| Colorectal cancer is the most common cancer and the third cause of cancer death. Surgeons in the cure of colon cancer cause substantial variability between the outcomes they achieve. Cancer chemotherapy often causes unwanted side effects due to the low selectivity of chemotherapeutic agents for cancer cells. Drug targeting, which could carry drugs directly to the target sites via specific ligands against receptors expressed on malignant cells, is one approach to overcome this problem, which makes it an attractive therapeutic choice. However, realization of targeting drug delivery was hampered by the difficulties in finding appropriate carrier molecules. Indeed, relatively few targeting carriers capable of effectively targeting solid tumors have been identified. Research about targeting drug delivery is now focus on finding new carriers with high selectivity on solid tumor and investigating their mechanism in vivo.Lectins are structurally diverse carbohydrate-binding proteins of nonimmune origin. All vertebrate cell surfaces are endowed with a glycocalyx, a carbohydrate coating composed of membrane glycoproteins, glycolipids, and glycoaminoglycans, together referred to as glycans or glycoconjugates. Cancerous cells often express different glycans, compared with their normal counterparts. Lectin recognizes glycans on cell surface with a high degree of specificity. By virtue of the high specificity of lectin-glycoconjugate interaction, lectins could serve as homing molecules against tumor-specific glycoconjugates or lectin receptors. An appropriate lectin may control the internalization and the intracellular routing of drug delivery system to realize cellular targeting. Therefore, lectins are proposed to be promising carrier molecules to target drugs specifically to different cells and tissues. However, there still need research about the lectin-mediated drug targeting system.The lectin of peanut agglutinin (PNA) is a tetramer, carbohydrate-free protein composed of four identical subunits exhibiting a molecular weight of 110 kDa. Exhibiting little toxic as nutrition, PNA does not agglutinate untreated erythrocytes of human ABO blood types and lymphocytes. At the same time, PNA resists proteolytic attack by pepsin, chymotrypsin, tripsin, elastase and pancreatin. PNA has beenextensively used as a probe to detect malignant phenotype in several tissues as it strongly recognizes the cancer specific T antigen (Gal-l-3GalNAc-). In addition, PNA receptors were highly expressed in gastric and intestinal (GI) cancer, but was not expressed or weakly expressed on normal tissues, showing excellent selectivity of PNA towards GI cancer. These data suggest the potential of PNA as a targeting carrier to be used in the drug delivery system for cancer chemotherapy.In order to take advantage of the lectin-sugar binding selectivity for targeting cytostatic agents to the colorectal cancer tissue, PNA was coupled by fixing its amino groups to the carbodiimide-activated carboxylic groups of 5-fluorouracil (5-Fu) derivative (Ni-substituted 5-Fu acetate) to form 5-Fu-PNA conjugate. Effects of many factors on the drug ratio and activity of the conjugate were investigated. The stability in vitro and the characteristics of SDS-PAGE of the conjugate were also investigated. Using the flow cytometry and fluorescence microscope, the mechanism and properties of the interaction between 5-Fu-PNA and cells were investigated adopting the human colorectal cancer cell line LoVo and the human normal liver cell line Chang as the model cell lines. The cytotoxicity, specificity and selectivity of 5-Fu-PNA were estimated using MTT assay on the same cell lines.The preparation of the conjugate was first investigated. How to protect the activity of the protein, to avoid the intramolecular crosslink of the lectin, to realize the binding of the drug to the lectin with high drug/lectin molar ratio and to maintain the activity of the lectin after conjugation are the key problems of this study. To avoid the intramolecular crosslink of the lectin, 5-Fu was first derived to obtain Ni-substituted 5-Fu acetate and then the derivative was activated by EDC/NHS to form an active ester. The active ester was cross-linked with PNA with protection of D-gal to form the conjugation.The affinity of the conjugate was tested using haemagglutination assay and the drug/lectin molar ratio was determined with TNBS method. Effects of the molar ratio of Ni-substituted 5-Fu acetate, EDC and NHS, the link reaction time of 12 h and 24 h and the amount of D-gal added on the drug/lectin molar ratio and activity of the conjugate were investigated using drug/lectin molar ratio and activity as the criteria for judgment. As a result, the conjugate with drug/lectin molar ratio of 38.42% and activity maintained completely was obtained under the condition of Ni-substituted 5-Fu acetate: EDC: NHS = 1:5:2 for 12 h with protection of D-gal [D-gal: PNA (w/w)= 2.5:1]. The objective of the investigation was obtained. In addition, the conjugate was characterized with SDS-PAGE and the stability of the conjugate wasalso investigated in vitro. There was a new bind with increased molecular of 3KDa in the picture of SDS-PAGE. The stability of the conjugate was tested after treatment. The results of stability showed that the conjugate was relatively stable when treated by lyophilization and SIF at 37°C. The conjugate was hydrolyzed at SGF at 37°C for 5 min.The mechanism and properties of the interaction between 5-Fu-PNA and the targeted cells LoVo and the non-targeted cells Chang were investigated using the flow cytometry and fluorescence microscope. Results showed that the conjugate adhered to LoVo cells specifically in a dose-dependent manner. In addition, this specific adherence displayed a temperature-dependent manner with stronger adherence at 4°C than that of 37 "C and could be inhibited by D-gal in a dose-dependent manner. However, the conjugate could hardly bind to the Chang cells. The above data showed that the conjugate could "recognize" its targeted cells.In order to examine the effectiveness of the targeting system in comparison to the free drug, the cytotoxicity, the cytotoxic selectivity and the specificity of the conjugate were assayed using MTT assay using LoVo cells and Chang cells as model. Firstly, the conjugate could inhibit the growth of LoVo cells in a dose-dependent and time-dependent manner as that of the free drug. Secondly, compared with the free drug, there was a time-delayed effect of the active conjugate. Tt took 96 h for the free drug at the concentration of 8 ^M to kill almost all the cells while the active conjugate took 120 h. Thirdly, lower cytotoxic effects on LoVo cells were observed for the non-active conjugate even at higher drug concentrations and the native PNA than that of the active conjugate, indicating the significance to maintain the affinity of lectin for lectin-mediated cytotoxicity. As a conclusion, the cytotoxic effect of conjugate was specific. Fourthly, 5-Fu-PNA conjugate exhibited much higher selectivity than the free drug since it killed colorectal cancer cells LoVo selectively and exhibited weak effect on the growth inhibition of the normal liver cells Chang. However, the free drug killed both. The above data indicated that the conjugate might selectively distribute the targeted tissue and cells thus reduce the toxicity inducing by unselective distribution in vivo. Still, the potential of 5-Fu-PNA conjugate as a targeting agent for colorectal cancer needs to be further investigated in vivo.In this study, lectin-mediated targeting system of 5-Fu-PNA was synthesized, which fulfilled the principal requirements for targeted systems such as binding specificity, low toxic effect on normal cells, etc. Based upon these optimistic results, 5-Fu-PNA conjugate may be a promising target system, which should be further investigated invivo for site-specific delivery to colon carcinoma. The investigation will enrich the content of targeted drag delivery system.
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