Polypeptide compound as well as preparation method and application thereof

Abstract

The invention relates to a polypeptide compound as well as a preparation method and application thereof, and belongs to the technical field of polypeptide medicines. The invention provides a polypeptide compound. The structural formula of the polypeptide compound is shown as a formula I, a drug carrier prepared from the polypeptide compound has very high biological safety, transfection efficiency and delivery efficiency, the transfection efficiency of the drug carrier to easily transfected cell lines reaches 85% or above, the delivery efficiency of the drug carrier to difficultly transfected cell lines reaches 70% or above, and the polypeptide compound has very high scientific research value and medical technology application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drugs, and particularly relates to a polypeptide compound, a preparation method thereof, and an application thereof. Background Art

[0002] Precision therapy is a new treatment method that uses a drug delivery system such as a drug carrier to accurately deliver drug molecules into cells and control their release in the cytoplasm, enabling them to directly act on target cells or kill diseased cells, thereby effectively treating tumors and metabolic diseases. Compared with traditional treatment methods, precision therapy has the advantages of high drug utilization rate, low toxic and side effects, strong specificity, and significant treatment effect.

[0003] However, precision therapy highly depends on the transport of drug molecules by the drug delivery system, which requires the drug delivery system to not only protect and clamp drug molecules during the transport process, but also be able to carry drug molecules to penetrate the cytoplasmic membrane and enter the cytoplasm after recognizing and reaching target cells or diseased cells, and successfully release drug molecules in the cytoplasm. Therefore, the drug delivery ability of the drug delivery system to drug molecules is an important factor affecting the effect of precision therapy.

[0004] Most of the existing technologies use nanoscale particle carriers to deliver drug molecules. However, the production process of nanoscale particle carriers is complex, the preparation cost is high, and the residual organic solvents in the preparation process may affect the biological activity of drug molecules and cause cytotoxicity to normal cells; the penetration ability to the cytoplasmic membrane is poor, resulting in a low delivery success rate of drug molecules, and thus a decline in the treatment effect; it has strong immunogenicity, can cause inflammatory reactions and may cause biological toxicity; in addition, the difference in the ability of nanoscale particle carriers to clamp drug molecules is large, some nanoscale particle carriers will cause drug molecule escape during transport, while some other nanoscale particle carriers are not easy to release drug molecules even when they enter the cytoplasm. Summary of the Invention

[0005] The purpose of the present invention is to provide a polypeptide compound, a preparation method thereof, and an application thereof, so as to solve the problems of insufficient biosafety of drug carriers, low transfection efficiency and delivery efficiency of drug molecules, and poor treatment effect in the prior art.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a polypeptide compound, and the structural formula of the polypeptide compound is shown in Formula I;

[0008]

[0009] The present invention also provides a method for preparing the polypeptide compound, comprising the following steps: mixing HBpep-Sp and cholesterol and reacting them to obtain the polypeptide compound.

[0010] Preferably, the molar ratio of the mixture of HBpep-Sp and cholesterol is 1:1 to 5.

[0011] Preferably, the reaction is an esterification reaction, the temperature of the reaction is 55 to 70 °C, and the reaction time is 6 to 14 h.

[0012] The present invention also provides the use of the polypeptide compound in the preparation of a nano-delivery system.

[0013] The present invention also provides a nano-delivery system, comprising the polypeptide compound and an acceptable excipient.

[0014] The present invention also provides the use of the polypeptide compound or the nano-delivery system in the preparation of a drug carrier.

[0015] The present invention also provides a drug carrier, which comprises the polypeptide compound or the nano-delivery system.

[0016] The present invention also provides the use of the polypeptide compound, the nano-delivery system or the drug carrier in the preparation of a product for delivering drug molecules.

[0017] Preferably, the drug molecules include proteins, nucleic acid molecules or compounds.

[0018] The present invention has the following technical effects and advantages:

[0019] (1) The polypeptide compound of the present invention is prepared by an esterification reaction of HBpep-Sp and cholesterol. The raw materials for preparation are easy to obtain, the preparation process is simple, and the preparation cost is low. Among them, HBpep-Sp is a polypeptide containing only 36 amino acids modified by NHS-SS-Ph, and NHS-SS-Ph is a degradable linker. The polypeptide compound of the present invention does not show toxic and side effects on normal and healthy cells, and can be degraded and absorbed and utilized after completing the delivery work of drug molecules, and has high biosafety;

[0020] (2) The drug carrier prepared by using the polypeptide compound of the present invention has high transfection efficiency and delivery efficiency. Its transfection efficiency for easily transfectable cell lines reaches more than 85%, and its delivery efficiency for difficult-to-transfect cell lines reaches more than 70%, both of which are significantly higher than the commercial transfection reagent Lipofectamine TM 2000, and has high scientific research value and medical technology application prospects. Description of the Drawings

[0021] Figure 1 The result of transfection of EGFP protein into HEK293 cell line by drug carrier B;

[0022] Figure 2 The result of delivering EGFP protein into HepG2 cell line by drug carrier B. Detailed implementation manners

[0023] The present invention provides a polypeptide compound, and the structural formula of the polypeptide compound is shown as Formula I;

[0024]

[0025]

[0026] In the present invention, the molecular formula of the polypeptide compound is C 177 H 233 N 39 O 37 S2, and the relative molecular mass is 3563.113.

[0027] The present invention also provides a preparation method of the polypeptide compound, including the following steps: mixing HBpep-Sp and cholesterol and reacting to obtain the polypeptide compound.

[0028] In the present invention, the structural formula of HBpep-Sp is Gly-His-Gly-Val-Tyr-Gly-His-Gly-Val-Tyr-Gly-His-Gly-Pro-Tyr-Lys(NHS-SS-Ph)-Gly-His-Gly-Pro-Tyr-Gly-His-Gly-Leu-Tyr-Trp, and the molecular formula is C 150 H 189 N 39 O 37 S2, the relative molecular mass is 3194.475, purchased from Zhongke Huayao (Jiangsu) Biotechnology Co., Ltd., and the purity is ≥95%;

[0029] The molecular formula of the cholesterol is C 27 H 46 O, the relative molecular mass is 386.654, purchased from Sangon Biotech (Shanghai) Co., Ltd., and the purity is ≥98%.

[0030] In the present invention, the molar ratio of the mixture of HBpep-Sp and cholesterol is 1:1 to 5, preferably 1:1.5.

[0031] In the present invention, the reaction is an esterification reaction, preferably an esterification reaction occurring through dehydration condensation between the α-carboxyl group of the tryptophan (Trp) residue in HBpep-Sp and the β-hydroxy group of cholesterol;

[0032] The temperature of the reaction is 55 - 70 °C, preferably 65 °C; the reaction time is 6 - 14 h, preferably 12 h.

[0033] The present invention also provides the use of the polypeptide compound in the preparation of a nano-delivery system.

[0034] The present invention also provides a nano-delivery system, comprising the polypeptide compound and an acceptable excipient.

[0035] The present invention also provides the use of the polypeptide compound or the nano-delivery system in the preparation of a drug carrier.

[0036] The present invention also provides a drug carrier, wherein the drug carrier comprises the polypeptide compound or the nano-delivery system.

[0037] The present invention also provides the use of the polypeptide compound, the nano-delivery system or the drug carrier in the preparation of a product for delivering a drug molecule.

[0038] In the present invention, the drug molecule comprises a protein, a nucleic acid molecule or a compound.

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] Among the reagents of the present invention, HBpep-Sp is purchased from Zhongke Huayao (Jiangsu) Biotechnology Co., Ltd., with a purity of ≥ 95%; cholesterol is purchased from Sangon Biotech (Shanghai) Co., Ltd., with a purity of ≥ 98%; MEM medium and the commercial transfection reagent Lipofectamine TM 2000 is purchased from Gibco, USA; the CCK-8 kit is purchased from Yeasen Biotech Co., Ltd. (Shanghai); the mRNA molecule expressing the EGFP protein is purchased from GenScript Biotech Corporation;

[0041] Among the cell lines of the present invention, the human embryonic kidney cell line HEK293 and the human hepatocellular carcinoma cell line HepG2 are purchased from Wuhan Shang'en Biotechnology Co., Ltd.

[0042] Example 1: Synthesis of the polypeptide compound

[0043] Mix 1 mol of HBpep-Sp and 1.5 mol of cholesterol, and carry out an esterification reaction dominated by dehydration condensation between the α-carboxyl group of the Trp residue in HBpep-Sp and the β-hydroxy group of cholesterol. React at 65 °C for 12 h to obtain a polypeptide compound. Use mass spectrometry to determine the actual relative molecular mass of the polypeptide compound, and use high-performance liquid chromatography technology to determine the purity of the polypeptide compound. The synthesis and index determination of the polypeptide compound are all entrusted to Zhongke Huayao (Jiangsu) Biotechnology Co., Ltd.

[0044] The results show that the structural formula of the polypeptide compound is as shown in Formula I, and the molecular formula is C 177 H 233 N 39 O 37 S2, the theoretical relative molecular mass is 3563.113, the actual relative molecular mass is 3562.857, and the purity reaches more than 95%;

[0045]

[0046]

[0047] Example 2: Preparation of drug carriers

[0048] Mix the polypeptide compound prepared in Example 1 with a 10 mmol / L acetic acid solution to prepare polypeptide solutions with final concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5 mg / mL respectively; drop the polypeptide solutions into RNA-free sterile PBS solutions respectively to prepare drug carriers A, B, C, and D, where the final concentrations of the polypeptide compound are 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5 mg / mL respectively. Use an enzyme-labeled instrument to measure the OD 600nm value, use RNA-free sterile PBS solution as the blank control, and calculate the relative turbidity (R). Use a laser particle size analyzer to measure the particle size of each drug carrier. The results are shown in Table 1;

[0049] The calculation formula for relative turbidity (R) is:

[0050] In the formula: T is the OD 600nm value of the drug carrier, and T0 is the OD 600nm value of the blank control.

[0051] Table 1 Physicochemical properties of drug carriers

[0052] Drug carrier Final concentration (mg / mL) Relative turbidity (%) Particle size (μm) A 0.5 58.20 0.74±0.13 B 1 70.45 0.88±0.20 C 2 85.26 1.16±0.12 D 5 91.33 1.20±0.19

[0053] The results showed that the relative turbidity of each drug carrier could reach up to 91.33%, and the particle size distribution range was 0.74±0.13 - 1.20±0.19 μm, indicating that the polypeptide compound of the present invention could be successfully prepared into a drug carrier.

[0054] Experimental Example 1: Biological Safety of Drug Carrier

[0055] Add 500 μL of MEM medium to each inoculation well of a 24-well plate, then inoculate the human embryonic kidney cell line HEK293 cells into the 24-well plate and incubate for 24 h. The inoculation amount is 1×10 6 cells / well. Then add 25 μL of each drug carrier prepared in Example 2 to each inoculation well, and use RNA-free sterile PBS solution as a control. After culturing for 4 h, use the CCK-8 kit to measure the survival rate of HEK293 cell line in each inoculation well. The results are shown in Table 2.

[0056] Table 2 Survival Rate of HEK293 Cell Line

[0057]

[0058]

[0059] The results showed that after co-culturing the HEK293 cell line with the drug carrier of the present invention for 4 h, the survival rate of the HEK293 cell line could reach more than 90%, and there was no significant difference compared with the control, indicating that the drug carrier of the present invention has good biological safety.

[0060] Experimental Example 2: Transfection Efficiency of Drug Carrier

[0061] Dissolve the mRNA molecule expressing EGFP protein in RNA-free sterile PBS to obtain an mRNA solution with an mRNA molecule concentration of 3.3 μg / mL. The specific steps refer to the operation instructions of the mRNA molecule; mix each drug carrier prepared in Example 2 with the mRNA solution until the final concentration of the mRNA molecule is 1 μg / mL. At this time, self-aggregation occurs, and the corresponding drug carrier solution loaded with EGFP is obtained (i.e., EGFP-drug carrier solutions A - D, where the final concentrations of the polypeptide compound are 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5 mg / mL respectively). Add 100 μL of MEM medium to a 96-well plate, then inoculate the human embryonic kidney cell line HEK293 cells into the 96-well plate and incubate for 24 h. The inoculation amount is 1×10 4 cells / well. Then add 10 μL of each EGFP-drug carrier solution to each inoculation well, and use the commercial transfection reagent Lipofectamine TM 2000 as a control. After culturing for 4 h, measure the transfection efficiency. The results are shown in Table 3.

[0062] Table 3 Transfection efficiency of drug carriers

[0063] Transfection reagent Transfection efficiency (%) EGFP-drug carrier solution A 74.20 EGFP-drug carrier solution B 86.76 EGFP-drug carrier solution C 69.03 EGFP-drug carrier solution D 58.36 <![CDATA[Lipofectamine TM 2000]]> 54.44

[0064] The results show that the drug carriers prepared with the polypeptide compounds of the present invention have higher transfection efficiencies for mRNA molecules than the control, and the drug carrier with a final concentration of 1 mg / mL of the polypeptide compound has the highest transfection efficiency for mRNA molecules.

[0065] Experimental Example 3: Delivery efficiency of drug carriers

[0066] The human hepatoma cell line HepG2 is a recognized difficult-to-transfect cell line, and neither EGFP protein nor mRNA molecules expressing EGFP protein can directly cross the cytoplasmic membrane of the HepG2 cell line. Co-culture the HepG2 cell line with each EGFP-drug carrier solution prepared in Experimental Example 2. The specific steps refer to the method described in Experimental Example 2, and use the commercial transfection reagent Lipofectamine TM 2000 as a control. After culturing for 4 h, measure the delivery efficiency. The results are shown in Table 4.

[0067] Table 4 Delivery efficiency of drug carriers

[0068] Transfection reagent Delivery efficiency (%) EGFP-drug carrier solution A 57.23 EGFP-drug carrier solution B 70.49 EGFP-drug carrier solution C 42.87 EGFP-drug carrier solution D 36.50 <![CDATA[Lipofectamine TM 2000]]> 40.33

[0069] The results show that the drug carriers prepared with the polypeptide compounds of the present invention can successfully deliver mRNA molecules into the HepG2 cell line, and the drug carrier with a final concentration of 1 mg / mL of the polypeptide compound has the highest delivery efficiency for mRNA molecules, and the delivery efficiency is significantly higher than the control.

[0070] As can be seen from the above examples, the present invention provides a polypeptide compound, its preparation method and application. The drug carrier prepared with the polypeptide compound of the present invention does not show toxic and side effects on normal cells, has a high transfection efficiency for easily transfectable cell lines, and can exert a high delivery efficiency for difficult-to-transfect cell lines.

[0071] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A polypeptide compound, characterized in that: The structural formula of the polypeptide compound is shown in Formula I; 2. The method for preparing the polypeptide compound according to claim 1, characterized in that: The method comprises the following steps: mixing HBpep-Sp and cholesterol, reacting them, and obtaining a polypeptide compound.

3. The preparation method according to claim 2, characterized in that: The molar ratio of the HBpep-Sp and cholesterol is 1:1-5.

4. The preparation method according to claim 3, characterized in that: The reaction is an esterification reaction, the reaction temperature is 55 to 70° C., and the reaction time is 6 to 14 hours.

5. Use of the polypeptide compound according to claim 1 in preparing a nano delivery system.

6. A nano delivery system, characterized in that: It comprises the polypeptide compound according to claim 1 and acceptable excipients.

7. Use of the polypeptide compound according to claim 1 or the nano delivery system according to claim 6 in the preparation of a drug carrier.

8. A drug carrier, characterized in that The drug carrier comprises the polypeptide compound according to claim 1 or the nano delivery system according to claim 6.

9. Use of the polypeptide compound according to claim 1, the nano delivery system according to claim 6 or the drug carrier according to claim 8 in the preparation of a product for delivering drug molecules.

10. The use according to claim 9, characterized in that: The drug molecules include proteins, nucleic acid molecules or compounds.

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