| Background:In recent years,with the improvement of living conditions,the incidence and mortality of colon cancer have shown an obvious upward trend.Chemotherapy is a common treatment for colon cancer.With the successive application of oxaliplatin,irinotecan and other drugs,the chemotherapeutic effect of colon cancer has been obviously improved.However,colon cancer is prone to develop drug resistance to commonly used chemotherapeutic drugs,which leads to local recurrence and metastasis of colon cancer.The 5-year survival rate of colon cancer patients with metastasis is less than10%.Therefore,new methods to inhibit the growth and metastasis of drug-resistant colon cancer are urgently needed.The uncontrolled proliferation and adhesion of colon cancer cells are the key factors that lead to the growth and metastasis of colon cancer.Clinical studies have found that the higher the content of galactin-3(Gal-3)in serum and cancer tissue of colon cancer patients,the higher the incidence of metastasis.In addition,Gal-3 also plays an important role in the migration,anti-anoikis and metastasis of cancer cells.Therefore,Gal-3 is considered as a new target to inhibit colon cancer metastasis.Studies indicated that small interfering RNA of Gal-3(si Gal-3)significantly reduced the migration,invasion and motility of oxaliplatin-resistant(hereafter referred to as drug-resistant)colon cancer cells,but the inhibitory effect of si Gal-3 on the proliferation of drug-resistant colon cancer cells was limited.We found that the saponin VII(PSⅦ)extracted from trillium significantly inhibited the proliferation of drug-resistant colon cancer cells,but its effect on the metastasis of drug-resistant colon cancer was not obvious,suggesting that the combined application of si Gal-3 and PSⅦ would synergically inhibit the growth and metastasis of drug-resistant colon cancer.Calcium phosphate nanoparticles are excellent delivery vectors for siRNA because of their high transfection efficiency,good biocompatibility and biodegradability.However,calcium phosphate nanoparticles are easy to aggregate during preparation and have no active targeting to colon cancer.Our previous study found that modified citrus pectin(MCP)could improve the stability of calcium phosphate nanoparticles.In addition,MCP is also a natural ligand of Gal-3.Thus,MCP-modified calcium phosphate nanoparticles can actively capture Gal-3 in the blood circulation.Through the interaction between Gal-3and integrin αvβ3,MCP-modified calcium phosphate nanoparticles actively target to drugresistant colon cancer cells on which the integrin αvβ3 is highly expressed.Objective:By using MCP and PSⅦ loaded alendronic acid-β-cyclodextrin inclusion complex(PSⅦ@ALN-β-CD)as stabilizers,calcium phosphate nanoparticles co-loaded with si Gal-3 and PSⅦ(SP@MCaP)were prepared through co-precipitation method.SP@MCaP delivered si Gal-3 and PSⅦ to drug-resistant colon cancer cells by actively capturing Gal-3 in blood.SP@MCaP inhibited the growth and metastasis of drug-resistant colon cancer by inhibiting proliferation and remodeling adhesion of drug-resistant colon cancer cells.It was expected to explore a new method for the treatment of drug-resistant colon cancer.Methods:1.By using MCP and PSⅦ@ALN-β-CD as stabilizers,SP@MCaP and SP@MCaP with Gal-3 corona(G-SP@MCaP)were prepared by co-precipitation method.Laser particle size analyzer,ultra-high performance liquid chromatograph(UPLC),transmission electron microscope(TEM),gel electrophoresis and ELISA were used to evaluate the particle size,Zeta potential,stability,appearance morphology,drug loading and in vitro drug release characteristics of the nanoparticles.2.MTT,cell clonal formation,dead/live cell staining and western blot were used to investigate the effect of G-SP@MCaP on the proliferation of HCT116/L cells and its mechanism.The migration experiment,invasion experiment and western blot were used to investigate the effects of G-SP@MCaP on the migration and invasion abilities of HCT116/L cells and their mechanisms.The effect of G-SP@MCaP on the adhesion ability of HCT116/L cells and its mechanism were investigated by fluorescent staining and western blot.3.The therapeutic effects of SP@MCaP on orthotopic drug-resistant colon cancer and liver metastasis of drug-resistant colon cancer in nude mice were investigated by in vivo imaging,H&E staining and immunofluorescence staining.The effect of SP@MCaP on immune microenvironment of drug-resistant colon cancer tissue was evaluated by immunofluorescence staining and ELISA.The safety of SP@MCaP in vivo was investigated by alanine aminotransferase activity,aspartate aminotransferase activity,lactate dehydrogenase activity,urea nitrogen content,creatinine content in serum and H&E staining.Results:1.The average particle size and Zeta potential of SP@MCaP was 141.5±3.7 nm and-14.9±1.5 m V,respectively.SP@MCaP showed a spherical appearance.The drug loading capacity of PSⅦ in SP@MCaP was(3.06±0.25)%.The results of gel electrophoresis and ELISA showed that SP@MCaP could actively capture Gal-3 to form G-SP@MCaP.The average particle size and Zeta potential of G-SP@MCaP was 151.2±3.1 nm and-4.3±0.6m V,respectively.G-SP@MCaP exhibited a spherical appearance.The drug loading capacity of PSⅦ in G-SP@MCaP was(3.02±0.27)%.G-SP@MCaP was stable within 7days and showed acid-responsive drug release properties.2.G-SP@MCaP significantly increased the expression of Bax,Cleaved caspase-3and E-cadherin and decreased the expression of Bcl-2,CD44,N-cadherin,MMP-9,Rho A,and Cdc42,which subsequently inhibited the proliferation,migration,invasion and movement of HCT116/L cells.G-SP@MCaP increased the adhesion between HCT116/L cells and decreased the adhesion between HCT116/L cells and extracellular matrix.At the same time,G-SP@MCaP also reduced the adhesion between HCT116/L cells and HUVEC cells.3.SP@MCaP could specifically accumulate in drug-resistant colon cancer tissue by actively capturing Gal-3 in the blood and inhibited the growth of orthotopic drug-resistant colon cancer in nude mice.SP@MCaP significantly increased the level of TNF-α and IFN-γ,and decreased the level of TGF-β and IL-10 in orthotopic drug-resistant colon cancer tissue by reducing the expression of Gal-3,indicating that SP@MCaP significantly ameliorated the immunosuppressive microenvironment of orthotopic drug-resistant colon cancer tissue.SP@MCaP also improved the number of NK cells in orthotopic drugresistant colon cancer tissue.In addition,SP@MCaP significantly decreased Gal-3 level in serum and Gal-3 expression in liver tissue in nude mice with liver metastasis of drugresistant colon cancer,thereby NK cells and B cells were recruited to kill drug-resistant colon cancer cells,which finally inhibited liver metastasis of drug-resistant colon cancer in nude mice.After injection of SP@MCaP to normal nude mice at therapeutic dose,the alanine aminotransferase activity,aspartate aminotransferase activity,lactate dehydrogenase activity,urea nitrogen content and creatinine content were all within the normal range,and no significant morphology changes were observed in brain,heart,liver,spleen,lung,and kidney tissue of nude mice,suggesting that therapeutic dose of SP@MCaP had good safety in vivo.Conclusions:G-SP@MCaP exhibited good stability and acid-responsive drug release characteristics.G-SP@MCaP selectively accumulated in HCT116/L cells with high integrin αvβ3 expression and inhibited the proliferation,migration,invasion and movement of HCT116/L cells.In addition,by actively capturing Gal-3 in the blood,SP@MCaP recognized drug-resistant colon cancer cells with high integrin αvβ3expression and specifically accumulated in drug-resistant colon cancer tissue in vivo.Furthermore,G-SP@MCaP reduced the metastasis of HCT116/L cells to the distal site by regulating their adhesion properties.Finally,under the synergistic effect of si Gal-3 and PSⅦ,SP@MCaP inhibited the growth and metastasis of drug-resistant colon cancer by reducing the proliferation of drug-resistant colon cancer cells,reconstructing the adhesion of drug-resistant colon cancer cells and ameliorating the immunosuppressive microenvironment of drug-resistant colon cancer tissue. |
PDF Full Text Request