阅读说明：本技术 双咪唑盐及载药体系在作为抗癌剂中的应用和抗癌制剂 (Application of biimidazole salt and drug-carrying system in serving as anticancer agent and anticancer preparation ) 是由 高印 栾雪 于 2021-10-22 设计创作，主要内容包括：本发明提供了双咪唑盐及载药体系在作为抗癌剂中的应用和抗癌制剂,涉及抗癌药技术领域。本发明提供作为糖基转移酶抑制剂的双咪唑盐C20/C22和包含所述双咪唑盐C20/C22的载药体系,研究了其针对人脐静脉内皮细胞系(HUVEC)和一系列癌细胞系的选择性细胞毒性；结果表明C20/C22及其载药体系具有作为新型抗癌剂的临床潜力,并且作为辅助性的抗癌药物值得进一步发展。(The invention provides application of a bisimidazole salt and a drug-loading system in serving as an anticancer agent and an anticancer preparation, and relates to the technical field of anticancer drugs. The invention provides a bisimidazole salt C20/C22 serving as a glycosyl transferase inhibitor and a drug-carrying system containing the bisimidazole salt C20/C22, and researches the selective cytotoxicity of the bisimidazole salt C20/C22 against a human umbilical vein endothelial cell line (HUVEC) and a series of cancer cell lines; the results show that C20/C22 and the drug-loading system thereof have clinical potential as a novel anticancer agent, and are worthy of further development as an auxiliary anticancer drug.)

1. The drug loading system with the anticancer effect and containing the bisimidazole salt is characterized in that the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and the structure of the bisimidazole salt is shown as a formula I:

wherein n is 20 or 22.

2. The use of claim 1, wherein the drug delivery system comprises encapsulating the bisimidazolium salt with albumin nanoparticles.

3. The use of claim 1 or 2, wherein the drug-loaded system has a drug loading of no less than 7%.

4. Use of a bisimidazolium salt or the drug-loaded system according to any one of claims 1 to 3 for the preparation of an anticancer agent, wherein the bisimidazolium salt comprises a positively charged bisimidazolium divalent salt having the structure shown in formula I:

wherein n is 20 or 22.

5. The use of claim 4, wherein the anti-cancer agent is resistant to metastatic cancer.

6. The use of claim 4 or 5, wherein the anti-cancer agent is against one or more of liver cancer, breast cancer, lung cancer, prostate cancer, colon cancer and cervical cancer.

7. An agent for inhibiting cancer cell proliferation, wherein the agent comprises a bisimidazolium salt or the drug delivery system of any one of claims 1 to 3, wherein the bisimidazolium salt comprises a positively charged bisimidazolium divalent salt, and has a structure represented by formula I:

wherein n is 20 or 22.

8. A preparation for inhibiting cancer cell migration and invasion, wherein the preparation comprises a bisimidazolium salt or the drug-carrying system according to any one of claims 1 to 3, the bisimidazolium salt comprises a positively charged bisimidazolium divalent salt, and the structure is shown as formula I:

wherein n is 20 or 22.

9. An agent for inducing apoptosis of cancer cells, wherein the agent comprises a bisimidazole salt or the drug delivery system of any one of claims 1 to 3, wherein the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and has a structure shown in formula I:

wherein n is 20 or 22.

10. An agent for inducing cancer cell cycle arrest, comprising a bisimidazolium salt or the drug delivery system of any one of claims 1 to 3, wherein the bisimidazolium salt comprises a positively charged bisimidazolium divalent salt, and has the structure shown in formula I:

wherein n is 20 or 22.

Technical Field

The invention belongs to the technical field of anticancer drugs, and particularly relates to application of a bisimidazole salt and a drug-loading system in serving as an anticancer agent and an anticancer preparation.

Background

Cancer is a life-threatening disease, most of the existing medicines for treating cancer are broad-spectrum chemotherapeutic medicines, and the existing medicines can seriously reduce the life quality of cancer patients due to the obvious killing effect on normal cells. Therefore, the discovery of novel anticancer drugs with selective cytotoxicity, new mechanism of action and less toxic side effects is urgently needed.

Protein glycosylation is the most important post-translational modification process, and by no means perfect statistics, more than half of human proteins are modified by glycosylation (PMID: 24339177). The abnormal folding of protein can cause the abnormal glycosylation due to the disturbance of organism biological function; aberrant glycosylation is closely associated with many diseases, and there is increasing evidence that glycosylation plays a major role in tumor formation and migration (PMID: 31550741). Cell surface glycoproteins are involved in many biological functions, including cell proliferation, differentiation, migration; cell-to-cell interaction, mutual recognition; the interaction of the extracellular matrix with host-pathogens; immune regulation; signal transduction, etc. (PMID: 19530676).

Glycosyltransferases are a large family of enzymes involved in glycan biosynthesis that catalyze highly specific glycosyltransfer reactions, some of which are aberrantly expressed in cancer cells (PMID: 19902428). The glycosyltransferase inhibitors currently on the market are as follows: deoxymannojirimycin Swainsonine (octahydroindolizinetriol) is an alpha 1, 2-mannosyltransferase inhibitor, alpha 1, 2-mannosyltransferase is a key N-glycosylation enzyme, and inhibition of its activity causes ER Stress to eventually cause apoptosis of cancer cells. GlcNAc β 1-2(4,6-di-O-methyl-) Man α 1-6Glc β -pnp (GlcNAc N-acetylglucosaminyltransferase V inhibitor) N-acetylglucosaminyltransferase catalyzes the branching of GIcNAc β 1-6 to N-glucans, and the final product of this enzyme catalysis promotes the migration and invasion of tumor cells. 2-deoxy-Man alpha 1-6(Gn beta 1-2Man alpha 1-3) Man beta-octyi mannose (alpha 1,3) glycoprotein beta 1,2-N acetylglucosaminyltransferase 2 inhibitors, which control the conversion of oligosaccharides to complex and hybrid asparagine-bound glycans. Man α 1-6(6-O-methyl-Man α 1-3) Man β -octyl: mannose (alpha 1,3) glycoprotein beta 1,2-N acetyl glucosyl transferase 1 inhibitor is substrate analogue, competitively inhibits enzyme activity, promotes apoptosis. However, there is still a risk in the current gene drugs for inhibiting glycosyltransferases, and there is still a technical difficulty in using them as anticancer agents because of the large molecular weight of competing glycosyltransferases, so that anticancer agents with small molecular weight are required.

Disclosure of Invention

In view of the above, the present invention aims to provide a bisimidazole salt and a drug carrier system for use as an anticancer agent and an anticancer preparation, wherein the small molecule bisimidazole salt (C20/C22) or the drug carrier system with the action of a glycosyltransferase inhibitor is used for anticancer, and can inhibit the growth of cancer cells and cause cell cycle arrest, and activate multiple apoptosis pathways to induce apoptosis of cancer cells.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a drug loading system with an anticancer effect and containing a bisimidazole salt, wherein the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and the structure of the bisimidazole salt is shown as a formula I:

wherein n is 20 or 22.

Preferably, the drug delivery system comprises the bisimidazole salt encapsulated by albumin nanoparticles.

Preferably, the drug loading rate of the drug-loading system is not less than 7%.

The invention also provides application of the bisimidazole salt or the drug-loaded system in preparation of an anticancer agent, wherein the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and the structure of the bisimidazole divalent salt is shown as the formula I:

wherein n is 20 or 22.

Preferably, the anti-cancer agent is resistant to metastatic cancer.

Preferably, the anticancer agent is against one or more of liver cancer, breast cancer, lung cancer, prostate cancer, colon cancer and cervical cancer.

The invention also provides a preparation for inhibiting cancer cell proliferation, which comprises a bisimidazole salt or the medicine carrying system, wherein the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and the structure is shown as the formula I:

wherein n is 20 or 22.

The invention also provides a preparation for inhibiting cancer cell migration and invasion, which comprises a bisimidazole salt or the medicine carrying system, wherein the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and the structure is shown as the formula I:

wherein n is 20 or 22.

The invention also provides a preparation for inducing cancer cell apoptosis, which comprises a bisimidazole salt or the medicine carrying system, wherein the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and the structure is shown as the formula I:

wherein n is 20 or 22.

The invention also provides a preparation for inducing the cancer cell cycle arrest, which comprises a bisimidazole salt or the medicine carrying system, wherein the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and the structure is shown as the formula I:

wherein n is 20 or 22.

Has the advantages that: the invention provides a bisimidazole salt C20/C22 (PMID: 23375091) as a glycosyl transferase inhibitor and a drug carrier system containing the bisimidazole salt C20/C22, and researches the selective cytotoxicity of the bisimidazole salt C20/C22 against a human umbilical vein endothelial cell line (HUVEC) and a series of cancer cell lines; and the antiproliferative and apoptotic properties of C20/C22 were studied in breast (MCF-7/MDA-MB-231) and liver cancer cells (HepG 2). The results show that C20/C22 has clinical potential as a novel anticancer agent, and is worthy of further development as an auxiliary anticancer drug.

In the embodiment of the invention, albumin nanoparticles are used for encapsulating the bisimidazole salt as a drug loading system (Am-C20/C22) for corresponding research, Am-C20/C22 shows a slow release effect and higher drug water solubility, allergy and immune rejection are reduced, and Am-C20/C22 shows higher cytotoxicity (p <0.001) on cancer cells compared with free C20/C22. The results of the examples show that treatment of cancer cells with C20/C22 and Am-C20/C22 causes G2/M phase arrest in cancer cells, promotes endoplasmic reticulum stress in cancer cells, and up-regulates the expression of TRAIL receptors DR4/DR5, down-regulates the expression of pro-survivin Bcl-2, and increases the expression of pro-apoptotic proteins CHOP, Bax, BaK, Caspase-3, Caspase-7, Caspase-8 and Caspase-9. Therefore, C20/C22 and Am-C20/C22 preferentially inhibit the growth of cancer cells, cause cell cycle arrest, can activate multiple apoptosis pathways to lead the apoptosis of the cancer cells, and can be used as an auxiliary drug of an anticancer drug.

Drawings

FIG. 1 shows the results of experiments conducted on cancer cells treated with C20/C22 in wells of a chamber covered with matrigel;

FIG. 2 shows the results of immunofluorescence analysis of control and C20/C22-treated cancer cells;

FIG. 3 shows the change in the adhesion ability of C20/C22-treated cancer cells to P-Selectin and E-Selectin;

FIG. 4 is a graph of the effect of C20/C22 on the reactive oxygen species production by cancer cells;

FIG. 5 is the effect of C20/C22 in the cell cycle;

FIG. 6 is a graph of the effect of C20/C22 on cancer cell apoptosis;

FIG. 7 is a graph showing the expression levels of apoptosis factors involved in receptor-independent apoptosis following C20/C22 treatment;

FIG. 8 shows the expression level of TRAIL signaling pathway DR4 and DR5 receptor in vitro and in cells after C20/C22 treatment.

Detailed Description

The invention provides a drug loading system with an anticancer effect and containing a bisimidazole salt, wherein the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and the structure of the bisimidazole salt is shown as a formula I:

wherein n is 20 or 22.

The drug-loading system (Am-C20/C22) of the invention preferably comprises the double imidazole salt (C20/C22) encapsulated by albumin nanoparticles. In the present invention, the albumin nanoparticle encapsulation preferably comprises: mixing Albumin nanoparticles (AmNps, abbreviated as Am) with the bisimidazole salt, wherein the weight ratio of the mixture of the Albumin nanoparticles and the bisimidazole salt is preferably (5-20): 1, more preferably 10: 1. The Encapsulation Efficiency (EE) of Am-C20/C22 according to the invention is related to the weight ratio of the blend when Am: the ratio of C20/C22(w/w) is 5: 1,10: 1 and 20: at 1, the EE values are 75.92%, 89.86% and 92.95%, respectively; when the medicine-fat ratio is 1: at 10, the drug loading was about 8.98%. The encapsulation of the present invention can enhance the solubility of C20/C22 in aqueous solution; and EE (%) decreased with time in saline and PBS containing 10% FBS, indicating that C20/C22 can be slowly released from Am-C20/C22 under physiological conditions to express its anti-cancer effect, such as cumulative release of C20/C22 at pH 5.5, 6.5 and 7.4, releasing more C20/C22 in acidic environment (e.g., tumor area).

The C20/C22 of the present invention has been disclosed in the literature (PMID: 23375091) as a small molecule glycosyltransferase inhibitor. In the present embodiment, the bisimidazolium salt is preferably a sulfite, and the structure detection data of C20 used is preferably as follows: 1H NMR (300MHz, DMSO-d6) d 1.15-1.40 (m,32H), 1.65-1.85 (m,4H),2.31(s,6H),3.85(s,6H),4.15(t, J ═ 9.2Hz,4H),7.71(s,2H),7.78(s,2H),9.14(s, 2H); 13C NMR (100MHz, DMSO-d6) d 25.5,28.4,28.8,28.9,29.00,29.04(3C),29.4,35.7, solvent peak, 48.7,122.2,123.6,136.6; HR (human HR)MS(ESI)[M-O₃SCH₃]And + calculating: c₂₉H₅₅N₄O₃539.3994 is the ratio of S to S; the actual measurement is 539.3991; the structure detection data for C22 used are as follows: 1H NMR (400MHz, DMSO-d6) d 1.12-1.42 (m,36H), 1.68-1.85 (m,4H),2.29(s,6H),3.84(s,6H),4.14(t, J ═ 7.2Hz,4H),7.70(s,2H),7.76(s,2H),9.11(s, 2H); 13CNMR (100MHz, DMSO-d6) d 25.5,28.3,28.8,28.9,29.0,29.1(4C),29.3,35.7, solvent peak, 48.7,122.2,123.6,136.5; HRMS (ESI) [ M-O ]₃SCH₃]+ calculate C₃₁H₅₉N₄O₃567.4307 is the ratio of S to S; it is actually measured that 567.4305.

The invention also provides application of the bisimidazole salt or the drug-loaded system in preparation of an anticancer agent, wherein the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and the structure of the bisimidazole divalent salt is shown as the formula I:

wherein n is 20 or 22.

The anticancer agent of the invention is preferably resistant to metastatic cancers, and more preferably resistant to liver cancer, breast cancer, lung cancer, prostate cancer, colon cancer and/or cervical cancer. In the present example, studies using liver cancer cell lines (SMMC-7721, HepG2 and Huh7), breast cancer cell lines (MCF-7 and MDA-MB-231), lung cancer cell line (A549), prostate cancer cell line (PC3), colon cancer cell line (SW480), cervical cancer cell line (Hela) and human umbilical vein endothelial cell line (HUVEC) were carried out to confirm the selective toxicity of the anticancer agent against cancer cells.

The invention also provides a preparation for inhibiting cancer cell proliferation, which comprises a bisimidazole salt or the medicine carrying system, wherein the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and the structure is shown as the formula I:

wherein n is 20 or 22.

In the embodiments of the present inventionHighest IC of C20/C22 was observed with normal HUVEC cell samples₅₀The value was 5. mu.M, while the lowest IC was observed with cancer cells₅₀The value was 2. mu.M. Under the condition of half inhibitory concentration of a cancer cell line sensitive to C20/C22, the inhibition rate of the cancer cell line on human HUVEC cells is less than 10%, and the inhibition rate of the cancer cell line on mouse hippocampal neuronal cells HT22 cells is less than 15%. The C20/C22 can be used as a specific cytotoxic agent within a certain treatment window range, has stronger functions of inhibiting proliferation and killing cancer cells, and has lower toxicity to normal cells.

The invention also provides a preparation for inhibiting cancer cell migration and invasion, which comprises a bisimidazole salt or the medicine carrying system, wherein the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and the structure is shown as the formula I:

wherein n is 20 or 22.

In the embodiment of the invention, the cancer cells treated by C20/C22 can obviously inhibit the migration of the cancer cells in a scratching experiment, and the inhibition effect is more obvious along with the increase of the administration concentration; the cancer cells treated by C20/C22 can obviously inhibit the invasion of the cancer cells in a small-chamber hole experiment which is covered by matrix glue, the invasion of the cancer cells is inhibited by C20 and C22, the inhibition effect is more obvious along with the increase of the administration concentration, and the result shows that the preparation can well inhibit the invasion of the cells and has potential clinical application value for preventing the cancer cells from migrating across endothelium and further generating cancer metastasis.

The invention also provides a preparation for inducing cancer cell apoptosis, which comprises a bisimidazole salt or the medicine carrying system, wherein the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and the structure is shown as the formula I:

wherein n is 20 or 22.

In the embodiment of the invention, the preparation can activate a plurality of ways to induce cancer cell apoptosis, for example, C20/C22 can cause mitochondrial stress and endoplasmic reticulum stress response of cancer cells to further cause apoptosis, and endogenous pro-apoptotic proteins Cytochrome C, CHOP, Bax, BAK and ER-stress marker proteins (GRP-78, IRE1 alpha, ATF6, PERK, P-JNK and the like) TRAIL apoptosis pathway DR4 in cells treated by C20/C22 has increased intracellular expression amount and cell surface distribution of R5 receptors. The expression levels of the promoters (Caspase-8 and Caspase-9) and effector caspases (Caspase-3, Caspase-6 and Caspase-7) were increased. Meanwhile, C20/C22 can reduce the expression level of the anti-apoptotic protein Bcl-2, and C20/C22 induces cancer cell apoptosis through multiple apoptosis paths. Therefore, C20/C22 all showed potential anticancer effects.

The invention also provides a preparation for inducing the cancer cell cycle arrest, which comprises a bisimidazole salt or the medicine carrying system, wherein the bisimidazole salt comprises a positively charged bisimidazole divalent salt, and the structure is shown as the formula I:

wherein n is 20 or 22.

In the present example, C20/C22 significantly increased the percentage of cancer cells in the G2/M phase (p <0.001), indicating that treatment with C20/C22 resulted in G2/M cell cycle arrest, which in turn caused endoplasmic reticulum stress leading to apoptosis.

The application of the bisimidazole salt and the drug carrier system provided by the present invention as an anticancer agent and an anticancer preparation are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

1. Experimental Material

Phosphate buffered saline (PBS, Thermo Fisher Scientific, shanghai, china) and trypsin-EDTA solution (neosemet, beijing, china).

Antibodies such as Bcl-2, Bax, Caspase-3, Caspace-8, and Caspace-9 were obtained from Affinity (Beijing, China).

anti-Lewis A, Lewis B, Sle^XAnd Lewis Y antibody was obtained from Abcam (USA).

TRAIL apoptosis-inducing factor was obtained from Santa Cruz Biotechnology, Inc (canada).

Super ECL detection reagent (Yeasen Biotech Co, Ltd), apoptosis detection kit (TransDetect Annexin V-FITC/PI, Yeasen Biotech, Shanghai, China) and cell cycle detection kit (PI staining, Shanghai, China).

2. Synthesis of C20/C22

The synthesis of C20/C22 was carried out according to the literature (PMID: 23375091), followed by¹H NMR and LC-ESI-MS analysis of compound structure, completed on the glycosyl transferase inhibition activity studies.

3. Cell culture

Liver cancer (SMMC-7721, HepG2 and Huh7), breast cancer (MCF-7 and MDA-MB-231), lung cancer (A549), prostate cancer (PC3), colon cancer (SW480), cervical cancer (Hela) and HUVEC cell lines, all obtained from the cell bank of the Committee for culture Collection of the national academy of sciences type (Shanghai, China).

Cell growth complete medium RPMI 1640 and DMEM (Thermo Fisher Scientific, Shanghai, China) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Shanghai, China) were used to grow the cells.

PC3, HUVEC and SW480 cell lines were cultured in RPMI 1640 medium. Meanwhile, MCF-7, MDA-MB-231, A549, SMMC-7721, HepG2 and Huh7 cell lines were cultured in DMEM medium. 5% CO at 37 ℃₂And (5) culturing in an aseptic incubator.

4. Cytotoxicity assays

Will be in logarithmic growth phase (1X 10)⁴) The cells were seeded in 96-well plates containing complete growth medium in CO₂Incubate in incubator for 24 h. Cells were then treated with varying concentrations of C20/C22 for 24h under serum-free conditions, with 0.01% DMSO as a blank. Then 10 μ LCCK8 solution (assist in san-Ching, Shanghai, China) was added to each well and incubated at 37 ℃ for 2h with an enzyme labelThe absorbance value (OD) at 450nm is detected by the instrument₄₅₀) And each measurement was repeated 6 times. Cell viability was calculated according to the following formula.

Cell viability (%) ═ (Experimental group OD)₄₅₀Blank OD₄₅₀) /(OD of control group)₄₅0-blank OD₄₅₀)×100％

5. Flow cytometry

Will grow from logarithmic phase (3X 10)⁵) The collected cells were plated onto 6-well plates overnight for incubation and C20/22 was added under serum-free media. After 24h incubation in medium containing C20/C22, cells were washed with Phosphate Buffered Saline (PBS) and trypsinized without EDTA.

The apoptosis and cell cycle detection kit (BD Biosciences, San Jose, CA) was described for staining cells, and flow cytometry was used to detect cellular reactive oxygen species production, apoptosis and cycle arrest.

6. Western blot analysis

Cells were incubated for 6, 12 and 24h in medium containing C20/C22, and then total protein was extracted and protein concentrations were measured using the BCA kit (assist in san-Ching, Shanghai, China). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Invitrogen). The membrane was incubated overnight at 4 ℃ with primary antibody, washed three times with TBST, and incubated at room temperature for 2h with secondary antibody (1000 Xdilution). The membranes were washed three times with TBST, and Enhanced Chemiluminescence (ECL) developing solution (Affinity, Biosciences) was added to the PVDF membrane, and the results were recorded and analyzed with Gapdh as a control.

7. Immunofluorescence assay

Placing TC-treated slide in 24-well plate, sterilizing by ultraviolet irradiation, and inoculating 1 × 10 glass per well⁵The cells were incubated overnight, half the inhibitory concentration of C20/C22 in serum-free medium was added, and the temperature was continued at 37 ℃ with 5% CO₂Incubating for 24h under the condition, discarding the supernatant, washing with precooled PBST for 3 times, fixing with 4% paraformaldehyde, washing with precooled PBST for 3 times, performing permeation treatment for detecting intracellular proteins, directly sealing cell surface proteins with 1% BSA at room temperature for 1h, discarding the sealing solution, and adding 1: 100 dilution of primary antibody, 4 ℃ overnight incubationCulturing, discarding supernatant, washing with pre-cooled PBST for 3 times, adding FITC labeled secondary antibody, incubating at room temperature in dark for 1h, washing with pre-cooled PBST in dark for 3 times, adding DAPI to stain nuclei, washing with PBST once, flaking, storing in dark at-20 deg.C, or directly detecting with confocal microscope.

8. Lectin detection glycogen expression assay

Lectin staining was performed according to the method described in the literature (PMID:22843320) to evaluate cell surface glycosylation. Inoculation of 1X 10 per well in 96-well plates⁵Cells were treated for 24h with C20/C22, with 0.01% DMSO as a control, fixed after dosing, and incubated with biotin-labeled lectin, alkaline phosphatase-labeled avidin, and a nitrobenzene-phosphate reaction substrate. OD measurement with microplate reader₄₀₅A color change is detected. Intensity normalized to 1.0 × 10⁵Strength of cells. The difference between the comparative mean values was examined using Student's t. In all assays, p<0.05 was considered statistically significant. Each sample was analyzed at least 6 times.

9. Analysis of cancer cell migration and invasion Capacity

9.1 scratch test for cell migration: pancreatin digestion of cells in a logarithmic growth phase, terminating digestion reaction by using a complete culture medium, abandoning the supernatant, suspending the cells by adding the complete culture medium, inoculating the cells into each hole of a six-hole plate, abandoning the supernatant after overnight incubation, adding 1mLPBS (multi-layered phosphate buffer) for washing once, quickly scratching by using a 10 muL gun head after washing, adding a C20/C22 solution with corresponding concentration after scratching, taking 0.1% DMSO as a blank control, respectively taking a picture of the scratch position after 0/24/48 hours after scratching, and taking a picture of the scratch position after 24/48 hours by subtracting 0 hour from the scratch width, namely the migration distance of each time point.

9.2 cell invasion capacity detection by perforation assay: serum-free medium culture was performed before cell experiments, and experiments were performed after starvation for 24 h. The matrigel was diluted with pre-cooled serum-free medium and added to the transwell chamber and sealed at 37 ℃. Dispensing: the dosage was 2 times the administration concentration. Sealing, performing cell treatment, digesting starved cells with pancreatin, re-suspending basic culture medium, counting, pre-mixing equal volume of cell suspension and C20/C22 solution, slightly blowing into a pre-sealed small chamber, completely culturing in a 24-pore plate at the outer side of the small chamber,37℃，5％CO₂incubate for 24h under conditions. Pouring the culture medium in the cell after culture, digging off the matrix glue, slightly wiping cells in the cell with an absorbent cotton swab, washing with precooled PBS twice, fixing the cell in ethanol, washing with PBS twice after fixing, slightly wiping the interior of the cell with the absorbent cotton swab after washing each time, completely volatilizing the ethanol, placing the cell in crystal violet for dyeing, slightly wiping the interior of the cell with the absorbent cotton swab after dyeing, and taking a picture after absorbing moisture to record a Transwell result.

9.3 selectin recognition experiments to detect cell invasion capacity:

and (2) carrying out selectin coating on an ELISA plate, keeping the temperature at 4 ℃ overnight, removing supernatant, washing twice, adding a sealing liquid, sealing at 4 ℃ overnight, removing the sealing liquid, washing twice, adding a C20/C22 treated cell suspension which is pretreated, incubating for 2 hours at 37 ℃, removing supernatant, adding 10% formaldehyde for fixation, carrying out crystal violet staining, washing the cells for 4 times by deionized water, taking a picture under a microscope, adding a methanol solution containing formic acid for dissolution, and detecting the absorbance at 590nm by an enzyme-labeling instrument.

9.4 lectin staining

Cell surface glycosylation was assessed by lectin staining according to the method described in the literature (PMID: 22843320): inoculation of 10 wells per 96-well plate⁵Cells were treated for 24h with C20/C22, with 0.01% DMSO as a control, fixed after dosing, and incubated with biotin-labeled lectin, alkaline phosphatase-labeled avidin, and a nitrobenzene-phosphate reaction substrate. OD measurement with microplate reader₄₀₅A color change is detected. Intensity normalized to 1.0 × 10⁵Strength of cells. The difference between the comparative mean values was examined using Student's t. In all assays, p<0.05 was considered statistically significant. Each sample was analyzed at least 6 times.

10. Statistical analysis

Statistical analysis was performed using Graphpad Prism 8 in the present invention and data are expressed as mean ± Standard Deviation (SD). Statistical significance was shown in our data as p <0.05, p <0.01, p <0.001 (using Student's t test and one-way ANOVA).

Second, result analysis

1. Structural analysis of C20/C22

The structure detection data for C20 is as follows: 1H NMR (300MHz, DMSO-d6) d 1.15-1.40 (m,32H), 1.65-1.85 (m,4H),2.31(s,6H),3.85(s,6H),4.15(t, J ═ 9.2Hz,4H),7.71(s,2H),7.78(s,2H),9.14(s, 2H); 13C NMR (100MHz, DMSO-d6) d 25.5,28.4,28.8,28.9,29.00,29.04(3C),29.4,35.7, solvent peak, 48.7,122.2,123.6,136.6; HRMS (ESI) [ M-O ]₃SCH₃]And + calculating: c₂₉H₅₅N₄O₃539.3994, 539.3991 is actually measured.

The structure detection data for C22 is as follows: 1H NMR (400MHz, DMSO-d6) d 1.12-1.42 (m,36H), 1.68-1.85 (m,4H),2.29(s,6H),3.84(s,6H),4.14(t, J ═ 7.2Hz,4H),7.70(s,2H),7.76(s,2H),9.11(s, 2H); 13C NMR (100MHz, DMSO-d6) d 25.5,28.3,28.8,28.9,29.0,29.1(4C),29.3,35.7, solvent peak, 48.7,122.2,123.6,136.5; HRMS (ESI) [ M-O ]₃SCH₃]+ calculate C₃₁H₅₉N₄O₃567.4307, 567.4305 is actually measured.

2. C20/C22 inhibit cancer cell proliferation

The cytotoxic effect of C20/C22 on cancer cells was evaluated by the CCK8 assay. Half maximal Inhibitory Concentration (IC) for all cell lines calculated by GraphPad Prism 8₅₀). Among the above cell lines, the highest IC of C20/C22 was observed with normal HUVEC cell samples₅₀Value of 5 μ M, and lowest IC observed with cancer cells₅₀The value was 2 μ M. Under the condition of half inhibitory concentration of a cancer cell line sensitive to C20/C22, the inhibition rate of the cancer cell line on human HUVEC cells is less than 10%, and the inhibition rate of the cancer cell line on mouse hippocampal neuronal cells HT22 cells is less than 15%. The C20/C22 can be used as a specific cytotoxic agent within a certain treatment window range, has stronger functions of inhibiting proliferation and killing cancer cells, and has lower toxicity to normal cells.

3. C20/C22 cancer cell migration and invasion inhibiting effect and mechanism analysis

The cancer cells treated by C20/C22 can obviously inhibit the migration of the cancer cells in a scratch test, for example, after MCF-7 cells are treated by C20 for 24 hours and 48 hours, after the MCF-7 cells are treated by C20, the average migration distance of a control group is about 0.4mm, the average migration distance of a C201 mu M group is about 0.2mm, the average migration distance of a C202 mu M group is about 0.18mm, after the MCF-7 cells are treated by 48 hours, the average migration distance of a control group is about 1.3mm, the average migration distance of a C201 mu M group is about 0.35mm, and the average migration distance of a C202 mu M group is about 0.3mm, and the result shows that the C20 has obvious cell migration inhibition effect on the MCF-7 cells and the inhibition effect is more obvious along with the increase of the administration concentration;

c22 treatment MCF-7 cells for 24h and 48h, after 24h of administration, the average migration distance of a control group is about 0.5mm, the average migration distance of a C221 mu M group is about 0.25mm, the average migration distance of a C222 mu M group is about 0.15mm, after 48h of administration, the migration distance of a control group is about 1.4mm, the average migration distance of a C221 mu M group is about 0.65mm, and the average migration distance of a C222 mu M group is about 0.5mm, and the result shows that C22 has obvious cell migration inhibition effect on the MCF-7 cells, and the migration inhibition effect is more obvious along with the increase of the administration concentration. The same inhibitory trend was shown in other cancer cell lines.

As shown in figure 1, cancer cells treated by C20/C22 can obviously inhibit cancer cell invasion in a small-chamber hole experiment covered by matrigel, C20 and C22 have an inhibition effect on the invasion of MCF-7 and other cancer cells, and the inhibition effect is more obvious with the increase of administration concentration, and the result shows that the drug can well inhibit the cell invasion and has potential clinical application value for preventing the cancer cells from migrating across endothelium and further generating cancer metastasis.

Lewis oligosaccharide Sle on surface of cancer cell^XThe cancer cells treated by the C20/C22 have the effect on the adhesion capacity of the selectin, and the C20/C22 treated cells remarkably inhibit the adhesion capacity of the cells to the selectin and the capacity of inhibiting the cancer cells from transferring to other tissues.

The results of immunofluorescence analysis are shown in FIG. 2, and the cancer cells treated with C20/C22 have cell surface Sle compared to the control group^XAnd the expression level of Lewis Y is obviously reduced, while the expression levels of Lewis A and Lewis B are slightly increased, probably because the beta 4GalNAT recognizes the same substrate, Sle^XAnd Lewis Y synthesis is catalyzed by beta 4GalNAT, and because the enzymatic activity is inhibited, the competitive enzyme beta 3GalNAT can recognize more substrates, thereby promoting the expression quantity increase of products of Lewis A and Lewis BAnd (4) adding. Sle^XAnd Lewis Y expression is important for cancer cell metastasis, and the reduction of the expression level of the two Lewis oligosaccharides can inhibit cancer cell metastasis and reduce the cancer cell malignancy, so that C20/C22 is expected to bring good news to patients with metastatic cancers.

As shown in FIG. 3, the cancer cells treated with C20/C22 showed reduced adhesion to both P-Selectin and E-Selectin, indicating that the inhibitors can inhibit Sle^XAnd Core2 synthesis reduces its ability to migrate across the endothelium.

4. Effect of C20/C22 on Reactive Oxygen Species (ROS) production by cancer cells

The cells after C20/C22 treatment were stained with DCFH-DA and assayed for ROS by flow cytometry, and the results are shown in FIG. 4, which caused an increase in ROS expression in cancer cells. This result suggests that C20/C22 may cause mitochondrial stress and endoplasmic reticulum stress in cancer cells and thus apoptosis.

5. C20/C22 inhibits cancer cell surface glycosylation process

The cancer cells treated with C20/C22 exhibited decreased expression levels of Gal recognized by RCA I, Gal β 4GlcNAc β 6 recognized by PHA-L and Gal β 4GlcNAc recognized by MALI (p < 0.01), while the expression level of GlcNAc recognized by WGA was increased.

6. Effect of C20/C22 in the cell cycle

The C20/C22 treated cancer cell lines were stained with PI and cell cycle analysis was performed by flow cytometry. As shown in FIG. 5, C20/C22 significantly increased the percentage of breast cancer cells MCF-7 and the like in the G2/M phase (p <0.001), indicating that treatment with C20/C22 results in G2/M cell cycle arrest. Which in turn causes endoplasmic reticulum stress leading to apoptosis.

7. Effect of C20/C22 on cancer cell apoptosis

As shown in FIG. 6, Annexin V-FITC and Propidium Iodide (PI) staining were used to determine the level of apoptosis in cancer cells such as breast cancer MCF-7, and the lower dosing concentration condition caused the cancer cells to undergo apoptosis to different degrees, and the proportion of apoptosis in the cancer cells increased with increasing dosing concentration, indicating that C20/C22 is a dose-dependent cytotoxic agent. The same cytotoxic effect is shown for other cancer cell lines such as liver cancer (HepG2) and the like.

8. Expression of anti-apoptotic factors, pro-apoptotic factors and caspase family molecules in cancer cells

The cell membrane and nuclear morphological changes of the AnnexinV-FITC and PI stained cancer cells were analyzed by flow cytometry, and the results showed that C20/C22 treatment could induce cancer cell apoptosis. To further reveal the apoptotic pathway triggered by C20/C22, the expression levels of apoptosis factors involved in receptor-independent apoptosis, the TRAIL apoptotic pathway and endoplasmic reticulum Stress (ER-Stress) profile were investigated by western blot analysis as shown in fig. 7. When cancer cells are treated with serum-free medium containing C20/C22, the expression level of Bcl-2 family molecules changes in a time-dependent manner, while the expression of pro-apoptotic factors Cytochrome C, Bax, BAK is increased in all cancer cells, the TRAIL signaling pathway DR4, DR5 receptor expression is increased, the expression levels of ER-Stress marker proteins GRP78, ATF6, IRE1 alpha, P-JNK and CHOP are increased; and the anti-apoptosis factor Bcl-2 is found to be inhibited. In addition, the expression of Caspase-8 and Caspase-9 increased with increasing incubation time of C20/C22, as was the case for Caspase-3(37 kDa). The immunofluorescence method analyzes that TRAIL signal channel DR4 and DR5 receptor are increased in expression inside and outside cells, as shown in figure 8, the apoptosis ratio of cancer cells is obviously increased by adding TRAIL ligand into cells treated by C20/C22. This analysis shows that C20/C22 stimulates apoptotic pathways in cancer cells (p <0.05), down-regulates anti-apoptotic factor Bcl-2, activates the apoptotic promoter Caspase-9, ultimately leading to activation of the effector Caspase-3 and increasing the level of cleaved Caspase-3 in a time-dependent manner, by up-regulating the pro-apoptotic protein factor Bax, BAK.

9. Properties of C20/C22 albumin nanoparticle

Preparation of a protein with a different albumin: albumin nanoparticles (AmNps) at a C20/C22(w/w) ratio to enhance the solubility of C20/C22 in aqueous solution and to analyze its physicochemical properties including particle size, polydispersity index (PDI), zeta potential, Encapsulation Efficiency (EE) and drug Loading Capacity (LC). The results show a higher Am: C20/C22(w/w) ratio with a small particle size distribution, e.g. 10:1 and 20: 1. further, when Am: the ratio of C20 to C22 is 10:1 also exhibits a smaller polydispersity index (PDI).

When Am: the ratio of C20/C22(w/w) is 1: 1,5: 1,10: 1 and 20: at 1, EE values were 58%, 75.92%, 89.86% and 92.95%, respectively. Finally, a selection is made of 10:1 Am-C20/C22 NPs were prepared for further analysis of stability and cytotoxicity.

Am-C20/C22 stability was measured by measuring the change in particle size over time in saline and PBS containing 10% FBS at 4 ℃ and 37 ℃. The scatter plot showed no significant change in particle size and could be maintained (no aggregation occurred) for at least one week. PDI data are plotted in the middle, which shows a narrow size distribution of Am-C20/C22.

EE (%) decreased with time in saline and PBS containing 10% FBS, indicating that C20/C22 can be slowly released from Am-C20/C22 under physiological conditions to express its anti-cancer effect. From the cumulative release of C20/C22 at pH 5.5, 6.5 and 7.4, it can be seen that more C20/C22 is released in acidic environment (e.g. tumor area).

10. Cytotoxicity of Am-C20/C22

Various concentrations of Am-C20/C22 complex (10: 1) were tested for 24h using the MTT method for cytotoxicity against cancer cell lines. Am-C20/C22 pairs of cancer cells IC after 24h of culture₅₀About 0.9. mu.M, significantly lower than the free C20/C22 group IC₅₀(2-3 μ M). Thus, Am-C20/C22 was shown to exhibit higher cytotoxicity (p) than free C20/C22 against all of the cancer cells tested<0.001)。

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.