Preparation and application of 10-hydroxycamptothecin modified polypeptide

Abstract

The invention relates to preparation of 10-hydroxycamptothecine modified polypeptide and a research on cytotoxicity of the 10-hydroxycamptothecine modified polypeptide. The method comprises the following steps: synthesizing SA-7 polypeptide by adopting an F-moc solid-phase synthesis method, and combining the SA-7 polypeptide with 10-hydroxycamptothecine through chemical coupling reaction to obtain the SA-7-HCPT compound. C18 reversed-phase high performance liquid chromatography (RP-HPLC) is used for purification, and the molecular weight is identified to be 1898Da by using mass spectrum. A further cytotoxicity test shows that the SA-7-HCPT shows relatively strong anticancer activity to HepG2, and the toxicity of the SA-7-HCPT to HepG2 is remarkably superior to that of 10-HCPT to HepG2. Meanwhile, the toxicity of SA-7-HCPT to normal liver cells LO2 is relatively low, and the SA-7-HCPT shows good selectivity. Therefore, the SA-7-HCPT has a relatively good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the preparation of a 10-hydroxycamptothecin modified polypeptide and its application in the treatment of liver cancer. Background Art

[0002] Liver cancer is a common malignant tumor worldwide. Existing treatment methods such as surgical resection, radiotherapy and chemotherapy, and targeted drugs have certain limitations. Therefore, it is of great significance to develop new, efficient and safe anti-liver cancer drugs. 10-Hydroxycamptothecin (10-HCPT) is a broad-spectrum anti-tumor drug that can target topoisomerase I and inhibit DNA reconnection, thereby causing apoptosis of tumor cells. However, the poor solubility, poor biocompatibility and large toxic side effects of 10-HCPT limit its clinical application. A large number of studies have shown that sialic acid has a high content on the surface of various tumor cells and can be used as a marker for tumor recognition. At the same time, increased sialic acid content can enhance the migration and infiltration ability of tumor cells and reduce their adhesion ability. Therefore, sialic acid can be used as a target for diagnosis and treatment of malignant tumors. SA-7 (AMDPRQSNRLYS) is a polypeptide that can target sialic acid, and the polypeptide has good targeting. The present invention obtains SA-7-HCPT by designing a form of 10-HCPT modified SA-7, which significantly improves its anti-tumor activity and selectivity. Summary of the invention

[0003] The present invention relates to the synthesis and purification of a drug SA-7-HCPT containing SA-7 polypeptide, as well as the study of its anticancer activity against HepG2 and its selective toxicity against normal liver cells LO2. Specifically, the following steps are included:

[0004] 1. Synthesis of SA-7: The present invention adopts the F-moc solid phase synthesis method to sequentially couple amino acids from the C-terminus to the N-terminus to the Rink Amide resin to prepare the SA-7 polypeptide.

[0005] 2. Synthesis of SA-7-HCPT: The synthesized SA-7 polypeptide was modified with anhydride and then connected with 10-hydroxycamptothecin through a chemical coupling reaction to synthesize the SA-7-HCPT complex.

[0006] 3. Purification of SA-7-HCPT: SA-7-HCPT was separated and purified using C18 reverse phase high performance liquid chromatography (RP-HPLC), and its molecular weight was identified as 1898 Da by mass spectrometry.

[0007] 4. Study on the cytotoxicity of SA-7-HCPT on liver cancer cells (HepG2): The inhibitory effect of SA-7-HCPT on HepG2 cells was tested by CCK-8 method. The results showed that SA-7-HCPT had significant anticancer activity. Its IC 50 The IC value was 1.388 μM, which was significantly better than that of 10-HCPT. 50 (3.837μM).

[0008] 5. Study on the selective toxicity of SA-7-HCPT to normal hepatocytes (LO2): In the experiment comparing normal hepatocytes LO2 and HepG2 cells, SA-7-HCPT showed low toxicity to LO2 cells, IC 50 greater than 20 μM, while 10-HCPT showed a higher toxicity (IC 50 This indicates that SA-7-HCPT has good selectivity and can effectively inhibit the growth of liver cancer cells, while having little toxicity to normal liver cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 SA-7-HCPT chromatographic separation and purification diagram

[0010] Figure 2 Mass spectrometry identification of purified SA-7-HCPT samples

[0011] Figure 3 Cytotoxicity of SA-7-HCPT and 10-HCPT to HepG2 cells

[0012] Figure 4 Cytotoxicity of SA-7-HCPT and 10-HCPT to LO2 cells DETAILED DESCRIPTION

[0013] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0014] Main materials and instruments

[0015] DMEM culture medium, fetal bovine serum and trypsin were purchased from Gibco, CCK8 kit was purchased from Bio-Time, and HepG2 and LO2 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0016] Example 1 Synthesis of SA-7

[0017] 1.1 Experimental procedures

[0018] SA-7 was synthesized using F-moc solid phase synthesis, with amino acids coupled to Rink Amide resin one by one in the order of C-terminus to N-terminus. The specific steps are as follows:

[0019] (1) Activation of resin: Weigh 0.1 mmol of resin, add 3 mL of DMF, pour the resin into the synthesis column, and place it in a mixer for activation for 1 h;

[0020] (2) Washing the resin: Wash the activated resin three times with DMF and drain;

[0021] (3) Deprotection: The dried resin was deprotected with 20% piperidine, the first reaction took 7 min and the second reaction took 8 min, then washed with DMF 8 times;

[0022] (4) Activation of amino acids: 0.5 mmol HOBT, 0.5 mmol HCTU and 0.4 mmol amino acid were dissolved in 3 mL 5% N-methylmorpholine solution and reacted for 15 min;

[0023] (5) First amino acid coupling: Add the activated amino acid to the resin and react for 1 h. After the reaction, drain the liquid and add DMF to wash 8 times;

[0024] (6) Second amino acid coupling: The amino acid coupled to the resin in (5) was deprotected with piperidine, and then the next activated amino acid was added and reacted for 1 h. After the reaction, the liquid was drained and washed with DMF 8 times;

[0025] (7) The subsequent amino acid coupling is carried out in the order of deprotection, activation, and coupling until the last amino acid coupling is completed.

[0026] Example 2 Synthesis of SA-7-HCPT

[0027] 2.1 Experimental procedures

[0028] When SA-7 is coupled to the last amino acid, the polypeptide is still on the resin. Take out 0.1mM of the peptide resin and put it into the synthesis column. Weigh 0.4mM glutaric anhydride, 0.5mM HCTU, and 0.5mM HOBT, dissolve in 5% N-methylmorpholine and mix with the resin, and react for 1h. After draining, wash with DMF 8 times, weigh 0.5mM DMAP, EDC and 0.4mM 10-hydroxycamptothecin, dissolve in pyridine and mix, then transfer to the synthesis column and react overnight to obtain the product. After the reaction is completed, wash the resin 8 times with DMF, drain the liquid, then wash it 8 times with anhydrous methanol, and drain the liquid. Finally, add the lysis solution, the formula of the lysis solution is 90% TFA, 2.5% anisole, 2.5% anisole thioether and 5% ethylene dimercaptoethane. The sample was lysed for 2.5 h, precipitated with ice ether, added with 30 mL of ether, and then mixed with the lysate, centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and naturally dried to obtain crude SA-7-HCPT.

[0029] Example 3 Purification of SA-7-HCPT

[0030] 3.1 Experimental procedures

[0031] The crude SA-7-HCPT was dissolved by mixing double distilled water and acetonitrile, and then filtered by a 0.22 μm filter head. The obtained supernatant was separated and purified by C18 RP-HPLC (Waters Company, Alliance HPLC & Millennium High Performance Liquid Chromatography Workstation, 2690 Solvent Delivery System, 996 PDA Detector, Separation Column: Reverse Phase Column, C18, 4.6 mmX250 mm; Eluents: A solution (0.1% TFA / H2O), B solution (0.1% TFA / ACN); Elution gradient, 10-60%), and the elution peak was collected by monitoring at 215 nm and freeze-dried.

[0032] 3.2 Experimental Results

[0033] The experimental results are as follows Figure 1 As shown, SA-7-HCPT was purified by RP-HPLC at 215 nm, and the peaks at different times were collected, a corresponding peak was collected in the first 5 minutes, and another peak was collected at 30.6 minutes. The molecular weight was then identified by mass spectrometry to determine which peak was the target.

[0034] Example 4 Mass Spectrometric Identification of SA-7-HCPT

[0035] 4.1 Experimental procedures

[0036] 1 μL of sample was spotted onto the mass spectrometry standard plate. After the liquid dried, 1 μL of the prepared CCA matrix (dissolved in 50% acetonitrile and 50% water, with 0.1% TFA added to the solution) was taken and covered onto the dried sample spot. After the sample dried, it was identified in the AB SCIEX5800MALDI TOF / TOFTM high-resolution system.

[0037] 4.2 Experimental Results

[0038] The sample peaks purified by chromatography were collected, 1 μL of the sample was spotted onto the mass spectrometry standard plate, and the sample was bombarded under laser conditions. Finally, we determined that the sample at 30.6 min was our target peak, and the molecular weight identified by mass spectrometry was 1898 Da.

[0039] Example 5 Toxicity of SA-7-HCPT to HepG2 Cells

[0040] 5.1 Experimental procedures

[0041] After HepG2 was digested with trypsin, the culture medium stopped digestion, centrifuged at 1000rpm for 3min, and the cell density was adjusted. 100μL (5000 cells) was added to each well of the 96-well plate. The added cells were cultured in the incubator for 24h and then the culture medium was aspirated. The experimental group was SA-7-HCPT and the control group was 10-HCPT. The corresponding prepared drugs were added to each well, and each group was repeated 3 wells. After the drug was incubated for 24h, the culture medium was aspirated and replaced with serum-free culture medium and 100μL was added to each well, and then 10μL of CCK-8 was quickly added to each well, and incubated for 1 to 5h. Finally, the absorbance value was measured at a wavelength of 450nm. The blank control was serum-free culture medium and the corresponding CCK-8 was added at the same time. Cell viability (%) = (absorbance value of the experimental group-absorbance value of the blank control) / (absorbance value of the experimental control group-absorbance value of the blank control).

[0042] 5.2 Experimental Results

[0043] After the isolated SA-7-HCPT was freeze-dried, the toxicity of the drug to HepG2 was tested. We prepared concentrations of 20μM, 10μM, 5μM, 2.5μM, 1.25μM, 0.625μM, 0.0625μM, and 0.0062μM. For the control group 10-HCPT, we also prepared concentrations of 20μM, 10μM, 5μM, 2.5μM, 1.25μM, 0.625μM, 0.0625μM, and 0.0062μM. Figure 3 As shown, after drug treatment for 24 hours, the inhibition of HepG2 by SA-7-HCPT at high concentration was more obvious than that of the control group. 50 The IC value of 10-HCPT on HepG2 was 1.388μM. 50The results showed that SA-7-HCPT had a better therapeutic effect.

[0044] Example 6: Cytotoxicity of SA-7-HCPT to LO2

[0045] 6.1 Experimental procedures

[0046] After LO2 was digested with trypsin, the culture medium was used to terminate digestion, centrifuged at 1000rpm for 3min, and the cell density was adjusted. 100μL (5000 cells) was added to each well of the 96-well plate. The added cells were cultured in the incubator for 24h and then the culture medium was removed. The experimental group was SA-7-HCPT and the control group was 10-HCPT. The corresponding prepared drugs were added to each well, and each group was repeated for 3 wells. After the drug was incubated for 24h, the culture medium was removed and replaced with serum-free culture medium and 100μL was added to each well, and then 10μL of CCK-8 was quickly added to each well, and the incubation continued for 1 to 5h. Finally, the absorbance value was measured at a wavelength of 450nm. The blank control was serum-free culture medium and the corresponding CCK-8 was added at the same time. Cell viability (%) = (absorbance value of the experimental group - absorbance value of the blank control) / (absorbance value of the experimental control group - absorbance value of the blank control).

[0047] 6.2 Experimental Results

[0048] In order to test whether SA-7-HCPT has better selectivity, we used LO2 cells to test whether the drug has obvious toxic side effects on normal liver cells. After the isolated SA-7-HCPT was lyophilized, the toxicity of the drug to LO2 was tested. We prepared concentrations of 20μM, 10μM, 5μM, 2.5μM, 1.25μM, 0.625μM, 0.0625μM, and 0.0062μM. For the control group 10-HCPT, we also prepared concentrations of 20μM, 10μM, 5μM, 2.5μM, 1.25μM, 0.625μM, 0.0625μM, and 0.0062μM. Figure 4 As shown, after drug treatment for 24 hours, the inhibition of LO2 by SA-7-HCPT at high concentration was weaker than that of the control group. 50 Greater than 20μM, 10-HCPT for LO2 IC 50 The results showed that SA-7-HCPT was less toxic to normal liver cells than 10-HCPT.

[0049] In conclusion, the effects of SA-7-HCPT on HepG2 IC 50 The IC value of 10-HCPT for HepG2 cells was 1.388 μM, which was greater than 20 μM for LO2 cells. 50The IC value for LO2 cells is 3.837 μM. 50 The results showed that SA-7-HCPT has a good effect on the treatment of HepG2, with strong selectivity and weak toxic side effects.

Claims

1. A method for synthesizing a drug SA-7-HCPT containing SA-7 polypeptide, characterized in that: The method comprises the following steps: (1) synthesizing a SA-7 polypeptide by using an F-moc solid phase synthesis method; (2) modifying the SA-7 polypeptide with glutaric anhydride and then connecting it with 10-hydroxycamptothecin by a chemical coupling reaction to obtain a SA-7-HCPT complex; (3) purifying the SA-7-HCPT by using C18 reverse phase high performance liquid chromatography (RP-HPLC), and identifying the molecular weight of the SA-7-HCPT as 1898 Da by mass spectrometry.

2. A method for testing the cytotoxicity of SA-7-HCPT drugs, characterized in that: The test method comprises: (1) using HepG2 cells and LO2 cells for drug treatment; (2) using CCK-8 method to test the inhibitory activity of SA-7-HCPT on HepG2 and LO2 cells; (3) calculating IC 50 The IC values ​​of SA-7-HCPT on HepG2 cells were obtained. 50 The value is 1.388 μM, and the IC value for LO2 cells 50 The value was greater than 20 μM.

3. The cytotoxicity test method according to claim 2, wherein the IC of SA-7-HCPT on HepG2 cells is 50 The IC value for LO2 cells is 1.388 μM. 50 Greater than 20μM, showing lower toxic side effects.

4. A SA-7-HCPT drug, characterized in that: The drug is synthesized by the method of claim 1 and has significant anti-liver cancer activity and lower toxic side effects on normal liver cells.

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