Polyamino acid composite material, preparation method thereof and antitumor drug

Abstract

The invention discloses a polyamino acid composite material, a preparation method thereof and an anti-tumor drug, and belongs to the technical field of tumor treatment. The polyamino acid composite material consists of a tetracarboxyl phenyl ferriporphyrin metal organic framework material and a block copolymer which is coated on the surface of the tetracarboxyl phenyl ferriporphyrin metal organic framework material and is shown as a formula 1, wherein x, y and z are polymerization degrees, x is selected from 1 to 20, y is selected from 1 to 10, and z is selected from 1 to 30. The polyamino acid composite material has excellent targeting effect and degradation function in a tumor microenvironment, and can realize synergistic interaction of photodynamic therapy and immunotherapy under illumination. The inhibition rate of the polyamino acid composite material in a 4T1 mouse breast cancer tumor in-situ model can reach 85.3%, and the polyamino acid composite material can significantly inhibit growth of far-end tumors in bilateral tumor treatment. In addition, the polyamino acid composite material has long-term anti-tumor immune response in vivo, does not have systemic toxicity, and is good in biological safety. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and in particular to a polyamino acid composite material, a preparation method thereof, and an anti-tumor drug. Background Art

[0002] Immunotherapy is a biological treatment strategy that uses immunological theories and methods to treat diseases, aiming to treat diseases by enhancing or suppressing the immune function of the body. There are many methods of immunotherapy, which are applicable to the treatment of various diseases. It can be divided into immunopotentiation therapy and immunosuppression therapy according to the impact on the body's immune function; specific immunotherapy and non-specific immunotherapy according to the specificity of treatment; active immunotherapy and passive immunotherapy according to the action characteristics of immunopreparations; and molecular therapy, cell therapy, and immunomodulator therapy according to the preparations used in treatment. In tumor treatment, immunotherapy aims to activate the human immune system and rely on the body's own immune function to kill cancer cells and tumor tissues. Among them, indoleamine-2,3-dioxygenase (IDO) inhibitors become immunotherapy targets by regulating the tryptophan content in the tumor microenvironment (TME), reducing activated effector T cells, and activating cytotoxic T lymphocytes (CTLs), thereby breaking tumor immune tolerance. Clinical studies have confirmed that IDO inhibitors can enhance the effect of anti-PD-1 / PD-L1 antibody therapy. However, due to reasons such as ineffective antigen presentation and insufficient anti-tumor host immune activation, the effective immune response against tumors generated by single immunotherapy is not ideal.

[0003] Photodynamic therapy is an advanced therapy based on the interaction between photosensitizing drugs and lasers. During the treatment process, patients first receive photosensitizing drugs by injection, and these drugs will specifically accumulate in the diseased tissues. Subsequently, doctors use lasers with specific wavelengths to irradiate the diseased area, and the laser energy activates the photosensitizing drugs, causing them to release energy and generate highly biotoxic reactive oxygen species (ROS). These active substances selectively damage the diseased tissues through photochemical reactions, thereby achieving the treatment purpose. This treatment method has the characteristics of strong targeting and small side effects, and shows good application prospects in the treatment of various diseases. Moreover, this method is selective and can precisely treat specific lesions with less damage to surrounding normal tissues.

[0004] The combination of immunotherapy and photodynamic therapy is a novel and promising cancer treatment method. This combination mainly combines tumor photodynamic therapy and immunotherapy, aiming to assist in enhancing the immune response ability to achieve the therapeutic effect. However, this combined treatment method is not effective for all patients. Some patients may be insensitive to photoimmunotherapy, or due to factors such as the location and depth of the tumor, the therapeutic effect is not good. In addition, although the combination of photodynamic therapy and immunotherapy has many advantages, further research and optimization are still needed to improve its efficacy and safety.

[0005] Therefore, it is of great significance to research and develop a new composite material for the combined immunotherapy and photodynamic therapy to treat tumors efficiently and safely. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a polyamino acid composite material, its preparation method, and an antitumor drug. The polyamino acid composite material has excellent targeting and degradation functions in the tumor microenvironment, and can achieve the synergistic enhancement of photodynamic therapy and immunotherapy under light irradiation.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a polyamino acid composite material, which is composed of a tetracarboxyphenyl iron porphyrin metal-organic framework material and a block copolymer shown in Formula 1 coated on its surface;

[0009]

[0010] Among them, x, y, and z are degrees of polymerization, x is selected from 1-20, y is selected from 1-10, and z is selected from 1-30.

[0011] The block copolymer (HA-PMTG, also known as HA-b-P(1-MDT-co- L -Glu)) shown in Formula 1 in the above composite nanomaterial has amphiphilicity, and it is coated on the surface of the tetracarboxyphenyl iron porphyrin metal-organic framework material (MOF TCPP-Fe ) through hydrophilic-hydrophobic, electrostatic, metal coordination and other interactions to form the composite nanomaterial (HA-PMTG@MOF TCPP-Fe ).

[0012] Moreover, the HA-PMTG@MOF TCPP-Fe has excellent targeting and degradation functions, enabling it to be efficiently taken up by tumor cells in vivo and degrade and release active ingredients in the tumor microenvironment to achieve targeted therapy.

[0013] In addition, under light illumination, the photosensitizer TCPP in the composite nanomaterial (HA-PMTG@MOF TCPP-Fe ) of the present invention can generate a large amount of reactive oxygen species (ROS) under laser irradiation. At the same time, Fe 3+ can synergistically generate more ROS through the Fenton reaction, effectively reverse the tumor hypoxic microenvironment, and reduce the intracellular glutathione (GSH) level, thereby leading to apoptosis of tumor cells. At the same time, the active ingredient 1-methyl-D-tryptophan (1-MDT) released after the degradation of HA-PMTG@MOF TCPP-Fe can inhibit the generation of indoleamine-2,3-dioxygenase, reduce the activation effect of T cells (Tregs), enhance the infiltration of cytotoxic T cells (CTLs) into tumor cells, break tumor immune tolerance, and induce immunogenic death of tumor cells, thereby realizing the synergistic enhancement of photodynamic therapy and immunotherapy.

[0014] The polyamino acid composite material HA-PMTG@MOF of the present invention TCPP-Fe has good therapeutic effects. The inhibition rate against 4T1 tumor cells can reach 85.3%, and it can significantly inhibit the growth of distal tumors in bilateral tumor treatment. At the same time, the polyamino acid composite material has a long-term anti-tumor immune response in vivo and no systemic toxicity, and has good biosafety.

[0015] Preferably, in the block copolymer shown in Formula 1 of the present invention, x is selected from 10-20, y is selected from 1-5, and z is selected from 1-15. In some specific embodiments of the present invention, x in the block copolymer shown in Formula 1 is selected from 10, y is selected from 4, and z is selected from 14.

[0016] Preferably, the polyamino acid composite material has a spindle-shaped structure and iron elements are evenly distributed on its surface.

[0017] Preferably, the hydrated particle size of the polyamino acid composite material is 200±100nm; more preferably 200±50nm; further preferably 200±9.5nm.

[0018] The present invention also provides a preparation method of the above polyamino acid composite material, including the following steps:

[0019] Mix the tetracarboxylphenyl iron porphyrin metal-organic framework material and the block copolymer shown in Formula 1, and obtain the polyamino acid composite material through electrostatic assembly, metal coordination, and hydrophilic-hydrophobic interaction.

[0020] Preferably, the mass ratio of the tetracarboxylphenyl iron porphyrin metal-organic framework material to the block copolymer shown in Formula 1 is 1:(1-20); more preferably 1:(1-15); further preferably 1:10.

[0021] The tetracarboxyphenylporphyrin-based metal-organic framework material (MOF TCPP-Fe ) is prepared by hydrothermal method from tetracarboxyphenylporphyrin (TCPP) and Fe 3+ .

[0022] Preferably, the block copolymer shown in Formula 1 is prepared by click chemical reaction from the random copolymer shown in Formula 2, α-alkyne hyaluronic acid and a copper catalyst in a basic environment;

[0023]

[0024] In Formula 2, m and n are degrees of polymerization, m is selected from 1-20, n is selected from 1-10, and p is selected from 1-30; in some specific embodiments of the present invention, m is selected from 10, n is selected from 4, and p is selected from 14.

[0025] The copper catalyst includes but is not limited to cuprous bromide, cuprous chloride, copper acetate, etc.

[0026] The basic environment is provided by triethylamine, pentamethyldiethylenetriamine, pyridine, etc.

[0027] In some specific embodiments of the present invention, the basic environment is provided by pentamethyldiethylenetriamine (PMDETA).

[0028] The random copolymer P(1-MDT-co- L -Glu) shown in Formula 2 is prepared by ring-opening polymerization of amino acids initiated by azidopropylamine.

[0029] The specific ring-opening polymerization of amino acids is as follows: BLG-NCA (L-glutamic acid benzyl ester N-carboxylic anhydride), 1-MDTNCA (1-methyl-D-tryptophan N-carboxylic anhydride) and azidopropylamine are mixed and reacted to obtain N3-P(1-MDT-co- L -Glu).

[0030] The solvent for the mixing reaction includes but is not limited to DMF (N,N-dimethylformamide), THF (tetrahydrofuran), DMSO (dimethyl sulfoxide), etc.

[0031] The temperature of the mixing reaction is preferably 20-30°C; more preferably 25°C.

[0032] The present invention also provides an anti-tumor drug, including the above-mentioned polyamino acid composite material or the polyamino acid composite material prepared by the above-mentioned preparation method.

[0033] Preferably, the anti-tumor drug releases 40%-60% of 1-methyl-D-tryptophan (1-MDT) under the action of protease; more preferably, it releases 50% of 1-MDT.

[0034] Preferably, the protease is selected from papain, trypsin or chymotrypsin.

[0035] Preferably, the anti-tumor drug inhibits the activity of tumor cells under laser irradiation;

[0036] Preferably, the conditions of the laser irradiation are as follows:

[0037] The wavelength of the laser is 500-700 nm;

[0038] The power density of the laser is 80-100 mW / cm -2 ;

[0039] The irradiation duration of the laser is 5-30 min.

[0040] More preferably, the wavelength of the laser is 635 nm;

[0041] More preferably, the power density of the laser is 100 mW / cm -2 ;

[0042] More preferably, the irradiation duration of the laser is 10 min.

[0043] In the present invention, the test conditions for the effect of the anti-tumor drug to be released in a responsive manner in the tumor microenvironment are as follows:

[0044] In an environment with a pH value of 5.5, incubate with 10.0 mM glutathione for 12 h.

[0045] Compared with the prior art, the polyamino acid composite material provided by the present invention is composed of a tetracarboxylphenyl iron porphyrin metal-organic framework material and a block copolymer shown in Formula 1 coated on its surface; wherein, x, y, and z are the degrees of polymerization, x is selected from 1-20, y is selected from 1-10, and z is selected from 1-30. The polyamino acid composite material has excellent targeting and degradation functions in the tumor microenvironment, and can achieve synergistic enhancement of photodynamic therapy and immunotherapy under light irradiation. The inhibition rate of the polyamino acid composite material in the 4T1 mouse breast cancer tumor orthotopic model can reach 85.3%, and it can significantly inhibit the growth of distal tumors in bilateral tumor treatment. In addition, the polyamino acid composite material has a long-term anti-tumor immune response in vivo and no systemic toxicity, and has good biosafety. Description of the Drawings

[0046] Figure 1 For HA-PMTG 11H NMR characterization diagram;

[0047] Figure 2 is the particle size distribution diagram of HA-PMTG@MOF TCCP-Fe ;

[0048] Figure 3 is the morphological characterization and element distribution diagram of HA-PMTG@MOF TCCP-Fe ;

[0049] Figure 4 is the TEM images of HA-PMTG@MOF TCPP-Fe in different environments, where A) is the TEM image of HA-PMTG@MOF TCPP-Fe under PBS (pH = 7.4, without GSH); B) is the TEM image of HA-PMTG@MOF TCPP-Fe under PBS (pH 5.5, containing 10 mM GSH);

[0050] Figure 5 is the absorbance of HA-PMTG@MOF TCPP-Fe measured by 9,10-anthracenediyl-bis(methylene)dicarboxylic acid (ABDA) in PBS at different pH values as a function of time under light (635 nm, 40 mW cm -2 ) irradiation. Among them, Figure A is pH = 7.4, GSH 0 mM; Figure B is pH = 5.5, GSH 0 mM; Figure C is pH = 5.5, GSH 10 mM; Figure D is pH = 7.4, GSH 10 mM;

[0051] Figure 6 is the curve of the release of 1-MDT from HA-PMTG over time at pH = 5.0;

[0052] Figure 7 is the fluorescence images of 4T1 and NH-3T3 cells taking up blank, HA-PMTG@MOF TCPP-Fe and free TCPP respectively;

[0053] Figure 8 is the cell viability images of 4T1 cells treated with TCPP + laser irradiation, HA-PMTG@MOF TCPP-Fe , HA-PMTG@MOF TCPP-Fe + laser irradiation respectively. The laser irradiation conditions are 635 nm, 100 mW cm -2 , 10 min;

[0054] Figure 9 are Control, 1-MDT, TCPP, MOF TCPP-Fe , HA-PMTG@MOF TCPP-FeComparison chart of the ability to consume GSH respectively;

[0055] Figure 10 For Control, 1-MDT, TCPP, MOF TCPP-Fe , HA-PMTG@MOF TCPP-Fe Comparison chart of the ability to consume H2O2 respectively;

[0056] Figure 11 For Control, TCPP, MOF TCPP-Fe , HA-PMTG@MOF TCPP-Fe Diagrams of inducing apoptosis of tumor cells respectively;

[0057] Figure 12 For Control, 1-MDT, TCPP, MOF TCPP-Fe , HA-PMTG@MOF TCPP-Fe Results diagrams of inducing ICD of tumor cells to cause CRT eversion respectively;

[0058] Figure 13 For Control, 1-MDT, TCPP, MOF TCPP-Fe , HA-PMTG@MOF TCPP-Fe Fluorescence intensity of HMGB1 in the cell nucleus after treating 4T1 cells respectively;

[0059] Figure 14 For HA-PMTG@MOF TCCP-Fe And diagrams of in vitro distribution experiments of TCPP after different times respectively;

[0060] Figure 15 For Control, 1-MDT, TCPP, MOF TCPP-Fe , HA-PMTG@MOF TCPP-Fe Comparison chart of tumor suppression effects, where A is the tumor suppression curve diagram and B is the tumor entity diagram;

[0061] Figure 16 For the effect diagram of immune cell activation, where (A) the proportion of CD4 + T in T cells in tumor tissue; (B) the proportion of CD8 + T in T cells in tumor tissue; (C) the ratio of Treg cells to CD4 + T in tumor tissue; (D) the proportion of central memory T cells (Tcm) in tumor tissue; (E) the proportion of effector memory T cells (Tem) in tumor tissue; (F) the proportion of CD4 + T in T cells in the spleen; (G) the ratio of Treg cells to CD4 +Ratio of T; (H) Ratio of central memory T cells (Tcm) in the spleen; (I) Ratio map of effector memory T cells (Tem) in the spleen

[0062] Figure 17 For inducing cytokine secretion map, where (A) Ratio of IFN-Υ in tumor tissue; (B) Ratio of TFN-α in tumor tissue; (C) Ratio of IL-6 in tumor tissue; (D) Ratio of central memory T cells (Tcm) in tumor tissue; (E) Ratio of IFN-Υ in plasma; (F) Ratio of TFN-β in plasma; (G) Ratio of IL-6 in plasma; (H) Ratio of IL-12 in plasma; (I) Ratio map of IL-12 in plasma

[0063] Figure 18 Is HA-PMTG@MOF TCCP-Fe Proximal (Figure A) and distal (Figure B) tumor suppression effect diagrams

[0064] Figure 19 Is HA-PMTG@MOF TCCP-Fe Tumor rechallenge experiment inhibition effect diagram, where Figure A shows the in vivo anti-cancer efficacy of rechallenged 4T1 tumor-bearing mice, and Figure B shows the survival of rechallenged experimental mice

[0065] Figure 20 Is the liver function marker diagram in serum, where Figure A shows the level of alanine aminotransferase (ALT); Figure B shows the level of aspartate aminotransferase (AST), and the renal function marker; Figure C shows the level of blood urea nitrogen (BUN); Figure D shows the level of creatinine (Cr) Detailed implementation mode

[0066] To further illustrate the present invention, the polyamino acid composite material provided by the present invention, its preparation method, and the anti-tumor drug will be described in detail below in combination with embodiments.

[0067] Example 1

[0068] I. Preparation of nano-assembly HA-PMTG@MOF TCPP-Fe Preparation

[0069] (1) Synthesis of random copolymer N3-P(1-MDT-co- L -Glu) initiated by azidopropylamine

[0070] BLG-NCA (2.104 g, 8.0 mmol) and 1-MDT NCA (0.49 g, 2.0 mmol) were placed in a dry ampoule together with azidopropylamine (10 mg, 0.1 mmol), and 50 mL of dry DMF was added to dissolve them. The reaction was carried out at 25 °C for three days. The reaction solution was precipitated with diethyl ether, and the resulting product was obtained by centrifugation. The product was dried under vacuum at room temperature for 24 h to obtain the unprotected polymer. Subsequently, 500 mg of the polymer was dissolved in 5 mL of trifluoroacetic acid, 3 mL of hydrogen bromide / acetic acid (HBr / HAc, 33 wt.%) was added, and the temperature was raised to 30 °C. After slowly stirring for 1 h, the product was precipitated three times in 20 mL of pre-cooled diethyl ether and washed twice with diethyl ether. After freeze-drying, N3-P(1-MDT-co- L -Glu) was obtained.

[0071] (2) Synthesis of hyaluronic acid block copolymer HA-b-P(1-MDT-co- L -Glu)

[0072] 0.5 g of the random copolymer N3-P(1-MDT-co- L -Glu), 0.5 g of α-alkyne hyaluronic acid, and pentamethyldiethylenetriamine (PMDETA, 16 μL, 78 μmol) were dissolved in 50 mL of dry DMSO. After stirring for 30 min, the mixture was degassed by repeated freeze-thaw cycles with liquid nitrogen three times. Purified copper(I) bromide (CuBr) was added, and the reaction was carried out for 72 h. Then, dialysis was performed with Milli-Q. Ethylenediaminetetraacetic acid (EDTA) was added to the solution for dialysis for four days, and then freeze-dried to obtain a white flocculent product, namely HA-b-P(1-MDT-co- L -Glu) (also known as HA-PMTG).

[0073] (3) Synthesis of nano-assembly HA-PMTG@MOF TCPP-Fe

[0074] 1 mL of MOF TCPP-Fe (DMF solution at 6 mg / mL) was added to 5 mL of HA-b-P(1-MDT-co-L-Glu) (2 mg / mL, Milli-Q) solution. The reaction was carried out at room temperature with stirring for 4 h. After the reaction, centrifugation was performed (10000 rpm, 20 min) to obtain the crude product, which was centrifugally washed three times with Milli-Q. Finally, the nano-complex HA-PMTG@MOF TCPP-Fe was obtained. HA-PMTG@MOF TCPP-Fe was dispersed in Milli-Q for subsequent characterization tests.

[0075] II. Nano-assembly HA-PMTG@MOF TCPP-FeStructural Characterization and Functional Verification

[0076] (1) Structural Characterization

[0077] The successful synthesis of the polymer was verified by nuclear magnetic resonance, Figure 1 for the 1 1H NMR characterization diagram of HA-PMTG, as Figure 1 shown, in the obtained polymer HA-PMTG, x is 10, y is 4, and z is 14. Figure 2 For the particle size distribution diagram of HA-PMTG@MOF TCCP-Fe as Figure 2 shown, the size of HA-PMTG@MOF was verified by dynamic light scattering (DLS) of the material, and its hydrated particle size is 200 ± 9.5 nm. TCPP-Fe

[0078] Figure 3 For the morphological characterization and elemental distribution diagram of HA-PMTG@MOF TCCP-Fe which proved by transmission electron microscopy (TEM) and elemental mapping that HA-PMTG@MOF TCPP-Fe presents a spindle-shaped structure and iron elements are evenly distributed on its surface.

[0079] The above Figures 1-3 fully proved the successful synthesis of HA-PMTG@MOF TCPP-Fe .

[0080] (2) Functional Verification

[0081] The performance of HA-PMTG@MOF TCPP-Fe was experimentally evaluated, and its pH and GSH responsiveness were verified by TEM. Figure 4 For the TEM images of HA-PMTG@MOF TCPP-Fe in different environments, where A) is the TEM image of HA-PMTG@MOF TCPP-Fe under PBS (pH = 7.4, 0 mM GSH); B) is the TEM image of HA-PMTG@MOF TCPP-Fe under PBS (pH 5.5, containing 10 mM GSH). The results show that after incubation with 10.0 mM GSH (glutathione) for 12 h at pH 5.5, HA-PMTG@MOF TCPP -Fe was completely decomposed, proving that they can be rapidly decomposed in the tumor microenvironment (TME) to release active ingredients.

[0082] Figure 5 For HA-PMTG@MOF measured by 9,10-anthracenediylbis(methylene)dipropanedioic acid (ABDA)TCPP-Fe Absorbance in PBS at different pH values as a function of time under laser (635 nm, 40 mW cm -2 ) irradiation to reflect its ability to generate singlet oxygen. Among them, A) is at pH = 7.4, GSH 0 mM; B) is at pH = 5.5, GSH 0 mM; C) is at pH = 5.5, GSH 10 mM; D) is at pH = 7.4, GSH 10 mM. Therefore, in the tumor microenvironment, HA-PMTG@MOF with dual responsiveness to acid and reducing agents can be rapidly disassembled, promoting the rapid generation of singlet oxygen.

[0083] Figure 6 Curve of the release of 1-MDT by HA-PMTG over time at pH = 5.0.

[0084] Through the degradation and release experiment, it was verified that the HA-PMTG component in HA-PMTG@MOF TCPP-Fe can be degraded under the action of protease. Under the condition of pH = 5.0, during the 125-h observation period, papain effectively decomposed HA-PMTG@MOF TCPP-Fe and released approximately 50% of 1-MDT.

[0085] Figure 7 Fluorescence images of cell uptake of HA-PMTG@MOF TCPP-Fe and free TCPP.

[0086] At the cellular level, the targeting of the material was verified by comparing the uptake levels of HA-PMTG@MOF TCPP-Fe by CD44 receptor-positive cell lines (4T1, mouse breast cancer cells) and CD44 receptor-negative cell lines (NIH-3T3, mouse embryonic fibroblasts). Stronger fluorescence signals of the material were observed in 4T1 cells, while no obvious fluorescence signals were observed in NIH-3T3 cells.

[0087] Figure 8 Cell viability graphs of 4T1 cells treated with TCPP + laser irradiation, HA-PMTG@MOF TCPP-Fe , HA-PMTG@MOF TCPP-Fe + laser irradiation, respectively, where the laser irradiation conditions were 635 nm, 100 mW cm -2 , 10 min.

[0088] By comparing HA-PMTG@MOF TCPP-Fe under laser irradiation conditions (i.e., the HA-PMTG@MOF TCPP-Fe + laser irradiation group) or in the dark (i.e., the HA-PMTG@MOF TCPP-FeThe cell viability under (a certain group) verified the cell killing ability of HA-PMTG@MOF TCPP-Fe under laser irradiation. Specifically, 4T1 cell viability was reduced to 19.42% under laser irradiation after treatment with HA-PMTG@MOF TCPP-Fe (containing 25 μg / mL of TCPP), which was the HA-PMTG@MOF TCPP-Fe + laser irradiation group), and was reduced to 37.02% without irradiation (i.e., the HA-PMTG@MOF TCPP-Fe group).

[0089] HA-PMTG@MOF TCPP-Fe showed the performance of high reactive oxygen species level generation, which could effectively reduce the intracellular glutathione (GSH) ( Figure 9 ) level, as well as consume hydrogen peroxide (H2O2) ( Figure 10 ) level through the Fenton reaction. And it induced apoptosis of tumor cells. Flow cytometry verified that the HA-PMTG@MOF TCPP-Fe group showed a higher apoptosis / necrosis rate, with an apoptosis rate of 46.5%, significantly higher than other groups ( Figure 11 ). At the same time, strong green fluorescence from calreticulin (CRT) was observed in the cells treated with HA-PMTG@MOF TCPP-Fe by confocal microscopy, and the CRT positive rate was 51.17%, indicating that HA-PMTG@MOF TCCP-Fe induced immunogenic cell death (ICD) manifested as CRT externalization ( Figure 12 ); in addition, the fluorescence intensities of high mobility group box 1 (HMGB1) in the cells treated with MOF TCPP-Fe and HA-PMTG@MOF TCPP-Fe decreased to 17.2% and <5% respectively, which indicated that HMGB1 was released from the nucleus to the cytoplasm after PDT ( Figure 13 ), also indicating that HA-PMTG@MOF TCPP-Fe induced immunogenic cell death of tumor cells.

[0090] Figure 14 This is the in vitro distribution experimental diagram of HA-PMTG@MOF TCCP-Fe .

[0091] Using BALB / c mice, an orthotopic breast cancer tumor model of mice was established. Monitoring the tumor volume size, when it reached 200 mm 3 , the mice were randomly divided into two groups, and free TCPP and HA-PMTG@MOF were injected into the mice by tail vein injection TCPP-Fe(200 μL / mouse, TCPP 3 mg / kg). Mice were sacrificed at different time points after injection (6, 12, 24, and 48 h). The tumors and major organs of the mice were removed intact, and fluorescence signals were obtained using an in vivo imaging system for ex vivo imaging analysis. After injecting HA-PMTG@MOF TCPP-Fe Strong fluorescence signals were detected in the tumor region 6 h after injection, reached a peak at 12 h, and the labeling increased 1.45-fold at 6 h, indicating optimal accumulation and persistence up to 48 h. In contrast, the free TCPP group showed a peak fluorescence at 6 h, followed by rapid systemic clearance, indicating poor tumor targeting.

[0092] Using BALB / c mice, an orthotopic breast cancer tumor model in mice was established by subcutaneous injection of 4T1 murine tumor cells. The volume of the tumor was monitored. When it reached 50 mm 3 At this time, the mice were randomly divided into 5 groups, with 10 mice in each group. The specific time for grouping the mice was set on the first day. PBS, 1-MDT, TCPP, MOF TCPP-Fe and HA-PMTG@MOF TCPP-Fe Five groups of mice were treated with a TCPP dose of 3 mg / kg and a 1-MDT dose of 1.0 mg / kg. 1-MDT was injected subcutaneously into the abdomen of the mice, and the materials of other groups were injected into the mice via the tail vein. Treatment was performed once every five days for a total of three times. 12 h after the injection of the materials, TCPP, MOF, and HA-PMTG@MOF TCPP-Fe were treated in situ with a 35 nm laser (100 mW / cm 2 ) for 10 min, and the treatment was performed 3 times on the 2nd, 7th, and 12th days respectively. The tumor volume and the body weight of the mice were monitored. The treatment effects are as Figure 15 shown. The tumor inhibition rates of HA-PMTG@MOF TCPP-Fe , MOF TCPP-Fe , TCPP, and 1-MDT were 85.3%, 57.1%, 37.7%, and 28.9% respectively. It can be seen that compared with other experimental groups, this material has good tumor treatment effects.

[0093] After the tumor inhibition experiment, we collected and post-processed the tumor tissues, spleens, and draining lymph nodes of the mice, and analyzed the immune cell levels by flow cytometry. The proportions of CD4 + T cells in T cells in the PBS treatment group and the 1-MDT group were 18.875% and 18.85% respectively. After photodynamic therapy, the three groups of TCPP, MOF TCPP-Fe , HA-PMTG@MOF TCPP-Fe The proportions of CD4 +The proportions of T cells were increased to 28.55%, 32.75% and 34.1% respectively; among the tumor-infiltrating lymphocytes, the proportions of CD8 + T cells in the T cells of the PBS treatment group and the 1-MDT treatment group were only 12.8% and 12.61%; after photodynamic therapy, in the TCPP, MOF TCPP-Fe and HA-PMTG@MOF TCPP-Fe groups, the proportions of CD8 + T cells were increased to 41.4%, 41.05% and 52.73%. Photodynamic therapy can increase the proportions of CD4 + T and CD8 + T cells in the tumor-infiltrating lymphocytes. In the PBS group, the proportion of Treg cells was the highest, at 32.03%. Compared with the PBS group, the proportion of Treg cells in the 1-DMT treatment group was reduced to 28.05%, confirming that 1-MDT inhibited the expression and activity of IDO enzyme to a certain extent and restricted the recruitment of Treg. After photodynamic therapy in the TCPP, MOF TCPP-Fe and HA-PMTG@MOF TCPP-Fe groups, the proportions of Treg were reduced to 28.63%, 23.75% and 11.83% respectively. The proportion of Treg in the tumor tissue after HA-PMTG@MOF TCPP-Fe treatment was reduced the most, indicating that the treatment with the synergistic small molecule 1-MDT effectively reversed the tumor immunosuppressive microenvironment and reduced the recruitment of Treg cells at the tumor site. Next, flow cytometry was used to detect the contents of central memory T cells (Tcm) and effector memory T cells (Tem) in the tumors of mice. Compared with the PBS group, the contents of both types of memory T cells in the HA-PMTG@MOF TCPP-Fe group were significantly increased. The data confirmed that HA-PMTG@MOF TCPP-Fe had a long-term anti-tumor immune response. ( Figure 16 ). After the anti-tumor experiment was completed, an ELISA kit was used to measure the concentrations of cytokines (TNF-α, IL-6, IFN-Υ, IL-2, IL-6, IL-12, TGF-β) in the sera of each treatment group. At the same time, for the tumor tissues, we also tested the cytokines IFN-γ, TNF-α, IL-6 in the tumor tissues ( Figure 17 ).

[0094] In addition, the HA-PMTG@MOF TCPP-FeTherapeutic potential. First, 4T1 cells were subcutaneously injected at the second pair of mammary glands on the right side of BALB / c mice to establish a 4T1 orthotopic tumor model. At the same time, 4T1 cells were subcutaneously injected at the second pair of mammary glands on the left side of the same mice to establish a 4T1 subcutaneous distal tumor model. When the tumor volume reached 100 - 150 mm 3 , the tumor-bearing mice were randomly divided into two groups for treatment: PBS, HA-PMTG@MOF TCPP-Fe . After 12 h of injection, the light irradiation group was treated with a 635 nm laser (100 mW / cm 2 ) for 10 min. For the orthotopic tumor, the treatment was performed once every five days for a total of three times. The volumes of the orthotopic tumor and the distal tumor were monitored, measured every two days, and their volume changes were calculated. In the Control group, around 22 days, the tumor volumes of the mice had all reached more than 1500 mm 3 , while the HA-PMTG@MOF TCPP-Fe group showed obvious inhibitory effects on the distal tumor, and the distal tumor volume was always maintained at around 150 mm 3 , significantly inhibiting the growth of the distal tumor. ( Figure 18 )

[0095] The immune memory effect was evaluated through tumor rechallenge experiments. 5.0×10 5 4T1 tumor cells were subcutaneously injected at the second pair of mammary glands on the right side of the mice, and the mice were treated with HA-PMTG@MOF TCPP-Fe . Four out of ten mice had their tumors completely eradicated after treatment. Thirty days after tumor clearance, the contralateral mammary glands of the cured BALB / c mice were challenged with 4T1 tumor cells again, while healthy mice were used as controls. The tumor volume changes of the mice were monitored and the tumor volumes were calculated. When the size of the right tumor in the Control group exceeded 2 cm 3 , the mice were sacrificed. The survival of the mice was detected while calculating the tumor volume. The results showed that the mice in the Control group began to die from 28 days; around 34 days, all the mice had died. While two completely cured mice in the HA-PMTG@MOF TCPP-Fe group had a survival time of up to 136 days. Based on the above analysis, HA-PMTG@MOF TCPP-Fe can cure mice and enable mice to have a long-term anti-tumor immune response. ( Figure 19 )

[0096] Figure 20 are graphs of liver function markers in serum. Among them, Figure A is the level of alanine aminotransferase (ALT); Figure B is the level of aspartate aminotransferase (AST), and renal function markers; Figure C is the level of blood urea nitrogen (BUN); Figure D is the level of creatinine (Cr). Through blood biochemical analysis, it was demonstrated that HA-PMTG@MOF TCPP-Fe has no systemic toxicity.

[0097] The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A polyamino acid composite material, characterized in that: It is composed of a tetracarboxylphenyl iron porphyrin metal organic framework material and a block copolymer shown in formula 1 coated on the surface thereof; Wherein, x, y, and z are the polymerization degrees, x is selected from 1-20, y is selected from 1-10, and z is selected from 1-30.

2. The polyamino acid composite material according to claim 1, characterized in that: In the block copolymer represented by Formula 1, x is selected from 10-20, y is selected from 1-5, and z is selected from 1-15.

3. The polyamino acid composite material according to claim 1, characterized in that: The nano composite material has a shuttle-shaped structure and the iron element is evenly distributed on the surface of the nano composite material.

4. The polyamino acid composite material according to claim 1, characterized in that: The hydrated particle size of the nanocomposite material is 200±100 nm.

5. The method for preparing the polyamino acid composite material according to any one of claims 1 to 4, characterized in that: The following steps are involved: The tetracarboxylphenyl iron porphyrin metal organic framework material and the block copolymer shown in Formula 1 are mixed, and the polyamino acid composite material is obtained through electrostatic assembly, metal coordination and hydrophilic and hydrophobic effects.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the tetracarboxylphenyl iron porphyrin metal organic framework material to the block copolymer shown in Formula 1 is 1:(1-20).

7. The preparation method according to claim 5, characterized in that: The block copolymer represented by formula 1 is prepared by a click chemistry reaction of a random copolymer represented by formula 2, α-alkyne hyaluronic acid and a copper catalyst in an alkaline environment; In Formula 2, m and n are the degree of polymerization, m is selected from 1-20, n is selected from 1-10, and p is selected from 1-30.

8. An anti-tumor drug, characterized in that: The invention comprises the polyamino acid composite material according to any one of claims 1 to 4 or the polyamino acid composite material prepared by the preparation method according to any one of claims 5 to 7.

9. The anti-tumor drug according to claim 8, characterized in that: The anti-tumor drug releases 40%-60% of 1-methyl-D-tryptophan under the action of protease; The protease is selected from papain, trypsin or chymotrypsin.

10. The anti-tumor drug according to claim 8, characterized in that: The anti-tumor drug inhibits the activity of tumor cells by using it under laser irradiation; The conditions of the laser irradiation are: The wavelength of the laser is 500-700nm; The power density of the laser is 80-100 mW cm -2 ; The laser irradiation time is 5-30 minutes.

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