In-situ self-assembled polypeptide bonding medicine, nano regulator, preparation method and application thereof

Abstract

The invention provides an in-situ self-assembled polypeptide bonding drug, a nano regulator, a preparation method and application thereof. The invention provides an in-situ self-assembled polypeptide bonding medicine with multiple response functions, which has a structure as shown in the following formula 1: in the formula 1, x is an integer from 10 to 145; the linking group A is active oxygen and a fibroblast activating protein (FAP enzyme) response peptide fragment; the linking group B does not exist or is a glutathione response fragment; the drug unit is from an indoleamine 2, 3-dioxygenase 1 inhibitor and a photosensitizer. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and relates to an in-situ self-assembling polypeptide bonded drug with multiple response functions and a formed nano regulator, as well as a preparation method and use thereof. Background Art

[0002] Although the emerging immune checkpoint blockade therapy has completely changed the treatment model for some tumor types, it has little effect in a considerable number of tumor types (such as pancreatic cancer, breast cancer and prostate cancer with highly fibrotic tumors). This is mainly attributed to the inherent immune tolerance mediated by the inherent factors of highly fibrotic tumors (such as tumor-associated fibroblasts) or the acquired immune tolerance caused by the evolution of tumors caused by immunotherapy. Among them, tumor-associated fibroblasts can form inherent immune tolerance by forming a dense extracellular matrix to hinder the infiltration of cytotoxic T cells, and secrete a variety of immune negative regulatory factors to hinder the activation of cytotoxic T cells and promote the proliferation and infiltration of immune tolerance cells (such as regulatory T cells). In the activation mechanism of tumor-associated fibroblasts, transforming growth factor signaling is one of its key signals. Targeting tumor-associated fibroblasts and inhibiting their transforming growth factor signaling is expected to overcome the inherent immune tolerance mediated by them. However, while single tumor-associated fibroblast targeted therapy enhances the function of cytotoxic T lymphocytes, the interferon γ secreted by them can reshape the tryptophan metabolism of tumor cells, leading to acquired immune tolerance and hindering the long-term effect of anti-tumor immunity. However, simply combining drugs that intervene in tumor-associated fibroblasts with tryptophan metabolism drugs has problems such as low bioavailability and serious toxic side effects caused by off-target effects. At the same time, the differences in kinetics of different drugs cannot coordinate with the dynamic changes of the tumor microenvironment. Therefore, how to conform to the changing laws of the tumor microenvironment, overcome inherent immune tolerance and acquired immune tolerance caused by treatment, and thus achieve effective and lasting anti-tumor immunity remains a daunting challenge.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide an in situ self-assembling polypeptide bonded drug with multiple response functions and a nano regulator formed therefrom, as well as a preparation method and use thereof.

[0005] On the one hand, the present invention provides an in situ self-assembling polypeptide bonded drug with multiple response functions, which has the following

[0006] The structure shown in formula 1:

[0007]

[0008] In Formula 1, x is an integer of 10-145, preferably an integer of 25-65, and more preferably 45;

[0009] The linker group A is a peptide segment responsive to reactive oxygen species and fibroblast activation protein (FAP enzyme);

[0010] Linker B is absent or is a glutathione responsive fragment;

[0011] The drug unit is derived from an indoleamine 2,3-dioxygenase 1 inhibitor and a photosensitizer;

[0012] When the drug unit is derived from an indoleamine 2,3-dioxygenase 1 inhibitor, one end of the linker group A is covalently bonded to a polyethylene glycol having a succinimide group at one end, the other end of the linker group A is covalently bonded to one end of the linker group B, and the other end of the linker group B is covalently bonded to the indoleamine 2,3-dioxygenase 1 inhibitor;

[0013] When the drug unit is derived from a photosensitizer, the linker B does not exist, one end of the linker A is covalently bonded to polyethylene glycol having a succinimide group at one end, and the other end of the linker A is covalently bonded to the photosensitizer.

[0014] In the present invention, the FAP enzyme and the reactive oxygen response peptide in the linker A may refer to a peptide comprising a proline and alanine linker fragment of the FAP enzyme response portion and a methionine peptide of the reactive oxygen response portion; wherein one end of the FAP enzyme response peptide is cysteine, which is bound to the succinimide on the polyethylene glycol (for example, by a click reaction); one end of the reactive oxygen response peptide is glutamic acid, which is covalently bonded to the linker B or the drug unit through an amino or carboxyl group. In some embodiments, the FAP enzyme response portion and the reactive oxygen response portion are connected by an amide bond. In a further preferred embodiment, the linker A is CAPGEMIMIE (N-terminal to C-terminal).

[0015] In the present invention, the glutathione responsive fragment in the linking group B may refer to a fragment having a specific chemical bond that can be cut by glutathione and its derivatives, preferably, selected from the group shown in the following formula:

[0016]

[0017] Wherein, M1 and M2 are each independently selected from an alkylene group having 1 to 4 carbon atoms; R1 and R2 are each independently selected from -C(=O)-, -NH-, -O-;

[0018] Further preferably, M1 and M2 are the same;

[0019] More preferably, the linking group B is selected from the group shown in the following chemical formula:

[0020]

[0021] Further preferably, R1 and R2 in the linking group B are independently derived from -COOH, -NCO, -NH2 or -OH.

[0022] In the present invention, the indoleamine 2,3-dioxygenase 1 inhibitor may refer to a small molecule inhibitor of indoleamine 2,3-dioxygenase 1 that inhibits the metabolic conversion of tryptophan into kynurenine, preferably, selected from the following compounds:

[0023]

[0024]

[0025] In the present invention, the photosensitizer refers to a molecule that can generate active oxygen, preferably, selected from the following compounds:

[0026]

[0027]

[0028] In some embodiments, when the drug unit is derived from an indoleamine 2,3-dioxygenase 1 inhibitor, one end (N-terminus) of the linker A is covalently bonded to a polyethylene glycol having a succinimide group at one end, the other end (C-terminus) of the linker A is covalently bonded to one end of a linker B, and the other end of the linker B is covalently bonded to the indoleamine 2,3-dioxygenase 1 inhibitor; when the drug unit is derived from a photosensitizer, the linker B is absent, one end (N-terminus) of the linker A is covalently bonded to a polyethylene glycol having a succinimide group at one end, and the other end (C-terminus) of the linker A is covalently bonded to the photosensitizer.

[0029] In some embodiments, the in situ self-assembling polypeptide bonding drug with multiple response functions is selected from:

[0030]

[0031]

[0032] On the other hand, the present invention also provides a method for preparing the above-mentioned in situ self-assembling polypeptide bonded drug, comprising the following steps:

[0033] One end of the linker group A is coupled to polyethylene glycol having a succinimide group at one end through a click reaction to obtain CH3O-PEG-linker group A;

[0034] When the drug unit is derived from an indoleamine 2,3-dioxygenase 1 inhibitor, the hydroxyl group of the indoleamine 2,3-dioxygenase 1 inhibitor is first reacted with the linker group B to obtain the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B, and then the reactive group of the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is reacted with the CH3O-PEG-linker group A to obtain the in situ self-assembling polypeptide bonded drug;

[0035] When the drug unit comes from a photosensitizer, the photosensitizer directly reacts with the CH3O-PEG-linking group A to obtain the in situ self-assembling polypeptide bonded drug.

[0036] In some embodiments, the preparation method specifically comprises the following steps:

[0037] A polyethylene glycol having a succinimide group at one end and a linker group A are dissolved in N,N-dimethylformamide (DMF), an organic base is added dropwise, and the mixture is stirred at room temperature for 12-24 hours. The product is dialyzed three times with an organic solvent and water in a dialysis bag, and lyophilized to obtain CH3O-PEG-linker group A;

[0038] When the drug unit is derived from an indoleamine 2,3-dioxygenase 1 inhibitor, the indoleamine 2,3-dioxygenase 1 inhibitor reacts with the linker B;

[0039] Wherein, when the reactive group of the linker group B is a carboxyl group, 4-dimethylaminopyridine (DMAP), N,N-diisopropylethylamine (DIEA) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) are added to activate the carboxyl group in anhydrous dichloromethane (DCM) and an ice bath for 1-2 hours, and then the indoleamine 2,3-dioxygenase 1 inhibitor is added and stirred at room temperature for 12-24 hours, and the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is purified by column chromatography; subsequently, the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is dissolved in a solvent (such as DMF or DCM), NHS and EDCI are added for activation for 1-6 hours, and then CH3O-PEG-linker group A is added to react at room temperature for 12-24 hours, and the product is dialyzed three times with an organic solvent and water in a dialysis bag, respectively, and lyophilized to obtain an in situ self-assembled polypeptide bonded drug;

[0040] When the reactive group of the linker group B is a hydroxyl group or an amino group, one end of the linker group B is reacted with tert-butyldimethylsilyl chloride (TBSCl) to protect the hydroxyl group or Fmoc to protect the amino group, then the indoleamine 2,3-dioxygenase 1 inhibitor is dissolved in DCM, activated with di(p-nitrobenzene) carbonate (NPC) and DMAP for 1-2 hours, and then the linker group B protected at one end is added to react to obtain the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B-protecting group, and the protecting group is removed to obtain the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B; then CH3O-PEG-linker group A is dissolved in DMF, NHS is added for activation for 1-6 hours, and then the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is added, and the product is dialyzed three times with an organic solvent and water in a dialysis bag, respectively, and lyophilized to obtain an in situ self-assembling polypeptide bonded drug;

[0041] When the reactive group of the linker group B is an isocyanate group, the linker group B and the indoleamine 2,3-dioxygenase 1 inhibitor are dissolved in N,N-dimethylformamide (DMF), reacted for 12-24 hours in the presence of an organic base, and purified by column chromatography to obtain the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B, and then the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is reacted with CH3O-PEG-linker group A, and the product is dialyzed three times with an organic solvent and water in a dialysis bag, respectively, and lyophilized to obtain an in situ self-assembled polypeptide bonded drug;

[0042] When the drug unit comes from a photosensitizer, the photosensitizer is dissolved in DMF, NHS is added for activation for 1-6 hours, and then CH3O-PEG-linker A is added to react at room temperature for 12-24 hours. The product is dialyzed three times with an organic solvent and water in a dialysis bag, and lyophilized to obtain an in situ self-assembled polypeptide bonded drug;

[0043] The organic base used in the preparation method may be DIEA and triethylamine (TEA), and the organic solvent used in dialysis may be N,N-dimethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide.

[0044] On the other hand, the present invention also provides a nano-regulator, which comprises two of the above-mentioned in situ self-assembling polypeptide bonded drugs, wherein the drug unit in one in situ self-assembling polypeptide bonded drug is derived from an indoleamine 2,3-dioxygenase 1 inhibitor, and the drug unit in the other in situ self-assembling polypeptide bonded drug is derived from a photosensitizer.

[0045] Preferably, the hydrodynamic radius of the nano-regulator is 30-80 nm, more preferably 50-60 nm.

[0046] The nano-regulator provided by the present invention has a hydrophobic core, and the hydrophobic core can further encapsulate a hydrophobic drug through hydrophobic interaction; the hydrophobic drug may include, but is not limited to, a tumor-associated fibroblast inhibitor, preferably selected from the following molecules:

[0047]

[0048] Also preferably, the tumor-associated fibroblast inhibitor encapsulated in the hydrophobic core of the nanoregulator may be one or two or more of the above-mentioned molecules.

[0049] In another aspect, the present invention also provides a method for preparing the nano-regulator, comprising the following steps:

[0050] The above-mentioned in situ self-assembled polypeptide bonded drug whose drug units are indoleamine 2,3-dioxygenase 1 inhibitor and photosensitizer respectively, and an optional hydrophobic drug (especially a tumor-associated fibroblast inhibitor) are dissolved in an organic solvent, mixed evenly, and then ultrasonically dispersed in water. The resulting solution is subjected to ultrafiltration or dialysis to obtain a nano-regulator.

[0051] In some embodiments, the mass ratio of the two in situ self-assembling polypeptide bonded drugs, wherein the drug units are indoleamine 2,3-dioxygenase 1 inhibitor and photosensitizer, is 1:0.8-1.2. In one embodiment, the mass ratio of the above in situ self-assembling polypeptide bonded drugs, wherein the drug units are indoleamine 2,3-dioxygenase 1 inhibitor and photosensitizer, is 1:1.

[0052] In a further embodiment, the mass ratio of the two in situ self-assembling polypeptide bonded drugs, wherein the drug units are indoleamine 2,3-dioxygenase 1 inhibitors and photosensitizers, to the hydrophobic drug (especially tumor-associated fibroblast inhibitors) is 1:0.8-1.2:0.2-0.4. In one embodiment, the mass ratio of the two in situ self-assembling polypeptide bonded drugs, wherein the drug units are indoleamine 2,3-dioxygenase 1 inhibitors and photosensitizers, to the tumor-associated fibroblast inhibitor is 1.5:1.5:0.4.

[0053] In some embodiments, the organic solvent is selected from one of N,N-dimethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide.

[0054] In some embodiments, the volume ratio of the aqueous phase to the organic solvent is 1:1 to 1:30, preferably, 1:5 to 1:10.

[0055] In some embodiments, the ultrafiltration process can be carried out by ultrafiltration tube centrifugal ultrafiltration or ultrafiltration machine ultrafiltration. The ultrafiltration tube or ultrafiltration membrane package used in the process can have a molecular weight cutoff of 1500D, 3kD, 5kD, or 10KD, and one of them can be reasonably selected.

[0056] In some embodiments, the molecular weight cutoff of the dialysis bag during dialysis can be 800, 1000, 3500, or 14000, and one of them can be reasonably selected. The solvent used for dialysis can be deionized water.

[0057] On the other hand, the present invention also provides a method for causing the first stage deformation of the nanoregulator, the method comprising the step of allowing the nanoregulator to reach highly fibrotic malignant tumor tissue through systemic circulation, and be responded to by the FAP enzyme of fibroblasts in the tumor tissue, thereby producing the first stage deformation to cause the nanoregulator to deform into a nanofiber in the first stage, and the nanofiber has a β-folded structure formed by the FAP enzyme and the reactive oxygen responsive peptide segment.

[0058] In another aspect, the present invention provides nanofibers obtained by the above method of causing the first stage deformation of the nanoregulator.

[0059] Preferably, the hydrodynamic diameter of the nanofibers is 5-10 nm and the length is 500-2000 nm.

[0060] On the other hand, the present invention also provides a method for causing the second stage deformation of the nano-regulator, which method includes the step of using laser and ultrasound to treat the above-mentioned nanofibers to obtain secondary nanoparticles.

[0061] On the other hand, the present invention also provides secondary nanoparticles obtained by the above method of causing the second stage deformation of the nanoregulator, characterized in that the secondary nanoparticles are formed by in situ self-assembly of the above nanofibers under the action of active oxygen caused by external conditions.

[0062] Preferably, the hydrodynamic radius of the secondary nanoparticles is 80-120 nm.

[0063] In another aspect, the present invention also provides a use of the nanoregulator in the preparation of a drug for treating highly fibrotic malignant tumors, wherein the highly fibrotic malignant tumors are preferably selected from pancreatic cancer, breast cancer, and prostate cancer.

[0064] The technical solution of the present invention has at least the following technical effects:

[0065] The nano-regulator of the present invention is a nanoparticle formed by self-assembly of in situ self-assembling polypeptide-bonded drugs. It can undergo two deformations in response to the tumor microenvironment, thereby improving the tissue distribution and pharmacokinetic properties of the loaded drug in the organism; it can selectively target the tumor site to achieve efficient accumulation and retention in the tumor tissue; it can also target different cell types in the tumor, and release the corresponding loaded drugs to different cells in a spatiotemporally controllable manner, thereby overcoming the inherent immune tolerance of the tumor and the acquired immune tolerance caused by treatment, and improving the efficacy of immunotherapy in highly fibrotic malignant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 The H NMR spectrum data and mass spectrum data of MFP are shown, wherein the upper figure a is the H NMR spectrum and the lower figure b is the mass spectrum.

[0067] Figure 2 The H NMR spectrum data and mass spectrum data of PPa-NHS are shown, wherein the upper a figure is the H NMR spectrum, and the lower b figure is the mass spectrum.

[0068] Figure 3 The H NMR spectrum data and mass spectrum data of PMFP are shown, wherein the upper a figure is the H NMR spectrum, and the lower b figure is the mass spectrum.

[0069] Figure 4 The H NMR spectrum data and mass spectrum data of NLG919-SS-COOH are shown, wherein the upper a figure is the H NMR spectrum, and the lower b figure is the mass spectrum.

[0070] Figure 5 The H NMR spectrum data and mass spectrum data of NLG919-SS-NHS are shown, wherein the upper a figure is the H NMR spectrum, and the lower b figure is the mass spectrum.

[0071] Figure 6 The NMR 1H spectrum data and mass spectrum data of NSMFP are shown, wherein the upper a figure is the NMR 1H spectrum, and the lower b figure is the mass spectrum.

[0072] Figure 7 The hydrodynamic particle size and transmission electron microscope image of the nano-regulator prepared in Example 4 of the present invention are shown. The left image is a hydrodynamic image, and the upper right image is a transmission electron microscope image.

[0073] Figure 8 The H NMR spectrum data and mass spectrum data of PMGP are shown, wherein the upper a figure is the H NMR spectrum, and the lower b figure is the mass spectrum.

[0074] Fig. 9The H NMR spectrum data and mass spectrum data of NCMFP are shown, wherein the upper a figure is the H NMR spectrum, and the lower b figure is the mass spectrum.

[0075] Fig.10 The ROS detection diagram of the nanofiber in Example 5 of the present invention is shown. The left figure is a flow cytometry diagram, the upper side of the flow cytometry diagram is the nanofiber plus illumination group, and the lower side of the flow cytometry diagram is the nanofiber group; the right figure is a flow cytometry quantitative diagram.

[0076] Fig.11 The transmission electron microscope images and circular dichroism spectra of the nanoregulator, nanofiber and secondary nanoparticles in Example 6 of the present invention are shown. In Figure A, the left side is the nanoregulator group, the middle is the nanofiber group, and the right side is the secondary nanoparticle group; Figure B is the circular dichroism spectrum.

[0077] Fig.12 The quantitative release diagram of the tumor-associated fibroblast inhibitor in Example 7 of the present invention is shown. The circular coordinate points represent the nano-regulator group, the square shape represents the FAP enzyme plus the nano-regulator group, and the triangle shape represents the FAP enzyme plus the control nanoparticle group.

[0078] Fig.13 The quantitative release graph of the indoleamine 2,3-dioxygenase 1 inhibitor in Example 8 of the present invention is shown.

[0079] Fig.14 The Western blotting diagram of markers related to the activation function of tumor-associated fibroblasts in Example 9 of the present invention (A) and the change diagram of the ratio of kynurenine to tryptophan (B) are shown.

[0080] Fig.15 The figure shows the tumor inhibition of PBS (#1), free CAF inhibitor + free IDO1 inhibitor (#2), and nano-modulator + light (#3) in female C57 BALB / c mice in Example 10 of the present invention. Wherein, A is the tumor volume, and B is the ratio of cytotoxic T cells / regulatory T cells.

[0081] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work. DETAILED DESCRIPTION

[0082] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0083] In order to solve the dynamic immune tolerance mechanism of inherent immune tolerance and acquired immune tolerance in highly fibrotic tumors, and to overcome the off-target effects, toxic and side effects, and drug dynamics mismatches in the combined use of small molecule drugs. In the embodiment of the present invention, a nano regulator with multiple response functions prepared based on in situ self-assembly polypeptide bonded drugs and its application are proposed. The nano regulator has good biocompatibility, can improve the enrichment of loaded drugs in tumors, and increase the selectivity of drugs for tumors, reducing the occurrence of toxic and side effects. The nano regulator can also respond to the tumor microenvironment, and the two deformation processes of nano regulator → nanofiber → secondary nanoparticles occur in sequence, so that the loaded drugs are released on demand, and the precise delivery of drugs to different types of cells in the tumor microenvironment is achieved, thereby overcoming the dynamic immune tolerance in highly fibrotic tumors.

[0084] The structure of the in situ self-assembling polypeptide bonded drug with multiple response functions provided by the embodiment of the present invention is shown in Formula 1 below:

[0085]

[0086] The in situ self-assembling polypeptide bonded drug comprises polyethylene glycol, a linking group A, a linking group B and a drug unit;

[0087] Wherein, x is an integer of 10-145, preferably an integer of 25-65, and more preferably 45;

[0088] The linker group A is the FAP enzyme and the reactive oxygen species responsive peptide segment;

[0089] Linker B is absent or is a glutathione responsive fragment;

[0090] The drug unit is derived from a group consisting of an indoleamine 2,3-dioxygenase 1 inhibitor and a photosensitizer;

[0091] When the drug unit is derived from an indoleamine 2,3-dioxygenase 1 inhibitor, one end of the linker group A is covalently bonded to a polyethylene glycol having a succinimide group at one end, the other end of the linker group A is covalently bonded to one end of the linker group B, and the other end of the linker group B is covalently bonded to the indoleamine 2,3-dioxygenase 1 inhibitor;

[0092] When the drug unit is a photosensitizer, the linking group B does not exist, one end of the linking group A is covalently bonded to polyethylene glycol having a succinimide group at one end, and the other end of the linking group A is covalently bonded to the photosensitizer.

[0093] In certain embodiments, the FAP enzyme response peptide and reactive oxygen in the linker A may refer to a peptide comprising a proline and alanine linker fragment of the FAP enzyme response portion and a methionine peptide of the reactive oxygen response portion; wherein one end of the FAP enzyme response peptide is cysteine, which is bound to the succinimide on the polyethylene glycol (for example, by a click reaction); one end of the reactive oxygen response peptide is glutamic acid, which is covalently bonded to the linker B or the drug unit through an amino or carboxyl group. In some embodiments, the FAP enzyme peptide and the reactive oxygen response peptide are connected by an amide bond. In a further preferred embodiment, the linker A is CAPGEMIMIE (N-terminus to C-terminus).

[0094] In some embodiments, the glutathione corresponding fragment in the linker group B is selected from the group shown in the following formula:

[0095]

[0096]

[0097] Wherein, M1 and M2 are each independently selected from an alkylene group having 1 to 4 carbon atoms; R1 and R2 are each independently selected from a carbonyl group -C(=O)-, an imino group -NH-, or -O-;

[0098] Further preferably, M1 and M2 are the same;

[0099] More preferably, the linking group B is selected from the group shown in the following chemical formula:

[0100]

[0101] Further preferably, R1 and R2 in the linking group B are independently derived from -COOH, -NCO, -NH2 or -OH.

[0102] In some embodiments, the indoleamine 2,3-dioxygenase 1 inhibitor is selected from the following molecules:

[0103]

[0104] In some embodiments, the photosensitizer can generate reactive oxygen species, preferably selected from the following molecules:

[0105]

[0106]

[0107] In some embodiments, when the drug unit is derived from an indoleamine 2,3-dioxygenase 1 inhibitor, one end (N-terminus) of the linker A is covalently bonded to a polyethylene glycol having a succinimide group at one end, the other end (C-terminus) of the linker A is covalently bonded to one end of a linker B, and the other end of the linker B is covalently bonded to the indoleamine 2,3-dioxygenase 1 inhibitor; when the drug unit is derived from a photosensitizer, the linker B is absent, one end (N-terminus) of the linker A is covalently bonded to a polyethylene glycol having a succinimide group at one end, and the other end (C-terminus) of the linker A is covalently bonded to the photosensitizer.

[0108] In some embodiments, the in situ self-assembling polypeptide bonding drug with multiple response functions is selected from:

[0109]

[0110] The present invention also provides a method for preparing the above-mentioned in situ self-assembling polypeptide bonded drug, comprising the following steps:

[0111] One end of the linker group A is coupled to polyethylene glycol having a succinimide group at one end through a click reaction to obtain CH3O-PEG-linker group A;

[0112] When the drug unit is derived from an indoleamine 2,3-dioxygenase 1 inhibitor, the hydroxyl group of the indoleamine 2,3-dioxygenase 1 inhibitor is first reacted with the linker group B to obtain the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B, and then the reactive group of the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is reacted with the CH3O-PEG-linker group A to obtain the in situ self-assembling polypeptide bonded drug;

[0113] When the drug unit comes from a photosensitizer, the photosensitizer directly reacts with the CH3O-PEG-linking group A to obtain the in situ self-assembling polypeptide bonded drug.

[0114] In some embodiments, the preparation method specifically comprises the following steps:

[0115] A polyethylene glycol with a succinimide group at one end and a linker group A are dissolved in DMF, an organic base is added dropwise, and the mixture is stirred at room temperature for 12-24 hours. The product is dialyzed three times with an organic solvent and water in a dialysis bag, and lyophilized to obtain CH3O-PEG-linker group A;

[0116] When the drug unit is an indoleamine 2,3-dioxygenase 1 inhibitor, the indoleamine 2,3-dioxygenase 1 inhibitor reacts with the linker B;

[0117] Wherein, when the reactive group of the linker group B is a carboxyl group, DMAP, DIEA and EDCI are added to activate the carboxyl group in anhydrous DCM and an ice bath for 1-2 hours, and then the indoleamine 2,3-dioxygenase 1 inhibitor is added and stirred at room temperature for 12-24 hours, and the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is purified by column chromatography; subsequently, the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is dissolved in a solvent (such as DMF or DCM), NHS and EDCI are added for activation for 1-6 hours, and then CH3O-PEG-linker group A is added to react at room temperature for 12-24 hours, and the product is dialyzed three times with an organic solvent and water in a dialysis bag, respectively, and lyophilized to obtain an in situ self-assembled polypeptide bonded drug;

[0118] When the reactive group of the linker group B is a hydroxyl group or an amino group, one end of the linker group B is reacted with TBSCl to protect the hydroxyl group or Fmoc to protect the amino group, then the indoleamine 2,3-dioxygenase 1 inhibitor is dissolved in DCM, activated with NPC and DMAP for 1-2 hours, and then the linker group B with one end protected is added to react to obtain the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B-protecting group, and the protecting group is removed to obtain the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B; then CH3O-PEG-linker group A is dissolved in DMF, NHS is added for activation for 1-6 hours, and then the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is added, and the product is dialyzed three times with an organic solvent and water in a dialysis bag, respectively, and lyophilized to obtain an in situ self-assembling polypeptide bonded drug;

[0119] When the reactive group of the linker group B is an isocyanate group, the linker group B and the indoleamine 2,3-dioxygenase 1 inhibitor are dissolved in N,N-dimethylformamide (DMF), reacted for 12-24 hours in the presence of an organic base, and purified by column chromatography to obtain the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B, and then the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is reacted with CH3O-PEG-linker group A, and the product is dialyzed three times with an organic solvent and water in a dialysis bag, respectively, and lyophilized to obtain an in situ self-assembled polypeptide bonded drug;

[0120] When the drug unit comes from a photosensitizer, the photosensitizer is dissolved in DMF, NHS is added for activation for 1-6 hours, and then CH3O-PEG-linker A is added to react at room temperature for 12-24 hours. The product is dialyzed three times with an organic solvent and water in a dialysis bag, and lyophilized to obtain an in situ self-assembled polypeptide bonded drug.

[0121] In some embodiments, the organic base used in the preparation method may be DIEA and TEA, and the organic solvent used in dialysis may be N,N-dimethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide.

[0122] An embodiment of the present invention also provides a nano-regulator, which comprises two of the above-mentioned in situ self-assembling polypeptide bonded drugs, wherein the drug unit in one in situ self-assembling polypeptide bonded drug is derived from an indoleamine 2,3-dioxygenase 1 inhibitor, and the drug unit in the other in situ self-assembling polypeptide bonded drug is derived from a photosensitizer.

[0123] Preferably, the hydrodynamic radius of the nano-regulator is 30-80 nm, more preferably 50-60 nm.

[0124] The nano-regulator has a hydrophobic core, and the hydrophobic core can further encapsulate a hydrophobic drug through hydrophobic interaction; the hydrophobic drug can be one or two or more selected from tumor-associated fibroblast inhibitors, selected from the following molecules:

[0125]

[0126] Preferably, the tumor-associated fibroblast inhibitor encapsulated in the hydrophobic core of the nanoregulator can be one or two or more of the above-mentioned molecules.

[0127] The present invention also provides a method for preparing the nano-regulator, comprising the following steps:

[0128] The drug units are respectively an indoleamine 2,3-dioxygenase 1 inhibitor and an in situ self-assembled polypeptide bonded drug of a photosensitizer, and an optional hydrophobic drug (especially a tumor-associated fibroblast inhibitor) are dissolved in an organic solvent, mixed evenly, and then ultrasonically dispersed in water. The resulting solution is subjected to ultrafiltration or dialysis to obtain a nano-regulator.

[0129] In some embodiments, the mass ratio of the two in situ self-assembling polypeptide bonded drugs, wherein the drug units are indoleamine 2,3-dioxygenase 1 inhibitor and photosensitizer, is 1:0.8-1.2. In one embodiment, the mass ratio of the above in situ self-assembling polypeptide bonded drugs, wherein the drug units are indoleamine 2,3-dioxygenase 1 inhibitor and photosensitizer, is 1:1.

[0130] In a further embodiment, the mass ratio of the two in situ self-assembling polypeptide bonded drugs, whose drug units are indoleamine 2,3-dioxygenase 1 inhibitor and photosensitizer, to the hydrophobic drug (such as tumor-associated fibroblast inhibitor) is 1:0.8-1.2:0.2-0.4. In one embodiment, the mass ratio of the two in situ self-assembling polypeptide bonded drugs, whose drug units are indoleamine 2,3-dioxygenase 1 inhibitor and photosensitizer, to the tumor-associated fibroblast inhibitor is 1.5:1.5:0.4.

[0131] In some embodiments, the organic solvent is selected from one of N,N-dimethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide.

[0132] In some embodiments, the volume ratio of the aqueous phase to the organic solvent is 1:1 to 1:30, preferably, 1:5 to 1:10.

[0133] In some embodiments, the ultrafiltration process can be carried out by ultrafiltration tube centrifugal ultrafiltration or ultrafiltration machine ultrafiltration. The ultrafiltration tube or ultrafiltration membrane package used in the process can have a molecular weight cutoff of 1500D, 3kD, 5kD, or 10KD, and one of them can be reasonably selected.

[0134] In some embodiments, the molecular weight cutoff of the dialysis bag during dialysis can be 800, 1000, 3500, or 14000, and one of them can be reasonably selected. The solvent used for dialysis can be deionized water.

[0135] An embodiment of the present invention also provides a method for causing the first stage deformation of the nanoregulator, the method comprising the steps of allowing the nanoregulator to reach highly fibrotic malignant tumor tissue through systemic circulation, and be responded to by the FAP enzyme of fibroblasts in the tumor tissue, thereby producing the first stage deformation to cause the nanoregulator to deform into a nanofiber in the first stage, wherein the nanofiber has a β-folded structure formed by the FAP enzyme and the reactive oxygen responsive peptide segment.

[0136] In another aspect, the present invention provides nanofibers obtained by the above method of causing the first stage deformation of the nanoregulator.

[0137] Preferably, the nanofiber has a hydrodynamic diameter of 5-10 nm and a length of 500-2000 nm.

[0138] An embodiment of the present invention also provides a method for causing the second stage deformation of the nano-regulator, which includes the step of using laser and ultrasound to process the above-mentioned nanofibers to obtain secondary nanoparticles.

[0139] An embodiment of the present invention also provides nanoparticles obtained by the above method of causing the second stage deformation of the nanoregulator, characterized in that the secondary nanoparticles are formed by in-situ self-assembly of the above nanofibers under the action of active oxygen caused by external conditions.

[0140] Preferably, the hydrodynamic radius of the secondary nanoparticles is 80-120 nm.

[0141] An embodiment of the present invention also provides a use of the nano regulator in the preparation of a drug for treating highly fibrotic malignant tumors, wherein the highly fibrotic malignant tumors are preferably selected from pancreatic cancer, breast cancer, and prostate cancer.

[0142] The nano-regulator of the present invention can simultaneously load intervention drugs for different types of cells, and has good tumor tissue accumulation and penetration capabilities. After reaching the tumor microenvironment, the tumor-associated fibroblasts in the tumor can induce the nano-regulator to undergo the first stage of deformation and produce nanofibers. The nanofibers target tumor-associated fibroblasts to release tumor-associated fibroblast inhibitors. Then, under the action of externally induced reactive oxygen species, the nanofibers can undergo the second stage of deformation and produce secondary nanoparticles. After the secondary nanoparticles are taken up by tumor cells, they can specifically release indoleamine 2,3-dioxygenase 1 inhibitors under the action of elevated glutathione in tumor cells, thereby achieving precise targeting of the drugs loaded in the nano-regulator to different cells, while overcoming the inherent immune tolerance mediated by tumor-associated fibroblasts and the acquired immune tolerance driven by treatment and caused by tumor cells, significantly improving the efficacy of immunotherapy in highly fibrotic tumors.

[0143] The present invention is further described below in conjunction with examples. It should be noted that the following examples are provided for illustrative purposes only and do not constitute a limitation on the scope of protection claimed for the present invention.

[0144] The reagents and instruments used in the following examples are as follows:

[0145] The pyropheophorbide a used in the examples was purchased from Dibai Chemical Technology Co., Ltd. Tumor-associated fibroblast (CAF) inhibitor (LY2157299) and indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor (NLG919) were purchased from Selleck. Proline endopeptidase FAP-α was purchased from Novoprotein. Glu-Ile-Met-Ile-Met-Glu-Gly-Pro-Ala-Cys (EC-10-SH, EIMIMEGPAC (C-terminus to N-terminus)) and Glu-Ile-Met-Ile-Met-Glu-Gly-Gly-Gly-Cys (EIMIMEGGGC (C-terminus to N-terminus)) were synthesized by GL Biochem. Methoxypolyethylene glycolamine (mPEG 2K-NH2) was purchased from Seebio Biotec Co., Ltd. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), 4-dimethylaminopyridine (DMAP), triethylamine (TEA), trifluoroacetic acid (TFA), N,N-diisopropylethylamine (DIEA), anhydrous dichloromethane (DCM), anhydrous ammonium (III) chloride hexahydrate, N,N-dimethylformamide (DMF) and anhydrous dimethyl sulfoxide (DMSO) were purchased from J&K Company, China. 2',7'-Dichlorofluorescein diacetate (DCFH-DA) was purchased from Sigma-Aldrich. RPMI 1640 cell culture medium and 6-diamino-2-phenylindole dihydrochloride (DAPI) were purchased from Dalian Meilun Biotechnology Co., Ltd., China. Lysotracker green was purchased from Life Technologies. Fetal bovine serum (FBS) was purchased from AusGeneX, Australia. Unless otherwise specified, all other reagents and solvents used were purchased from Sinopharm Group (Shanghai) Chemical Reagent Co., Ltd.

[0146] Panc02 cells and NIH / 3T3 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Female C57 (6-8 weeks, 18-20 g) were purchased from the Shanghai Experimental Animal Center.

[0147] The hydrodynamic particle size of the nano-regulator was measured by a Malvern laser particle size analyzer (ZEN3690, Malven, USA); the morphology of the nanoparticles was investigated by a 120 kV transmission electron microscope (Talos L120C, FEI, USA). Electronic balance (Quintix 224-1, Sartorius Germany); rotary evaporator (B-40, Buchi, Switzerland); constant temperature magnetic stirrer (DF-101S, Shanghai Yuhua Instrument Co., Ltd.); nuclear magnetic resonance spectrometer (Mercury Plus 400, Varian, USA); Waters 2695 high performance liquid chromatograph (waters e2695 chromatographic pump, Xbridge C18 5μm19*250mm chromatographic column, Waters 2998 UV detector). Flow cytometry data were measured by a BD FACS Fortessa flow cytometer. Unless otherwise specified, the equipment and test methods used are conventional equipment and methods in the field.

[0148] Example 1: Synthesis of MFP

[0149]

[0150] EC-10-SH (327 mg, 0.30 mmol) and Mal-PEG2K (500 mg, 0.25 mmol) was dissolved in anhydrous DMF (20 mL), and DIEA (131 μL, 0.75 mmol) was slowly added. After 24 h of reaction, the reaction solution was transferred to a 2000 Da dialysis bag, dialyzed twice with DMF and three times with deionized water, and finally freeze-dried to obtain a white powder (700 mg, yield 90.5%), which was MFP.

[0151] The MFP was characterized and the obtained H NMR spectrum and mass spectrum data were as follows: Figure 1 As shown, it indicates that MFP was successfully synthesized.

[0152] Example 2: Synthesis of PMFP

[0153]

[0154] Accurately weigh pyropheophorbide-a (PPa) (100 mg, 0.19 mmol) in a 25 mL flask, add 2 mL of anhydrous DCM to dissolve. Under argon protection, add N-hydroxysuccinimide (NHS) (34.4 mg, 0.30 mmol) and EDCI (57.4 mg, 0.30 mmol) to the reaction solution and react at room temperature for 5 h. After the reaction, the reaction solution was washed three times with saturated sodium chloride solution and sodium bicarbonate solution, and dried over anhydrous sodium sulfate to obtain a black powder (110 mg, yield 93.2%), which is activated PPa (PPa-NHS).

[0155] Accurately weigh MFP (70 mg) and activated PPa (18 mg) in a 25 mL round-bottom flask, dissolve in 4 mL anhydrous DMF, and slowly drop TEA (9 μL). After 24 h of reaction under argon protection, the reaction solution was transferred to a 2000 Da dialysis bag, dialyzed twice with DMF and three times with deionized water. Freeze-dried to obtain a dark green powder (69 mg, yield 83.2%), which is PMFP.

[0156] PPa-NHS was characterized, and the obtained H NMR spectrum and mass spectrum data were as follows: Figure 2 As shown, it shows that PPa-NHS was successfully synthesized.

[0157] PMFP was characterized, and the obtained H NMR spectrum and mass spectrum data were as follows: Figure 3 As shown, it shows that PMFP was successfully synthesized.

[0158] Example 3: NSMFP (NLG919-SS-EIMIMEGPAC-PEG 2K )

[0159]

[0160] Accurately weigh 4,4-dithiodibutyric acid (HOOC-SS-COOH, 508 mg, 2.13 mmol), DMAP (260 mg, 2.13 mmol), EDCI (408 mg, 2.13 mmol) and DIEA (275 μL, 2.13 mmol) and dissolve in 2 mL of anhydrous DCM. Stir for 2 h in an ice bath at 0 ° C under argon protection to activate the carboxyl group. Subsequently, NLG919 (200 mg, 0.71 mmol) was added and the reaction continued at room temperature for 24 h. The reaction solution was washed three times with ammonium chloride and dried over anhydrous magnesium sulfate. Column chromatography purification (DCM: MeOH = 30: 1) gave a light yellow powder (310 mg, yield 87.3%), which was NLG919-SS-COOH.

[0161] Accurately weigh NLG919-SS-COOH (50 mg, 0.10 mmol) and dissolve it in anhydrous 1 mL DCM. Then add NHS (18 mg, 0.16 mmol) and EDCI (30 mg, 0.16 mmol) and react at room temperature for 5 h under argon protection. After the reaction is completed, the reaction solution is washed three times with saturated sodium chloride solution and sodium bicarbonate solution, dried over anhydrous sodium sulfate, and spin-dried to obtain a light yellow powder (55 mg, yield 94.8%). The obtained yellow powder is NLG919-SS-NHS.

[0162] Accurately weigh MFP (70 mg) and NLG919-SS-NHS (17 mg), place in a 25 mL round-bottom flask, dissolve in 3 mL anhydrous DMF, and slowly drop TEA (6 μL). After 24 h of reaction under argon protection, the reaction solution was transferred to a 2000 Da dialysis bag, dialyzed twice with DMF and three times with deionized water. Freeze-dried to obtain a light yellow powder (71 mg, yield 84.5%), which is NSMFP.

[0163] NLG919-SS-COOH was characterized, and the obtained H NMR spectrum and mass spectrum data were as follows: Figure 4 As shown, it shows that NLG919-SS-COOH was successfully synthesized.

[0164] NLG919-SS-NHS was characterized, and the obtained H NMR spectrum and mass spectrum data are as follows Figure 5 As shown, it was shown that NLG919-SS-NHS was successfully synthesized.

[0165] The NSMFP was characterized and the obtained H NMR spectrum and mass spectrum data were as follows: Figure 6 As shown, it indicates that NSMFP was successfully synthesized.

[0166] Example 4: Preparation of Nano-regulator

[0167] Under the condition that the feeding mass ratio of PMFP, NSMFP and tumor-associated fibroblast inhibitor (LY2157299) was 1.5:1.5:0.4, 100 μL of organic phase was obtained, slowly dripped into 900 μL of aqueous phase, and then the nano-regulator was obtained under ultrasound.

[0168] The results are as follows Figure 7 As shown, the nano-regulator appears as an ellipse in a transmission electron microscope, and its hydrodynamic radius is 52.9±1.1 nm and its PDI is 0.19±0.02 as measured by a Malvern particle size analyzer.

[0169] PMGP that is not responsive to MGP and FAP enzymes was prepared by the same synthesis method as in Example 1-2 except that EC-10-SH was replaced by a small peptide having an amino acid sequence of EIMIMEGGGC, and characterized (the results are shown in Figure 8 As shown) and the tumor-associated fibroblast inhibitor (LY2157299) were added at a feed mass ratio of 1.5:0.4 to obtain 100 μl of organic phase, which was slowly dripped into 900 μl of aqueous phase, and then the FAP enzyme non-responsive control nanoregulator in Example 7 was obtained under ultrasound.

[0170] The glutathione-insensitive NCMFP was prepared by the same synthesis method as in Example 3 except that 4,4-dithiodibutyric acid was replaced by sebacic acid and characterized (the results are shown in Fig. 9 As shown) and dissolved in 100 μl of organic phase, slowly dripped into 900 μl of aqueous phase, and then under ultrasound, the glutathione non-responsive control secondary nanoparticles in Example 8 were obtained.

[0171] Example 5: Testing of ROS generation capability of nanoregulators

[0172] The nanoregulator in Example 4 was mixed with a FAP enzyme (50 μg mL -1 ) and pH 7.4 PBS buffer, and the nanofibers produced by the first stage of deformation were obtained after slow shaking at 37°C for 12 hours. To monitor the generation of ROS, Panc02 cells were incubated with nanofibers for 6 hours. After the cells were washed with PBS solution, 10 μM DCFH-Da was added and incubated for 20 minutes in the dark, and then the light was irradiated at a wavelength of 671 nm and an intensity of 100 mW / cm 2 The cells were irradiated with laser for 5 minutes (nanofiber + light irradiation group), while the nanofiber group was not irradiated. The cells were digested with trypsin, washed three times with PBS, and analyzed by flow cytometry (Ex / Em=480 / 550nm).

[0173] Test results such as Fig.10As shown, an increase in reactive oxygen species was significantly observed in the nanofiber + light group compared with the nanofiber group, which indicates that nanofibers can produce reactive oxygen species under light conditions.

[0174] Example 6: Nano-regulator deformation test

[0175] In order to study the deformation ability of the nanoregulator, the nanoregulator in Example 4 was mixed with a FAP enzyme (50 μg mL -1 ) and pH 7.4 PBS buffer, and then slowly shaken at 37°C for 12 hours to obtain the nanofibers produced by the first stage of deformation. 2 After the nanofibers were irradiated with laser for 5 minutes, the secondary nanoparticles were obtained by the second stage deformation. The nanoregulator, nanofibers and secondary nanoparticles were characterized by transmission electron microscopy and circular dichroism.

[0176] Test results such as Fig.11 As shown, under the transmission electron microscope, it can be observed that the nano-regulator produces nanofibers and secondary nanoparticles under specific conditions, which indicates that the nano-regulator can undergo two stages of deformation in sequence under specific conditions. The circular dichroism experiment shows that after the nano-regulator is treated with FAP enzyme, a positive peak appears at a wavelength of 206nm and a negative peak appears at a wavelength of 222nm, indicating the formation of nanofibers, verifying that the nano-regulator can undergo a deformation process from nanoparticles to nanofibers under the FAP enzyme; and under the action of ROS generated after laser irradiation, the aforementioned characteristic peak disappears, indicating a deformation process from nanofibers to nanoparticles. This shows that the nano-regulator can undergo two stages of deformation in sequence under specific conditions.

[0177] Example 7: Release test of tumor-associated fibroblast inhibitor

[0178] The release amount of tumor-associated fibroblast inhibitor in the nano regulator and FAP enzyme unresponsive control nano regulator in Example 4 was determined by in vitro dialysis. 100 μL of nano regulator and FAP enzyme unresponsive control nano regulator solution was transferred to a dialysis bag with a molecular cutoff of 3500Da, and then dialyzed in PBS containing 0.5% Tween 80 and 50 μg / mL FAP-α. Among them, another group of nano regulator solutions was dialyzed in PBS containing 0.5% Tween 80 and PBS, which was a nano regulator group. Protect from light and shake continuously at 37 ° C. At predetermined time intervals (0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h), 1mL of solution was taken out. The release of tumor-associated fibroblast inhibitor was analyzed by high performance liquid chromatography with 0.1% TFA (v / v=95% / 5%) in methanol / water as the mobile phase, a flow rate of 0.5 mL / min, a column temperature of 25°C, and a wavelength of 254 nm.

[0179] Test results such as Fig.12 As shown, no obvious release of tumor-associated fibroblast inhibitors from the nanoparticles was observed in the nanoregulator group and the FAP enzyme + FAP enzyme non-responsive control nanoregulator group under HPLC detection, while the release of tumor-associated fibroblast inhibitors in the FAP enzyme + nanoregulator group increased significantly with the extension of incubation time, which indicates that the nanoregulator can release tumor-associated fibroblast inhibitors under the action of FAP enzyme, and the release amount increases with time.

[0180] Example 8: Release test of indoleamine 2,3-dioxygenase 1 inhibitor

[0181] In order to investigate the release of indoleamine 2,3-dioxygenase 1 inhibitor under reducing conditions, the secondary nanoparticles in Example 6 and the glutathione non-responsive control secondary nanoparticles prepared in Example 4 were incubated with a mixed solution of PBS and MeOH with a volume ratio of 1:1 containing / not containing 10mM glutathione at 37°C. The release of indoleamine 2,3-dioxygenase 1 inhibitor was monitored by high performance liquid chromatography, and 50μL of the solution was taken for analysis at predetermined time points (0h, 0.5h, 1h, 2h, 4h,, 6h, 12h, 18h, 24h). The release of indoleamine 2,3-dioxygenase 1 inhibitor was analyzed by high performance liquid chromatography, with 0.1% TFA (v / v=70% / 30%) methanol / water as the mobile phase, a flow rate of 0.5mL / min, a column temperature of 25°C, and a wavelength of 254nm.

[0182] Test results such as Fig.13As shown, no significant increase in the release of indoleamine 2,3-dioxygenase 1 inhibitor was observed in the glutathione non-responsive control secondary nanoparticle group, the glutathione non-responsive control secondary nanoparticle group + glutathione, and the secondary nanoparticle group at different time points, but the release of indoleamine 2,3-dioxygenase 1 inhibitor in the secondary nanoparticle + glutathione group increased significantly with the extension of incubation time, which indicates that the secondary nanoparticles release indoleamine 2,3-dioxygenase 1 inhibitor under the action of glutathione, and the release amount increases with time.

[0183] Example 9: Evaluation of the inhibitory effect of nano-regulators on tumor-associated fibroblasts and tumor cell tryptophan metabolism

[0184] In order to investigate the functional effects of nanoregulators on tumor-associated fibroblasts, the nanoregulators in Example 4, the FAP enzyme non-responsive control nanoregulator group, and the free CAF inhibitor were co-incubated with tumor-associated fibroblasts pretreated with transforming growth factor (10 ng / ml) (Selleck Chem Co., Ltd (Shanghai, China)) for 24 hours at an equal dose of 5 μM. After 24 hours, protein immunoblotting was used to detect markers related to the activation function of tumor-associated fibroblasts (including α-actin, FAP enzyme and type III collagen).

[0185] In order to investigate the effect of the nano-regulator on tryptophan metabolism in tumor cells, the secondary nanoparticle regulator and the glutathione non-responsive control secondary nanoparticles in Example 6 were co-incubated with tumor cells pretreated with interferon γ (10 ng / ml) for 24 hours, and tumor cells pretreated with interferon γ (10 ng / ml) were set as the control group. The culture supernatant was collected and the kynurenine and tryptophan contents were detected by high performance liquid chromatography using 8% sodium acetate / acetonitrile in a volume ratio of 95% / 5% as the mobile phase, a flow rate of 0.5 mL / min, a column temperature of 25°C, and a wavelength of 254 nm, and the kynurenine / tryptophan ratio was calculated.

[0186] Test results such as Fig.14 As shown in A and B, protein immunoblotting technology detection suggests that the nano-regulator can significantly inhibit the expression of α-actin, FAP enzyme and type III collagen, indicating that the nano-regulator can significantly inhibit the function of tumor-associated fibroblasts. Compared with the control group and the glutathione non-responsive control secondary nanoparticle group, the kynurenine / tryptophan ratio of the indoleamine 2,3-dioxygenase 1 inhibitor (positive control group) and the secondary nanoparticle group decreased, indicating that the secondary nanoparticles can inhibit the tryptophan metabolism of tumor cells.

[0187] Example 10: Evaluation of the effect of nano-regulators on tumor growth inhibition and anti-tumor immune activation

[0188] 2.5 × 10 6 Panc02 cells and 2.5×10 6 Primary tumor-associated fibroblasts (NIH / 3T3) were cultured to establish a subcutaneous tumor model. When the tumor volume reached 150 mm 3 At about 1:10 pm, mice were randomly divided into PBS (#1), free CAF inhibitor + free IDO1 inhibitor (#2), and nano-regulator in Example 4 + light (#3). The mice were given an equal dose of 10 mg / kg CAF inhibitor via the tail vein. 8 hours after the administration, the #3 group was given a light with an intensity of 100 mW / cm 2 , irradiation with a laser of 671nm wavelength for 5 minutes. The above prescription was performed once every other day, for a total of three times. During the process, the tumor volume and weight of the mice were measured every other day. The calculation method of the mouse tumor volume is as follows (L is the longest diameter, W is the shortest diameter, in millimeters):

[0189] V=L×W×W / 2

[0190] 14 days after the last treatment, mouse tumor tissues were collected, and after removing surface blood stains, skin, hair and other tissues, they were cut into pieces and added into tumor tissue digestion solution (containing 0.2 mg / ml IV collagenase, 0.2 mg / ml IV hyaluronidase and 0.1 mg / ml DNA nuclease). After grinding with a tissue grinder, the tissues were incubated on a shaker at 37 degrees for 2 hours, and then the digestion was terminated. After washing and lysing red blood cells, the tumor tissue cell suspension was obtained. Flow cytometry was used to detect the proportion of cytotoxic T cells and regulatory T cells in the tumor tissue, and the ratio was statistically calculated to determine the activation of anti-tumor immunity.

[0191] Test results such as Fig.15 As shown, the nano-regulator + light group (#3) showed the best tumor inhibition effect compared with the PBS group (#1) and the free CAF inhibitor + free IDO1 inhibitor group (#2), which shows that the nano-regulator can effectively inhibit tumor growth. Cytotoxic T cells and regulatory T cells in tumor tissues were monitored by flow cytometry. The ratio of cytotoxic T cells / regulatory T cells in the nano-regulator + light group (#3) was significantly increased compared with the PBS group (#1) and the free CAF inhibitor + free IDO1 inhibitor group (#2), which shows that the nano-regulator can effectively activate the anti-tumor immune effect of mice.

[0192] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An in situ self-assembling polypeptide bonded drug with multiple response functions, which has a structure as shown in the following formula 1: In Formula 1, x is an integer of 10-145, preferably an integer of 25-65, and more preferably 45; The linker group A is a peptide segment responsive to reactive oxygen species and fibroblast activation protein (FAP enzyme); Linker B is absent or is a glutathione responsive fragment; The drug unit is derived from an indoleamine 2,3-dioxygenase 1 inhibitor and a photosensitizer; When the drug unit is derived from an indoleamine 2,3-dioxygenase 1 inhibitor, one end of the linker group A is covalently bonded to a polyethylene glycol having a succinimide group at one end, the other end of the linker group A is covalently bonded to one end of the linker group B, and the other end of the linker group B is covalently bonded to the indoleamine 2,3-dioxygenase 1 inhibitor; When the drug unit is derived from a photosensitizer, the linker B does not exist, one end of the linker A is covalently bonded to polyethylene glycol having a succinimide group at one end, and the other end of the linker A is covalently bonded to the photosensitizer.

2. The in situ self-assembling polypeptide bonded drug with multiple response functions according to claim 1, in, The linking group A is CAPGEMIMIE; and / or Wherein, the linking group B is selected from the group shown in the following formula: Wherein, M1 and M2 are each independently selected from an alkylene group having 1 to 4 carbon atoms; R1 and R2 are each independently selected from -C(=O)-, -NH-, -O-; further preferably, M1 and M2 are the same; more preferably, the linking group B is selected from the following groups:

3. The in situ self-assembling polypeptide bonded drug with multiple response functions according to claim 1 or 2, wherein: The indoleamine 2,3-dioxygenase 1 inhibitor is selected from the following compounds: and / or Wherein, the photosensitizer is selected from the following compounds:

4. The in situ self-assembling polypeptide bonded drug with multiple response functions according to claim 1, wherein: The in situ self-assembling polypeptide bonding drug with multiple response functions is selected from:

5. A method for preparing the in situ self-assembling polypeptide bonded drug according to claim 1, comprising the following steps: One end of the linker group A is coupled to polyethylene glycol having a succinimide group at one end through a click reaction to obtain CH3O-PEG-linker group A; When the drug unit is derived from an indoleamine 2,3-dioxygenase 1 inhibitor, the hydroxyl group of the indoleamine 2,3-dioxygenase 1 inhibitor is first reacted with the linker group B to obtain the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B, and then the reactive group of the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is reacted with the CH3O-PEG-linker group A to obtain the in situ self-assembling polypeptide bonded drug; When the drug unit comes from a photosensitizer, the photosensitizer directly reacts with the CH3O-PEG-linking group A to obtain the in situ self-assembling polypeptide bonded drug. In particular, the following steps are included: A polyethylene glycol having a succinimide group at one end and a linker group A are dissolved in N,N-dimethylformamide (DMF), an organic base is added dropwise, and the mixture is stirred at room temperature for 12-24 hours. The product is dialyzed three times with an organic solvent and water in a dialysis bag, and lyophilized to obtain CH3O-PEG-linker group A; When the drug unit is derived from an indoleamine 2,3-dioxygenase 1 inhibitor, the indoleamine 2,3-dioxygenase 1 inhibitor reacts with the linker B; Wherein, when the reactive group of the linker group B is a carboxyl group, 4-dimethylaminopyridine (DMAP), N,N-diisopropylethylamine (DIEA) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) are added to activate the carboxyl group in anhydrous dichloromethane (DCM) and an ice bath for 1-2 hours, and then the indoleamine 2,3-dioxygenase 1 inhibitor is added and stirred at room temperature for 12-24 hours, and the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is purified by column chromatography; subsequently, the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is dissolved in a solvent (such as DMF or DCM), NHS and EDCI are added for activation for 1-6 hours, and then CH3O-PEG-linker group A is added to react at room temperature for 12-24 hours, and the product is dialyzed three times with an organic solvent and water in a dialysis bag, respectively, and lyophilized to obtain an in situ self-assembled polypeptide bonded drug; When the reactive group of the linker group B is a hydroxyl group or an amino group, one end of the linker group B is reacted with tert-butyldimethylsilyl chloride (TBSCl) to protect the hydroxyl group or Fmoc to protect the amino group, then the indoleamine 2,3-dioxygenase 1 inhibitor is dissolved in DCM, activated with di(p-nitrobenzene) carbonate (NPC) and DMAP for 1-2 hours, and then the linker group B protected at one end is added to react to obtain the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B-protecting group, and the protecting group is removed to obtain the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B; then CH3O-PEG-linker group A is dissolved in DMF, NHS is added for activation for 1-6 hours, and then the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is added, and the product is dialyzed three times with an organic solvent and water in a dialysis bag, respectively, and lyophilized to obtain an in situ self-assembling polypeptide bonded drug; When the reactive group of the linker group B is an isocyanate group, the linker group B and the indoleamine 2,3-dioxygenase 1 inhibitor are dissolved in N,N-dimethylformamide (DMF), reacted for 12-24 hours in the presence of an organic base, and purified by column chromatography to obtain the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B, and then the indoleamine 2,3-dioxygenase 1 inhibitor-linker group B is reacted with CH3O-PEG-linker group A, and the product is dialyzed three times with an organic solvent and water in a dialysis bag, respectively, and lyophilized to obtain an in situ self-assembled polypeptide bonded drug; When the drug unit comes from a photosensitizer, the photosensitizer is dissolved in DMF, NHS is added for activation for 1-6 hours, and then CH3O-PEG-linker A is added to react at room temperature for 12-24 hours. The product is dialyzed three times with an organic solvent and water in a dialysis bag, and lyophilized to obtain an in situ self-assembled polypeptide bonded drug; The organic base used in the preparation method may be DIEA and triethylamine (TEA), and the organic solvent used in dialysis may be N,N-dimethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide.

6. A nano-regulator comprising two in situ self-assembling polypeptide bonded drugs according to any one of claims 1 to 4, wherein: The drug unit in one in situ self-assembling peptide bonded drug is derived from an indoleamine 2,3-dioxygenase 1 inhibitor, and the drug unit in another in situ self-assembling peptide bonded drug is derived from a photosensitizer; In particular, the nano-regulator has a hydrophobic core, and the hydrophobic core can further encapsulate a hydrophobic drug through hydrophobic interaction; preferably, the hydrophobic drug is a tumor-associated fibroblast inhibitor, more preferably, one or more selected from the following molecules:

7. A nanofiber or secondary nanoparticle obtained from the nanoregulator according to claim 6, wherein: The hydrodynamic diameter of the nanofiber is 5-10 nm, and the length is 500-2000 nm; the hydrodynamic radius of the secondary nanoparticle is 80-120 nm.

8. A method for preparing the nano-regulator as claimed in claim 6, comprising the following steps: The in situ self-assembling polypeptide bonded drug, whose drug units are indoleamine 2,3-dioxygenase 1 inhibitor and photosensitizer, and an optional hydrophobic drug (especially a tumor-associated fibroblast inhibitor) are dissolved in an organic solvent, mixed evenly, and then ultrasonically dispersed in water. The resulting solution is subjected to ultrafiltration or dialysis to obtain a nano-regulator.

9. The method according to claim 8, in, The organic solvent is selected from N,N-dimethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide; and / or Wherein, the volume ratio of the aqueous phase to the organic solvent is 1:1 to 1:30, preferably 1:5 to 1:10; and / or The ultrafiltration process is carried out by ultrafiltration tube centrifugal ultrafiltration or ultrafiltration machine ultrafiltration, and the ultrafiltration tube or ultrafiltration membrane package used in the process has a molecular weight cutoff of 1500D, 3kD, 5kD or 10KD; and / or The molecular weight cutoff of the dialysis bag during the dialysis process is 800, 1000, 3500 or 14000.

10. Use of the nanoregulator as claimed in claim 6 in the preparation of a drug for treating highly fibrotic malignant tumors, preferably, the highly fibrotic malignant tumors are preferably selected from pancreatic cancer, breast cancer, and prostate cancer.

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