Supramolecular polypeptide coupling medicine as well as preparation method and application thereof

Abstract

The invention relates to a supramolecular polypeptide coupled drug as well as a preparation method and application thereof. The supramolecular polypeptide coupled drug comprises a conjugate and an anti-tumor functional unit, wherein the conjugate is formed by sequentially connecting tumor targeting peptide, an enzyme response connexon and cucurbit [7] uril, and the anti-tumor functional unit is loaded onto the cucurbit [7] uril through non-covalent host-guest interaction. In the supramolecular polypeptide coupled drug, an anti-tumor functional unit is loaded on cucurbit [7] uril through non-covalent host-guest interaction, chemical modification of small-molecular drugs in a traditional method is avoided, and cucurbit [7] uril as a carrier can be compatible with various small-molecular hydrophobic drugs; due to the introduction of the tumor targeting peptide, the efficient enrichment of the medicine in tumor tissues can be realized; the unique design of the enzyme response connexon can realize rapid and efficient release of the anti-tumor functional unit in tumor cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a supramolecular polypeptide conjugate, its preparation method, and its application. Background Technology

[0002] Malignant tumors are among the major diseases that seriously threaten human health, and their high incidence and mortality rates make them a major public health problem worldwide. Traditional cancer treatments such as surgery, radiotherapy, and chemotherapy, while widely used, still have certain limitations, such as limited efficacy, significant side effects, and high rates of recurrence and metastasis.

[0003] Antibody-drug conjugates (ADCs) and peptide-drug conjugates (PDCs) have emerged as some of the fastest-growing drug categories in the field of targeted cancer therapy in recent years. These two types of drugs consist of tumor-specific monoclonal antibodies or targeting peptides conjugated to small-molecule cytotoxic agents via linkers. Leveraging the targeting ability of the antibody or peptide, the cytotoxic payload is delivered directly to tumor cells, overcoming the limitations of traditional chemotherapy drugs' non-specific toxicity and the insufficient efficacy of antibody drugs. They are hailed as "biological missiles" in cancer treatment. To date, 19 antibody-drug conjugates and 2 peptide-drug conjugates have been launched on the market.

[0004] Currently, small molecule cytotoxins are mainly covalently coupled to antibodies or peptides for targeted therapy. However, this strategy has several limitations: First, the toxin molecules often require chemical modification before coupling, inevitably altering their physicochemical properties and affecting efficacy. Second, drug release after entering the cell lacks precise control, easily leading to insufficient or premature release, resulting in decreased efficacy and increased systemic toxicity. Third, this strategy relies on specific toxin structures, making it difficult to rapidly extend to small molecule drugs with diverse structures and mechanisms of action, thus severely limiting the universality and scalability of the conjugation platform. Therefore, there is an urgent need to develop a novel drug conjugation strategy that avoids chemical modification of small molecule drugs, is applicable to a variety of small molecule drugs, and establishes an efficient and controllable release mechanism to ensure precise activation of small molecule drugs within cells. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a supramolecular polypeptide conjugate drug, and more particularly to provide a supramolecular polypeptide conjugate drug, its preparation method, and its application.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a supramolecular polypeptide conjugate drug, the supramolecular polypeptide conjugate drug comprising: a conjugate formed by sequentially linking a tumor-targeting peptide, an enzyme-responsive linker, and cucurbit[7]urea, and an antitumor functional unit loaded onto cucurbit[7]urea through non-covalent host-guest interactions.

[0008] Preferably, the tumor-targeting peptide is any one or a combination of at least two of the following: a peptide targeting HER2, a peptide targeting EGFR, and a peptide targeting integrins.

[0009] Preferably, the polypeptide sequence targeting HER2 is selected from YCDGFYACYMDV.

[0010] Preferably, the EGFR-targeting polypeptide sequence is selected from YHWYGYTPQNVI.

[0011] Preferably, the polypeptide sequence of the targeting integrin is selected from RGD.

[0012] Preferably, the enzyme-responsive linker is a polypeptide sequence that can be cleaved by cathepsin B.

[0013] The supramolecular polypeptide conjugate designed in this invention utilizes the specific binding of tumor-targeting peptides to tumor surface receptors to deliver anti-tumor functional units to tumor cells, which then enter the lysosomes of tumor cells via endocytosis. The cathepsin B, which is highly expressed in the lysosomes, can cleave the enzyme-responsive linker, exposing the N-terminal phenylalanine residues that have a strong host-guest interaction with cucurbita[7]urea, thereby replacing the anti-tumor functional units originally loaded in cucurbita[7]urea, achieving specific targeted delivery and efficient intracellular release of the drug.

[0014] Preferably, the polypeptide sequence of the enzyme-responsive linker is selected from GLFG and / or FKFL.

[0015] Preferably, the enzyme-responsive linker is linked to the tumor-targeting peptide via a hydrophobic alkyl chain.

[0016] Preferably, the enzyme-responsive linker is linked to cucurbita[7]urea via a hydrophobic alkyl chain and an azide group-modified lysine.

[0017] Preferably, the hydrophobic alkyl chain is an alkyl chain containing 2 to 12 carbon atoms.

[0018] The specific point values ​​from 2 to 12 can be selected as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0019] Preferably, the ratio of the number of cucurbit[7]urea coupled to the enzyme-responsive linker is 1:1.

[0020] Preferably, the antitumor functional unit is an antitumor drug.

[0021] Preferably, the antitumor drug is selected from any one or a combination of at least two of oxaliplatin, paclitaxel, or camptothecin.

[0022] In a second aspect, the present invention provides a method for preparing a supramolecular polypeptide-conjugated drug as described in the first aspect, the method comprising:

[0023] Using resin as a carrier and lysine modified with azide groups as the starting amino acid, azide-modified lysine, hydrophobic alkyl chain, enzyme-responsive linker, hydrophobic alkyl chain, and tumor-targeting peptide were sequentially linked using a polypeptide solid-phase synthesis method to prepare azide-modified tumor-targeting peptide-enzyme-responsive linker-lysine conjugate.

[0024] Mono-alkynyl-substituted cucurbit[7]urea was prepared and coupled with an azide-modified tumor-targeting peptide-enzyme-responsive linker-lysine conjugate to obtain a polypeptide-cucurbit[7]urea conjugate.

[0025] Antitumor functional units were loaded into polypeptide-cucurbita[7]urea conjugates to obtain supramolecular polypeptide conjugates.

[0026] Thirdly, the present invention provides the use of supramolecular polypeptide-conjugated drugs as described in the first aspect in the preparation of medicaments for the diagnosis, prevention or treatment of tumors.

[0027] Preferably, the tumor is selected from any one of colon cancer, breast cancer, stomach cancer, leukemia, bladder cancer, cervical cancer, or nasopharyngeal carcinoma.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention creatively constructs a supramolecular polypeptide-conjugated drug that utilizes the specific binding of tumor-targeting peptides to tumor surface receptors to deliver anti-tumor functional units to tumor cells and enter the lysosomes of tumor cells through endocytosis. The cathepsin B highly expressed in the lysosomes can cleave the enzyme-responsive linker, exposing the N-terminal phenylalanine residues that have strong host-guest interactions with cucurbita[7]urea, thereby replacing the anti-tumor functional units originally loaded in cucurbita[7]urea, achieving specific targeted delivery and efficient intracellular release of the drug.

[0030] The antitumor functional unit is loaded onto cucurbita[7]urea through non-covalent host-guest interactions, avoiding the chemical modification of small molecule drugs in traditional methods; the introduction of tumor-targeting peptides can achieve efficient enrichment of drugs in tumor tissues; the unique design of enzyme-responsive linkers can achieve rapid and efficient release of antitumor functional units in tumor cells. Compared with traditional peptide-coupled drugs, this supramolecular peptide-coupled drug does not chemically modify the drug and can be widely adapted to a variety of hydrophobic drugs, with broad application prospects. Attached Figure Description

[0031] Figure 1 A schematic diagram illustrating the structure and working principle of supramolecular peptide-conjugated drugs;

[0032] Figure 2 The diagram shows the molecular structure of the polypeptide-cucurbit[7]urea involved in each preparation example of the present invention, wherein (1) to (8) correspond to the molecular structure of the polypeptide-cucurbit[7]urea prepared in the corresponding preparation examples 1 to 8 respectively.

[0033] Figure 3 These are the mass spectra of "polypeptide-cucurbit[7]urea" in each preparation example of the present invention, wherein (1) to (8) correspond to the mass spectra of "polypeptide-cucurbit[7]urea" prepared in preparation examples 1 to 8 respectively;

[0034] Figure 4 The liquid chromatogram of the "polypeptide-cucurbita[7]urea" obtained in Preparation Example 1 of the present invention;

[0035] Figure 5 The figures show the drug release curve of the supramolecular polypeptide conjugate obtained in Example 1 of the present invention in the presence of cathepsin B (Figure a) and the mass spectrometry characterization of the main product of the supramolecular polypeptide conjugate after enzyme cleavage (Figure b).

[0036] Figure 6 This is a tumor enrichment analysis diagram of the supramolecular polypeptide conjugate and oxaliplatin obtained in Example 1 of the present invention in a tumor-bearing mouse model.

[0037] Figure 7 The image shows the antitumor activity of the supramolecular polypeptide conjugate obtained in Example 1 of this invention in a tumor-bearing mouse model (Figure a) and the changes in body weight of the mice during treatment (Figure b). Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be considered as specific limitations thereof.

[0039] Where specific techniques or conditions are not specified in the preparation and testing examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0040] All animal experiments involved in this invention have been reviewed and approved by the Laboratory Animal Ethics Committee of the National Center for Nanoscience and Technology, and comply with the requirements of laboratory animal welfare and ethics and relevant laws and regulations.

[0041] Preparation Example 1

[0042] The tumor-targeting peptide used in this preparation example is the EGFR-targeting polypeptide sequence YHWYGYTPQNVI; the enzyme-responsive linker polypeptide sequence is FKFL; the hydrophobic alkyl chains are all alkyl chains containing 2 carbon atoms, and their molecular structures are as follows: Figure 2 As shown in (1).

[0043] In this preparation example, a "polypeptide-cucurbita[7]urea" structure was constructed and prepared by the following method:

[0044] 1) Preparation of azide-modified tumor-targeting peptide-enzyme-responsive linker-lysine conjugate using solid-phase peptide synthesis: Rink amide AM resin (amino acid loading rate 0.35 mmol / g) was used. The first coupled amino acid was an azide-substituted Fmoc-protected lysine, i.e., Fmoc-Lys(N3)-OH. Subsequent steps involved repeated deprotection of Fmoc and coupling with the next amino acid. Finally, the peptide was cleaved from the resin and purified using preparative liquid chromatography to obtain the azide-modified tumor-targeting peptide-enzyme-responsive linker-lysine conjugate.

[0045] 2) Synthesis of mono-alkynyl-substituted cucurbit[7]urea: 5.0 g (4.3 mmol) of cucurbit[7]urea was weighed and dissolved in 500 mL of deionized water. Under nitrogen protection, 2.2 g of ammonium persulfate was added, and the mixture was stirred at 85 °C for 12 h. The reaction solution was lyophilized and purified by preparative liquid chromatography to obtain 1.1 g (0.75 mmol) of monohydroxy-substituted cucurbit[7]urea. The obtained monohydroxy-substituted cucurbit[7]urea was dissolved in a mixture of 10 mL of dry DMF and 30 mL of dry DMSO. Under nitrogen protection, 0.56 g (14 mmol) of sodium hydride was added, and the mixture was stirred at room temperature for 4 h. Then, 6 mL (10 mmol) of bromopropyne was added under ice-water bath conditions, and the reaction was continued for 4 h. After the reaction was completed, a large amount of methanol was added to the reaction solution to fully precipitate the product. The product was centrifuged, washed three times with methanol, and dried overnight in a vacuum drying oven. Finally, 500 mg (0.33 mmol) of mono-alkynyl-substituted cucurbita[7]urea was obtained.

[0046] 3) Synthesis of “peptide-cucurbita[7]urea” conjugate: 100 mg of mono-alkynyl-substituted cucurbita[7]urea (0.065 mmol) was weighed, dissolved in 4 mL of deionized water, and an equimolar proportion of azide-modified tumor-targeting peptide-enzyme-lysine conjugate was added. Nitrogen gas was then introduced to remove oxygen. Subsequently, 2 mg (0.008 mmol) CuSO4·5H2O and 5 mg (0.025 mmol) sodium ascorbate were added, and the mixture was stirred at 55 °C for 12 h. After the reaction was completed, the mixture was purified by preparative liquid chromatography to obtain a white powder, which is the “peptide-cucurbita[7]urea” conjugate.

[0047] Preparation Example 2

[0048] The tumor-targeting peptide used in this preparation example is the EGFR-targeting polypeptide sequence YHWYGYTPQNVI; the enzyme-responsive linker polypeptide sequence is GLFG; the hydrophobic alkyl chains are all alkyl chains containing 2 carbon atoms, and their molecular structures are as follows: Figure 2 As shown in (2).

[0049] In this preparation example, a "polypeptide-cucurbita[7]urea" structure was constructed. The difference between the preparation method and the preparation example 1 is that the enzyme response linker in step (1) was replaced with the polypeptide sequence of GLFG.

[0050] Preparation Example 3

[0051] The tumor-targeting peptide used in this preparation example is the EGFR-targeting polypeptide sequence YHWYGYTPQNVI; the enzyme-responsive linker polypeptide sequence is FKFL, and the hydrophobic alkyl chains are all alkyl chains containing 4 carbon atoms, with the molecular structure as follows: Figure 2 As shown in (3).

[0052] In this preparation example, a "polypeptide-cucurbita[7]urea" structure was constructed. The difference between the preparation method and the preparation example 1 is that in step (1), the hydrophobic alkyl chain connecting the tumor-targeting peptide and the enzyme-responsive linker, the linker of the enzyme-responsive linker and the lysine modified by the azide group were all replaced with an aliphatic chain containing 4 carbon atoms.

[0053] Preparation Example 4

[0054] The tumor-targeting peptide used in this preparation example is the EGFR-targeting polypeptide sequence YHWYGYTPQNVI; the enzyme-responsive linker polypeptide sequence is GLFG; the hydrophobic alkyl chains are all alkyl chains containing 4 carbon atoms, and their molecular structures are as follows: Figure 2 As shown in (4).

[0055] In this preparation example, a "polypeptide-cucurbita[7]urea" structure was constructed. The difference between the preparation method and the preparation example 1 is that the enzyme response linker in step (1) was replaced with the polypeptide sequence of GLFG; the hydrophobic alkyl chain connecting the tumor-targeting peptide and the enzyme response linker, and connecting the enzyme response linker and the lysine modified by the azide group in step (1) were all replaced with an aliphatic chain containing 4 carbon atoms.

[0056] Preparation Example 5

[0057] The tumor-targeting peptide used in this preparation example is the HER2-targeting polypeptide sequence YCDGFYACYMDV; the enzyme-responsive linker polypeptide sequence is FKFL; the hydrophobic alkyl chains are all alkyl chains containing 2 carbon atoms, and their molecular structures are as follows: Figure 2 As shown in (5).

[0058] In this preparation example, a "peptide-cucurbita[7]urea" structure was constructed. The difference between the preparation method and the preparation example 1 is that the tumor-targeting peptide connected in step (1) was replaced with the polypeptide sequence YCDGFYACYMDV that targets HER2.

[0059] Preparation Example 6

[0060] The tumor-targeting peptide used in this preparation example is the HER2-targeting polypeptide sequence YCDGFYACYMDV; the enzyme-responsive linker polypeptide sequence is GLFG; the hydrophobic alkyl chains are all alkyl chains containing 2 carbon atoms, and their molecular structures are as follows: Figure 2 As shown in (6).

[0061] In this preparation example, a "polypeptide-cucurbita[7]urea" structure was constructed. The difference between the preparation method and the preparation example 1 is that the tumor-targeting peptide in step (1) was replaced with the HER2-targeting polypeptide sequence YCDGFYACYMDV; and the enzyme-response linker in step (1) was replaced with the GLFG polypeptide sequence.

[0062] Preparation Example 7

[0063] The tumor-targeting peptide used in this preparation example is the peptide sequence RGD, which targets integrin; the peptide sequence of the enzyme-responsive linker is FKFL, and the hydrophobic alkyl chains are all alkyl chains containing 2 carbon atoms, and their molecular structures are as follows. Figure 2 As shown in (7).

[0064] In this preparation example, a "peptide-cucurbita[7]urea" structure was constructed. The difference between the preparation method and that of Preparation Example 1 is that the tumor-targeting peptide linked in step (1) was replaced with the peptide sequence RGD targeting integrin.

[0065] Preparation Example 8

[0066] The tumor-targeting peptide used in this preparation example is the polypeptide sequence RGD, which targets integrin; the polypeptide sequence of the enzyme-responsive linker used is GLFG, and the hydrophobic alkyl chain is an alkyl chain containing 2 carbon atoms, and its molecular structure is as follows. Figure 2 As shown in (8).

[0067] In this preparation example, a "polypeptide-cucurbita[7]urea" structure was constructed. The difference between the preparation method and the preparation example 1 is that the tumor-targeting peptide in step (1) was replaced with the polypeptide sequence RGD targeting integrin; and the enzyme-response linker in step (1) was replaced with the polypeptide sequence GLFG.

[0068] Test Example 1

[0069] The molecular weight of the "peptide-cucurbita[7]urea" conjugate was determined by electrospray ionization mass spectrometry (ESI-MS). The specific method is as follows: The "peptide-cucurbita[7]urea" conjugate was dissolved in a mixture of ultrapure water, mass spectrometry grade methanol, and mass spectrometry grade acetonitrile (the volume ratio of ultrapure water, mass spectrometry grade methanol, and mass spectrometry grade acetonitrile was 1:1:1), and the concentration was set to 5 μM. The ESI-MS was used for testing.

[0070] like Figure 3 As shown, the molecular weight characterization mass spectra of the "polypeptide-cucurbit[7]urea" conjugates were obtained. (1) to (8) correspond to the mass spectra of the "polypeptide-cucurbit[7]urea" conjugates obtained in Preparation Examples 1 to 8, respectively.

[0071] Test Example 2

[0072] The purity of the "peptide-cucurbita[7]urea" conjugate obtained in Preparation Example 1 was tested by high performance preparative liquid chromatography (HPLC). The specific method is as follows: The "peptide-cucurbita[7]urea" conjugate was dissolved in a mixture of ultrapure water and mass spectrometry grade acetonitrile (the volume ratio of ultrapure water to mass spectrometry grade acetonitrile was 1:1), and the concentration was set to 1 mg / mL. The concentration was then tested by HPLC. Figure 4 The liquid chromatogram of the "polypeptide-cucurbit[7]urea" conjugate obtained in Example 1 was prepared. The results showed that the "polypeptide-cucurbit[7]urea" conjugate had no obvious large impurity peaks, and the purity was 98%, with good retention and separation.

[0073] Test Example 3

[0074] Affinity test of polypeptide-cucurbita[7]urea conjugate with small molecule drugs

[0075] In this test example, the affinity of the polypeptide-cucurbita[7]urea conjugate obtained in Preparation Example 1 to the small molecule drug oxaliplatin was tested. In the first step, the AutoDock Vina software was used for calculation simulation. The binding energy of the optimal structure obtained by calculation was less than or equal to -8 kcal / mol, which can preliminarily confirm that the polypeptide-cucurbita[7]urea conjugate and the small molecule drug can form a stable supramolecular polypeptide conjugate drug.

[0076] The second step involved using an isothermal titration calorimeter to determine the actual binding constant between the polypeptide-cucurbita[7]urea conjugate and the small molecule drug. The experimental value was greater than or equal to 1×10⁻⁶. 6 M -1 Therefore, it is determined that the "polypeptide-cucurbita[7]urea" conjugate has the ability to form a stable supramolecular polypeptide conjugate with small molecule drugs.

[0077] The results showed that the simulated binding energy of the "polypeptide-cucurbita[7]urea" conjugate to oxaliplatin was -8.1 kcal / mol, while the experimentally measured value was 2 × 10⁻⁶ kcal / mol. 6 M -1 Therefore, oxaliplatin can be identified as a suitable candidate drug for supramolecular peptide conjugates.

[0078] Example 1

[0079] This embodiment provides a supramolecular polypeptide-conjugated drug, and the specific preparation method is as follows:

[0080] The polypeptide-cucurbita[7]urea conjugate obtained in Preparation Example 1 was dissolved in DMSO, and the small molecule drug oxaliplatin was dissolved in pure water at an equimolar ratio, with a concentration of 5 mM for both. Nine times the volume of 5% glucose injection was added to make their respective concentrations 500 μM. The mixture was vigorously mixed for 1 minute at a speed of 3000 rpm using a vortex mixer to obtain a supramolecular polypeptide conjugate drug solution.

[0081] Test Example 4

[0082] Enzyme-responsive drug release of supramolecular peptide-conjugated drugs

[0083] The enzyme-responsive drug release was performed using the supramolecular polypeptide-conjugated drug obtained in Example 1, as follows:

[0084] (1) Dissolve 2 U of human cathepsin B in 100 μL of ultrapure water.

[0085] (2) Weigh 15.4 mg (0.1 mmol) dithiothreitol and 14.6 mg (0.05 mmol) ethylenediaminetetraacetic acid and dissolve them in 1 mL of PBS buffer at pH=6.

[0086] (3) Mix 24 μL of human cathepsin B solution with 6 μL of dithiothreitol / ethylenediaminetetraacetic acid solution and incubate at 37 °C for 15 min.

[0087] (4) Add 20 μL of 500 μM supramolecular peptide-conjugated drug and 50 μL of pH 6 PBS, mix well, and incubate at 37 °C for 15, 30, and 60 min. The enzyme-responsive drug release was tested by HPLC and ESI-MS.

[0088] Experimental results are as follows Figure 5As shown in the figure, under the action of cathepsin B, the drug release efficiencies of supramolecular peptide-conjugated drugs at 20, 40, and 60 min were 62%, 79%, and 85%, respectively (Figure a). Furthermore, it was determined that at 60 min, 90% of the supramolecular peptide-conjugated drugs contained intramolecular supramolecular cyclic structures in the product after the reaction with the enzyme, which could efficiently replace the drug (Figure b).

[0089] Test Example 5

[0090] Tumor enrichment analysis of supramolecular peptide-conjugated drugs in tumor-bearing mouse models was conducted to evaluate their in vivo delivery characteristics and tumor targeting capabilities.

[0091] The supramolecular polypeptide conjugate obtained in Example 1 was used to perform tumor enrichment analysis in a tumor-bearing mouse model. The specific method is as follows:

[0092] (1) Establishment of tumor-bearing model: 6-8 week old female BALB / c nude mice were selected, and HCT-116 tumor cell suspension was subcutaneously injected into the right axilla to establish a tumor-bearing model. The cell concentration was 1×10⁻⁶. 6 One per tumor; until the tumor volume grows to approximately 100 mm. 3 At that time, random groups were used for subsequent experiments.

[0093] (2) Tumor-bearing mice were randomly divided into a supramolecular peptide-conjugated drug (SPD) group and an oxaliplatin control group, with 3 mice in each group. The SPD and oxaliplatin were dissolved in 5% glucose injection and administered via tail vein injection at a dose of 4 mg / kg (the dose of the SPD was calculated based on the amount of oxaliplatin contained in it). Twenty-four hours after administration, the tumor-bearing mice were sacrificed, and the major organs (heart, liver, spleen, lung, and kidney) and tumors were harvested.

[0094] (3) Drug distribution detection: The drug concentration in each tissue was quantitatively analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) and inductively coupled plasma mass spectrometry (ICP-MS). Among them, for supramolecular peptide conjugates, the platinum content was also determined by ICP-MS to determine the distribution of the drug in each tissue.

[0095] Experimental results showed that, compared to oxaliplatin, supramolecular peptide-conjugated drugs increased enrichment in tumor tissues of tumor-bearing mice by 2.2 times ( Figure 6 The distribution of the drug in the kidneys of tumor-bearing mice decreased by 45%. These results indicate that supramolecular peptide-conjugated drugs possess good in vivo stability and tumor-targeting delivery capabilities, effectively improving drug accumulation efficiency at tumor sites and providing a basis for subsequent anti-tumor efficacy studies.

[0096] Test Example 6

[0097] Evaluation of the antitumor effects of supramolecular peptide-conjugated drugs in tumor-bearing mouse models.

[0098] The antitumor effect of the supramolecular polypeptide-conjugated drug obtained in Example 1 in a tumor-bearing mouse model was evaluated using the following specific method:

[0099] Select an established tumor-bearing mouse model (tumor volume approximately 100 mm). 3 Mice were randomly divided into three groups of four each. The groups were administered supramolecular peptide-conjugated drugs (SPDs), oxaliplatin, and saline, respectively, via tail vein injection. Both the SPD group (based on oxaliplatin dosage) and the oxaliplatin group received 4 mg / kg of medication every two days for a total of five administrations. During treatment, mouse weight and tumor volume were measured periodically. Tumor volume was calculated using the formula V = 0.5 × a × b² (where a is the major axis and b is the minor axis). At the end of the experiment, the animals were sacrificed, and the tumor tissue was dissected and weighed. The tumor inhibition rate (TIR) ​​was calculated as [1 - W]. t / W n ]×100%, where W t and W n (The average tumor weights of the treatment group and the control group are respectively).

[0100] Experimental results are as follows Figure 7 As shown in the figure, the supramolecular peptide-conjugated drug significantly inhibited tumor growth, with a significantly smaller tumor volume compared to the control group, achieving a tumor inhibition rate of 80% (Figure a). Animal weight remained stable (Figure b), indicating that the supramolecular peptide-conjugated drug had no significant toxic side effects. Compared to the oxaliplatin group, the supramolecular peptide-conjugated drug also exhibited a better tumor inhibition rate and had less impact on animal weight.

[0101] In summary, this invention provides a supramolecular peptide-conjugated drug composed of a tumor-targeting peptide, an enzyme-responsive linker, cucurbita[7]urea, and its loaded anti-tumor functional unit. This supramolecular peptide-conjugated drug can significantly increase the enrichment efficiency of the drug in tumor tissues, can efficiently release the loaded small molecule toxic drug in tumor cells, and exhibits good anti-tumor activity and in vivo safety in tumor-bearing mouse models, proving that it has excellent targeted delivery and therapeutic potential. Compared with traditional peptide-conjugated drugs, this supramolecular peptide-conjugated drug does not chemically modify the drug, can be widely adapted to a variety of hydrophobic drugs, and has broad application prospects.

[0102] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A supramolecular polypeptide-conjugated drug, characterized in that, The supramolecular polypeptide conjugate includes: a conjugate formed by sequentially linking a tumor-targeting peptide, an enzyme-responsive linker, and cucurbit[7]urea, and an antitumor functional unit loaded onto cucurbit[7]urea through non-covalent host-guest interactions.

2. The supramolecular polypeptide conjugate drug according to claim 1, characterized in that, The tumor-targeting peptide is any one or a combination of at least two of the following: a peptide targeting HER2, a peptide targeting EGFR, and a peptide targeting integrins. Preferably, the polypeptide sequence targeting HER2 is selected from YCDGFYACYMDV; Preferably, the EGFR-targeting polypeptide sequence is selected from YHWYGYTPQNVI; Preferably, the polypeptide sequence of the targeting integrin is selected from RGD.

3. The supramolecular polypeptide conjugate drug according to claim 1 or 2, characterized in that, The enzyme-responsive linker is a polypeptide sequence that can be cleaved by cathepsin B.

4. The supramolecular polypeptide conjugate drug according to any one of claims 1 to 3, characterized in that, The polypeptide sequence of the enzyme-responsive linker is selected from GLFG and / or FKFL; Preferably, the enzyme-responsive linker is linked to the tumor-targeting peptide via a hydrophobic alkyl chain; Preferably, the enzyme-responsive linker is linked to cucurbita[7]urea via a hydrophobic alkyl chain and an azide group-modified lysine.

5. The supramolecular polypeptide conjugate drug according to any one of claims 1 to 4, characterized in that, The hydrophobic alkyl chain is an alkyl chain containing 2 to 12 carbon atoms.

6. The supramolecular polypeptide conjugate drug according to any one of claims 1 to 5, characterized in that, The ratio of the number of cucurbit[7]urea coupled to the enzyme-responsive linker is 1:

1.

7. The supramolecular polypeptide conjugate drug according to any one of claims 1 to 6, characterized in that, The anti-tumor functional unit is an anti-tumor drug; Preferably, the antitumor drug is selected from any one or a combination of at least two of oxaliplatin, paclitaxel, or camptothecin.

8. The method for preparing a supramolecular polypeptide-conjugated drug according to any one of claims 1 to 7, characterized in that, The preparation method includes: Using resin as a carrier and lysine modified with azide groups as the starting amino acid, azide-modified lysine, hydrophobic alkyl chain, enzyme-responsive linker, hydrophobic alkyl chain, and tumor-targeting peptide were sequentially linked using a polypeptide solid-phase synthesis method to prepare azide-modified tumor-targeting peptide-enzyme-responsive linker-lysine conjugate. Mono-alkynyl-substituted cucurbit[7]urea was prepared and coupled with an azide-modified tumor-targeting peptide-enzyme-responsive linker-lysine conjugate to obtain a polypeptide-cucurbit[7]urea conjugate. Antitumor functional units were loaded into polypeptide-cucurbita[7]urea conjugates to obtain supramolecular polypeptide conjugates.

9. The use of supramolecular polypeptide-conjugated drugs according to any one of claims 1 to 7 in the preparation of drugs for the diagnosis, prevention or treatment of tumors.

10. The application according to claim 9, characterized in that, The tumor is selected from any one of colon cancer, breast cancer, stomach cancer, leukemia, bladder cancer, cervical cancer, or nasopharyngeal carcinoma.

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