A polypeptide conjugate, a hydrogel, a use and an antitumor drug

Abstract

The application belongs to the technical field of biotechnology, and discloses a polypeptide conjugate, which comprises a polypeptide and a chemotherapeutic drug; the polypeptide and the chemotherapeutic drug are connected through a covalent bond; the polypeptide has at least one D-type amino acid; and the polypeptide is formed by connection of a self-assembling polypeptide and a cell-penetrating peptide. Researches of the present application find that when D-type amino acids are used to replace corresponding amino acids in polypeptides with cell-penetrating and self-assembling functions, the water solubility and antitumor activity of the chemotherapeutic drug with poor water solubility can be effectively improved. Meanwhile, the application also provides a hydrogel, application of the conjugate and a medicine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a polypeptide conjugate, a hydrogel, a use and an antitumor drug. BACKGROUND

[0002] According to the International Agency for Research on Cancer of the World Health Organization, 1 in 5 people in the world will suffer from cancer in their lifetime, and the prevention and treatment of cancer has become one of the most important public health challenges in the 21st century. The main advantage of chemotherapy is fast-acting, and chemotherapy drugs can kill a large number of tumor cells, but they have poor specificity and often cause serious side effects, which is called palliative chemotherapy. Therefore, patients are afraid of chemotherapy, which reduces patient compliance and is not conducive to recovery. Therefore, it is urgent to develop new tumor treatment strategies to efficiently and specifically target tumor cells, thereby improving tumor treatment effects and reducing toxicity to normal tissues.

[0003] CN102552929A discloses a method for improving the targeting selectivity of a drug delivery system by modifying a cell-penetrating peptide, which is described as follows: the drug delivery system is composed of an activatable cell-penetrating peptide capable of improving the targeting selectivity and an anti-cancer and / or tracer and / or biologically active substance and / or carrier. The activatable cell-penetrating peptide includes a shielding peptide sequence, an enzymatic substrate peptide sequence, and a cell-penetrating peptide sequence, and the enzymatic substrate peptide sequence is between the shielding peptide sequence and the cell-penetrating peptide sequence. It is further described that the anti-cancer drugs include doxorubicin, paclitaxel, docetaxel, mitomycin, daunorubicin, cisplatin, carboplatin, camptothecin, hydroxycamptothecin, vincristine, bleomycin, 5-fluorouracil, cyclophosphamide, gemcitabine, methotrexate, capecitabine, lomustine, etoposide, the tracers include fluorescent substances, radioisotopes, quantum dots, and the biologically active substances include DNA, RNA, proteins or peptide substances.

[0004] The scheme discloses that the cell-penetrating peptide is combined with paclitaxel to achieve targeted delivery to target cells.

[0005] In Example 5, Example 6, and Example 7 of the case, the liposome and the micelle form are used to encapsulate the liposome, and the essential reason is that the case only finds that the cell-penetrating peptide has a cell delivery effect on paclitaxel, and does not solve the problem of water solubility optimization before delivery, so it must rely on the liposome or the micelle to achieve the purpose of the scheme.

[0006] Moreover, the polypeptide of the case has good water solubility, which further proves that the polypeptide of the case has no substantial contribution to the water solubility optimization of paclitaxel.

[0007] The present applicant has found in further research that some chemotherapy drugs such as paclitaxel and camptothecin have poor water solubility, resulting in low cell uptake rate and inability to fully exhibit the antitumor activity of the chemotherapy drugs.

[0008] Therefore, the technical problem solved by the present application is how to improve the cell uptake rate and anti-tumor activity of a poorly water-soluble chemotherapeutic drug. SUMMARY

[0009] In view of the deficiencies of the prior art, the present application aims to provide a polypeptide conjugate. The present application has found that when a D-type amino acid is used to replace a corresponding amino acid in a polypeptide having a membrane-penetrating and self-assembling function, the water solubility and anti-tumor activity of a poorly water-soluble chemotherapeutic drug can be effectively improved.

[0010] Meanwhile, the present application also provides a hydrogel, an application of the conjugate and a medicament.

[0011] To achieve the object of the present application, the present application adopts the following technical solution: a polypeptide conjugate comprising a polypeptide and a chemotherapeutic drug; the polypeptide and the chemotherapeutic drug are connected by a covalent bond; the polypeptide has at least one D-type amino acid; and the polypeptide is formed by connecting a self-assembling polypeptide and a membrane-penetrating peptide.

[0012] In the polypeptide conjugate described above, the amino acid sequence of the polypeptide is as shown in SEQ ID No. 1.

[0013] The polypeptide as shown in SEQ ID No. 1 described in the present application is discussed in detail in the applicant's prior application CN116253805B;

[0014] The prior application comprehensively discusses the polypeptide from the aspects of characterization, stability, toxicity, cell uptake rate, anti-tumor mechanism, etc. It has been found through research that the cell uptake rate of polypeptide WDL is 158.3 times that of polypeptide WLL, and the cell uptake rate of polypeptide WDD is 541.9 times that of polypeptide WLL after 24 hours. The prior application can prove that the D-type amino acid has a greater contribution to the cell uptake rate.

[0015] In the present application, the polypeptide and the chemotherapeutic drug are connected by a covalent bond, and it is surprisingly found that this not only significantly optimizes the cell uptake rate, but also significantly improves the water solubility of the poorly water-soluble chemotherapeutic drug, thereby achieving the purpose of improving the anti-tumor activity of the chemotherapeutic drug.

[0016] Preferably, all the amino acids in the self-assembling polypeptide and / or the membrane-penetrating peptide are D-type amino acids.

[0017] Preferably, the chemotherapeutic drug is paclitaxel, camptothecin, navelbine, SN38 or exatecan.

[0018] Meanwhile, the present application also discloses a hydrogel containing the polypeptide conjugate as described above.

[0019] The polypeptide conjugate is used in the preparation of an antitumor drug.

[0020] Finally, the application also discloses an antitumor drug containing the polypeptide conjugate.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] The application adds D-type amino acids into the synthesis of the polypeptide, which can significantly enhance the stability and improve the cell uptake rate, and covalently connects the polypeptide containing D-type amino acids with camptothecin to form PDC, which has significantly enhanced water solubility, significantly improved cell uptake rate, and significantly enhanced antitumor activity in vivo compared with camptothecin and the peptide alone.

[0023] The polypeptide conjugate drug group of the application can significantly slow down the growth of tumors, has better curative effect than the chemotherapy drug camptothecin, and has important research value. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The molecular structure of the polypeptide conjugate provided for the embodiment 1 of the application;

[0025] Figure 2 The serum stability test results of the polypeptide conjugate drug synthesized by the full D-type amino acid;

[0026] Figure 3 The lipophilicity test results of the polypeptide conjugate drug;

[0027] Figure 4 The water solubility test results of the polypeptide conjugate drug;

[0028] Figure 5 The cell uptake rate test results of the polypeptide conjugate drug;

[0029] Figure 6 The cell uptake rate test results of the polypeptide conjugate drug;

[0030] Figure 7 The cytotoxicity test results of the polypeptide conjugate drug;

[0031] Figure 8 The mouse antitumor test results of the polypeptide conjugate drug. DETAILED DESCRIPTION

[0032] The technical solutions of the application are further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the application and should not be regarded as specific limitations on the application.

[0033] The specific experimental steps or conditions are not specified in the examples, which can be carried out according to the conventional experimental steps described in the literature or the operation or conditions. The reagents or instruments used are not specified by the manufacturer, which are conventional reagent products that can be obtained by purchase.

[0034] Example 1

[0035] Drug synthesis

[0036] The synthesis is carried out by using dichloride resin as a carrier. The required amino acid, small molecule drug, HBTU and DIEA are respectively weighed and dissolved, and then put into different synthesis bottles. The synthesis is set according to the operation of the automatic synthesis instrument. The resin is cut and precipitated to obtain the crude polypeptide. The corresponding polypeptide conjugated drug lyophilized powder can be obtained by purification and lyophilization by high performance liquid chromatograph.

[0037] The structure of the synthesized drug is as shown in Figure 1

[0038] Among them, CPT1-DL represents: camptothecin and polypeptide 1-DL are connected by covalent bond, in which FFKLV is D type amino acid; RRVR is L type amino acid.

[0039] CPT1-DD represents: camptothecin and polypeptide DL are connected by covalent bond, in which FFKLVRRVR is all D type amino acid.

[0040] CPTFF1-DD represents: camptothecin and polypeptide FF1-DD are connected by covalent bond, in which FFKLVRRVR is all D type amino acid.

[0041] Cbl-1LL represents: vinblastine and polypeptide LL are connected by covalent bond, in which FFKLVRRVR is all L type amino acid.

[0042] 1DD represents: FFKLVRRVR is all D type, and the N terminal is covalently connected with naphthalene acetic acid.

[0043] 1DL represents: FFKLV is D type amino acid; RRVR is L type amino acid, and the N terminal is covalently connected with naphthalene acetic acid.

[0044] Example 2

[0045] Stability of polypeptide conjugated drug

[0046] The polypeptide conjugated drug is configured into a 20mM stock solution, diluted to 300μM using PBS buffer containing 10% FBS, and then sampled at different time points to detect the degradation of polypeptide by analytical HPLC, and the degradation curve is drawn by calculation.

[0047] Results are referred to Figure 2 ​; The polypeptide conjugated drug synthesized by all D-type amino acids has higher serum stability.

[0048] Since CPT1-LL has poor stability, subsequent tests were performed using 1-DL, 1-DD, CPT1-DL, CPT1-DD, and CPTFF1-DD.

[0049] Example 3

[0050] Lipophilicity of polypeptide conjugated drug

[0051] The molecular formulas of camptothecin (CPT), polypeptides (1-DL and 1-DD), and polypeptide conjugated drugs (CPT1-DL and CPT1-DD) were drawn by ChemDraw 18.0, and then each molecular formula was selected, and the values could be directly read in the Chemical Properties Window in ChemDraw 18.0.

[0052] Results reference Figure 3 The lipophilicity of camptothecin (CPT), polypeptides (1-DL and 1-DD), and polypeptide conjugated drugs (CPT1-DL and CPT1-DD) was simulated and calculated by ChemDraw 18.0, and the results showed that the hydrophilicity of the polypeptide conjugated drug was significantly stronger than that of camptothecin and polypeptides, proving that the polypeptide conjugated drug can enhance the drugability of camptothecin.

[0053] Example 4

[0054] Solubility test of polypeptide conjugated drug in PBS buffer

[0055] 2 mg of sample was added to 100 μL of water, and then ultrasonic-assisted dissolution was performed, followed by the addition of 100 μL of 2xPBS, and then incubation was performed at 37°C in a constant temperature incubator for 12 h.

[0056] Figure 4 It is shown that polypeptides 1-DL and 1-DD both form hydrogels, camptothecin CPT forms a turbid liquid, and polypeptide conjugated drugs CPT1-DL and CPT1-DD are both clear and transparent solutions, and after changing the sequence, CPTFF1-DD still forms a hydrogel, indicating that the polypeptide sequence-specific conjugated drugs CPT1-DL and CPT1-DD can significantly improve the water solubility of camptothecin, and thus enhance its drugability.

[0057] Example 5

[0058] Cellular uptake rate of polypeptide conjugated drug

[0059] The qualitative analysis of cell uptake was carried out by fluorescence confocal microscopy. The polypeptide CPT1-DD, CPT1-DL, CPT and cells were incubated for different time. The fluorescence confocal imaging showed that the cell uptake rate of polypeptide CPT1-DD was the highest, while the uptake efficiency of CPT was the lowest. With the increase of time, the content of polypeptide CPT1-DDL in cells increased, and the uptake efficiency of CPT was far lower than that of polypeptide conjugated drug CPT1-DD within 12h (see Figure 5 ). The qualitative analysis by flow cytometry showed that the cell uptake rate of polypeptide CPT1-DD was 6 times that of CPT after 4h (see Figure 6 ), which further confirmed that polypeptide conjugated drugs could affect the cell uptake rate.

[0060] Reference Figure 5 Using different compounds to treat HCT-116 cells, it was found that polypeptide conjugated drugs could significantly improve the internalization of CPT in cells, and D-type amino acid conjugated drug CPT1-DD had the best cell uptake rate, indicating that D-type polypeptide not only improved the water solubility of CPT, but also promoted the internalization of polypeptide.

[0061] Reference Figure 6 The quantitative analysis of cells treated with drugs was carried out by flow cytometry. The experimental results were the same as those of fluorescence confocal microscopy. Polypeptide conjugated drugs could improve the internalization of CPT in cells, and D-type polypeptide conjugated drugs significantly improved the internalization efficiency of CPT in cells.

[0062] Example 6

[0063] Cytotoxicity of polypeptide conjugated drugs

[0064] The polypeptide conjugated drugs and CPT were configured into a 20mM stock solution, which was gradiently diluted and added to the 96-well plate with HCT116 cells pre-plated, 100μL per well, three replicate wells for each concentration, the treatment time was 3 days, and then the OD value was measured at 572nm by MTT, and the obtained value was calculated.

[0065] Reference Figure 7 Using CPT and polypeptide conjugated drugs of CPT and polypeptide to test cytotoxicity, it was found that polypeptide conjugated drugs did not reduce the toxicity of CPT, but the solubility of polypeptide conjugated drugs of CPT greatly changed the solubility of CPT, which could enhance the possibility of clinical use.

[0066] Example 7

[0067] Effect of polypeptide conjugated drugs on mice

[0068] HCT-116 tumor cells were injected subcutaneously into nude mice, and after the tumors grew to a certain size, different solutions were injected around the tumors of the nude mice. The solutions were divided into polypeptide drug 1-DD group (20 mg / kg), CPT1-DD (20 mg / kg), anticancer drug CPT group, PBS group, and the mice were given the drugs once every two days, 100 μL each time, 6 mice in each group, the body weight of the nude mice was measured twice a day, and the tumor volume of the nude mice was measured using a vernier caliper, and the nude mice were sacrificed after 10 days (attached Figure 7 , the first behavior is the PBS group, the second behavior is the 20 mg / kg 1-DD group; the third behavior is the 20 mg / kg CPT1-DD group, and the fourth behavior is the camptothecin.

[0069] Reference Figure 8 The polypeptide conjugate drug group can significantly slow down the growth of tumors, has better efficacy than the chemotherapeutic drug camptothecin, and has important research value.

Claims

1. A polypeptide conjugate, characterized in that, The polypeptide and the chemotherapeutic drug are connected by a covalent bond; the polypeptide contains at least one D-type amino acid; the polypeptide is formed by a self-assembling polypeptide and a cell-penetrating peptide, and the amino acid sequence of the polypeptide is shown as SEQ ID No. 1, and the chemotherapeutic drug is camptothecin.

2. The polypeptide conjugate of claim 1, wherein, All the amino acids in the self-assembling polypeptide and / or the cell-penetrating peptide are D-type amino acids.

3. A hydrogel, characterized in that, The polypeptide conjugate as claimed in claim 1 or 2.

4. Use of the polypeptide conjugate as claimed in claim 1 or 2 in the preparation of an antitumor drug.

5. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The polypeptide conjugate as claimed in claim 1 or 2.

Images