Preparation of an anticancer lipopeptide and its application in antitumor treatment

Abstract

The present application relates to an anticancer lipopeptide C8H 15 Preparation of O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu and its application in antitumor treatment, belonging to the field of biological medicine. The anticancer lipopeptide is prepared based on the key biological characteristics (positive charge, alpha-helix structure and amphiphilicity) of membrane lytic peptide. The present application provides a solid-phase synthesis method of the anticancer lipopeptide. The present anticancer lipopeptide has the characteristics of small hydrophobic moment, low hemolytic activity and good serum stability, can effectively make up for the defects of most anticancer peptides with high hemolytic toxicity, and solve the problem of poor in-vivo stability of peptide drugs. In-vitro anticancer experiments and in-vivo antitumor experiments prove that the present anticancer lipopeptide has good antitumor effect and good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the preparation of an artificially synthesized anticancer lipopeptide with excellent in vitro and in vivo anticancer activity and its application in antitumor therapy. Background Technology

[0002] Despite significant advancements in cancer treatment, it remains one of the leading causes of death worldwide. Currently, chemotherapy and radiotherapy are the primary treatments for cancer; however, these treatments often come with severe side effects and multidrug resistance. Anticancer peptides (ACPs) offer advantages such as high selectivity, good biocompatibility, and high tumor penetration. Most ACPs are α-helical cationic amphiphilic peptides, and their activity is primarily determined by net charge, hydrophobicity, and helicity. Compared to normal cells, cancer cell membranes carry a large number of anionic components such as phosphatidylserine and phosphatidylinositol, giving the cancer cell surface a net negative charge. Most anticancer peptides contain multiple lysine and arginine residues, resulting in a large positive charge. These positively charged anticancer peptides can be attracted to the negatively charged cancer cell membrane through electrostatic interactions, further forming an α-helical conformation. They then insert into the lipid bilayer, creating channels, disrupting membrane integrity, and ultimately leading to cancer cell death. Arginine can interact with negatively charged components on the cell membrane surface through side-chain guanidinium ions to generate numerous hydrogen bonds, thereby achieving a transition from water-soluble to membrane-soluble. Simultaneously, the membrane-penetrating ability of oligomeric arginine increases with the number of consecutive arginine residues and the peptide concentration. Furthermore, increasing the hydrophobicity of peptides can enhance their anticancer activity; however, high hydrophobicity often leads to high hemolytic activity. Introducing hydrophilic residues on the hydrophobic side of α-helical amphiphilic peptides reduces the hydrophobic torque, thereby decreasing hemolytic activity.

[0003] Peptide drugs have limited clinical application due to their short half-life, low bioavailability, poor stability, and susceptibility to protease hydrolysis. Fatty acid modification can increase the membrane permeability of peptides and enhance their anticancer activity. Furthermore, fatty acid-modified therapeutic agents (such as proteins, peptides, and siRNAs) can prolong in vivo circulation time and avoid protease hydrolysis. Therefore, this invention designs and synthesizes a novel arginine-rich cationic amphiphilic anticancer lipopeptide. Arginine (Arg) provides a positive charge and binds to a large number of negatively charged components on the surface of cancer cell membranes via electrostatic interactions. In addition, tryptophan (Trp) and leucine (Leu) are introduced to increase the hydrophobicity of the peptide chain and improve membrane permeability. Octanoic acid modification increases the stability of the peptide chain and enhances its anticancer effect. When the inventors searched and compared the full-sequence amino acid structure of the anticancer lipopeptide of this invention using the NCBI protein database, no identical polypeptides were found. The anticancer lipopeptide of this invention exhibits good in vitro and in vivo anticancer activity and can provide new ideas for cancer treatment. Summary of the Invention

[0004] The purpose of this invention is to synthesize an anticancer lipopeptide with excellent anticancer activity through solid-phase synthesis technology and to apply this anticancer lipopeptide to antitumor therapy.

[0005] To achieve the objectives of this invention, the following technical solution is provided:

[0006] The amino acid sequence of the anticancer lipopeptide of this invention is: C8H 15 The technical solution for O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu is as follows:

[0007] (1) Design of anticancer lipopeptides: First, based on the positive charge and amphiphilicity of cationic anticancer peptides, positively charged arginine (Arg) and hydrophobic amino acids such as tryptophan (Trp) and leucine (Leu) are introduced. The hydrophobic torque of the peptide chain is reduced through a reasonable arrangement of amino acids to decrease hemolytic toxicity. Simultaneously, octanoic acid (C8H2O) is used at the N-terminus. 16 O2) modification was used to improve the stability of the peptide, and a cationic amphiphilic anticancer lipopeptide was finally designed.

[0008] (2) The anticancer lipopeptide was prepared by solid-phase synthesis. The preparation method is summarized as follows: Amino acids with Fmoc protecting groups and side-chain protecting groups were deprotected, and then each amino acid was sequentially coupled to Wang resin from the C-terminus to the N-terminus using a catalyst. Octanoic acid was then coupled to the N-terminus. Finally, the resin was cleaved with trifluoroacetic acid to remove the side-chain protecting groups, yielding the anticancer lipopeptide. The structural formula of the anticancer lipopeptide of this invention is as follows:

[0009]

[0010] (3) The in vitro anticancer activity of anticancer lipopeptides was detected by MTT assay and flow cytometry: the cytotoxicity of anticancer lipopeptides to HepG2 cells was tested by MTT assay, and the effect of anticancer lipopeptides on HepG2 cell apoptosis was detected by flow cytometry using Annexin V-FITC / PI apoptosis detection kit.

[0011] (4) Detection of the secondary structure of anticancer lipopeptides by circular dichroism (CD): Water was used to simulate the hydrophilic environment, and SDS solution was used to simulate the hydrophobic environment such as the cell membrane. The changes in the secondary structure of anticancer lipopeptides in aqueous solution and SDS solution were detected by circular dichroism.

[0012] (5) Study on the membrane-breaking activity of anticancer lipopeptides: After co-culturing anticancer lipopeptides with HepG2 cells for 3 hours, the effect of anticancer lipopeptides on cell membrane morphology was observed under a microscope. The ability of anticancer lipopeptides to lyse cancer cell membranes was quantitatively determined by lactate dehydrogenase (LDH) release assay.

[0013] (6) Study on hemolysis and serum stability of anticancer lipopeptides: The anticancer lipopeptides were co-cultured with fetal bovine serum (FBS) and then the stability was assessed by MTT assay of the changes in anticancer activity after FBS pretreatment; the hemolytic activity was assessed by the effect of the anticancer lipopeptides on the morphological changes of mouse erythrocytes.

[0014] (7) Study on the in vivo antitumor activity of anticancer lipopeptides: Tumor-implanted mice were selected as experimental models. Anticancer lipopeptides were injected into the tail vein every other day. The weight and tumor volume of the mice were measured. The tumor tissue of the mice was dissected and tissue sections and HE staining were performed to detect the in vivo antitumor activity of the anticancer lipopeptides. Attached Figure Description

[0015] Figure 1 MTT assay was used to detect the anticancer activity of anticancer lipopeptides on HepG2 cells. (a) Relative cell viability after co-culturing HepG2 cells with different concentrations of anticancer lipopeptides for 24 h. (b) Relative cell viability after co-culturing HepG2 cells with 24 μM anticancer lipopeptides for different time periods.

[0016] Figure 2 CD spectra of anticancer lipopeptides in pure water and SDS solution;

[0017] Figure 3 Apoptosis was detected by FITC-Annexin V / PI double staining. Data on the effects of lipopeptides on HepG2 cell apoptosis, measured by flow cytometry, were obtained from blank control (a), 4 μM (b), 8 μM (c), and 16 μM (d).

[0018] Figure 4 Membrane-permeable activity of anticancer lipopeptides. The morphology of HepG2 cells was observed under a fluorescence microscope (400×) after co-incubation with blank control (a), 8 μM (b), and 16 μM (c) lipopeptides for 3 h. (d) Lactate dehydrogenase release rate of HepG2 cells after co-culturing with different concentrations of lipopeptides for 24 h.

[0019] Figure 5Hemolytic activity and serum stability of anticancer lipopeptides. Two hours after tail vein injection of physiological saline (a) and 2.25 mg / mL anticancer lipopeptide (b), blood was collected and erythrocyte morphology was observed under a microscope (400×). (c) The anticancer lipopeptide was incubated with PBS containing 10% serum and serum-free PBS at 37°C for 24 h and 48 h, respectively, and the changes in anticancer activity of the anticancer lipopeptide against HepG2 cells were detected by the MTT assay.

[0020] Figure 6 In vivo antitumor activity of anticancer lipopeptides. (a) Change in body weight over time in tumor-transplanted mice after tail vein injection of anticancer lipopeptides every other day. (b) Change in relative tumor volume over time in tumor-transplanted mice. (c) Tumor tissue section 11 days after injection of saline every other day. (d) Tumor tissue section (400×) 11 days after injection of anticancer lipopeptides every other day. Detailed Implementation

[0021] The following provides specific embodiments of the present invention to further illustrate the structure of the present invention, but it is not intended that the present invention be limited to the embodiments described below.

[0022] Example 1: Preparation method of anticancer lipopeptides

[0023] The anticancer lipopeptide of this invention is synthesized using an Fmoc-protected solid-phase peptide synthesis method. The sequence of the anticancer lipopeptide is C8H. 15 The specific synthesis steps for O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu are as follows:

[0024] (1)Fmoc-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf) -Synthesis of Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-WangResin.

[0025] Weigh a certain amount of Fmoc-Leu-Wang Resin with a loading of 0.535 mmol / g and place it in a beaker. Add anhydrous DMF to swell the resin for 30 min. Take a small amount of the above resin for ninhydrin detection. If it is colorless, continue. Add piperidine solution at a ratio of DMF:piperidine = 4:1, stir magnetically for 30 min to remove Fmoc groups. After the reaction is complete, filter the solution and wash the resin three times each with DMF, DCM, and DMF in sequence. Add anhydrous DMF again to swell for 30 min. Add two molar amounts of Fmoc-Arg(Pbf)-OH, 2.6 molar amounts of DCC, HOBT, and DIEA to a beaker and react magnetically at room temperature for at least 48 hours. After the reaction is complete, wash the resin five times each with DMF, DCM, and DMF, and finally wash it multiple times with anhydrous ethanol. After filtration, transfer the sample to a dialysis bag (MW: 8000-14000). Change the dialysis buffer every 30 minutes and dialyze at least 20 times. Freeze-dry the dialyzed sample to obtain Fmoc-Arg(Pbf)-Leu-Wang Resin. Repeat the above steps to couple amino acids until Fmoc-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gl is synthesized. y-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin, where Pbf is the side-chain protecting group of Arg, Boc is the side-chain protecting group of Trp, tBu is the side-chain protecting group of Ser, and OtBu is the side-chain protecting group of As p;

[0026] (2)C8H 15 Synthesis of O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu.

[0027] First, combine Fmoc-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin was added to anhydrous DMF to swell the resin for 30 min. Piperidine was then added at a DMF:piperidine ratio of 4:1. The mixture was magnetically stirred for 30 min to remove the Fmoc group. After the reaction was complete, the resin was filtered and washed three times each with DMF, DCM, and DMF. The resin was then freeze-dried to obtain Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Leu-Wang Resin. Two molar amounts of C8H... 16 O2, 2.5 molar amounts of NHS, and 2.5 molar amounts of EDC were added to anhydrous DMF and magnetically stirred for 5 hours to activate the carboxyl groups. Then, Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-WangResin was added and reacted for 48 hours. After the reaction was completed, the resin was washed 5 times each with DMF, DCM, and DMF in sequence. Finally, it was washed multiple times with anhydrous ethanol. After filtration, the sample was transferred to a dialysis bag (MW: 8000-14000). The dialysis solution was changed every 30 minutes, and dialysis was performed more than 20 times. The dialysis sample was then freeze-dried to obtain C8H. 15 O-As p(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin was added to the cleavage solution at a ratio of trifluoroacetic acid (TFA):water (H2O):triisopropylsilane (Tis) = 95:2.5:2.5. The mixture was magnetically stirred for 1.5 h to remove the resin and all side-chain protecting groups. After the reaction was completed, the filtrate was collected, concentrated by rotary evaporation, and precipitated by adding ice-cold diethyl ether. The precipitate was centrifuged and washed five times with diethyl ether, and then freeze-dried to obtain the lipopeptide C8H. 15O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Ar g-Arg-Leu.

[0028] Example 2: In vitro anticancer activity assay of anticancer lipopeptides

[0029] The in vitro anticancer activity of anticancer lipopeptides was tested using the MTT assay. HepG2 cells in logarithmic growth phase were seeded into 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. After the cells had filled the wells, the old culture medium was removed, and the lipopeptides were prepared into different concentrations using DMEM medium. 100 μL of the drug-containing medium was added to each well. To ensure the reliability of the experimental results, five replicates were set up for each concentration. After 24 hours of incubation, 20 μL (5 mg / mL) of the drug-containing medium was added to each well. -1 Incubate the MTT solution in a cell culture incubator for 4 hours. Discard the supernatant and add 150 μL of DMSO to each well to dissolve the blue-purple MTT crystals. Wrap the 96-well plate in aluminum foil to protect it from light and place it on a shaker at 170 rpm. -1 Shake for 7 minutes to fully dissolve the crystals in the wells. After dissolution, transfer to an ELISA reader for detection at a wavelength of 485 nm. Measure the absorbance of each well and calculate the cell viability.

[0030] Example 3: Determination of the secondary structure of anticancer lipopeptides

[0031] The secondary structure of lipopeptides in water and SDS solution was detected using circular dichroism spectroscopy. Lipopeptides were prepared into an aqueous solution with a final concentration of 150 μM and a sodium dodecyl sulfate (SDS) solution (30 mM) with a final concentration of 150 μM. The aqueous solution simulated a hydrophilic environment, and the SDS solution simulated a hydrophobic environment such as a cell membrane. The solutions were placed in quartz cuvettes (optical path length 0.5 mm) and tested using a circular dichroism spectroscopy instrument with a wavelength range of 180-260 nm. The obtained spectra were converted into average residue ellipticity using the following formula:

[0032] θ M =θ obs ×1000 / cln

[0033] Where θ M It is the average residue ellipticity; θ obs The ellipticity is obtained by correcting with buffer at a given wavelength; c represents the sample concentration (mM); l represents the optical path length of the quartz cuvette (mm), which is 0.5 mm here; n is the number of amino acid residues in the peptide sample.

[0034] Example 4: Effects of anticancer lipopeptides on cancer cell apoptosis

[0035] Apoptosis was detected using the Annexin V-FITC / PI apoptosis detection kit. Healthy HepG2 cells were digested with trypsin, and 2 mL of cell suspension (5 × 10⁶ cells per well) was added to each well of a 6-well plate. 5 Cells were incubated in a CO2 incubator for no more than 24 hours. After cell attachment, the old culture medium was aspirated, and the cells were washed twice with PBS. Then, DMEM medium containing various concentrations of lipopeptide solutions was added, and the cells were cultured for another 24 hours. The old culture medium from each well was then collected, digested with trypsin (without EDTA), centrifuged, and the supernatant was removed. The cells were washed twice with PBS, and then mixed with the previously collected old culture medium. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS and centrifuged and washed once more. The cells were resuspended in 100 μL of Annexin V-FITC binding buffer, and 5 μL of Annexin V-FITC and 10 μL of PPI were added. The cells were incubated at room temperature in the dark for 15 min. After staining, the cells were placed on ice in the dark and the staining was performed by flow cytometry within 1 hour. Annexin V-FITC showed green fluorescence, and PPI showed red fluorescence.

[0036] Example 5: Study on the membrane-permeable activity of anticancer lipopeptides against cancer cells

[0037] To investigate the cell membrane permeation activity of anticancer lipopeptides, 8 μM and 16 μM anticancer lipopeptides were co-cultured with HepeG2 cells for 3 h, and the effects of the anticancer lipopeptides on cell membrane morphology were observed under a microscope (400×).

[0038] Lactate dehydrogenase release assay was used to detect cell membrane lysis activity. When the cell membrane structure is disrupted, intracellular lactate dehydrogenase is released, and measuring the amount of lactate dehydrogenase released is an important method for studying cell membrane lysis. First, HepG2 cells were seeded in 96-well plates, with a blank negative control group, a drug group, and a positive control group (1% Tritium X-100 was added to represent 100% release). After culturing for 24 hours, different concentrations of peptide solutions (without serum) were added, and the cells were cultured for another 24 hours. The positive control group was incubated with Tritium X-100 one hour earlier and cultured for another 1 hour. Then, the cells were centrifuged at 400g for 5 minutes. 60 μL of detection working solution was added to each well according to the LDH cytotoxicity Assay Kit (Beyotime, China), and the cells were incubated in the dark on a shaker for 30 minutes. The absorbance was measured at 490 nm, and the LDH release rate was calculated using the following formula.

[0039] LDH release rate (%) = (OD test - OD negative) / (OD positive - OD negative) × 100%

[0040] Example 6: Serum stability and hemolytic activity test of anticancer lipopeptides

[0041] To assess the serum stability of the anticancer lipopeptides, they were co-cultured with fetal bovine serum (FBS) and then the changes in anticancer activity of the FBS-pretreated lipopeptides were detected by MTT assay to evaluate their stability. First, the anticancer lipopeptides were pre-incubated with 10% FBS and PBS for 24 h and 48 h, respectively. When HepG2 cells reached approximately 90% confluence in culture dishes, they were digested with trypsin and seeded into 96-well plates (approximately 2000 cells per well). The cells were cultured in a cell culture incubator (37℃, 5% CO2) for 24 h. The old culture medium was aspirated, and the pre-incubated lipopeptides were added, followed by another 24 h of culture. After 24 h, 20 μL (5 mg / mL) of the pre-incubated lipopeptides was added to each well. -1 Incubate in MTT solution in the dark for 4 hours, then aspirate the supernatant and add 150 μL of DMSO to each well. Place in a shaker (160 rpm) in the dark. -1 After 8 minutes, the absorbance was measured using an ELISA reader (485nm) to calculate the HepG2 survival rate.

[0042] In the hemolytic activity assay, two female mice (30-35g) were randomly selected, and physiological saline and 2.25mg / mL (15mg·kg⁻¹) of saline solution were added. -1 The lipopeptide was injected into mice via tail vein injection. Two hours later, two drops of fresh blood were collected from the eyeball and placed into an anticoagulant tube. The blood was then diluted with physiological saline and the morphology of the red blood cells was observed under a fluorescence microscope.

[0043] Example 7: In vivo antitumor activity of anticancer lipopeptides

[0044] An in vivo tumor-bearing mouse model was established to test the in vivo antitumor activity of lipopeptides. All mice used in the experiment were female Kunming mice (30–40 g) and were strictly fed according to the "Guidelines for the Care and Use of Animals in Experiments". 1×10 6 One H22 cell was subcutaneously injected into the left forelimb axilla of mice. When the tumor volume was approximately 150-220 mm, 3 Mice were randomly divided into two groups of five each: a saline group and a lipopeptide group. 0.2 mL (15 mg / kg) of saline solution was administered to each group. -1 That is, 2.25 mg / mL -1 The drug was administered via tail vein injection every other day. Mice were weighed and tumor size was measured before administration to calculate tumor volume. A total of 5 injections were given. After the experiment, mice were sacrificed by cervical dislocation. The tumors of each group of mice were dissected and fixed in 4% paraformaldehyde solution for more than 24 hours. Finally, the tumor tissue sections were prepared and stained with hematoxylin and eosin (HE).

[0045] This invention prepares anticancer lipopeptides with excellent biomedical properties:

[0046] (1) This anticancer lipopeptide has excellent in vitro anticancer activity.

[0047] The in vitro anticancer activity of anticancer lipopeptides was detected by the MTT assay. Figure 1 As shown in Figure a, the relative cell viability of HepG2 cells gradually decreased with increasing lipopeptide concentration, and the relative cell viability was only 44.68% at 16 μM, indicating that lipopeptides exhibit concentration-dependent killing effects on cancer cells and possess strong in vitro anticancer effects. Figure 1 As shown in b, the relative survival rate of HepG2 cells decreased rapidly within 0-4 hours over time, indicating that the anticancer lipopeptide can exert a highly efficient anticancer ability. Therefore, the anticancer lipopeptide of this invention has excellent in vitro anticancer activity.

[0048] (2) This anticancer lipopeptide exhibits an α-helix structure in a cell membrane simulated environment.

[0049] The secondary structure of the anticancer lipopeptide in water and SDS solution was detected by circular dichroism spectroscopy. Figure 2 As shown, the solid line represents the aqueous solution, simulating the hydrophilic environment encountered by the peptide, while the dashed line represents the SDS solution, used to simulate the hydrophobic environment of the cell membrane. The results show that the anticancer lipopeptide in SDS solution exhibits a positive peak at 195 nm and negative peaks at 208 and 222 nm, displaying a distinct α-helix structure. However, in aqueous solution, most of the anticancer lipopeptides exhibit a random coil structure, and their double negative peaks are not obvious. This indicates that most anticancer lipopeptides do not exhibit an α-helix structure in water, but will show a distinct α-helix structure when encountering a hydrophobic environment. Therefore, it can be inferred that when the anticancer lipopeptide encounters the cell membrane, it will insert into the cell membrane with an α-helix structure, exerting a membrane-breaking effect.

[0050] (3) This anticancer lipopeptide has excellent ability to induce apoptosis in cancer cells.

[0051] The effects of different concentrations of anticancer lipopeptides on apoptosis in HepG2 cells were investigated using an Annexin V-FITC / PI apoptosis assay kit. The eversion of phosphatidylserine residues on the inner cell membrane is a marker of early apoptosis. FITC-labeled Annexin V can bind to phosphatidylserine residues on the cell membrane surface to detect early apoptosis; PI can only enter cells with damaged cell membranes. Late apoptosis cells, due to cell membrane disruption, can be simultaneously penetrated by both FITC-Annexin V and PI, thus allowing for the detection of late apoptosis. Figure 3As shown in (ad), the rate of late apoptosis (UR quadrant) significantly increased with increasing concentration, indicating that the degree of cancer cell membrane rupture gradually increased with increasing peptide concentration. Furthermore, the rate of early apoptosis also increased with increasing concentration. The total apoptosis rate was calculated by summing early and late apoptosis. The apoptosis rates in the control group, the 4μM group, the 8μM group, and the 16μM group were 4.9%, 7.1%, 20.2%, and 42.4%, respectively, showing a dose-dependent effect, and the results were consistent with the MTT assay. Therefore, the anticancer lipopeptide induces apoptosis in a dose-dependent manner.

[0052] (4) This anticancer lipopeptide has a strong ability to lyse cancer cell membranes.

[0053] To investigate the cell membrane permeabilizing activity of anticancer lipopeptides, HepeG2 cells were co-cultured with 8 μM and 16 μM anticancer lipopeptides for 3 h. The effects of the anticancer lipopeptides on cell membrane morphology were observed under a microscope. Figure 4 As shown in ac, the group without medication ( Figure 4 a) The cell membrane morphology was intact, the cell condition was good, and the 8μM anticancer lipopeptide group ( Figure 4 b) Cells begin to shrink, and a small number of cell membranes rupture; 16μM anticancer lipopeptide group ( Figure 4 c) Most cells shrink and become rounded, and the cell membrane is severely damaged, and the cell morphology is no longer regular. Therefore, it can be seen that the anti-cancer lipopeptide destroys the cancer cell membrane in a dose-dependent manner, thereby exerting an anti-tumor effect.

[0054] When cell membrane structure is disrupted, intracellular LDH (lactate dehydrogenase) is released extracellularly. The ability of anticancer lipopeptides to cleave cancer cell membranes is quantitatively determined using an LDH release assay. For example... Figure 4 As shown in d, the LDH release increases with the increase of the anticancer lipopeptide concentration, and the LDH release reaches 67.04% at 16 μM, indicating that this anticancer lipopeptide has a good ability to lyse cancer cell membranes.

[0055] (5) This anticancer lipopeptide has high serum stability and low hemolytic toxicity.

[0056] The hemolytic activity of peptides is an important indicator for evaluating their in vivo safety. This study assessed the hemolytic activity of anticancer lipopeptides by examining their effects on mouse erythrocyte morphology. Figure 5 As shown in ab, the morphological changes of erythrocytes in mice in the saline group and the anticancer lipopeptide group were not significant, both exhibiting normal biconcave disc shape. Therefore, this anticancer lipopeptide did not show obvious hemolytic toxicity and had high biocompatibility.

[0057] The poor in vivo stability of most peptides is the biggest obstacle to their clinical application. 24 μM anticancer lipopeptides were pre-incubated in 10% FBS and PBS for 24 h and 48 h, respectively, and then the serum stability of the peptides was evaluated by detecting cell viability. Figure 5As shown in c, the cell viability of the anticancer lipopeptide after pretreatment with 10% FBS and PBS for 24 h was 40% and 36%, respectively, with little change in its anticancer activity. Meanwhile, the anticancer activity of the lipopeptide pretreated with 10% FBS for 48 h only decreased slightly. Therefore, this anticancer lipopeptide has high serum stability.

[0058] (6) This anticancer lipopeptide has a good in vivo antitumor effect.

[0059] To investigate the in vivo antitumor activity of anticancer lipopeptides, a tumor-bearing mouse model was established, and H22 cells (approximately 1 × 10⁻⁶) were injected under the armpits of the mice. 6 The tumor grew to 150-220 mm in size. 3 The mice were injected with the drug every other day, and the tumor volume was measured and weighed. After the experiment, the tumor tissue was dissected, fixed with 4% paraformaldehyde, and then sectioned and stained with hematoxylin and eosin (HE). Figure 6 As shown in Figure a, the body weight of tumor-bearing mice in both the saline group and the anticancer lipopeptide group did not change significantly with increasing administration frequency and duration, while the relative tumor volume of mice in the anticancer lipopeptide group ( Figure 6 (b) The tumor size was significantly reduced compared to the saline group, indicating that the anticancer lipopeptide can significantly inhibit tumor growth without affecting the mouse's body weight. To further investigate the antitumor activity of the anticancer lipopeptide, the mouse tumor tissue was dissected after the experiment, fixed with 4% paraformaldehyde, and then sectioned and stained with hematoxylin and eosin (HE). Figure 6 As shown in cd, NS group ( Figure 6 c) Tumor cells are densely packed with intact nuclei, and contain anticancer lipopeptides ( Figure 6 d) The tumor cell density was lower than that of the NS group, and the number of necrotic cells was significantly higher than that of the NS group. Therefore, this anticancer lipopeptide can exert a significant in vivo antitumor effect.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Any non-essential modifications, alterations, or substitutions made based on the present invention should be included within the scope of protection of the present invention. sequence list <110> Binzhou Medical College <120> Preparation of an anticancer lipopeptide and its application in antitumor therapy <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <400> 1 Asp Ser Asp Val Trp Trp Gly Gly Arg Arg Leu Leu Arg Arg Leu Arg 1 5 10 15 Leo Arg

Claims

1. An anticancer lipopeptide, characterized in that, The sequence from N-terminus to C-terminus is C8H. 15 O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu, where Asp is aspartic acid, Ser is serine, Val is valine, Trp is tryptophan, Gly is glycine, Arg is arginine, and Leu is leucine, C8H 15 O represents caprylic acid (C8H) 16 The molecular formula of the anticancer lipopeptide after dehydration condensation with the N-terminal Asp amino group (O2) is as follows: 。 2. Anti-cancer lipopeptide C8H 15 The preparation method of O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu is characterized by, Includes the following steps: (1) First, Fmoc-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp-(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin was synthesized by solid-state synthesis. A certain amount of Fmoc-Leu-Wang Resin (Fmoc stands for 9-fluorenylmethoxycarbonyl, used here as an amino protecting group) was weighed and added to DMF (N,N-dimethylformamide) to swell the resin for 30 min. Then, piperidine was added at a ratio of DMF:piperidine = 4:1, and the reaction was allowed to proceed for 30 min to remove the Fmoc group. After the reaction was completed, the resin was filtered and washed repeatedly with DMF, DCM (dichloromethane), and DMF in sequence. Anhydrous DMF was then added again to swell the resin for 30 min. Add two molar amounts of Fmoc-Arg(Pbf)-OH (Pbf being the side-chain protecting group of Arg), 2.6 molar amounts of DCC (dicyclohexylcarbodiimide), HOBT (1-hydroxybenzotriazole), and DIEA (N,N-diisopropylethylamine), and react at room temperature for 48 hours. After the reaction is complete, wash the resin repeatedly with DMF, DCM, and DMF in sequence, then transfer it to a dialysis bag (MW: 8000-14000) and dialyze it with anhydrous ethanol for purification. Freeze-dry the dialyzed sample to obtain Fmoc-Arg(Pbf)-Leu-Wang. Resin, repeat the above steps to sequentially couple amino acids until Fmoc-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin is synthesized, where Boc is the Trp side chain protecting group, tBu is the Ser side chain protecting group, and OtBu is the Asp side chain protecting group; (2)C8H 15 Synthesis of O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu; First, Fmoc groups were removed from Fmoc-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Leu-WangResin according to step (1) to obtain Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-WangResin. Resin; 2 molar amounts of C8H 16 O2 (octanoic acid), 2.5 molar amounts of NHS (N-hydroxysuccinimide) and 2.5 molar amounts of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) were added to anhydrous DMF and magnetically stirred for 5 hours to activate C8H. 16 The carboxyl group of O2 was removed, and then Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin was added and reacted for 48 h. The mixture was then filtered and transferred to a dialysis bag (MW: 8000-14000), purified by dialyzing with anhydrous ethanol, and freeze-dried to obtain C8H. 15 O-Asp(OtBu)-Ser(tBu)-Asp(OtBu)-Val-Trp(Boc)-Trp(Boc)-Gly-Gly-Arg(Pbf)-Arg(Pbf)-Leu-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Arg(Pbf)-Leu-Wang Resin was prepared as a cutting fluid according to the ratio of trifluoroacetic acid (TFA):water (H2O):triisopropylsilane (Tis) = 95:2.5:2.

5. This fluid was mixed with the lyophilized resin and magnetically stirred for 1.5 h to remove the resin and all side-chain protecting groups. After the reaction was complete, the filtrate was collected, concentrated by rotary evaporation, and precipitated by adding ice-cold diethyl ether. The precipitate was then centrifuged and washed repeatedly with diethyl ether, and finally lyophilized to obtain the lipopeptide C8H. 15 O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu.

3. The anticancer lipopeptide C8H according to claim 1 15 Application of O-Asp-Ser-Asp-Val-Trp-Trp-Gly-Gly-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg-Leu in the preparation of anti-hepatocellular carcinoma drugs.

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