR820 / IMDQ-PEG-Chol) NPs + laser. After 12 h, the tumor area was directly irradiated with a 660 nm laser at 1000 mW / cm 2 for 2 min. During irradiation, thermal infrared images were captured using an infrared thermal imaging camera (Testo-ComSoft-Basic-5).

[0137] In vivo antigenic tumor efficacy:

[0138] Male BALB / c mice (3 - 5 weeks old) were subcutaneously injected with 1 * 10 6 CT26.WT (mouse colon cancer cells) in the left area of each mouse. When the tumor volume reached 100 mm 3At that time, the mice were randomly divided into 9 groups and received intratumoral injections with different formulations according to the experimental design. The drug was administered once every 4 days for 5 consecutive times, for a total of 28 consecutive days. After 12 h, the tumor area was directly irradiated with a 660 nm laser at 1000 mW / cm 2 for 2 min. For 28 consecutive days, the tumor volume, tumor weight and their inhibition rates were measured every 2 days. On the 28th day, the mice were euthanized, and all tumors were dissected, weighed and photographed.

[0139] Tumor volume = (D length × D width 2 ) / 2

[0140] Tumor inhibition rate (%) = [(Wc - Wt) / Wc] × 100%

[0141] where Wc and Wt are the average tumor weights of the PBS group and each treatment group, respectively.

[0142] Efficacy of anti-metastatic tumor in vivo:

[0143] Male BALB / c mice (3 - 5 weeks old) were subcutaneously injected with 1*10 6 CT26.WT (mouse colon cancer cells) in the left area of each mouse. When the tumor volume reached 100 mm 3 , the mice were randomly divided into 9 groups and received the first intratumoral injection with different formulations according to the experimental design. At the same time, an equal amount of CT26 cells was subcutaneously injected into the right flank. Then, an in vivo efficacy experiment of the primary tumor was carried out on the mice. According to the experimental design, the bilateral tumor volumes and inhibition rates were measured every 2 days.

[0144] (Ce6 / IR820 / IMDQ-PEG-Chol) NPs promote immunotherapy in vivo:

[0145] After the in vivo anti-tumor efficacy analysis, the spleen, draining lymph nodes and tumors were collected, and mononuclear lymphocytes and tumor cells were isolated respectively. Mononuclear lymphocytes were labeled with different immunofluorescent antibodies, and their subsets were analyzed by flow cytometry. The data were processed using the FlowJo software package.

[0146] Evaluation of the regulation effect of tumor-associated macrophage polarization: Isolate tumor-associated macrophages, label them with immunofluorescent antibodies, and evaluate the proportion of macrophages and the regulation effect of polarization by flow cytometry (anti-CD45, fixable viability dye, anti-F4 / 80, anti-CD206, anti-iNOS).

[0147] Activation of DCs: Flow cytometry was used to detect the proportion of DC subsets (anti-CD45, fixable viability dye, and anti-CD11c), as well as the expression of related activation factors such as anti-CD86, anti-IL-12, anti-MHC-II, and anti-PD-L1.

[0148] Activation of CD8+ T cells: Flow cytometry was used to detect the proportion of CD8 + T cell subsets (anti-CD45, fixable viability dye, and anti-CD8), as well as the expression of related activation factors such as anti-TNF-α, anti-IFN-γ, anti-ICOS, and anti-Tim-3.

[0149] Activation of CD4+ T cells: Flow cytometry was used to detect the proportion of CD4 + T cell subsets (anti-CD45, fixable viability dye, and anti-CD4), as well as the expression of related activation factors such as anti-TNF-α, anti-IFN-γ, anti-ICOS, and anti-Tim-3.

[0150] Activation of NK cells: Flow cytometry was used to detect the proportion of NK subsets (anti-CD45, fixable viability dye, and anti-NK), as well as the expression of related activation factors anti-CD107a, anti-IFN-γ, and anti-Tim-3.

[0151] In vivo immunofluorescence staining analysis: Mice were sacrificed 28 days after the first administration, and the main organs (such as tumors, heart, liver, spleen, lung, and kidney) were isolated and fixed in 4% paraformaldehyde solution. Hematoxylin-eosin (H&E), Ki67, and TUNEL staining were used to evaluate the morphological and pathological characteristics, cell proliferation intensity, and apoptosis of each tumor. H&E staining was used to evaluate the morphological and pathological characteristics of the main organs.

[0152] Statistical analysis: t-tests and one-way ANOVA were used to evaluate the differences between different treatment groups. Data were expressed as mean ± standard deviation (mean ± SD).

[0153] Result:

[0154] (Ce6-PEG-Chol) NPs and (IR820-PEG-Chol) NPs in CT26 tumor-bearing BALB / c mice, the tissue distribution and tumor accumulation were directly measured by the IVIS in vivo imaging system for the infrared fluorescence of Ce6 and IR820 ( Figure 11)。The treatment method is intravenous injection or direct intratumoral injection. 48 h after injection, the mice were euthanized, and the tumors and livers were analyzed by flow cytometry to quantify the cellular association of Ce6 and IR820, respectively. In the case of Ce6-PEG-Chol and Ce6, intravenous injection led to strong accumulation in the liver, and no signal was detected in the tumors. In contrast, intratumoral injection enabled the detection of significant Ce6 signals in both the tumors and the livers, suggesting that the compound could enter the systemic circulation after local administration. In the case of IR820-PEG-Chol and IR820, intravenous injection produced similar results to those observed for Ce6. However, intratumoral injection of IR820-PEG-Chol produced strong and long-lasting signals in the tumors with limited systemic distribution, while IR820 could enter the systemic circulation and accumulate in the liver. Further analysis by flow cytometry showed that neither tail vein injection nor intratumoral injection could result in the distribution of (Ce6-PEG-Chol) NPs and (IR820-PEG-Chol) NPs in the adjacent lymph nodes and tumor-infiltrating lymphocytes. Based on these results, we selected intratumoral injection for further in vivo experiments.

[0155] Under the guidance of near-infrared fluorescence real-time imaging, 12 h after administration, the in vivo photothermal conversion performance of (Ce6 / IR820 / IMDQ-PEG-Chol) NPs + laser and the control group was evaluated using CT26 tumor-bearing BALB / c mice. Under the irradiation of a 660 nm single-wavelength near-infrared laser at 1.0 W / cm 2 , the temperature of the (Ce6 / IR820 / IMDQ-PEG-Chol) NPs group increased from 37 °C to 45 °C within 2 minutes ( Figure 12 A). (IR820-PEG-Chol) NPs and (IR820 / IMDQ-PEG-Chol) NPs showed similar photothermal conversion in vivo. Meanwhile, the temperature increase in other groups was negligible. Generally, photothermal heating to a high temperature of 50 °C or above can effectively ablate tumors but also damage normal tissues. In the absence of oxygen participation, mild photothermal temperature ~45 °C can promote tumor cell apoptosis or necrosis, enhance tumor immunogenicity, and create a tumor microenvironment conducive to immune responses. These results indicate that (Ce6 / IR820 / IMDQ-PEG-Chol) NPs can slightly improve the photothermal conversion efficiency of local tumor hyperthermia.

[0156] Subsequently, repeated administration was carried out in CT26 tumor-bearing BALB / c mice to observe its therapeutic effect on the primary tumor. The application treatment plan is shown in Scheme 1. The doses of (IMDQ-PEG-Chol) NPs, (Ce6-PEG-Chol) NPs, and (IR820-PEG-Chol) NPs were 0.1 mg / kg, 0.4 mg / kg, and 0.4 mg / kg, respectively. The tumor volume, weight, and growth inhibition rate were detected every 2 days. Free Ce6, free IR820, and the phototherapy group were not irradiated with laser because of the rapid clearance in the body, poor tumor accumulation, and limited tumor growth inhibition rate. 12 h after administration, a single-wavelength 660 nm near-infrared laser was used to irradiate the mice for 2 min. As 2 shown in Figures 11B and 12C, the tumor volume in the PBS group and the (PBS + laser) group increased rapidly. Due to the separate treatment of the control group with (IMDQ-PEG-Chol) NPs, (Ce6-PEG-Chol) NPs + laser, and (IR820-PEG-Chol) NPs + laser, the tumor growth rate was relatively low ( Figure 12 Figure 11D). The (Ce6 / IR820 / IMDQ-PEG-Chol) NPs + laser group showed the highest growth inhibition efficiency due to its outstanding retention ability at the tumor site. Consistent with expectations, the (Ce6 / IR820 / IMDQ-PEG-Chol) NPs + laser group had the lowest average tumor weight, the highest tumor inhibition rate (99.21 ± 2.33%), and the longest median survival time ( Figure 12 Figure 11C, 12D, and 12E). It is worth noting that the photothermal, photodynamic, and immune triple therapy induced by (Ce6 / IR820 / IMDQ-PEG-Chol) NPs had good therapeutic effects and could completely inhibit tumor growth, which could be further confirmed by visually observing the excised tumor tissues of each group ( Figure 12 Figure 11B and 12C). In addition, during the experiment, there were no obvious changes in the body weights of each treatment group, indicating that this treatment had good safety and low systemic toxicity ( Figure 12 Figure 12E1). Figure 12 E1).

[0157] In these images, the cell nuclei in each laser treatment group were significantly reduced and shrunk, indicating the occurrence of apoptosis and necrosis ( Figure 12 Figure 11F). In addition, for the (IR820 / IMDQ-PEG-Chol) NPs and (Ce6 / IR820 / IMDQ-PEG-Chol) NPs groups, large blank areas could be seen in the pictures under laser irradiation. H&E staining was used to analyze the main organs of each group. As Figure 15As shown, no obvious lesions or physiological abnormalities were observed in each group, indicating that the systemic toxicity of nanoparticles to healthy tissues can be ignored. The (Ce6 / IR820 / IMDQ-PEG-Chol)NPs + laser group significantly inhibited tumor cell proliferation, and the Ki67 staining intensity was the lowest among all experimental groups. In addition, the TUNEL fluorescence staining images (FITC) showed that the apoptosis level of tumor cells in this group was the highest. In summary, the triple therapy of photothermal, photodynamic, and immunotherapy mediated by a single-wavelength 660 nm near-infrared laser and (Ce6 / IR820 / IMDQ-PEG-Chol)NPs showed good therapeutic effects in vivo.

[0158] To obtain an animal model with stable immune function, male BALB / c mice were subcutaneously injected with 1*10 6 CT26.WT (mouse colon cancer cells) in the left area of each mouse. When the tumor volume reached 100 mm 3 , the mice were randomly divided into 9 groups and intratumorally injected with different formulations according to the previous experimental design. At the same time, the same number of CT26 cells (1*10 6 ) were subcutaneously injected into the right wing area to establish a metastatic tumor model. Then, the mice were treated in the same way as before for the primary tumor efficacy experiment, and the tumors on both sides of the mice were monitored for their volume and growth inhibition rate. Interestingly, a growth-decline-clearance trend of vulvar metastatic tumors was observed in the (Ce6 / IR820 / IMDQ-PEG-Chol)NPs + laser group, indicating that both the primary and vulvar tumors in this treatment group were completely inhibited ( Figure 12 C3 and Figure 15 ).

[0159] After the in vivo anti-tumor efficacy analysis, tumors, draining lymph nodes, and spleens were collected, converted into single-cell suspensions, stained with different fluorescently labeled antibody panels, and analyzed by flow cytometry. Since the tumors in the (Ce6 / IR820 / IMDQ-PEG-Chol)NPs + laser treatment group were too small to isolate tumor-infiltrating lymphocytes (TILs), the draining lymph node data were selected for local immune analysis of the primary tumor site. In addition, spleen data were selected for systemic peripheral immune analysis. On the one hand, as Figure 13 shown, there were no obvious changes in M2 macrophages (F4 / 80 + CD206 + ) in both local and systemic immune detections; on the other hand, M1 macrophages (F4 / 80 + INOS +) Significantly increased. Compared with other groups, the M1 / M2 ratio also increased significantly, which was consistent with our in vitro analysis data. In addition, we found that in the local immune detection at the primary site, the percentage of macrophages increased, while there was no difference in the systemic peripheral immune detection, which might be due to the long-term immune recovery mechanism and immune compensatory response. As shown above, (Ce6 / IR820 / IMDQ-PEG-Chol) NPs + laser treatment could effectively repolarize the TAM polarization phenotype, promote the M1 polarization-mediated inflammatory response, and relieve the tumor immunosuppressive microenvironment ( Figure 13 ).

[0160] (Ce6 / IR820 / IMDQ-PEG-Chol) NPs + laser group showed a significant increase in the proportion of DCs in the local immune analysis at the primary tumor site ( Figure 14 A). For the systemic peripheral immune analysis, we observed a similar trend, but relatively mild, which was beneficial to immune renormalization. This FCC system also showed strong DC activation ability, such as upregulating the expression of costimulatory factors CD86, IL-12, and MHC-II, and downregulating the expression of PD-L1, which was beneficial to the presentation of tumor associated antigens (TAAs) and enhanced the anti-tumor immune response. Dendritic cells, as the link between innate immunity and adaptive immunity, can activate T cells and trigger a systemic anti-tumor immune response. As Figure 14 B and 14C, the proportion of CD8 + T cells in the lymph nodes at the primary site and CD4 + T cells in the spleen periphery increased significantly. At the same time, the expression of related T cell activation factors TNF-α, IFN-γ, and ICOS was upregulated, and the expression of the immunosuppressive molecule Tim-3 was relatively stable at the primary site, while decreased in the systemic immune organ spleen. Tim-3 was considered to inhibit anti-tumor immunity by mediating T cell exhaustion. At the same time, downregulating the expression of Tim-3 could effectively increase the invasive memory precursor CD8 + T cells and effector T cells in tumors and enhance the antigen-specific immune response. Since NK cells are the main effector cells of the natural immune system against tumors, we then analyzed the percentage and activation of NK cells after treatment. Interestingly, we found that the proportion of NK cells in the (Ce6 / IR820 / IMDQ-PEG-Chol) NPs + laser treatment group increased significantly, and at the same time, the production of IFN-γ and CD107 increased, and the expression of the immunosuppressive molecule Tim-3 decreased in the primary tumor site ( Figure 14 D1). A similar phenomenon also occurred in the spleen ( Figure 14 D2).

[0161] Matters not covered by this invention are well-known techniques.

[0162] The above embodiments are only used to illustrate the technical concept and features of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. It is not intended to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A micelle with triple photo-immunological functions, characterized in that, The micelles include amphiphilic cholesterol polyethylene glycol, and the amphiphilic cholesterol polyethylene glycol is modified with functional small molecules; The functional small molecules include photosensitizers and TLR7 / 8 agonists; The photosensitizers include Ce6 and IR820; The TLR7 / 8 agonist is IMDQ.

2. The micelle according to claim 1, characterized in that, The micelles with triple photo-immunological functions are nanoparticles, with a spherical morphology and fluorescence characteristics.

3. The preparation method of the micelle with triple photo-immunological function according to any one of claims 1-2, characterized in that, The preparation method includes: Amphiphilic cholesterol polyethylene glycol modified with photosensitizers and TLR7 / 8 agonists is synthesized separately. The above raw materials are dissolved in DMSO, and then added to water and stirred. Micelles are obtained by self-assembly.

4. The preparation method according to claim 3, characterized in that, The specific method for "separately synthesizing amphiphilic cholesterol polyethylene glycol modified with photosensitizers and TLR7 / 8 agonists" includes: IR820-PEG-Chol, Ce6-PEG-Chol and IMDQ-PEG-Chol are synthesized separately; Among them, the specific synthesis method of IR820-PEG-Chol includes: Dissolve NH2-PEG-cholesterol in anhydrous DMF under an inert atmosphere; Dissolve IR820 in the above anhydrous DMF containing NH2-PEG-cholesterol; then mix the obtained solution with anhydrous N,N-diisopropylethylamine and stir overnight at a high temperature; after removing DMF, dissolve the obtained polymer in a water / methanol mixture, dialyze, and freeze-dry to obtain; The specific temperature control for the overnight stirring is 70-90 °C; The specific dialysis method is: Dialyze in a 0.1% ammonium hydroxide solution by volume for one day, and dialyze in methanol for 2-3 days. The dialysis membrane has a cut-off molecular weight of 0.5-5 kDa.

5. The preparation method according to claim 4, characterized in that, The mass ratio of NH2-PEG-cholesterol to IR820 is 3-6:1-3; The specific temperature control for the overnight stirring is 80 °C; The dialysis membrane has a cut-off molecular weight of 1 kDa.

6. The preparation method according to claim 5, characterized in that, The mass ratio is 4.57:1.

62.

7. The preparation method according to any one of claims 4 to 6, characterized in that, The specific synthesis method of Ce6-PEG-Chol includes: Dissolve NH2-PEG-cholesterol in anhydrous DMF under an inert atmosphere; Dissolve 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate in the above anhydrous DMF containing NH2-PEG-cholesterol, then mix with anhydrous N,N-diisopropylethylamine and add Ce6, stir overnight and remove the solvent DMF. Dissolve the obtained polymer in a water / methanol mixture, dialyze, and freeze-dry to obtain.

8. The preparation method according to claim 7, characterized in that, The specific dialysis method is: Dialyze in a 50:50 water and methanol mixture by volume for one day, and dialyze in methanol or acetonitrile for 2-3 days.

9. Use of the micelles according to any one of claims 1-2 in the preparation of an anti-tumor delivery system.

10. An anti-tumor delivery system, characterized in that, The anti-tumor delivery system has an active ingredient comprising the micelles according to any one of claims 1-2.

11. An anti-tumor system, characterized in that, The anti-tumor system includes: a) The micelles according to any one of claims 1-2 or the anti-tumor delivery system according to claim 10; and, b) A laser irradiation device.

12. The anti-tumor system according to claim 11, characterized in that, The light source emitted by the laser irradiation device is a near-infrared light source.

13. The anti-tumor system according to claim 12, wherein, The wavelength of the light source is 660 nm.

Images