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Title: | Wheat germ agglutinin-conjugated poly-dl-lactic-co-glycolic acid (PLGA) nanoparticles for enhanced uptake and retention of paclitaxel by colon cancer cells | Authors: | WANG CHUNXIA | Keywords: | WGA, PLGA nanoparticles, paclitaxel, colon cancer | Issue Date: | 29-Oct-2009 | Citation: | WANG CHUNXIA (2009-10-29). Wheat germ agglutinin-conjugated poly-dl-lactic-co-glycolic acid (PLGA) nanoparticles for enhanced uptake and retention of paclitaxel by colon cancer cells. ScholarBank@NUS Repository. | Abstract: | The purpose of this project was to confirm the hypothesis that conjugation of WGA to PLGA nanoparticles loaded with paclitaxel (WNP) could improve the delivery of paclitaxel to colonic cancer cells. Glycosylation patterns of representative colon cancer cells (Caco-2 and HT-29 cells) and normal cells (colon fibroblasts, CCD-18Co cells) were first investigated. Our results confirmed the higher expression levels of WGA-binding glycoproteins (N-acetylglucosamine and sialic acid) in the Caco-2 and HT-29 cells, than in the CCD-18Co cells. Most of the WGA-recognizable glycoproteins in the Caco-2 and HT-29 cells had molecular weight > 75 kDa, whereas the CCD-18Co cells showed an apparent lack of expression of such large proteins. The ranking order of expression of WGA-recognizable glycoproteins in the three cell lines was HT-29 > Caco-2 > CCD-18Co cells. In vitro cytotoxicity and cellular uptake studies were then carried out to evaluate the potential of WGA for targeted activity against the colon cell models. WGA exhibited different degrees of cytotoxicity against the Caco-2, HT-29 and CCD-18Co cells, but showed anti-proliferative activity in all 3 cell lines at concentrations ¿ 50 µg/ml. Cellular uptake of fWGA ranked in the order of Caco-2 > HT-29 > CCD-18Co cells. The higher binding of fWGA to the malignant cells may be associated with the greater expression of WGA-recognizable glycoproteins in these cells relative to the colon fibroblast cells. WGA was conjugated onto the surface of paclitaxel-loaded PLGA nanoparticles to prepare the WNP formulation. Cellular uptake and cytotoxicity of WNP were evaluated in the three colon cell lines. In vitro anti-proliferation studies suggested that the incorporation of WGA enhanced the cytotoxicity of the paclitaxel-loaded PLGA nanoparticles against the cancerous Caco-2 and HT-29 cells. Paclitaxel uptake from the WNP formulation at 2h incubation was the highest in the Caco-2 cells compared to the other two cell lines. Caco-2 and HT-29 cells showed preferential uptake of WNP compared to PNP, suggesting that WGA conjugation to the PLGA nanoparticles was advantageous in facilitating the nanoparticle uptake by the cultured colon cancer cells. The greater efficacy of WNP correlated well with the higher cellular uptake and sustained intracellular retention of paclitaxel associated with the formulation, which again might be attributed to the over-expression of N-acetyl-D-glucosamine- containing glycoprotein on the colon cell surface. About 30% of the endocytosed WNP was observed in the late endo-lysosomes, but fluorimetric measurements indicated successful escape of the WNP from the endo-lysosome compartment into the cytosol with increasing incubation time. To evaluate the effect of mucin glycoprotein on the cellular uptake of WNP, the uptake experiments were repeated using a mucin-secreting cell line, LS174T. The LS174 cells showed no significant differences in paclitaxel uptake from the WNP formulation on day 3 of culture compared to day 6 of culture, despite the significantly higher production of mucin by day 6. The presence of mucin may therefore not be a barrier for the cellular uptake of WGA-conjugated nanoparticles. FRAP results showed that fWNP was capable of diffusing in the mucin layer, although the diffusion rate was slowed down by the viscous mucin. This diffusion capability allowed the fWNP to remain mobile in the mucin layer and enabled the particles to be transported into the cells. Interaction between the mucin glycoprotein and WGA is postulated to serve as a bridge between WNP and the colon cancer cells under the mucin layer. On the basis of these results, it may be concluded that WNP has the potential to be applied as a targeted delivery platform for paclitaxel in the treatment of colon cancer. | URI: | http://scholarbank.nus.edu.sg/handle/10635/17993 |
Appears in Collections: | Ph.D Theses (Open) |
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