DESIGN AND METHODS: The activity of DPD was measured using 5-[2- (14)C]Fluorouracil (5-[2-(14)C]FUra) followed by separation of substrate and product 5-[2-(14)C]FUraH(2) with a 15 x 4.6 mm I.D., 5 microm particle size (d(p)) porous graphitic carbon (PGC) column (Hypercarb(R)) and HPLC with online detection of the radioactivity. This was standardized using the protein concentration of the cytosol (NanoOrange(R) Protein Quantitation).
RESULTS: Complete baseline separation of 5-[2-(14)C]Fluorouracil (5-[2-(14)C]FUra) and 5-[2-(14)C]Fluoro-5,6-dihydrouracil (5-[2-(14)C]FUraH(2)) was achieved using a porous graphitic carbon (PGC) column. The detection limit for 5-[2-(14)C]FUraH(2) was 0.4 pmol.
CONCLUSIONS: By using linear gradient separation (0.1% Trifluoroacetic acid [TFA] in water to 100% Methanol) protocols in concert with PGC columns (Hypercarb(R)), we have demonstrated that a PGC column has a distinct advantage over C-18 reverse phase columns in terms of column stability (pH 1-14). This method provides an improvement on the specific assay for DPD enzyme activity. It is rapid, reproducible and sensitive and can be used for routine screening for healthy and cancer patients for partial and profound DPD deficiency before treatment with 5- FUra.
METHODS: In the present study, graphene oxide-polyethylene glycol (GOPEG) nanocarrier is designed and loaded with two anticancer drugs; Protocatechuic acid (PCA) and Chlorogenic acid (CA). The designed anticancer nanocomposite was further coated with folic acid to target the cancer cells, as their surface membranes are overexpressed with folate receptors.
RESULTS: The particle size distribution of the designed nanocomposite was found to be narrow, 9-40 nm. The release profiles of the loaded drugs; PCA and CA was conducted in human body simulated PBS solutions of pH 7.4 (blood pH) and pH 4.8 (intracellular lysosomal pH). Anticancer properties were evaluated against cancerous cells i.e. liver cancer, HEPG2 and human colon cancer, HT-29 cells. The cytocompatbility was assessed on normal 3T3 fibroblasts cells.
CONCLUSION: The size of the final designed anticancer nanocomposite formulation, GOPEG-PCACA-FA was found to be distributed at 9-40 nm with a median of 8 nm. The in vitro release of the drugs PCA and CA was found to be of sustained manner which took more than 100 h for the release. Furthermore, the designed formulation was biocompatible with normal 3T3 cells and showed strong anticancer activity against liver and colon cancer cells.