Tyrosinase responsive cyclic peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of tumor drug preparation, and particularly relates to tyrosinase responsive cyclopeptide as well as a preparation method and application thereof. The invention innovatively provides an in-situ self-assembly nano-response system based on enzyme response type cyclopeptide and by utilizing the tyrosinase overexpression characteristic, and the in-situ self-assembly nano-response system specifically targets the cytoskeleton of melanoma. The tyrosinase responsive cyclopeptide containing a tyrosine motif is subjected to specific oxidation crosslinking under the catalytic action of tyrosinase to form a beta-folding dominant nanofiber network, the process does not need exogenous carrier intervention, and the migration invasion ability of melanoma cells is selectively inhibited by physically destroying the dynamic balance of F-actin. Compared with a traditional enzyme response system, self-supply type activation of the response module is achieved, the tumor targeting property is improved, meanwhile, the system toxicity is remarkably reduced, and a new technical path is provided for solid tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor drug preparation, and particularly relates to a tyrosinase-responsive cyclic peptide, a preparation method thereof, and an application thereof. Background Art

[0002] Melanoma, as the most aggressive cutaneous malignancy, has a high tendency of metastasis and treatment resistance. Although targeted / immunotherapy represented by MAPK pathway inhibitors and PD-1 / CTLA-4 antibodies has achieved clinical breakthroughs, their applications are still limited by dose-dependent systemic toxicity (such as immune-related adverse events), acquired drug resistance (BRAF inhibitor resistance occurs in about 50% of patients), and off-target effects caused by tumor microenvironment heterogeneity, which urges the urgent development of new treatment paradigms with tumor-specific activation mechanisms in the field.

[0003] Studies have found that melanoma cells specifically overexpress tyrosinase (TYR), and this rate-limiting enzyme drives melanin anabolism by catalyzing the hydroxylation of L-tyrosine and the oxidation reaction of dopaquinone. Technicians have attempted to develop enzyme-responsive prodrug systems using tyrosinase activity. For example: 1) Nitrogen mustard prodrugs activated by tyrosinase oxidation, and drug release is achieved through quinone-thiol adducts; 2) Phenol-modified nanoparticles loaded with chemotherapeutic drugs, and intratumoral aggregation is achieved through enzymatic polymerization.

[0004] However, such schemes rely on the delivery of exogenous cytotoxins, resulting in systemic defects such as low drug loading efficiency (usually <10%) and poor biocompatibility of the carrier. Summary of the Invention

[0005] The purpose of the present invention is to provide a tyrosinase-responsive cyclic peptide, a preparation method thereof, and an application thereof. The tyrosinase-responsive cyclic peptide provided by the present invention does not require the intervention of an exogenous carrier.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a tyrosinase-responsive cyclic peptide, and its structure is shown in Formula I:

[0008]

[0009] The present invention also provides a preparation method of the tyrosinase-responsive cyclic peptide described in the above scheme, including the following steps:

[0010] Mix a linear peptide precursor, a cyclizing reagent, and a solvent for cyclization reaction to obtain the tyrosinase-responsive cyclic peptide; the amino acid sequence of the linear peptide precursor is Arg-Gly-Asp-Lys-Tyr-Gln (RGDKYQ).

[0011] Preferably, the purity of the linear peptide precursor is ≥95%.

[0012] Preferably, the cyclizing reagent is phthalaldehyde.

[0013] Preferably, the solvent includes alcohol and water.

[0014] Preferably, the volume ratio of alcohol to water is 1:1.

[0015] Preferably, the volume ratio of the cyclizing reagent to the solvent is 1 - 5:1 - 50.

[0016] Preferably, the linear peptide precursor, the cyclizing reagent and the solvent are mixed to obtain a linear peptide precursor reaction solution, and the concentration of the linear peptide precursor in the linear peptide precursor reaction solution is 1 - 10 mg / mL.

[0017] Preferably, the mixing is magnetic stirring; the rotation speed of the magnetic stirring is 100 - 500 rpm, the mixing time is 10 - 50 min, the mixing temperature is 20 - 30 °C, and the pressure is normal pressure.

[0018] The present invention also provides the application of the tyrosinase-responsive cyclic peptide described in the above scheme or the tyrosinase-responsive cyclic peptide obtained by the preparation method described in the above scheme in the preparation of drugs targeting melanoma.

[0019] The present invention provides a tyrosinase-responsive cyclic peptide. The present invention innovatively proposes an enzyme-responsive cyclic peptide, utilizes the overexpression characteristic of tyrosinase, constructs an in-situ self-assembled nano-responsive system, and specifically targets the cytoskeleton of melanoma. The tyrosinase-responsive cyclic peptide containing a tyrosine motif of the present invention undergoes specific oxidative cross-linking under the catalytic action of tyrosinase to form a nanofiber network dominated by β-sheets. This process does not require the intervention of exogenous carriers and selectively inhibits the migration and invasion ability of melanoma cells by physically disrupting the dynamic balance of F-actin. Compared with the traditional enzyme-responsive system, the present invention realizes the "self-supply" activation of the response module, significantly reduces the system toxicity while improving the tumor targeting property, and provides a new technical path for the treatment of solid tumors. Specifically, as Figure 1 shown, the tyrosinase-responsive cyclic peptide provided by the present invention has the following advantages:

[0020] 1) High tumor selectivity, with the targeting property improved by ≥2 times: the survival rate of normal cells is ≥95% (about 62% in the existing prodrug system), and the apoptosis rate of tumor cells is ≥90% (about 45% in the existing prodrug system); the in vivo treatment window is expanded by 5 times, and the maximum tolerated dose (MTD) is increased from 10 mg / kg to 50 mg / kg.

[0021] This is because of the enzyme-triggered self-assembly mechanism and cytoskeleton targeting of the cyclic peptides of the present invention: The tyrosinase-responsive cyclic peptides are only oxidized to quinone structures in the tumor microenvironment with overexpressed tyrosinase, enabling in-situ nanofiber formation and avoiding non-specific aggregation in normal tissues; the nanofibers specifically bind to integrins on the surface of tumor cells through the RGD motif, selectively inhibiting the Arp2 / 3 complex (IC 50 = 5 μM), with no significant interference with the actin network of normal cells.

[0022] 2) The therapeutic effect is persistent, and the tumor inhibition rate is increased by 87.5%. This is because of the supramolecular nanostructure stability and cell motility blockade of the cyclic peptides of the present invention: The quinone nanofibers form a rigid structure through π-π stacking (Zeta potential = -25 mV), resisting lysosomal degradation and prolonging the retention time in the tumor; the inhibition of actin branching leads to a decrease in the migration rate of tumor cells by ≥80%, blocking the metastasis pathway at the root.

[0023] 3) Good safety, with a 60% reduction in toxicity: The cardiac toxicity marker (cTnI) is normal (the doxorubicin prodrug will increase by 3 times), the immunogenicity is negative, and the levels of IL-6 and TNF-α detected by ELISA have no difference from the blank group. This is because of the absence of exogenous drug loading and biocompatibility design of the cyclic peptides of the present invention: The therapeutic function is directly activated through an endogenous enzymatic reaction, eliminating the off-target leakage risk of traditional chemotherapeutic drugs (such as doxorubicin); the cyclic peptide backbone is composed of natural amino acids, and no endotoxin residue (≤0.1 EU / mg) is verified by HPLC.

[0024] The present invention also provides a preparation method of the tyrosinase-responsive cyclic peptides described in the above solution. The preparation method provided by the present invention has simple steps, high efficiency, convenient operation, high feasibility, good safety and stability, specifically as Figure 1 shown.

[0025] The present invention also provides the application of the tyrosinase-responsive cyclic peptides described in the above solution or the tyrosinase-responsive cyclic peptides obtained by the preparation method described in the above solution in the preparation of drugs targeting melanoma. The tyrosinase-responsive cyclic peptides provided by the present invention do not require the intervention of exogenous carriers, have high drug-loading efficiency, good biocompatibility, high tumor targeting, and low toxicity, and are suitable for use in the preparation of drugs targeting melanoma. Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0027] Figure 1Preparation and mechanism of action diagram of tyrosinase-responsive cyclic peptide;

[0028] Figure 2 Results diagram of blank control of B16 melanoma cells, linear peptide RGDKYQ and tyrosinase-responsive cyclic peptide acting for 24 h;

[0029] Figure 3 Scratch migration results diagram of blank control of B16 melanoma cells, linear peptide RGDKYQ and tyrosinase-responsive cyclic peptide acting for 24 h;

[0030] Figure 4 Bar chart of different cell lines B16, L929, U2OS added with different concentrations of tyrosinase-responsive cyclic peptide for 24 h;

[0031] Figure 5 Physical diagram and line chart of different cell lines B16, L929, U2OS added with different concentrations of tyrosinase-responsive cyclic peptide for 24 h;

[0032] Figure 6 Infrared spectrum diagram (upper left), fluorescence spectrum diagram (upper right), particle size diagram (lower left) and potential diagram (lower right) of Test Example 1. Detailed implementation mode

[0033] The present invention provides a tyrosinase-responsive cyclic peptide, and the structure is shown in Formula I:

[0034]

[0035] The tyrosinase-responsive cyclic peptide provided by the present invention is an o-phthalaldehyde-mediated cyclic peptide structure, and includes:

[0036] (a) RGD motif: endows tumor blood vessel targeting function;

[0037] (b) Tyrosine residue: serves as a specific recognition site for tyrosinase;

[0038] (c) Quinone reaction group: triggers molecular self-assembly through enzymatic oxidation.

[0039] The tyrosinase-responsive cyclic peptide provided by the present invention is an enzyme-triggered self-assembly system, and the mechanism of cytoskeleton disruption is as follows: After tyrosinase-catalyzed oxidation, the tyrosinase-responsive cyclic peptide undergoes a quinone structure transformation, triggering intermolecular π-π stacking and hydrogen bonding to form supramolecular nanofibers with a rigid β-sheet structure (diameter 20-50 nm, length 200-500 nm); the self-assembly process has pH-responsive characteristics (optimal pH value 6.5-7.4), achieving specific activation in the tumor microenvironment; the supramolecular nanofibers interfere with actin dynamics through the following pathways: i) competitively binding to the F-actin binding domain (Kd = 3.8 ± 0.5 nM); ii) inhibiting actin branching mediated by the Arp2 / 3 complex (inhibition rate > 82%); iii) activating the RhoA / ROCK signaling pathway to induce the formation of abnormal stress fibers.

[0040] In vitro experimental results show that: the tyrosinase-responsive cyclic peptide at a concentration of 50 μM can inhibit the migration of > 90% of melanoma cells (scratch assay); in a B16 tumor-bearing mouse model, injecting a dose of 5 mg / kg around the tumor every 3 days can reduce the tumor volume by 76.3 ± 8.2% (p < 0.001), and no abnormal liver function indicators were observed.

[0041] The present invention also provides a preparation method of the tyrosinase-responsive cyclic peptide described in the above solution, including the following steps:

[0042] Mix the linear peptide precursor, cyclizing reagent, and solvent for cyclization reaction to obtain the tyrosinase-responsive cyclic peptide; the amino acid sequence of the linear peptide precursor is Arg-Gly-Asp-Lys-Tyr-Gln (RGDKYQ), denoted as Sequence 1.

[0043] In the present invention, the purity of the linear peptide precursor can be ≥ 95%; the linear peptide precursor can be chemically synthesized by solid-phase synthesis or purchased commercially.

[0044] In the present invention, the cyclizing reagent can be o-phthalaldehyde (OPA); the purity of the o-phthalaldehyde can be analytical grade. The present invention can also use cyclizing reagents such as 1,1'-carbonyldiimidazole and dicyclohexylcarbodiimide to cyclize the linear peptide precursor.

[0045] In the present invention, the solvent can include alcohol and water (H2O); the alcohol can be absolute ethanol (EtOH); the water can be deionized water; the resistivity of the deionized water can be ≥ 18 MΩ·cm.

[0046] In the present invention, the volume ratio of the alcohol to the water can be 1:1.

[0047] In the present invention, the volume ratio of the cyclizing reagent to the solvent can be 1 to 5:1 to 50, specifically it can be 1:1, 3:1, 5:1, 1:5, 3:5, 1:10, 3:10, 5:10, 1:20, 3:20, 5:20, 1:35, 3:35, 5:35, 1:50 or 3:50.

[0048] On the one hand, the present invention optimizes the solvent ratio, enabling phthalaldehyde to efficiently mediate intramolecular cyclization under mild conditions and avoiding side reactions (by-products ≤ 2%); on the other hand, it designs a special sequence for the linear peptide: the spatial arrangement of the side chains of tyrosine (Y) and lysine (K) in the RGDKYQ sequence, enabling OPA to preferentially react with the N-terminal amino group and the adjacent thiol group to achieve site-directed cyclization. Ultimately, the yield of the preparation method of the present invention is increased by 185%: the yield of the single-step cyclization reaction is ≥ 85%, while the total yield of the three-step reaction of the traditional polypeptide modification technology is ≤ 30%; the preparation cycle is shortened by 80%: the cyclization is completed in 10 minutes, while the traditional method takes more than 6 hours.

[0049] In the present invention, the linear peptide precursor, the cyclizing reagent and the solvent are mixed to obtain a linear peptide precursor reaction solution, and the concentration of the linear peptide precursor in the linear peptide precursor reaction solution can be 1 to 10 mg / mL, specifically it can be 3 mg / mL, 5 mg / mL, 7 mg / mL or 9 mg / mL.

[0050] In the present invention, the mixing can be magnetic stirring; the rotation speed of the magnetic stirring can be 100 to 500 rpm, specifically it can be 200 rpm, 300 rpm or 400 rpm, the mixing time can be 10 to 50 minutes, specifically it can be 20 minutes, 30 minutes or 40 minutes, the mixing temperature can be room temperature (20 to 30 °C, specifically it can be 23 °C, 25 °C or 27 °C), and the pressure can be atmospheric pressure. The magnetic stirring mixing in the present invention helps the intramolecular cyclization reaction mediated by phthalaldehyde.

[0051] In the present invention, after the cyclization reaction, it may further include purifying the obtained reaction product; the purification can be: removing the solvent from the reaction product.

[0052] In the present invention, the solvent removal can be vacuum drying; the temperature of the vacuum drying can be 37 °C, and the pressure can be ≤ 0.1 MPa; the equipment for the vacuum drying can be a vacuum drying oven.

[0053] The present invention also provides the use of the tyrosinase-responsive cyclic peptide described in the above scheme or the tyrosinase-responsive cyclic peptide obtained by the preparation method described in the above scheme in the preparation of a drug targeting melanoma.

[0054] The tyrosinase-responsive cyclic peptide provided by the present invention can be used for the development of a treatment system for tumors with high expression of tyrosinase, especially melanoma or metastatic skin cancer, and is adapted to the dosage form design of local administration patches, tumor microenvironment-responsive hydrogels, etc.

[0055] To further illustrate the present invention, the solutions of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0056] Example 1

[0057] In this example, a tyrosinase-responsive cyclic peptide was prepared, including the following steps:

[0058] 1. Composition and source of raw materials:

[0059] Linear peptide precursor: The amino acid sequence is Arg-Gly-Asp-Lys-Tyr-Gln (RGDKYQ), with a purity of ≥95%, chemically synthesized by solid-phase synthesis or commercially customized polypeptide.

[0060] Cyclization reagent: o-phthalaldehyde (OPA), analytical pure.

[0061] Solvent: Absolute ethanol (EtOH) and deionized water, with the resistivity of deionized water ≥18 MΩ·cm; volume ratio of OPA:EtOH:H2O = 1:1:1.

[0062] 2. Preparation of tyrosinase-responsive cyclic peptide:

[0063] (1) Cyclization reaction:

[0064] Dissolve the linear peptide precursor RGDKYQ in the OPA / EtOH / H2O mixed reagent, control the concentration of the linear peptide precursor to be 3 mg / mL, and magnetically stir at 200 rpm for 10 min at room temperature (25 ± 2 °C) and normal pressure to carry out the o-phthalaldehyde-mediated cyclization reaction to obtain a reaction solution.

[0065] (2) Product purification:

[0066] Transfer the reaction solution to a vacuum drying oven at 37 °C, with a pressure ≤0.1 MPa, and dry until the solvent is completely removed to obtain a powdery tyrosinase-responsive cyclic peptide. Detected by HPLC (C18 chromatographic column, gradient elution with acetonitrile / water), the purity of the tyrosinase-responsive cyclic peptide is ≥98%.

[0067] Example 2

[0068] In this example, a tyrosinase-responsive cyclic peptide was prepared to verify the efficiency of the cyclization reaction and the product structure, including the following steps:

[0069] (1) Raw material preparation: Prepare linear peptide precursor RGDKYQ (molecular weight: 849.95 Da, solid-phase custom synthesis, HPLC purity ≥ 95%), cyclization reagent and solvent (phthalaldehyde mixed with EtOH / H2O in a volume ratio of 1:1:1).

[0070] (2) Cyclization reaction: Dissolve the linear peptide precursor in the mixed solvent of the cyclization reagent and solvent. The final concentration of the linear peptide precursor is 3 mg / mL (3.53 mM). Carry out the cyclization reaction under normal pressure, at 25 °C, with magnetic stirring at 200 rpm for 10 min, and then dry under vacuum at 37 °C to remove the solvent, obtaining a powdery tyrosinase-responsive cyclic peptide (denoted as RGDKYQ-OPA).

[0071] Test Example 1

[0072] 1. Mechanism of action and application method:

[0073] (1) Tumor microenvironment-triggered self-assembly:

[0074] Dissolve the tyrosinase-responsive cyclic peptide in physiological saline to prepare a 1 mM stock solution. Administer it by intravenous injection or intratumoral injection to mice. The tyrosinase-responsive cyclic peptide is catalytically oxidized by overexpressed tyrosinase (activity ≥ 50 U / mg) in melanoma tissue to generate a quinone-type active intermediate.

[0075] (2) Formation of supramolecular nanostructures:

[0076] The quinone-type active intermediate self-assembles into nanofibers with a particle size of 50 - 100 nm (measured by dynamic light scattering method) through π-π stacking and hydrophobic interactions. After the local concentration reaches the critical micelle concentration (CMC = 0.2 mM), a phase transition is triggered.

[0077] (3) Targeted disruption of the cytoskeleton:

[0078] The nanofibers selectively bind to actin microfilaments, inhibit actin branching mediated by the Arp2 / 3 complex (IC 50 = 5 μM), resulting in a decrease in the cell migration rate of ≥ 80% (measured by scratch assay), and induce apoptosis of tumor cells through the mitochondrial pathway (apoptosis rate ≥ 90%, AnnexinV / PI double staining method).

[0079] 2. Characterization of key process parameters:

[0080] (1) Cyclization efficiency: Confirm the cyclic structure (molecular weight error ≤ 0.1%) through infrared spectra, fluorescence spectra, particle size analysis, and potential analysis. The results are as Figure 6 shown. According to Figure 6 It can be seen that the cyclic peptide structure was successfully prepared in this invention, that is, the tyrosinase-responsive cyclic peptide was prepared;

[0081] (2) Enzyme-responsive specificity: It triggers self-assembly only in cells with high tyrosinase activity (B16 melanoma), and normal fibroblasts (NIH / 3T3) show no response.

[0082] (3) Therapeutic window: The maximum tolerated dose in the mouse model is ≥50 mg / kg, and the tumor growth inhibition rate (TGI) reaches 75% (compared with the control group, p < 0.001).

[0083] Test Example 2

[0084] The purity of the tyrosinase-responsive cyclic peptide of Example 2 was analyzed by HPLC (C18 column, gradient elution with acetonitrile / water), and the molecular weight was confirmed by MALDI-TOF mass spectrometry. The results are shown in Table 1.

[0085] Table 1 Test results of the tyrosinase-responsive cyclic peptide of Example 2

[0086] Index Data Prior Art (Prodrug System in Background Art) Cyclization Yield 89％±2％ 30% ± 5% (Total Yield of 3-step Reaction) Product Purity (HPLC) 98.5％ 85％ Molecular Weight Error +0.03 Da (Theoretical Value: 848.92 Da) +2.1 Da (Coupling By-Product)

[0087] It can be seen from Table 1 that the preparation method provided by the present invention is significantly superior to the prior art in terms of cyclization efficiency, can obtain the target cyclic peptide more efficiently, has higher purity, reduces the generation of by-products, and is beneficial to the stability and reliability of subsequent applications (such as drug development or biomedical research).

[0088] Test Example 3

[0089] In this test example, in vitro verification of tyrosinase-triggered self-assembly was carried out to prove the formation of nanostructures of the tyrosinase-responsive cyclic peptide under the action of tyrosinase. The method is as follows:

[0090] The tyrosinase-responsive cyclic peptide of Example 2 (1 mM) was incubated with tyrosinase (50 U / mL) in PBS (pH 6.8) (37 °C, 1 h), and the group without tyrosinase or using a tyrosinase inhibitor (kojic acid, 1 mM) was used as a control.

[0091] Characterization means: The particle size of the nanoparticles was measured by dynamic light scattering (DLS); the morphology was observed by transmission electron microscopy (TEM); the generation of quinone structures was detected by fluorescence spectroscopy (excitation wavelength 320 nm, emission wavelength 420 nm). The test results are shown in Table 2.

[0092] Table 2 Test results of the tyrosinase-responsive cyclic peptide of Example 2

[0093]

[0094] As can be seen from Table 2, the tyrosinase-responsive cyclic peptide undergoes specific responsive changes under the action of tyrosinase. Kojic acid can effectively inhibit the activity of tyrosinase, preventing the responsive changes of the tyrosinase-responsive cyclic peptide and the formation of nanostructures.

[0095] Test Example 4

[0096] In this test example, in vitro cytotoxicity and selectivity were evaluated to verify the specific killing of melanoma cells by the tyrosinase-responsive cyclic peptide. The method is as follows:

[0097] Cell lines: B16 (mouse melanoma, high tyrosinase activity); L929 (mouse fibroblasts, low tyrosinase activity); U2OS (human osteosarcoma cells, other tumor cells).

[0098] Treatment conditions: Treatment with the tyrosinase-responsive cyclic peptide of Example 2 (0 - 200 μM) for 24 h.

[0099] Detection index: CCK8, and the detection results are as Figure 4 shown.

[0100] According to Figure 4 it can be seen that as the concentration of RGDKYQ-OPA increases, the survival rate of B16 cells decreases significantly, while the survival rates of L929 cells and U2OS cells do not decrease significantly, indicating that the cyclic peptide of the present invention shows a specific killing effect on B16 cells within the test concentration range. Combining its tyrosinase responsiveness, the cyclic peptide of the present invention can be used to develop a targeted drug delivery system or biosensor for melanoma.

[0101] Test Example 5

[0102] In this test example, the in vivo anti-tumor effect was evaluated (mouse model) to verify the in vivo safety and tumor inhibitory effect of the tyrosinase-responsive cyclic peptide. The method is as follows:

[0103] Model construction: C57BL / 6 mice were subcutaneously inoculated with B16 cells (5×10 5 / mouse), and grouped when the tumor volume reached 100 mm 3 (n = 6).

[0104] Drug administration plan: Experimental group: The tyrosinase-responsive cyclic peptide of Example 2 (50 mg / kg, peritumoral injection, once every 3 days); Control group: Normal saline as a blank control, and DOX-Prodrug as a control of existing drugs (10 mg / kg), with the same frequency.

[0105] Detection index: Tumor volume (measured with a vernier caliper, formula: V = 0.5 × length × width 2 );Survival period (observed for 60 days). The test results are as Figure 5As shown in Table 3.

[0106] Table 3 Test Results of Tyrosinase-Responsive Cyclic Peptide in Example 2

[0107] Group Tumor Growth Inhibition Rate (TGI) Median Survival Time (days) cTnI (ng / mL) Tyrosinase-Responsive Cyclic Peptide 75％±6％ >60 0.8±0.2 DOX-Prodrug 40％±5％ 35 3.2±0.5 Normal Saline - 28 0.7±0.1

[0108] According to Figure 5 and Table 3, it can be seen that the tyrosinase-responsive cyclic peptide has significant advantages in inhibiting tumor growth. This may be due to its specific response to tyrosinase, which enables it to release active ingredients more effectively in the tumor microenvironment and can also significantly prolong the survival period of tumor-bearing animals. The low level of the cTnI marker indicates that the tyrosinase-responsive cyclic peptide has less toxicity to the heart and higher safety, showing potential clinical application value.

[0109] Test Example 6

[0110] In this test example, the mechanism of cell migration inhibition was studied to analyze the targeting effect of tyrosinase-responsive cyclic peptide nanofibers on the actin cytoskeleton. The method is as follows:

[0111] Cell treatment: B16 cells were co-incubated with tyrosinase-responsive cyclic peptide (10 μM) for 6 h. A blank control of B16 melanoma cells and a linear peptide RGDKYQ control were set.

[0112] Detection technique: Actin microfilaments were stained with phalloidin-TRITC, and confocal microscopy imaging was performed. The detection results are as Figures 2 - 3 shown in Table 4.

[0113] Table 4 Test Results of Tyrosinase-Responsive Cyclic Peptide in Example 2

[0114] Index Tyrosinase-Responsive Cyclic Peptide Treatment Group Control Group Actin Branch Density Decrease by 85% ± 7% (p < 0.001) Normal Reticular Structure

[0115] According to Figures 2 - 3 and Table 4, it can be seen that the tyrosinase-responsive cyclic peptide can significantly reduce the actin branch density, indicating its potential application value in regulating the dynamics of the cytoskeleton. The cyclic peptide of the present invention may be used to study cytoskeleton-related biological processes, such as cell migration and morphological changes, and may even be used to develop treatment strategies for certain diseases (such as cancer metastasis).

[0116] Test Example 7

[0117] In this test example, the performance of the present invention (tyrosinase-triggered self-assembly system of tyrosinase-responsive cyclic peptide) and traditional tyrosinase-responsive systems was compared, including cytotoxicity, synthesis complexity, and in vivo tumor inhibition rate. The test method is as follows:

[0118] Cytotoxicity (CCK8 method): The CCK-8 (Cell Counting Kit-8) method was used to detect cell viability. Normal cells (L929) and tumor cells (B16) were respectively inoculated into 96-well plates, different concentrations of the compound of the present invention or control compounds were added, after culturing for a certain time, CCK8 reagent was added, and the absorbance at 450 nm was measured by an enzyme-linked immunosorbent assay (ELISA) reader to calculate the cell viability and apoptosis rate.

[0119] Synthesis complexity: The synthesis complexity was evaluated by comparing chemical synthesis routes. The synthesis steps, reaction conditions, purification methods and total yield required by the prior art and the present invention were recorded. Nuclear magnetic resonance (NMR), mass spectrometry (MS) and other techniques were used to characterize the structures of intermediates and final products to verify the synthesis efficiency and product purity.

[0120] Tumor inhibition rate in vivo: A mouse tumor model was used to evaluate the tumor inhibition effect in vivo. Tumor cells (such as B16) were inoculated subcutaneously in mice. After the tumors grew to a certain volume, they were randomly grouped and injected with the compound of the present invention or control compounds respectively. The tumor volume was measured regularly, and the tumor growth inhibition rate (TGI) was calculated. At the same time, the body weight and survival status of the mice were monitored to evaluate the maximum tolerated dose (MTD) and toxicity. The test results are shown in Table 5.

[0121] Table 5 Comparison between the present invention and traditional tyrosinase-responsive systems

[0122]

[0123] It can be seen from Table 5 that the present invention significantly improves the selective killing effect on tumor cells, while reducing the toxicity to normal cells, and has better biosafety. The synthesis steps are simplified, the yield is greatly improved, the production cost is reduced, and it has higher potential for industrial application. The maximum tolerated dose and tumor inhibition effect of the drug are significantly improved, the treatment window is expanded, and it has stronger clinical application prospects.

[0124] Test Example 8

[0125] This test example tested the effect of reaction temperature on the cyclization reaction efficiency. Tyrosinase-responsive cyclic peptides were prepared at different temperatures. The preparation method referred to Example 2, and the cyclization yield test method referred to General Principles 0512 (High Performance Liquid Chromatography) in Part IV of the Chinese Pharmacopoeia 2020 Edition or International Standard ISO 13845:2017 (HPLC Analysis Method for Peptide Compounds). The cell apoptosis rate test method referred to General Principles 1401 (Flow Cytometry) in Part IV of the Chinese Pharmacopoeia 2020 Edition or International Standard ISO 10993-5:2009 (Biological Evaluation of Medical Devices - Cytotoxicity Test). The results are shown in Table 6.

[0126] Table 6 Effect of reaction temperature on the cyclization reaction efficiency

[0127]

[0128] As can be seen from Table 6, the tyrosinase-responsive cyclic peptide prepared by the method of the present invention has a higher yield and better activity.

[0129] It can be known from the above examples that the tyrosinase-responsive cyclic peptide provided by the present invention does not require the intervention of an exogenous carrier, has a high drug-loading efficiency, good biocompatibility, high tumor targeting, and low toxicity, and is suitable for preparing drugs targeting melanoma.

[0130] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments can also be obtained based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tyrosinase-responsive cyclic peptide, characterized in that: The structure is shown in Formula I:

2. The method for preparing the tyrosinase-responsive cyclic peptide according to claim 1, characterized in that: The following steps are involved: A linear peptide precursor, a cyclization agent and a solvent are mixed to carry out a cyclization reaction to obtain the tyrosinase-responsive cyclic peptide; the amino acid sequence of the linear peptide precursor is Arg-Gly-Asp-Lys-Tyr-Gln.

3. The preparation method according to claim 2, characterized in that: The purity of the linear peptide precursor is ≥95%.

4. The preparation method according to claim 2 or 3, characterized in that: The cyclization reagent is o-phthalaldehyde.

5. The preparation method according to claim 2, characterized in that: The solvents include alcohol and water.

6. The preparation method according to claim 5, characterized in that: The volume ratio of the alcohol to water is 1:

1.

7. The preparation method according to claim 2 or 5, characterized in that: The volume ratio of the cyclization reagent to the solvent is 1-5:1-50.

8. The preparation method according to claim 2 or 3, characterized in that: The linear peptide precursor, cyclization reagent and solvent are mixed to obtain a linear peptide precursor reaction solution, wherein the concentration of the linear peptide precursor in the linear peptide precursor reaction solution is 1-10 mg / mL.

9. The preparation method according to claim 2, characterized in that: The mixing is magnetic stirring; the rotation speed of the magnetic stirring is 100-500 rpm, the mixing time is 10-50 min, the mixing temperature is 20-30° C., and the pressure is normal pressure.

10. Use of the tyrosinase-responsive cyclic peptide according to claim 1 or the tyrosinase-responsive cyclic peptide obtained by the preparation method according to any one of claims 2 to 9 in the preparation of a drug targeting melanoma.

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