Effects Of Peptide Modified Drug Delivery System On Liver Metastasis Of Colon Cancer

Liver metastasis is a leading cause of death in colon cancer.Liver resection and chemotherapy are the standard methods for liver metastatic therapy.However,liver resection is not suitable for all patients and therapeutic effects of chemotherapy are not satisfactory as a result of off-target toxicities.Therefore,it is vital to provide a key strategy for improving the survival rate and quality of life for patients with liver metastatic CRC.In fact,the pathological changes in metastatic cells,including strong invasive abilities,receptor numbers and tortuous vasculature,contribute to the off-target effect.This study aims to find the different receptor numbers between metastatic cells and original cells from orthotopic tumors and then a new targeting drug delivery system was established.The anti-metastatic effects of colon cancer could be achieved by enhanced drug concentrations in metastatic cells.?5?1 integrin is a transmembrane glycoprotein receptor that is highly expressed on invasive colorectal cells.It has been reported that ?5?1 integrin closely associated with liver metastasis.In this study,higher ?5?1 integrin expression in metastatic cells was found when compared with original cells.The wound healing percentage and invasive ability of metastatic cells were stronger than that of original cells.In western Blot assay,the amount of MMP-9 and VEGF in metastatic HT29 cells increased in response to the highly invasive ability of metastatic cells.Proliferative potential of tumor cells was evaluated by splenic injection in vivo.The results showed that metastatic cells largely diffused in the liver and parenchyma was decreased when compared with original cells in the same period of time.All the results indicated that liver metastatic cells had higher?5?1 integrin and highly invasive ability,which probably provides the guidance for designing a new targeting drug delivery system.In this study,low molecular weight of chitosan oligosaccharide(CSO),a marine biological material,was applied to synthesize chitosan oligosaccharide-g-stearic acid(CSOSA)in the presence of EDC.RPM peptide modified chitosan oligosaccharide-g-stearic acid(RPM-CSOSA)was synthesized by amide reactions in the presence of EDC/NHS and DSC.CSOSA and RPM-CSOSA conjugates are capable of forming micelles in aqueous solution.The critical micelle concentration(CMC)of CSOSA and RPM-CSOSA was 79.4 ?g/mL and 94.6 ?g/mL,respectively.The average particle size of CSOSA and RPM-CSOSA was 68.5±0.5 nm and 76.4±2.9 nm,respectively.The morphologies of RPM-CSOSA and CSOSA were determined by TEM;both were spherical with average sizes.Doxorubicin(DOX),a potent anthracycline-based cytotoxic drug,is widely used for several cancers.Curcumin(CUR),polyphenols derived from Curcuma longa,exerts its anti-cancer effects as chemosensitizer.It has been reported that combination DOX and CUR can increase effectively drug concentrations in tumor cells,inhibit multi-drug resistance and enhance the anti-tumor effects.DOX and CUR were chosen as model drugs and was loaded in RPM-CSOSA micelles to form RPM-CSOSA/DOX and RPM-CSOSA/CUR drug delivery system by dialysis method,respectively.The encapsulated efficiency of DOX was 86.5%,while the encapsulated efficiency of CUR was 45.9%.The released patterns of drug delivery system were studied in PBS 5.0.Compared with CSOSA/DOX and CSOSA/CUR,there were about 54.7%accumulative DOX release in RPM-CSOSA,while the CUR release percentage was 53.2%up to 72 h.The cellular internalization was assessed by laser scanning confocal microscope.The results showed that RPM-CSOSA micelles enhance internalization in HT29 cells and HUVEC cells by ?s?1 integrin-mediated pathway.Furthermore,modification by RPM peptide resulted in the enhanced internalization of RPM-CSOSA in HT29 cells,which decreased the cytotoxicity to normal cells.To exert the best effects,an optimal ratio of free DOX and CUR chosen by MTT assay was 1:2(w/w)in vitro.The combined targeting drug delivery system based on the optimal ratio(RPM-CSOSA/DOX&RPM-CSOSA/CUR)was established to evaluate the anti-tumor effects.The IC50 values in HT29 cells and HUVEC cells was 0.81?g/mL and 2.62?g/mL respectively,which was lower than combined drug delivery system(CSOSA/DOX&CSOSA/CUR)and DOX&CUR groups.The results in cell cycle indicated that combination DOX and CUR could induce G2/M phase arrest,and the combined targeting drug delivery system had the most significant effects.Wound healing and Transwell assays showed that the combined targeting drug delivery system could inhibit the migratory and invasive abilities of tumor cells more effectively compared with other groups.Liver metastatic models were established by splenic injection.Distribution of CSOSA micelles in liver metastatic lesions was enhanced by RPM modification.The therapeutic anti-metastasis potential of drug delivery system was further examined in HT29 metastatic models.The results showed that the inhibition rate of metastatic sites in combined targeting drug delivery system was 87.0%,which was 1.3-fold higher than that of the combined drug delivery system.The immunohistochemical staining of liver tissue indicated that combined targeting drug delivery system could significantly inhibited tumor vessels when compared with DOX,DOX&CUR and the combined drug delivery system.The results in this study indicated that synthesized RPM peptide modified drug delivery system based on the different receptor numbers in orthotopic and metastatic cells had satisfactory targeting ability,enhanced the drug concentrations in tumor cells,exerted anti-metastatic effects.Meanwhile,the combination of DOX and CUR further inhibited the liver metastasis of colorectal cancer.The active targeting drug delivery system may provide a new method for the treatment of metastatic colorectal cancer.