Tumor-targeting photosensitizer chimeric peptide and applications thereof

Abstract

The application provides a tumor-targeting photosensitizer chimeric peptide and application thereof. 30 The tumor-targeting photosensitizer chimeric peptide has the structure shown in the following formula (I), wherein R1 is selected from C6-C12 alkyl; R2 is a residue of a photosensitizer after removing one hydroxyl group; R3 is a polypeptide capable of targeting endoplasmic reticulum in tumors; and n is an integer selected from 4-12. The tumor-targeting photosensitizer chimeric peptide can specifically target endoplasmic reticulum in tumors, realize accumulation of the photosensitizer at the tumor site, has a better in-vivo tumor inhibition effect compared with the photosensitizer alone, and can be used as a drug carrier to wrap an antitumor drug, so that a synergistic antitumor effect is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a tumor-targeting photosensitizer chimeric peptide and application thereof. BACKGROUND

[0002] Cancer is the second leading cause of death worldwide, seriously threatening human health. The clinical tumor chemotherapy drugs, immunotherapy drugs, targeted therapy drugs, etc. are delivered into the body, due to the clearance rate of the organism itself, the drugs are metabolized by the liver or kidney, and the drugs reaching the tumor site are significantly reduced, which makes the bioavailability of antitumor drugs poor. And the antitumor drugs have relatively large toxic and side effects on the organism, such as gastrointestinal reactions, bone marrow suppression, etc. Therefore, targeted therapy for cancer has become a research hotspot. At present, nano-targeted drugs are mainly passively targeted to tumor sites through the EPR effect, compared with simple drug injection, nano-targeted drugs increase the accumulation in tumor sites, improve the utilization rate of drugs, and reduce the toxic and side effects of drugs. However, the nano-drugs passively target the tumor site for a limited time, the body metabolizes the drugs quickly, and the nano-drugs accumulated in the tumor are still metabolized quickly, and the utilization rate of the nano-drugs still has room for improvement. In order to improve the accumulation and retention time of drugs in the tumor, it is difficult to achieve the active targeting of the tumor.

[0003] Photodynamic therapy (PDT) is a method of directly treating the tumor site, and is a new means widely used for treating cancer. Due to its safety and non-invasiveness, it has been used for the treatment of skin cancer, breast cancer, esophageal cancer, bladder cancer and other cancerous lesions. The photosensitizer has been certified by FDA. Photodynamic therapy is to irradiate the photosensitizer with light of a specific wavelength, and to produce phototoxicity killing reaction through two forms, type I is to generate oxygen free radicals (ROS) by the bioelectronic transfer reaction of the excited photosensitizer and oxygen in the cell, to destroy the organelles and cell structure. Type II is to transfer the energy of the excited photosensitizer to molecular oxygen to form singlet oxygen, to further make the cell apoptosis and necrosis. Before treatment, the photosensitizer needs to be injected first, so that the photosensitizer can be accumulated at the tumor site, and then the tumor is locally irradiated by infrared light, so that the photosensitizer produces reactive oxygen species (ROS) and singlet oxygen, to promote the apoptosis and necrosis of tumor cells. The photosensitizer nano-drug can be passively targeted to the tumor site through the ERP effect, which enables the photosensitizer and the combined drug to be targeted and accumulated in the tumor to exert the photodynamic therapy effect, to achieve the effect of necrosis and apoptosis of tumor cells. However, the targeted drug loaded with the photosensitizer is retained in the tumor site for a limited time, and is cleared by the metabolism of the organism. How the photosensitizer can be retained in the tumor site for a long time becomes a difficult problem. In addition, photodynamic therapy is also affected by light, photosensitizer and oxygen. Due to the limitations of light transmittance, hypoxic environment of tumor, metabolism and clearance of photosensitizer, the photodynamic anti-tumor treatment effect is greatly reduced, and it has not been widely used in clinical practice. SUMMARY

[0004] To solve the problems of the prior art, one of the purposes of the present application is to provide a tumor-targeting photosensitizer chimeric peptide, which can specifically target the endoplasmic reticulum in tumors, achieve the accumulation of photosensitizers at tumor sites, have better in vivo tumor inhibition effect compared to photosensitizers alone, and can be used as a drug carrier to wrap anti-tumor drugs to achieve synergistic anti-tumor effect.

[0005] Specifically, the present application includes the following technical solutions.

[0006] A tumor-targeting photosensitizer chimeric peptide has the following structure shown in formula (I):

[0007]

[0008] wherein R1 is C6-C 30 alkyl; R2 is the residue after the photosensitizer loses one hydroxyl group; R3 is a polypeptide capable of targeting the endoplasmic reticulum of tumors; and n is an integer selected from 4-12.

[0009] For example, the tumor-targeting photosensitizer chimeric peptide has the following structure:

[0010]

[0011] The second purpose of the present application is to provide the use of the tumor-targeting photosensitizer chimeric peptide, including the following technical solutions.

[0012] The use of the tumor-targeting photosensitizer chimeric peptide as an active ingredient in the preparation of an anti-tumor drug.

[0013] The use of the tumor-targeting photosensitizer chimeric peptide as a drug carrier in the preparation of an anti-tumor drug.

[0014] The third purpose of the present application is to provide an anti-tumor nano-drug, including the following technical solutions.

[0015] An anti-tumor nano-drug is prepared by wrapping an anti-tumor drug with the tumor-targeting photosensitizer chimeric peptide.

[0016] The fourth purpose of the present application is to provide a preparation method of the anti-tumor nano-drug, including the following technical solutions.

[0017] A preparation method of the anti-tumor nano-drug includes the following steps:

[0018] The anti-tumor drug and the tumor-targeting photosensitizer chimeric peptide are respectively dissolved with a cosolvent, mixed, and then water is added to prepare an aqueous solution, and ultrasonic treatment is performed to make the tumor-targeting photosensitizer chimeric peptide micelles wrap the anti-tumor drug, so that the anti-tumor nanodrug is obtained.

[0019] The fifth object of the present application is to provide an application of the nanodrug, which comprises the following technical solution.

[0020] The application of the nanodrug as an active ingredient in the preparation of an anti-tumor drug.

[0021] The sixth object of the present application is to provide an anti-tumor pharmaceutical composition, which comprises the following technical solution.

[0022] An anti-tumor pharmaceutical composition is prepared from an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the tumor-targeting photosensitizer chimeric peptide and / or the anti-tumor nanodrug.

[0023] The tumor-targeting photosensitizer chimeric peptide of the present application has the following beneficial effects:

[0024] The tumor-targeting photosensitizer chimeric peptide prepared by the present application has specific recognition function for tumors, has good targeting effect, and can specifically target the endoplasmic reticulum of tumor cells. After the photosensitizer bonded by the polypeptide reaches the tumor site, it is attached to the tumor site, promotes tumor necrosis and apoptosis, improves the photodynamic anti-tumor effect, and has better in vivo tumor inhibition effect compared with the photosensitizer alone.

[0025] The tumor-targeting photosensitizer chimeric peptide can also be used as a drug carrier to load other anti-tumor drugs, realizing co-delivery of photosensitizer and synergistic drugs. This not only increases the attachment of photosensitizer in the tumor and improves the utilization rate of photosensitizer, but also reduces the loss of co-loaded drugs during the process of reaching the tumor site, prolongs the residence time of drugs in the tumor, thereby greatly improving the bioavailability of co-loaded drugs, making the anti-tumor drugs such as chemotherapy, targeted therapy, and immunotherapy play the maximum anti-tumor effect, so that good photodynamic effect of the tumor and synergistic anti-tumor effect of other anti-tumor drugs can be achieved.

[0026] In addition, the tumor-targeting photosensitizer chimeric peptide has good biocompatibility, small toxic and side effects on the organism, and has the effect of specific, precise, and efficient targeted therapy of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The tumor-targeting photosensitizer chimeric peptide prepared in Example 1 is subjected to ESI-MS mass spectrometry to obtain the main peak and the impurity peak of the related polypeptide.

[0028] Figure 2The polypeptide main peak of the tumor-targeting photosensitizer chimeric peptide prepared in Example 1 is screened by ESI-MS mass spectrometry and polypeptide molecular weight.

[0029] Figure 3 The particle size, stability and electron microscope morphology of the nanomedicine formed by the tumor-targeting photosensitizer chimeric peptide wrapping the anti-tumor drug are determined.

[0030] Figure 4 The targeting of the tumor-targeting photosensitizer chimeric peptide in the endoplasmic reticulum, mitochondria, lysosomes and nucleus of tumor cells is tested.

[0031] Figure 5 The effect of the tumor-targeting photosensitizer chimeric peptide on cell activity in the simple polypeptide light group (E+), the simple polypeptide dark treatment group (E), the polypeptide drug wrapping light group (EN+), the polypeptide drug wrapping dark treatment group (EN), the free photosensitizer light group (Ce6+), the free photosensitizer dark treatment group (Ce6), the simple drug group (NLG919) and the blank control group (Blank) is determined.

[0032] Figure 6 The imaging results of the polypeptide drug wrapping (EN) of the tumor-targeting photosensitizer chimeric peptide in a mouse tumor model are determined.

[0033] Figure 7 The 4T1 tumor volume change chart after the tumor-targeting photosensitizer chimeric peptide and its drug wrapping are used for photodynamic therapy in tumor mice.

[0034] Figure 8 The 4T1 tumor mass statistical chart after the tumor-targeting photosensitizer chimeric peptide and its drug wrapping are used for photodynamic therapy in tumor mice. DETAILED DESCRIPTION

[0035] The experimental methods not specified in the following examples of the present application are generally carried out according to the conventional conditions, or according to the conditions recommended by the manufacturers. The various common chemical reagents used in the examples are all commercially available products.

[0036] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0037] The terms "comprising" and "having" and any variations thereof in the present application are intended to cover non-exclusive inclusion. For example, a process, method, device, product or equipment including a series of steps is not limited to the listed steps or modules, but optionally further includes steps not listed, or optionally further includes other steps inherent to these processes, methods, products or equipment.

[0038] The "multiple" mentioned in the present application refers to two or more than two. The "and / or" describes the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B can mean that A exists alone, A and B exist together, and B exists alone. The character " / " generally represents that the front and rear associated objects are in an "or" relationship.

[0039] In one embodiment of the present application, a tumor-targeting photosensitizer chimeric peptide is provided, which has the following structure shown in formula (I):

[0040]

[0041] wherein R1 is C6-C 30 alkyl; R2 is the residue after removing one hydroxyl group from a photosensitizer; R3 is a polypeptide capable of targeting the endoplasmic reticulum in tumor; and n is an integer selected from 4-12.

[0042] In some preferred embodiments, R1 is C 10 -C 20 alkyl.

[0043] In some more preferred embodiments, R1 is C 13 -C 17 alkyl.

[0044] In some more preferred embodiments, R1 is C 15 alkyl.

[0045] The photosensitizer in the tumor-targeting photosensitizer chimeric peptide of the present application includes but is not limited to a chlorin or a porphyrin.

[0046] The polypeptide capable of targeting the endoplasmic reticulum in tumor in the tumor-targeting photosensitizer chimeric peptide of the present application can have the amino acid sequence of phenylalanine-phenylalanine-lysine-aspartic acid-glutamic acid-leucine.

[0047] In some preferred embodiments, n is selected from 6, 7, 8, 9, 10, and more preferably 8.

[0048] In some preferred embodiments, the tumor-targeting photosensitizer chimeric peptide of the present application has the following structure:

[0049]

[0050] In one embodiment of the present application, the tumor-targeting photosensitizer chimeric peptide is also provided for use as an active ingredient in the preparation of an antitumor drug.

[0051] In one embodiment of the present application, the tumor-targeting photosensitizer chimeric peptide is also provided for use as a drug carrier in the preparation of an antitumor drug.

[0052] The tumor includes, but is not limited to, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, cholangiocarcinoma, skin cancer, head and neck cancer, bladder cancer, etc.

[0053] In one embodiment of the present application, an anti-tumor nano-drug is also provided, which is prepared by wrapping an anti-tumor drug with the tumor-targeting photosensitizer chimeric peptide.

[0054] The anti-tumor drug includes, but is not limited to, NLG919, BMS-1, BMS-202, JQ1, DOX, MAC, etc.

[0055] In some preferred embodiments, the molar ratio of the tumor-targeting photosensitizer chimeric peptide to the anti-tumor drug in the anti-tumor nano-drug is 1.5-2.5:1.

[0056] In some preferred embodiments, the average particle size of the anti-tumor nano-drug is 150-300 nm.

[0057] In one embodiment of the present application, a preparation method of the anti-tumor nano-drug is also provided, which comprises the following steps:

[0058] The anti-tumor drug and the tumor-targeting photosensitizer chimeric peptide are dissolved with a cosolvent respectively, then mixed, and water is added to prepare an aqueous solution, and the tumor-targeting photosensitizer chimeric peptide micelles wrap the anti-tumor drug, so that the anti-tumor nano-drug is obtained.

[0059] In some preferred embodiments, in the aqueous solution, the concentration of the anti-tumor drug is 0.4-0.6 mg / ml, and the concentration of the tumor-targeting photosensitizer chimeric peptide is 0.8-1.2 mg / ml.

[0060] In some preferred embodiments, the ultrasonic conditions include: KQ3200E ultrasonic cleaner, temperature 30-45°C, power 40-60 W, and ultrasonic time 2-10 min.

[0061] In some preferred embodiments, the cosolvent is dimethyl sulfoxide.

[0062] In one embodiment of the present application, the application of the nano-drug as an active ingredient in the preparation of an anti-tumor drug is also provided.

[0063] The tumor includes, but is not limited to, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, cholangiocarcinoma, skin cancer, head and neck cancer, bladder cancer, etc.

[0064] In one embodiment of the present application, there is also provided an anti-tumor pharmaceutical composition prepared from an active ingredient and a pharmaceutically acceptable adjuvant, wherein the active ingredient comprises the tumor-targeting photosensitizer chimeric peptide and / or the anti-tumor nano-drug.

[0065] That is, the tumor-targeting photosensitizer chimeric peptide and / or the anti-tumor nano-drug of the present application can also be prepared into a pharmaceutical preparation for preventing and / or treating tumors together with a pharmaceutically acceptable adjuvant. The pharmaceutical preparation can be administered to a patient by various routes of administration, including but not limited to oral, transdermal, intramuscular, subcutaneous, and intravenous injection.

[0066] Polypeptides are a class of compounds composed of specific amino acids arranged in a certain sequence, which have good biocompatibility and physiological activity and specific functions, and are mainly divided into responsive peptides and functional peptides. Polypeptides have powerful performance and specificity, and can be applied to tumor targeting, molecular recognition, nano-drug loading, etc. Due to its wide biological activity, diverse modifiability and selectivity, it can be applied to the fields of chemical modification, chemical analysis, biological analysis, etc. In the past, polypeptides were selected from biological proteins, and now they can be chemically synthesized, and the synthesis method is simple and has strong modifiability.

[0067] Clinical tumor chemotherapy drugs, immunotherapy drugs, targeted therapy drugs, etc. After being delivered into the body, due to the clearance rate of the organism itself, the drugs are metabolized by the liver or kidney, and the drugs reaching the tumor site are significantly reduced, which makes the bioavailability of the anti-tumor drugs poor. And the anti-tumor drugs have relatively large toxic and side effects on the organism, such as gastrointestinal reactions, bone marrow suppression, etc. In addition, photodynamic therapy is mainly affected by light, photosensitizer, and oxygen. Due to the reasons of light transmittance, tumor hypoxic environment, and metabolic clearance of photosensitizer, the effect of photodynamic anti-tumor therapy is greatly discounted. In order to overcome these problems, the present application prepares a tumor-targeting photosensitizer chimeric peptide by bonding a photosensitizer with a polypeptide and introducing a hydrophobic alkyl chain and a hydrophilic polyethylene glycol chain according to a specific connection order. Among them, by bonding the photosensitizer with the polypeptide, the solubility and biocompatibility of the photosensitizer for photodynamic therapy are significantly improved, and the dark toxicity of the photosensitizer is reduced; the introduction of the tumor endoplasmic reticulum targeting peptide realizes the accumulation of the photosensitizer at the tumor site; the R1 alkyl part is a lipophilic substance, which can better chimeric into the cell membrane and better realize the transmembrane transport; the hydrophilic polyethylene glycol can increase the hydrophilicity of the targeting peptide photosensitizer bond, so that the drug can stably exist in the solution or body environment, reduce the loss of the drug during transportation, and improve the drug transportation efficiency. Under the synergistic cooperation of each structural unit as a whole, the tumor-targeting photosensitizer chimeric peptide prepared by the present application can greatly prolong the accumulation and retention time of the photosensitizer at the tumor site, and enhance the effect of photodynamic anti-tumor therapy.

[0068] In addition, the prepared targeted peptide photosensitizer bonding compound can be used as a nanocarrier to load antitumor drugs, such as chemotherapeutic drugs, targeted therapeutic drugs, immunotherapeutic drugs and the like; the targeted peptide bonding compound itself can be self-assembled into micelles through hydrophobic force, π-π conjugation and the like, and the targeted peptide photosensitizer bonding compound after self-assembly into micelles can wrap antitumor drugs to form stable nanomedicines. These nanomedicines can be passively targeted or actively targeted to tumor sites through the EPR effect, greatly improving the accumulation of drugs at tumor sites and enhancing the efficacy of photodynamic therapy, immunotherapy, targeted therapy, chemotherapy and other antitumor therapies, so as to achieve a combined antitumor effect. It can be seen that the polypeptide nanocarrier platform of the present application solves the above problems well, not only improves the bioavailability of the drug, but also reduces the toxic and side effects of the drug on the organism. The tumor targeting peptide can be passively targeted to tumor cells through the EPR effect, greatly improving the antitumor effect of the drug. After chemical modification of the polypeptide sequence, the tumor targeting peptide can specifically target tumors, increase tumor targeting activity, and further improve the utilization rate of the drug.

[0069] Therefore, the tumor targeting photosensitizer chimeric peptide of the present application has the following beneficial effects:

[0070] The tumor targeting photosensitizer chimeric peptide prepared by the present application has specific recognition function for tumors, has good targeting effect, and can specifically target tumors and endoplasmic reticulum of tumor cells. After the polypeptide bonded photosensitizer reaches the tumor site, it is attached to the tumor site, promotes tumor necrosis and apoptosis, improves the photodynamic antitumor effect, and has better in vivo tumor inhibition effect than the photosensitizer alone.

[0071] The tumor targeting photosensitizer chimeric peptide can also be used as a drug carrier to load other antitumor drugs, realizing co-delivery of photosensitizer and synergistic drugs. This not only increases the attachment of photosensitizer in tumors and improves the utilization rate of photosensitizer, but also reduces the loss of co-loaded drugs during the process of reaching the tumor site, prolongs the residence time of drugs in tumors, greatly improves the bioavailability of co-loaded drugs, makes chemotherapeutic drugs, targeted therapeutic drugs, immunotherapeutic drugs and other antitumor drugs play the maximum antitumor effect, so as to achieve good synergistic antitumor effect of photodynamic therapy and other antitumor drugs.

[0072] In addition, the tumor targeting photosensitizer chimeric peptide has good biocompatibility, small toxic and side effects on organisms, and has the effect of specific, precise and efficient targeted therapy of tumors.

[0073] The tumor targeting photosensitizer chimeric peptide of the present application adopts solid-phase polypeptide synthesis technology, and the preparation method is simple and the purification process is simple.

[0074] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application.

[0075] Example 1

[0076] Synthesis of the positively charged tumor targeting photosensitizer chimeric peptide (C16-lysine (chlorin e6 (Ce6))-PEG8-phenylalanine-phenylalanine-lysine-aspartic acid-glutamic acid-leucine) (also known as the targeted peptide photosensitizer key 5 compound). Its structural formula is as follows:

[0077]

[0078] (1) A reactor containing 10 mL of redistilled N,N-dimethylformamide was added with 0.3 g of 2-chloro-trityl chloride resin (1.102 mmol / g), and after the 2-chloro-trityl chloride resin was swelled in N,N-dimethylformamide for 2 h, the N,N-dimethylformamide was removed.

[0079] (2) Fmoc-protected leucine (3 equivalents of the resin active site), N,N- diisopropyl ethylamine (3 equivalents of the amino acid) were dissolved in 10 mL of N,N-dimethylformamide, and then added to the reactor, and reacted at room temperature for 5 h to bond leucine to the resin, and the solvent was removed, and the N,N-dimethylformamide was washed 5 times.

[0080] (3) A solution of methanol / N,N-dimethylformamide / N,N-diisopropyl ethylamine in a ratio of 1:7:2 (V / V / V) 10 mL was added to the reactor, and the unreacted active sites on the resin were capped by reacting at room temperature for 30 min, and the solvent was removed, and the resin was washed with N,N-dimethylformamide 5 times.

[0081] (4) 20% (V / V) piperidine / N,N-dimethylformamide (i.e. the volume ratio of piperidine to N,N-dimethylformamide is 2:8) solution 10 mL was added to the reactor, and after reacting at room temperature for 15 min, the solvent was removed; the piperidine / N,N-dimethylformamide solution was repeatedly added to react to remove the Fmoc protecting group, and after the reaction was completed, the solvent was removed, and the resin was washed with N,N-dimethylformamide 5 times.

[0082] (5) Fmoc-protected amino acid (glutamic acid) (3 equivalents of the active sites of the resin), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.6 equivalents of the active sites of the resin), 1-hydroxybenzotriazole (3.6 equivalents of the active sites of the resin), N,N-diisopropyl ethylamine (6 equivalents of the active sites of the resin) were dissolved in N,N-dimethylformamide, and added to the reactor. Glutamic acid was linked to the resin by reacting at room temperature for 2 h. The solvent was removed, and the resin was washed with N,N-dimethylformamide 5 times.

[0083] (6) Other amino acids (phenylalanine-phenylalanine-lysine-aspartic acid) were linked one by one according to steps (4) and (5).

[0084] (7) 10 mL of 20% (V / V) piperidine / N,N-dimethylformamide (i.e. the volume ratio of piperidine to N,N-dimethylformamide is 2:8) solution was added to the reactor, and after reacting at room temperature for 15 min, the solvent was removed. The reaction was repeated by adding piperidine / N,N-dimethylformamide solution to remove the Fmoc protecting group. After the reaction was completed, the solvent was removed, and the resin was washed with N,N-dimethylformamide 5 times.

[0085] (8) N-Fluorenylmethoxycarbonyl-octaglutamic acid (PEG8) (3 equivalents of the active sites of the resin), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.6 equivalents of the active sites of the resin), 1-hydroxybenzotriazole (3.6 equivalents of the active sites of the resin), N,N-diisopropyl ethylamine (6 equivalents of the active sites of the resin) were dissolved in N,N-dimethylformamide, and added to the reactor. N-Fluorenylmethoxycarbonyl-octaglutamic acid (PEG8) was linked to the resin by reacting at room temperature for 4 h. The solvent was removed, and the resin was washed with N,N-dimethylformamide 5 times.

[0086] (9) 10 mL of 20% (V / V) piperidine / N,N-dimethylformamide (i.e. the volume ratio of piperidine to N,N-dimethylformamide is 2:8) solution was added to the reactor, and after reacting at room temperature for 15 min, the solvent was removed. The reaction was repeated by adding piperidine / N,N-dimethylformamide solution to remove the Fmoc protecting group. After the reaction was completed, the solvent was removed, and the resin was washed with N,N-dimethylformamide 5 times.

[0087] (10) Fmoc-protected lysine (Fmoc-Lys(Dde)-OH) (3 equivalents of resin active sites), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.6 equivalents of resin active sites), 1-hydroxybenzotriazole (3.6 equivalents of resin active sites), N,N-diisopropyl ethylamine (6 equivalents of resin active sites) were dissolved in N,N-dimethylformamide and added to the reactor, and lysine (Fmoc-Lys(Dde)-OH) was bonded by reacting at room temperature for 2 h. The solvent was removed, and the resin was washed with N,N-dimethylformamide 5 times.

[0088] (11) 10 mL of 20% (V / V) piperidine / N,N-dimethylformamide (i.e. the volume ratio of piperidine to N,N-dimethylformamide is 2:8) solution was added to the reactor, and after reacting at room temperature for 15 min, the solvent was removed. The reaction was repeated by adding piperidine / N,N-dimethylformamide solution to cut off the Fmoc protecting group. After the reaction was completed, the solvent was removed, and the resin was washed with N,N-dimethylformamide 5 times.

[0089] (12) Palmitic acid C16 (3 equivalents of resin active sites), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.6 equivalents of resin active sites), 1-hydroxybenzotriazole (3.6 equivalents of resin active sites), N,N-diisopropyl ethylamine (6 equivalents of resin active sites) were dissolved in N,N-dimethylformamide and added to the reactor, and palmitic acid C16 was bonded by reacting at room temperature for 2 h. Constant temperature heating was required during the reaction to prevent palmitic acid from precipitating. The solvent was removed, and the resin was washed with N,N-dimethylformamide 5 times.

[0090] (13) Chlorin e6 (Ce6) (3 equivalents of resin active sites), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.6 equivalents of resin active sites), 1-hydroxybenzotriazole (3.6 equivalents of resin active sites), N,N-diisopropyl ethylamine (6 equivalents of resin active sites) were dissolved in 10 mL of N,N-dimethylformamide and added to the reactor, and chlorin e6 (Ce6) was bonded by reacting at room temperature for 24 h. The solvent was removed, and the resin was washed with N,N-dimethylformamide, methanol, dichloromethane 5 times in turn.

[0091] (14) A solution consisting of the following components by volume percentage was added to the reactor to cut off the polypeptide bonded substance and side groups on the 2-chloro-trityl chloride resin at room temperature for 2 h: 83% trifluoroacetic acid, 4.6% water, 4.6% benzyl mercaptan, 2.1% ethanedithiol, 5.7% phenol.

[0092] (15)Collecting the cutting liquid, rotary evaporation, vacuum drying to obtain the tumor targeting photosensitizer chimeric peptide, light storage in the drying tower. The mass spectrum is shown in Figure 1 and Figure 2

[0093] Example 2:

[0094] The anti-tumor drug (IDO inhibitor NLG919) was dissolved in dimethyl sulfoxide (DMSO) by ultrasonic dissolution to obtain a DMSO solution of the anti-tumor drug with a concentration of 10 mg / ml.

[0095] The targeting peptide photosensitizer bond compound prepared in Example 1 was dissolved in DMSO to obtain a DMSO solution of the targeting peptide photosensitizer bond compound with a concentration of 100 mg / ml.

[0096] According to the ratio of 2:1 of the targeting peptide photosensitizer bond compound to the anti-tumor drug, the DMSO solution of the anti-tumor drug was added to the DMSO solution of the targeting peptide photosensitizer bond compound, and distilled water was added to prepare a 1 ml aqueous solution, wherein the concentration of the anti-tumor drug was 0.5 mg / ml, and the concentration of the targeting peptide photosensitizer bond compound was 1 mg / ml. The anti-tumor drug was wrapped by the targeting peptide photosensitizer bond compound micelles by ultrasonic (KQ3200E ultrasonic cleaner, 37℃, power 50w, time 5min) to form a new stable nano drug.

[0097] The prepared nano drug (diluted 5 times) was measured for particle size and stability (PDI) by Malvern particle size analyzer.

[0098] The results are shown in Figure 3 The particle size and stability test of the formed nano drug showed that the particle size and dispersion coefficient of the nano drug were stable within 7 days. Transmission electron microscopy showed that the anti-tumor drug could be wrapped by the targeting peptide photosensitizer bond compound to form a nano drug.

[0099] Example 3:

[0100] 4T1 cells were seeded into three confocal dishes, and EN materials (nano drug prepared in Example 2) were endocytosed for 6h. After the culture medium was discarded, PBS was washed 3 times, fresh culture medium containing nuclear dye (Hoechst 33342), mitochondrial green fluorescent dye (Mito Tracker Green), endoplasmic reticulum green fluorescent dye (ERTracker Green) and lysosome green fluorescent dye (Lyso-Tracker Green) was added, and incubation was performed for 20min, 20min, 30min and 30min, respectively. The staining culture medium was discarded, PBS was washed 3 times, fresh culture medium was added, and the fluorescence of the cells was observed and analyzed by CLSM. The corresponding concentration of Ce6 in the EN material was 10ug / ml.​

[0101] Results are shown in Figure 4 The fluorescence of the tumor-targeting photosensitizer chimeric peptide overlapped with the fluorescence of the endoplasmic reticulum, but no obvious fluorescence overlap was observed in the mitochondria, lysosomes, and nucleus, indicating that the tumor-targeting photosensitizer chimeric peptide can specifically target the endoplasmic reticulum of tumor cells.

[0102] Example 4

[0103] The experimental groups of this example are: NLG919 group, Ce6 light group, Ce6 non-light group, E light group, E non-light group, EN light group, and EN non-light group. Among them, E refers to the tumor-targeting photosensitizer chimeric peptide prepared in Example 1, and EN refers to the nanodrug prepared in Example 2 using the tumor-targeting photosensitizer chimeric peptide as a carrier.

[0104] 4T1 cells were seeded into 7 96-well plates, and after adhering for 24 h, a series of concentration gradients of each material were added. The highest material concentration of E was Ce6: 5 ug / ml; the highest material concentration of EN was Ce6: 5 ug / ml; the highest material concentration of NLG919 was 1.9 ug / ml. A two-thirds gradient method was used for dilution. After 6 h, the Ce6 light group, E light group, and EN light group were irradiated for 3 min at an intensity of 30% and a power of 68 W, and the dark treatment group was not irradiated. After 24 h, 20 uL of MTT (5 mg / ml ultrapure water) was added to each well, and the incubation was continued for 4 h, after which the solution was discarded, and 150 ul of DMSO was used to dissolve uniformly, and a 570 nm microplate reader was used for detection.

[0105] Results are shown in Figure 5 In the NLG919 group, Ce6 light group, Ce6 non-light group, E light group, E non-light group, EN light group, and EN non-light group (in which the initial concentration of Ce6 was 5 ug / ml, the initial concentration of Ce6 in E and EN was 5 ug / ml, the initial concentration of NLG919 was 1.875 ug / ml, and the dilution was carried out in proportion to the 2 / 3 concentration gradient), the tumor-targeting photosensitizer chimeric peptide E and its drug-loaded EN did not produce obvious dark toxicity to 4T1 cells, while the E light group and the EN light group produced strong cytotoxicity to 4T1 cells after photodynamic therapy, which was more obvious than the effect of free photosensitizer Ce6. This indicates that the tumor-targeting photosensitizer chimeric peptide has small dark toxicity and good photodynamic therapy effect.

[0106] Example 5

[0107] 4-6 week old balb / c female mice were inoculated with 4T1 cells on the right leg back, and when the tumor volume reached 100 mm 3Afterwards, EN (Ce6: 2.25 mg / kg; NLG919: 0.85 mg / kg) and Ce6 (Ce6: 2.25 mg / kg) were injected through the tail vein; the drug accumulation in the tumor was observed under the in vivo imaging instrument at 0, 2, 4, 6, 8, 12 hours, respectively.

[0108] Results are shown in Figure 6 The tumor-targeting photosensitizer chimeric peptide drug-loaded EN can be accumulated in the tumor at 2, 4, 6, 8, 12 hours, and the drug can be retained in the tumor for a long time. The free photosensitizer Ce6 has a small dose of accumulation in the tumor and weak targeting. It is shown that the tumor-targeting photosensitizer chimeric peptide can increase the accumulation of the drug in the tumor, prolong the retention time of the drug, improve the utilization rate of the drug, and enhance the photodynamic anti-tumor effect.

[0109] Example 6

[0110] 4-6 weeks old BALB / C female mice were inoculated with 4T1 cells on the right leg back. When the tumor volume of the tumor-bearing mice reached 100 mm 3 , 200 μl of 4T1 cells (2 million) were injected through the tail vein, and the mice were randomly divided into 8 groups. The experimental groups were: Blank group (blank control group), NLG919 group, Ce6 light group, Ce6 non-light group, E non-light group, E light group, EN light group and EN non-light group. Among them, E refers to the tumor-targeting photosensitizer chimeric peptide prepared in Example 1, and EN refers to the nanodrug with the tumor-targeting photosensitizer chimeric peptide as the carrier prepared in Example 2. NLG919: 0.85 mg / kg; Ce6: 2.25 mg / kg; E containing Ce6: 2.25 mg / kg; EN containing Ce6: 2.25 mg / kg and NLG919: 0.85 mg / kg were injected through the tail vein, respectively, and laser irradiation treatment was performed for 10 min after 8 hours, the intensity was 30%, the power was 68W, and the dark treatment group was not irradiated. The treatment was performed once every two days, a total of 4 times, and the tumor volume and tumor-bearing mouse weight were monitored every other day. After 4 times of photodynamic treatment, on the 29th day, the experimental mice were dissected, the blank control group, the dark treatment group and the light treatment group of nude mice were sacrificed, and the tumors were peeled off, and the corresponding tumor weights were measured.

[0111] Results are shown in Figure 7 , Figure 8After treatment, the average tumor volume and mass of the tumor-targeting photosensitizer chimeric peptide E light group and the drug-coated EN light group were lower than those of the blank control group and the dark treatment group. And the average tumor mass of the tumor-targeting photosensitizer chimeric peptide E light group and the drug-coated EN light group was lower than that of the free photosensitizer Ce6 group. It shows that the photodynamic therapy effect of the tumor-targeting photosensitizer chimeric peptide E light group and the drug-coated EN light group is good. At the same time, it also shows that the tumor-targeting photosensitizer chimeric peptide can enhance the tumor accumulation effect of the photosensitizer and improve the bioavailability of the photosensitizer.

[0112] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, all possible combinations of the technical features in the following embodiments are not described, but as long as the combinations of the technical features do not exist contradictions, they should be considered as the scope of the present disclosure.

[0113] The above-described embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent of the present application. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. An antitumor nanomedicine, characterized in that, It was prepared by encapsulating an anti-tumor drug with a tumor-targeting photosensitizer chimeric peptide; The structural formula of the tumor-targeting photosensitizer chimeric peptide is as follows: ； The anti-tumor drug is NLG919.

2. The antitumor nanomedicine according to claim 1, characterized in that, In the antitumor nanomedicine, the molar ratio of the tumor-targeting photosensitizer chimeric peptide to the antitumor drug is 1.5-2.5:

1.

3. The antitumor nanomedicine according to any one of claims 1-2, characterized in that, Its average particle size is 150nm-300nm.

4. A method for preparing an antitumor nanomedicine according to any one of claims 1-3, characterized in that, Includes the following steps: The antitumor drug and the tumor-targeting photosensitizer chimeric peptide are dissolved separately in a cosolvent, mixed, and then water is added to prepare an aqueous solution. The solution is then sonicated to encapsulate the antitumor drug in micelles of the tumor-targeting photosensitizer chimeric peptide, thus obtaining the antitumor nanomedicine.

5. The method for preparing antitumor nanomedicine according to claim 4, characterized in that, In the aqueous solution, the concentration of the antitumor drug is 0.4 mg / ml-0.6 mg / ml, and the concentration of the tumor-targeting photosensitizer chimeric peptide is 0.8 mg / ml-1.2 mg / ml; and / or, The ultrasonic conditions include: a KQ3200E ultrasonic cleaner, a temperature of 30℃-45℃, a power of 40W-60W, and an ultrasonic treatment time of 2-10 minutes; and / or, The co-solvent is dimethyl sulfoxide.

6. The use of the nanomedicine according to any one of claims 1-3 as an active ingredient in the preparation of an antitumor drug, wherein the tumor is breast cancer.

7. An antitumor pharmaceutical composition, characterized in that, It is prepared from an active ingredient and an acceptable excipient in a drug, wherein the active ingredient includes the antitumor nanomedicine according to any one of claims 1-3.

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