Tumor microenvironment enzyme response type nanoparticles, preparation method and application thereof

Abstract

The invention relates to the field of biological science and drug carriers, in particular to tumor microenvironment enzyme response type nano-particles, a preparation method and application thereof, and the tumor microenvironment enzyme response type nano-particles comprise at least two structural domains a which are sequences of matrix metalloproteinase substrate peptides; at least one structural domain b which is a sequence of melittin; the structural domain a is connected to the C tail end and the N tail end of the structural domain b; the tumor microenvironment enzyme response type melittin can slowly release melittin in a tumor microenvironment under enzyme response to achieve an anti-cancer effect, can also be used for preparing a nano-drug, and can deliver a ferroptosis small-molecule accelerant RSL3 to the tumor microenvironment in a targeted manner, so that the drug can infiltrate into tumor cells and induce tumor cell death, and the tumor microenvironment enzyme response type melittin has a good anti-cancer effect. The antigens in tumor cells are released, so that tumor immunotherapy and tumor metastasis treatment are realized, and the drug resistance of liver metastatic tumors is eliminated.

Description

Technical Field

[0001] The present invention relates to the fields of bioscience and drug carriers, and particularly to a tumor microenvironment enzyme-responsive nanoparticle, a preparation method thereof, and an application thereof. Background Art

[0002] Liver cancer has a high degree of malignancy, rapid disease progression, and relatively high incidence and mortality rates. The liver is relatively close to sites prone to tumors such as the colon, and the blood flow velocity in the hepatic sinusoids is slow. Factors such as the immune tolerance and inhibitory microenvironment of the liver under normal conditions make the liver prone to becoming an organ for tumor metastasis and settlement. Currently, the most common methods for treating liver metastases are through surgical resection, radiofrequency ablation, etc., or in combination with radiotherapy, chemotherapy, and liver transplantation. However, the multi-site prone characteristics of liver metastases result in poor prognosis. At the same time, liver metastases can utilize the host's peripheral immune tolerance mechanism to cause acquired immune therapy resistance through CD8+ T cell deficiency; the drug resistance problems caused by treatment methods mainly based on targeted drugs, chemotherapy drugs, and combination drugs are a major challenge in current liver cancer treatment. Long-term chemotherapy also leads to tolerance to first-line and second-line treatment drugs in 25% of patients. Therefore, developing new treatment strategies for tumor liver metastases will provide an effective basis for the comprehensive treatment of drug-resistant tumor liver metastases and has great social demand.

[0003] Exogenous drugs induce ferroptosis in tumor cells, break the barrier function of cell membranes to release intracellular antigenic substances, enabling intracellular antigens to be recognized and presented by APC cells, activating the body's T cell immunity, eliminating the drug resistance of liver metastases, reversing the tumor immunosuppressive microenvironment, and thus playing a role in cure. The ferroptosis small molecule promoter RSL3, as a small molecule compound, is a direct inhibitor of glutathione peroxidase 4 (GPX4) and can effectively induce cellular lipid peroxidation to produce ROS. However, the effective delivery of small molecule drugs and their low uptake rate by tumor cells have always been difficult points. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a tumor microenvironment enzyme-responsive nanoparticle, a preparation method thereof, and an application thereof. The tumor microenvironment enzyme-responsive melittin can slowly release melittin under enzyme response in the tumor microenvironment to achieve an anti-cancer effect, and can also be used to prepare nano-drugs, which can target and deliver the ferroptosis small molecule promoter RSL3 to the tumor microenvironment, enabling the drug to infiltrate into tumor cells, induce tumor cell death, release intracellular antigens of tumor cells, and achieve tumor immunotherapy, treatment of tumor metastasis, and elimination of drug resistance of liver metastases.

[0005] To this end, the present invention provides the following technical solutions:

[0006] A tumor microenvironment enzyme-responsive melittin, comprising:

[0007] At least two domain a, with sequences of matrix metalloproteinase substrate peptides;

[0008] At least one domain b, with a sequence of melittin;

[0009] Domain a is connected to the C-terminus and N-terminus of domain b.

[0010] Optionally, the amino acid sequence of the matrix metalloproteinase substrate peptide is at least one of PLGMWSR, PLGLWA, and PQGIAGQ;

[0011] and / or

[0012] The sequence of the melittin is as positions 8 - 34 in SEQ ID NO.1, or a sequence having at least 85% identity thereto and having a cytolytic effect of forming pores in the cell membrane.

[0013] Optionally, the C-terminus and / or N-terminus of the tumor microenvironment enzyme-responsive melittin further comprises a His-tag sequence;

[0014] Optionally, the tumor microenvironment enzyme-responsive melittin sequentially includes domain a, domain b, domain a, and His-tag sequence from the N-terminus to the C-terminus. Optionally, the His-tag sequence is a His-tag sequence composed of 6 His amino acids. Optionally, the amino acid sequence of the tumor microenvironment enzyme-responsive melittin is as shown in any one of SEQ ID NO.1 - 3.

[0015] A biomaterial, including any one of the following:

[0016] A1, a nucleic acid molecule encoding the tumor microenvironment enzyme-responsive melittin;

[0017] A2, a recombinant vector containing the nucleic acid molecule in A1;

[0018] A3, a host cell containing the recombinant vector in A2.

[0019] An enzyme-responsive nanocarrier targeting the tumor microenvironment, comprising: the tumor microenvironment enzyme-responsive melittin; an organic compound self-assembled from polylactic acid, porphyrin or porphyrin derivative, and Co 2+ ion conjugate; lipid.

[0020] An enzyme-responsive nano-drug targeting the tumor microenvironment, comprising: the enzyme-responsive nanocarrier targeting the tumor microenvironment; the drug is a ferroptosis small molecule promoter; the ferroptosis small molecule promoter includes RSL3, Fin56, or ML210.

[0021] Optionally, it includes: polylactic acid, porphyrin or porphyrin derivative, and Co 2+ The organic compound self-assembled by the ion conjugate has a core-shell structure, with a ferroptosis small molecule promoter in the core, lipids wrapped on the core-shell, and the tumor microenvironment enzyme-responsive melittin loaded on the surface of the core-shell;

[0022] Optionally, the tumor microenvironment enzyme-responsive melittin is connected to Co in the organic compound through a histidine tag 2+ by an ionic coordination bond;

[0023] Optionally, the porphyrin derivative is a porphyrin derivative containing only one carboxyl group;

[0024] Optionally, the porphyrin derivative is pyropheophorbide-α;

[0025] Optionally, the molecular weight of the polylactic acid is 1900 - 2200;

[0026] Optionally, the lipid is PEG-phospholipid, preferably DSPE-PEG2000;

[0027] Optionally, the average particle size of the nanocarrier is 120 - 280 nm.

[0028] A preparation method of the enzyme-responsive nanodrug targeting the tumor microenvironment described above includes the following steps:

[0029] S1. React polylactic acid with porphyrin or porphyrin derivative, and carry out a condensation reaction between the hydroxyl group at the end of polylactic acid and the carboxyl group of porphyrin or porphyrin derivative, so that the porphyrin or porphyrin derivative is bonded to the end of the polylactic acid polymer chain;

[0030] S2. Add Co 2+ ions to the product of step S1, and embed Co 2+ ions into the porphyrin ring to obtain an organic compound self-assembled by polylactic acid, porphyrin or porphyrin derivative, and Co 2+ ion conjugate;

[0031] S3. Dissolve the organic compound, lipid, and ferroptosis small molecule promoter prepared in step S2 in an organic solvent respectively, drop them into physiological saline, and evaporate the organic solvent to obtain a solution in which nanoparticles containing cobalt ion porphyrin rings are evenly distributed in physiological saline;

[0032] S4. Incubate the tumor microenvironment enzyme-responsive melittin connected with a histidine tag overnight with the solution prepared in step S3 to obtain the enzyme-responsive nanodrug targeting the tumor microenvironment.

[0033] Optionally, it is characterized in that

[0034] In step S3, the evaporation temperature is 37 - 48°C, optionally 40°C;

[0035] And / or, in step S3, the concentration of the organic compound solution after dissolving the organic compound is 1 - 20 mg / ml, the concentration of the lipid solution after dissolving the lipid is 1 - 20 mg / ml, and the concentration of the ferroptosis small molecule promoter solution after dissolving the ferroptosis small molecule promoter is 1 - 20 mg / ml;

[0036] And / or, in step S3, the volume ratio of the organic compound solution, the lipid solution, the ferroptosis small molecule promoter solution and the physiological saline solution is (0.5 - 1.5):(0.5 - 1.5):(0.5 - 1.5):(2 - 10);

[0037] And / or, in step S4, the mass - volume ratio of the tumor microenvironment enzyme - responsive melittin and the solution of the nanoparticle containing a cobalt - ion porphyrin ring is 0.1 - 0.5:1, mg / ml; the concentration of the solution of the nanoparticle containing a cobalt - ion porphyrin ring is 1 - 10 mg / ml;

[0038] And / or, in step S4, the overnight incubation temperature is 2 - 8°C, optionally 4°C;

[0039] And / or, in step S4, an ultrafiltration step is further included, and the molecular weight of the ultrafiltration tube is 10 - 30 KD, optionally 10000 Dal.

[0040] The tumor microenvironment enzyme - responsive melittin, the enzyme - responsive nanomedicine targeting the tumor microenvironment or the enzyme - responsive nanomedicine targeting the tumor microenvironment prepared by the preparation method has the following uses:

[0041] (1) Use in the preparation of tumor immunotherapy drugs;

[0042] (2) Use in the preparation of drugs for treating tumor metastasis; optionally, use in the preparation of drugs for treating liver metastasis of tumors.

[0043] The technical solution of the present invention has the following advantages:

[0044] 1. A tumor microenvironment enzyme-responsive melittin provided by the present invention comprises: at least two domain a, which are sequences of matrix metalloproteinase substrate peptides; at least one domain b, which is a sequence of melittin; domain a is connected to the C-terminus and N-terminus of domain b; Melittin (MLT) has shown excellent anti-cancer effects in preclinical cell and animal experiments. However, due to its non-specific cytotoxicity, easy degradation, hemolytic activity and other problems, its applicability to tumor treatment is relatively low. Through the research of the present invention, matrix metalloproteinases (MMP), especially MMP2 and MMP9, are generally present in the tumor microenvironment and are highly expressed in almost all types of tumor tissues, which is closely related to the development and metastasis of tumors. By fusing the substrate peptides of MMPs at both ends of melittin, the activity of MLT can be temporarily shielded. When reaching the tumor microenvironment, MMP specifically cleaves the peptide bond between the substrate peptide of MMPs and MLT, exposing MLT to exhibit the biological activity of punching holes in the cell membrane, achieving the effect of anti-tumor cells, thereby constructing a tumor microenvironment enzyme-responsive nano-drug delivery system, which can safely deliver MLT. At the same time, when the fusion protein of the substrate peptide of MMPs and melittin is used to prepare nano-drugs, it can be assembled with ferroptosis small molecule promoters (RSL3, Fin56 or ML210). When in the tumor microenvironment, after being cleaved by matrix metalloproteinase MMP2 / 9, MLT is exposed to play a role in punching holes in the cell membrane, which can promote the infiltration of the slowly released RSL3 into tumor cells, induce ferroptosis of tumor cells, thereby releasing tumor intracellular antigens, promoting immunogenic death of tumors and selective elimination of M2 macrophages, releasing more intracellular antigens, and presenting them to T cells through antigen-presenting cells (APCs), activating more body immune cells to participate in killing tumors, realizing tumor immunotherapy, treating tumor metastasis such as liver metastasis of tumors, and eliminating the drug resistance of liver metastatic tumors.

[0045] 2. The present invention provides an enzyme-responsive nano-drug targeting the tumor microenvironment, comprising: an organic compound self-assembled from polylactic acid, porphyrin or porphyrin derivative and Co 2+ ion conjugate has a core-shell structure, the core contains a ferroptosis small molecule promoter (including RSL3, Fin56 or ML210), the core-shell is wrapped with lipids, and the surface of the core-shell is loaded with the tumor microenvironment enzyme-responsive melittin; in the above nano-drug, polylactic acid (PLA) is a new type of bio-based and renewable biodegradable material, non-toxic, with good biocompatibility and encapsulation performance, and is widely used in pharmaceuticals and medical engineering materials and has obtained US FDA certification. Based on this, an organic compound self-assembled from polylactic acid, porphyrin or porphyrin derivative and Co 2+ ion conjugate has a core-shell structure, and the Co conjugated on the core-shell structure 2+Ions have multiple coordination bonds and can be coupled with tumor microenvironment enzyme-responsive melittin containing His tags to form a stable structure, and the tumor microenvironment enzyme-responsive melittin is loaded on the surface of the core-shell; small molecule drugs (RSL3, Fin56 or ML210) are insoluble in water, and their interaction with lipids can be encapsulated inside the core-shell structure, thereby obtaining an enzyme-responsive nano-drug targeting the tumor microenvironment. When the nano-drug reaches the tumor microenvironment, tumor cells and M2 macrophages highly express MMP2 / 9. The highly expressed MMP2 / 9 cleaves the MMP substrate peptide linked to the tail end of MLT, and the exposed MLT punches holes in the cell membrane, promoting the uptake of RSL3 by tumor cells and M2 macrophages, thereby promoting the immunogenic death of tumors and the selective elimination of M2 macrophages, releasing more intracellular antigens, and presenting them to T cells through APC cells, activating more body immune cells to participate in killing tumors. In summary, the nano-drug can target the tumor microenvironment, achieve the slow release of MLT in the tumor microenvironment, and promote the accumulation of small molecule drugs (RSL3, Fin56 or ML210) in tumor cells and M2 cells. MLT and small molecule drugs (RSL3, Fin56 or ML210) can synergistically treat tumors and tumor metastases, achieve tumor immunotherapy, and reverse the drug resistance of metastatic tumors, such as the treatment of tumor liver metastases. Brief Description of the Drawings

[0046] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0047] Figure 1 It is the result of the hydrodynamic diameter of the nanoparticle NP obtained in step (3) detected by a dynamic light scattering (DLS) system in Example 2 of the present invention;

[0048] Figure 2 It is the potential result of the nanoparticle NP obtained in step (3) detected by a Zeta potential detector in Example 2 of the present invention;

[0049] Figure 3 It is the ultraviolet absorption spectrum of the nanoparticle NP obtained in step (3) in Example 2 of the present invention;

[0050] Figure 4 It is the SDS-page electrophoresis diagram of the serum stability experiment of the enzyme-responsive nano-drug targeting the tumor microenvironment detected in Example 2 of the present invention and the nanoparticle after being destroyed by Triton-X100;

[0051] Figure 5 This is the fast protein liquid chromatography map of MLT in the enzyme-responsive nanodrug for detecting the tumor microenvironment in Example 2 of the present invention;

[0052] Figure 6 This is the high performance liquid chromatography map of RSL3 in the enzyme-responsive nanodrug for detecting the tumor microenvironment in Example 2 of the present invention;

[0053] Figure 7 This is the result map of the enzyme-responsive nanodrug for targeting the tumor microenvironment in Example 2 of the present invention under transmission electron microscope imaging;

[0054] Figure 8 This is the result map of the enzyme-responsive nanodrug for targeting the tumor microenvironment in Example 2 of the present invention under scanning electron microscope imaging;

[0055] Figure 9 This is the whole blood incubation hemolysis experiment of the enzyme-responsive nanodrug for targeting the tumor microenvironment in Experimental Example 1 of the present invention;

[0056] Figure 10a This is the confocal fluorescence image of the in vitro targeted uptake of the enzyme-responsive nanodrug for targeting the tumor microenvironment in Experimental Example 1 of the present invention by lung cancer tumor cells (scale bar is 20 μm);

[0057] Figure 10b This is the confocal fluorescence image of the in vitro targeted uptake of the enzyme-responsive nanodrug for targeting the tumor microenvironment in Experimental Example 1 of the present invention by melanoma tumor cells (scale bar is 20 μm);

[0058] Figure 11 This is the flow cytometry analysis map of the in vitro targeted uptake of the enzyme-responsive nanodrug for targeting the tumor microenvironment in Experimental Example 1 of the present invention by tumor cells;

[0059] Figure 12 This is the confocal fluorescence image of the in vitro targeted uptake of the enzyme-responsive nanodrug for targeting the tumor microenvironment in Experimental Example 1 of the present invention by M2-type tumor cells;

[0060] Figure 13a This is the confocal fluorescence image of the enzyme-responsive nanodrug for targeting the tumor microenvironment in Experimental Example 1 of the present invention generating ROS after incubation with in vitro cells;

[0061] Figure 13b This is the confocal fluorescence image of the control NP-Only generating ROS after incubation with in vitro cells in Experimental Example 1 of the present invention;

[0062] Figure 14 This is the result of the cytotoxicity proliferation experiment of the enzyme-responsive nanodrug for targeting the tumor microenvironment in Experimental Example 1 of the present invention;

[0063] Figure 15 This is the immunofluorescence confocal imaging of frozen sections of the livers of normal mice with the enzyme-responsive nanomedicine targeting the tumor microenvironment in Experimental Example 1 of the present invention;

[0064] Figure 16 This is the experimental result of the whole-body organ distribution of the enzyme-responsive nanomedicine targeting the tumor microenvironment after intravenous administration to mice in Experimental Example 2 of the present invention;

[0065] Figure 17 This is the analysis of the blood protein corona mass spectrum components of tumor cells of the enzyme-responsive nanomedicine targeting the tumor microenvironment in Experimental Example 2 of the present invention;

[0066] Figure 18 This is the fluorescence image of the therapeutic effect of the enzyme-responsive nanomedicine targeting the tumor microenvironment on liver metastasis of tumors in mice in Experimental Example 2 of the present invention;

[0067] Figure 19 This is the targeting effect diagram of the enzyme-responsive nanomedicine targeting the tumor microenvironment on liver metastasis of tumors in mice in Experimental Example 2 of the present invention;

[0068] Figure 20 This is the HE section image of the liver tissue after the treatment of liver metastasis of tumors in mice with the enzyme-responsive nanomedicine targeting the tumor microenvironment in Experimental Example 2 of the present invention;

[0069] Figure 21 This is the detection result of the liver tissue weight after the treatment of liver metastasis of tumors in mice with the enzyme-responsive nanomedicine targeting the tumor microenvironment in Experimental Example 2 of the present invention;

[0070] Figure 22 This is the detection result of the proportion of liver tumor burden after the treatment of liver metastasis of tumors in mice with the enzyme-responsive nanomedicine targeting the tumor microenvironment in Experimental Example 2 of the present invention;

[0071] Figure 23 This is the survival percentage after the treatment of liver metastasis of tumors in mice with the enzyme-responsive nanomedicine targeting the tumor microenvironment in Experimental Example 2 of the present invention;

[0072] In the above-mentioned drawings, * involved indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed implementation manners

[0073] The following embodiments are provided to better further understand the present invention. It is not limited to the best implementation manner, and does not limit the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other existing technologies falls within the protection scope of the present invention.

[0074] For those embodiments where specific experimental procedures or conditions are not specified, the operations or conditions of the conventional experimental procedures described in the literature in this field can be followed. For reagents or instruments whose manufacturers are not specified, they are all conventional reagent products that can be obtained commercially.

[0075] PLA organic compounds: polylactic acid, porphyrin or porphyrin derivatives, and Co 2+ Organic compounds self-assembled with ion conjugates. The PLA organic compounds in the following embodiments are PLA organic compounds of porphyrin combined with cobalt ions prepared by the method in (Steps 1 - 3) of Example 1 in Chinese Patent Document CN113476598A.

[0076] The small molecule drug RSL3 is a glutathione peroxidase 4 inhibitor and a cell ferroptosis inducer, with the molecular formula: C 23 H 21 C l N2O5, and the molecular weight is: 440.88.

[0077] Example 1 Tumor Microenvironment Enzyme-Responsive Melittin

[0078] This example provides a tumor microenvironment enzyme-responsive melittin. The specific sequence is shown in the following table. To facilitate the preparation of enzyme-responsive nanodrugs targeting the tumor microenvironment, a histidine tag is connected to one end of the sequence.

[0079]

[0080] The above-mentioned tumor microenvironment enzyme-responsive melittin can be prepared by conventional methods. In the present invention, it is prepared and synthesized by entrusting Hefei Guotai Biotechnology Co., Ltd.

[0081] Example 2 Enzyme-Responsive Nanodrug Targeting Tumor Microenvironment

[0082] This example provides a preparation method for an enzyme-responsive nanodrug targeting the tumor microenvironment, including the following steps:

[0083] (1). Dissolve 10 mg of PLA organic compound in 1 mL of absolute ethanol, place it in a test tube to obtain the first liquid phase; subsequently, dissolve 10 mg of DSPE-PEG2000 phospholipid and 0.1 mg of small molecule drug RSL3 in 1 mL of absolute ethanol respectively. After complete dissolution, place them in test tubes to obtain the second liquid phase and the third liquid phase respectively, where both the DSPE-PEG2000 phospholipid and RSL3 are purchased from commercial reagents.

[0084] (2) Pipette 100 μL each from the first liquid phase, the second liquid phase, and the third liquid phase, and drop them into normal saline (500 μL) at 40 °C one by one. Rotate and evaporate ethanol at 40 °C for 4 hours using a rotor to obtain a normal saline solution containing nanoparticles NP.

[0085] (3) Centrifuge the normal saline solution containing nanoparticles NP in step (2) at 3000 rpm for 20 min using a 30KD ultrafiltration centrifuge tube to obtain a normal saline solution containing nanoparticles NP with a high concentration (concentration is 1 - 10 mg / ml, and 10 mg / ml is selected in this example).

[0086] (4) Incubate 0.5 mg of MMP - MLT - 1 polypeptide with the solution containing nanoparticles NP (1 ml) overnight at 4 °C, and then ultracentrifuge through an ultrafiltration tube (molecular weight is 10000 Dal) to obtain nanoparticles (NP - R - M) conjugated with MMP - MLT - 1 polypeptide. Purify using FPLC to obtain the enzyme - responsive nanodrug NP - R - M targeting the tumor microenvironment.

[0087] Use a dynamic light scattering (DLS) system to test the particle size of the nanoparticles NP obtained in step (3) of this example. The results are as Figure 1 shown, showing d = 108 ± 15.6 nm. At the same time, the electron microscopy results show that the particle size of the nanoparticles is uniform, the dispersion is good, and there is no aggregation phenomenon.

[0088] Use a Zeta potential detector to test the potential of the nanoparticles NP obtained in step (3) of this example. The results are as Figure 2 shown, and the nanoparticles carry a negative charge of about -7 mv.

[0089] Detect the ultraviolet absorption spectrum of the nanoparticles NP obtained in step (3) of this example. The results are as Figure 3 shown, and the nanoparticles have absorption peaks at 430 nm and 660 nm.

[0090] Detect the stability of the enzyme - responsive nanodrug NP - R - M targeting the tumor microenvironment prepared in this example. The method is as follows: Mix the nanodrug NP - R - M (containing 0.1 mg of MLT) with the dye DiD (1 μL, concentration is 10 mg / ml) to obtain the nanodrug NP - R - M encapsulating the dye DiD; then mix the nanodrug NP - R - M encapsulating the dye DiD (20 μL) with mouse serum (180 μL). The mouse serum contains 10% fetal bovine serum FBS or 5% tween - 20 or no FBS. After incubating at 37 °C for different times, use SDS page gel electrophoresis to detect the stability of the nanodrug NP - R - M, and detect the integrity of the nanodrug through whole - body fluorescence imaging.

[0091] The results are as follows Figure 4 shown. In the figure, EX represents the laser excitation spectrum, and EM represents the laser emission spectrum. The results show that the enzyme-responsive nanodrug NP-R-M targeting the tumor microenvironment has good stability;

[0092] For MLT in the enzyme-responsive nanodrug NP-R-M targeting the tumor microenvironment using a fast protein liquid chromatography system (detecting the peptide bond spectral absorption of polypeptides at a wavelength of 280 nm), the results are as follows Figure 5 shown, indicating that the nanodrug successfully loaded the MLT polypeptide;

[0093] For RSL3 in the enzyme-responsive nanodrug NP-R-M targeting the tumor microenvironment using high performance liquid chromatography, the method is as follows: HPLC detection conditions: C18 reverse phase chromatography column (4.6×250 mm, 5 μm), flow rate 1 ml / min, injection volume 10 μl, detection wavelength 254 nm, mobile phase 100% methanol. The results are as follows Figure 6 shown, indicating that the nanodrug NP-R-M successfully loaded the small molecule drug RSL3;

[0094] For imaging of the enzyme-responsive nanodrug NP-R-M targeting the tumor microenvironment using a transmission electron microscope, the results are as follows Figure 7 shown. The particle size of the nanodrug is about 100 nm, which is in good agreement with the DLS results ( Figure 1 );

[0095] For imaging of the enzyme-responsive nanodrug targeting the tumor microenvironment using a scanning electron microscope, the results are as follows Figure 8 shown. The particle size of the nanodrug is about 100 nm, which is in good agreement with the DLS results ( Figure 1 );

[0096] Example 3

[0097] The difference between this example and Example 1 is that in step (1), the concentration of the dissolution solution obtained by dissolving the PLA organic compound in absolute ethanol is 1 mg / ml to obtain the first liquid phase; the concentration of the dissolution solution obtained by dissolving the DSPE-PEG2000 phospholipid in absolute ethanol is 20 mg / ml to obtain the second liquid phase; the concentration of the dissolution solution obtained by dissolving the small molecule drug RSL3 in absolute ethanol is 1 mg / m to obtain the third liquid phase;

[0098] In step (2), 50 μL is respectively pipetted from the first liquid phase, the second liquid phase and the third liquid phase and dropped into physiological saline (1000 μL) at a temperature of 37 °C drop by drop;

[0099] In step (4), 0.1 mg of MMP-MLT-2 polypeptide was mixed with a solution containing nanoparticles NP (concentration 1 mg / ml, 1 ml), incubated overnight at 2 °C, and then ultracentrifuged through an ultrafiltration tube (molecular weight 10,000 Dal).

[0100] Example 4

[0101] The difference between this example and Example 1 is that in step (1), the PLA organic compound was dissolved in absolute ethanol to obtain a solution with a concentration of 20 mg / ml to obtain the first liquid phase; the DSPE-PEG2000 phospholipid was dissolved in absolute ethanol to obtain a solution with a concentration of 1 mg / ml to obtain the second liquid phase; the RSL3 small molecule drug was dissolved in absolute ethanol to obtain a solution with a concentration of 20 mg / m to obtain the third liquid phase;

[0102] In step (2), 150 μL was separately pipetted from the first liquid phase, the second liquid phase, and the third liquid phase and slowly dropped into normal saline (200 μL) at a temperature of 48 °C;

[0103] In step (4), 0.3 mg of MMP-MLT-3 polypeptide was mixed with a solution containing nanoparticles NP (concentration 10 mg / ml, 1 ml), incubated overnight at 8 °C, and then ultracentrifuged through an ultrafiltration tube (molecular weight 30,000 Dal).

[0104] Experimental Example 1 In vitro experiment of the enzyme-responsive nanodrug NP-R-M targeting the tumor microenvironment

[0105] 1. Experimental sample: The concentration of the polypeptide in the enzyme-responsive nanodrug NP-R-M targeting the tumor microenvironment prepared in Example 2 was determined by the Lorry method.

[0106] 2. Experimental methods and results

[0107] 2.1 Biosafety experiment and results

[0108] The method was as follows: 1) The protein content of the MLT polypeptide in NP-R-M was detected by the lowry method (using MLT to make a standard curve); 2) MLT and NP-R-M were sequentially added to the diluted blood cells (mouse blood diluted 5-fold) at the same concentration of MLT, with 1% Triton×100 added as a control, and left to stand at 37 °C for 1 - 3 h, and the hemolysis of red blood cells was observed and imaged;

[0109] The results were as Figure 9As shown, MLT was added to the first row. It can be seen that hemolysis occurred in blood cells corresponding to 5 - 20 μM of MLT, while no hemolysis occurred in red blood cells corresponding to the enzyme-responsive nanomedicine NP-R-M containing the same concentration of MLT added to the second row, indicating the biosafety of the nanomedicine NP-R-M.

[0110] 2.2 In vitro cell targeting experiment

[0111] The method is as follows: 100,000 LLC-mCherry (lung cancer) and B16-mCherry (melanoma) tumor cells were respectively added to a confocal imaging dish and cultured for 24 hours; 2) After adding the nanomedicine NP-R-M to a final concentration of 5 μM and incubating for 4 hours, Hoechst 33324 staining was given to the cell nuclei; 3) Observation was carried out using a confocal microscope, and the confocal fluorescence images are as Figure 10a 、 10b shown. The enzyme-responsive nanomedicine targeting the tumor microenvironment has good targeting ability.

[0112] Analysis was carried out using a flow cytometer. The flow cytometry analysis images are as Figure 11 shown. The enzyme-responsive nanomedicine NP-R-M targeting the tumor microenvironment has good targeting ability.

[0113] 2.3 In vitro M2 macrophage targeting experiment

[0114] The method is as follows: 100,000 bone marrow-derived induced M2 macrophages were added to a confocal imaging dish and cultured for 24 hours; 2) After adding the nanomedicine NP-R-M to a final concentration of 5 μM and incubating for 4 hours, Hoechst 33324 staining was given to the cell nuclei; 3) Observation was carried out using a confocal microscope, and the confocal fluorescence images are as Figure 12 shown (NP in the figure corresponds to NP-R-M). The enzyme-responsive nanomedicine targeting the tumor microenvironment has good targeting ability to tumor-related M2 macrophages.

[0115] 2.4 Ferroptosis effectiveness

[0116] The method is as follows: An enzyme-responsive nanomedicine targeting the tumor microenvironment was used, and the nanomedicine without loading the RSL3 small molecule drug was used as a control NP-Only (prepared according to Example 2, with the difference that the RSL3 small molecule drug was not added). 100,000 LLC tumor cells were cultured in a confocal dish, and NP-R-M and NP-only with a final concentration of 5 μM were respectively added and incubated. Subsequently, a ROS detection kit was used for staining to detect the intracellular ROS content, and the ROS content was used as a marker of cell ferroptosis.

[0117] Observed by confocal microscopy, the confocal fluorescence images of the enzyme-responsive nanodrug NP-R-M targeting the tumor microenvironment are as follows Figure 13a shown, and the confocal fluorescence images of the control are as follows Figure 13b shown. It can be concluded that the enzyme-responsive nanodrug NP-R-M targeting the tumor microenvironment has ferroptosis efficacy.

[0118] 2.5 Cytotoxicity experiment

[0119] The method is as follows: The CCK8 method was used to detect the cytotoxicity proliferation of nanodrugs at different concentrations. MF represents the scrambled state of MLT and serves as a negative control (prepared according to Example 2, where the MLT sequence is replaced with the scrambled MLT, see SEQ ID NO.4), while MT represents the forward state and represents bioactive MLT, that is, the nanodrug NP-R-M prepared according to Example 2. At the same time, a drug-free group was set. Then, the conventional CCK8 method experiment was carried out as follows:

[0120] (1) Collect cells in the logarithmic growth phase (LLC cells), adjust the cell suspension concentration, add 100 μl to each well of the 96-well plate, so that the density of the cells to be tested is 1000 - 10000 cells / well.

[0121] (2) Incubate the cells at 37 °C with 5% CO2 for 24 hours (the incubation time is adjusted according to the type of cells and the number of cells in each well).

[0122] (3) Add 10 μl of the test substances at different concentrations (nanodrug NP-R-M or negative control), and incubate the culture plate in the incubator for 24 hours.

[0123] (4) Add 10 μl of CCK-8 solution to each well. If the initial culture volume is 200 μl, then 20 μl of CCK-8 solution needs to be added, and so on for other cases.

[0124] (5) Continue to incubate in the cell incubator for 4 hours.

[0125] (6) Measure the absorbance of each well at 450 nm with an enzyme-labeled instrument.

[0126] Cell viability % = absorbance of the experimental group / absorbance of the drug-free group.

[0127] The results are as follows Figure 14 shown. The enzyme-responsive nanodrug targeting the tumor microenvironment has strong killing ability against tumor cells.

[0128] 2.6 Targeting experiment of M2 macrophages in liver tissue

[0129] Method: After injecting 200 μl of the nano-drug NP-R-M with a standard MLT concentration of 0.5 mg / ml into mice via the tail vein, the liver tissues of the mice were taken 24 hours later. After frozen sectioning, immunohistochemical antibody staining was performed (F4 / 80 antibody was used to label M2 macrophages), and immunofluorescence confocal imaging was adopted. The results are as Figure 15 shown (in the figure, NP represents the nano-drug NP-R-M). The enzyme-responsive nano-drug targeting the tumor microenvironment has strong targeting ability to M2 macrophages in the mouse liver.

[0130] Experimental Example 2 Animal Experiment of the Enzyme-Responsive Nano-Drug NP-R-M Targeting the Tumor Microenvironment

[0131] 1. Experimental sample: The concentration of the MLT polypeptide in the enzyme-responsive nano-drug NP-R-M targeting the tumor microenvironment prepared in Example 2 was determined by the Lorry method, and the concentration of the MLT polypeptide was adjusted to 0.5 mg / ml.

[0132] 2. Experimental methods and results

[0133] The efficacy of the drug was verified in mice with splenic liver metastases (spleen to liver). On the 3rd, 5th, 8th, and 21st days, 200 μL of the drug was administered to each mouse via the tail vein. The experimental design is as Figure 18 The first row. The specific method is as follows: 1) The mice with splenic liver metastases were randomly divided into 5 groups (control group, NP group, NP-R group, NP-M group, NP-R-M group), with 5 mice in each group. The drug administration conditions for each group are as follows:

[0134] The control group was given PBS;

[0135] The NP group was given the nanoparticle NP (prepared according to steps (1)-(3) in Example 2, and the RSL3 small molecule drug was not added in step (1));

[0136] The NP-R group was given the nanoparticle NP containing the RSL3 small molecule drug (prepared according to steps (1)-(3) in Example 2, and the concentration of the RSL3 small molecule drug solution was 20 mg / ml);

[0137] The NP-M group was given the nanoparticle NP containing MLT (prepared according to Example 2, the RSL3 small molecule drug was not added in step (1), and the MLT concentration was 1 mg / ml);

[0138] The NP-R-M group was given the nano-drug NP-R-M prepared in Example 2 (MLT concentration was 0.5 mg / ml).

[0139] On the 0th day, 5×10 6pcs;

[0140] On the 1st day, corresponding drugs were injected respectively (200 μL of the drug was administered to each mouse via the tail vein).

[0141] On the 3rd day, each mouse in each group was injected with the corresponding drug respectively (200 μL of the drug was administered to each mouse via the tail vein). Then, the whole-body organ distribution experiment of the enzyme-responsive nanodrug NP-R-M targeting the tumor microenvironment was detected 24 hours after intravenous administration in mice. The results are as Figure 16 shown, indicating that the nanodrug NP-R-M has the ability of liver targeting.

[0142] On the 5th day, each mouse in each group was injected with the corresponding drug respectively (200 μL of the drug was administered to each mouse via the tail vein). The venous blood of the mice in the NP-R-M group was collected (the blood containing circulating tumor cells (CTCs) was collected), and the protein corona mass spectrometry composition analysis was carried out. The results are as Figure 17 shown, indicating that the nanodrug NP-R-M has the ability of tumor homing.

[0143] On the 8th day, each mouse in each group was injected with the corresponding drug respectively (200 μL of the drug was administered to each mouse via the tail vein).

[0144] On the 21st day, each mouse in each group was injected with the corresponding drug respectively (200 μL of the drug was administered to each mouse via the tail vein). At this time, the corresponding drug injected in each group was the corresponding drug encapsulated with DiD dye. The method for encapsulating the drug with DiD dye was: mixing the nanodrug NP-R-M, NP-R, NP-M or NP with the dye DiD (1 μL, concentration of 10 mg / ml) to obtain the nanodrug encapsulated with dye DiD.

[0145] The overall fluorescence image of the mouse liver dissected on the 23rd day is as shown in the second row of figures in Figure 18 . The stronger the fluorescence intensity, the larger the tumor volume represents, indicating that the tumor grows faster and the treatment effect of the drug on the tumor is worse. The liver without fluorescence indicates that it does not contain tumor tissue, indicating that the treatment effect of the drug is better. As can be seen from the figure, the nanodrug NP-R-M has no fluorescence, indicating that it has a good treatment effect on tumor liver metastasis.

[0146] The targeting effect diagram of the nanodrug for tumor liver metastasis treatment was obtained by immunohistochemistry method, as shown in Figure 19 (in the figure, NP corresponds to the nanodrug NP-R-M). The tumor tissue is yellow, and the nanodrug NP-R-M is red. The red drug is only taken up in the yellow tumor tissue, indicating that the nanodrug NP-R-M has good targeting ability for the tumor lesion area.

[0147] The liver tissue of the mouse was taken for HE section asFigure 20 As shown, it is shown that the nano-drug NP-R-M has a significant effect on treating liver metastasis of tumors. The liver weights of mice in each group were weighed, and the results are as Figure 21 shown. It can be seen that the liver weight after treatment with the nano-drug NP-R-M is comparable to that of normal mice, with no statistical difference, indicating that the nano-drug NP-R-M has a significant effect on treating liver metastasis of tumors. The tumor burden in the livers of mice in each group was detected, and the results are as Figure 22 shown, indicating that the nano-drug NP-R-M has a significant effect on treating liver metastasis of tumors, which is significantly better than NP-R and NP-M, indicating that the melittin and RSL3 small molecule drugs in the nano-drug NP-R-M of the present invention have a synergistic effect on treating liver metastasis of tumors.

[0148] Survival rate detection: Operate according to the above experimental method (6 mice in each group), with the difference that the mice are not dissected on the 23rd day, and the mice are raised normally, and the survival rates of mice in each group are detected. The results are as Figure 23 shown. The survival rate is high after treatment with the nano-drug NP-R-M, indicating that the nano-drug NP-R-M has a significant effect on treating liver metastasis of tumors.

[0149] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of the present invention.

Claims

1. A tumor microenvironment enzyme-responsive melittin, characterized in that, Comprising: At least two domain a, which are sequences of matrix metalloproteinase substrate peptides; At least one domain b, which is a sequence of melittin; Domain a is connected to the C-terminus and N-terminus of domain b.

2. The tumor microenvironment enzyme-responsive melittin according to claim 1, wherein The amino acid sequence of the matrix metalloproteinase substrate peptide is at least one of PLGMWSR, PLGLWA, and PQGIAGQ; And / or The sequence of melittin is from positions 8 to 34 in SEQ ID NO.1, or a sequence having at least 85% identity thereto and having a cytolytic effect of forming pores in the cell membrane.

3. The tumor microenvironment enzyme-responsive melittin according to any one of claims 1-2, characterized in that The C-terminus and / or N-terminus of the tumor microenvironment enzyme-responsive melittin further comprises a His-tag sequence; Optionally, the tumor microenvironment enzyme-responsive melittin sequentially comprises domain a, domain b, domain a, and His-tag sequence from the N-terminus to the C-terminus.

4. A biological material, characterized in that, Including any one of the following: A1. A nucleic acid molecule encoding the tumor microenvironment enzyme-responsive melittin according to any one of claims 1-3; A2. A recombinant vector containing the nucleic acid molecule described in A1; A3. A host cell containing the recombinant vector described in A2.

5. An enzyme-responsive nanocarrier targeting the tumor microenvironment, characterized in that, Comprising: the tumor microenvironment enzyme-responsive melittin according to any one of claims 1-3; an organic compound self-assembled from polylactic acid, porphyrin or porphyrin derivative, and Co 2+ ion conjugate; lipid.

6. An enzyme-responsive nanomedicine targeting the tumor microenvironment, characterized in that, Comprising: The enzyme-responsive nanocarrier targeting the tumor microenvironment according to claim 5; The drug is a ferroptosis small molecule promoter; The ferroptosis small molecule promoter includes RSL3, Fin56, or ML210.

7. The enzyme-responsive nanomedicine targeting the tumor microenvironment according to claim 6, characterized in that, Comprising: an organic compound self-assembled from polylactic acid, porphyrin or porphyrin derivative, and a Co 2+ ion conjugate has a core-shell structure, with a ferroptosis small molecule promoter in the core, lipids wrapped around the core-shell, and the tumor microenvironment enzyme-responsive melittin loaded on the surface of the core-shell; Optionally, the tumor microenvironment enzyme-responsive melittin is interconnected with Co in the organic compound through a histidine tag 2+ by an ionic coordination bond; Optionally, the porphyrin derivative is a porphyrin derivative containing only one carboxyl group; Optionally, the porphyrin derivative is pyropheophorbide-α; Optionally, the molecular weight of the polylactic acid is 1900-2200; Optionally, the lipid is PEG-phospholipid, preferably DSPE-PEG2000; Optionally, the average particle size of the nanocarrier is 120-280 nm.

8. A method for preparing an enzyme-responsive nanomedicine targeting the tumor microenvironment as described in claim 6 or 7, characterized in that, Including the following steps: S1. React polylactic acid with porphyrin or porphyrin derivative, subject the hydroxyl group at the end of polylactic acid and the carboxyl group of porphyrin or porphyrin derivative to a condensation reaction, and bond the porphyrin or porphyrin derivative to the end of the polylactic acid polymer chain; S2. Add Co to the product of step S1 2+ ions to embed Co 2+ ions into the porphyrin ring, obtaining an organic compound self-assembled from a combination of polylactic acid, porphyrin or porphyrin derivatives, and Co 2+ ions S3. Dissolve the organic compound, lipid, and ferroptosis small molecule promoter prepared in step S2 respectively with an organic solvent, drop them into physiological saline, and evaporate the organic solvent to obtain a solution in which nanoparticles containing cobalt ion porphyrin rings are uniformly distributed in physiological saline; S4. Mix and incubate overnight the tumor microenvironment enzyme-responsive melittin linked with a histidine tag with the solution prepared in step S3 to obtain an enzyme-responsive nanodrug targeting the tumor microenvironment.

9. The method for preparing the enzyme-responsive nanodrug targeting the tumor microenvironment according to claim 8, wherein In step S3, the evaporation temperature is 37-48 °C, optionally 40 °C; And / or, in step S3, the concentration of the organic compound solution after dissolving the organic compound is 1-20 mg / ml, the concentration of the lipid solution after dissolving the lipid is 1-20 mg / ml, and the concentration of the ferroptosis small molecule promoter solution after dissolving the ferroptosis small molecule promoter is 1-20 mg / ml; And / or, in the step S3, the volume ratio of the organic compound solution, the lipid solution, the ferroptosis small molecule promoter solution and the physiological saline solution is (0.5 - 1.5):(0.5 - 1.5):(0.5 - 1.5):(2 - 10); And / or, in the step S4, the mass-volume ratio of the tumor microenvironment enzyme-responsive melittin and the solution of the nanoparticle containing a cobalt ion porphyrin ring is 0.1 - 0.5:1, mg / ml; the concentration of the solution of the nanoparticle containing a cobalt ion porphyrin ring is 1 - 10 mg / ml; And / or, in the step S4, the incubation temperature is 2 - 8°C, optionally 4°C; And / or, in the step S4, it further includes a step of ultrafiltration using an ultrafiltration tube, and the molecular weight of the ultrafiltration tube is 10 - 30 KD, optionally 10000 Dal.

10. The tumor microenvironment enzyme-responsive melittin according to claim 1 or 2, the enzyme-responsive nano-drug targeting the tumor microenvironment according to claim 6 or 7, or the enzyme-responsive nano-drug targeting the tumor microenvironment prepared by the preparation method according to claim 8 or 9 has the following uses: (1) Use in the preparation of tumor immunotherapy drugs; (2) Use in the preparation of drugs for treating tumor metastasis; optionally, use in the preparation of drugs for treating liver metastasis of tumors.

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