The application of layer-by-layer (LbL) approach on nanoparticle surface coating improves the colon-specific drug delivery of insoluble drugs. Here, we aimed to formulate a self-assembled cysteamine-based disulphide cross-linked sodium alginate with LbL self-assembly to improve the delivery of paclitaxel (PCX) to colonic cancer cells. Cysteamine was conjugated to the backbone of oxidized SA to form a core of self-assembled disulphide cross-linked nanospheres. P3DL was selected for PCX loading and fabricated LbL with poly(allylamine hydrochloride) (PAH) and poly(4-styrenesulfonic acid-co-maleic acid) sodium salt (PSSCMA) resulting from characterization and drug release studies. P3DL-fabricated PCX-loaded nanospheres (P3DL/PAH/PSSCMA) exhibited an encapsulation efficiency of 77.1% with cumulative drug release of 45.1%. Dynamic light scattering analysis was reported at 173.6 ± 2.5 nm with polydispersity index of 0.394 ± 0.105 (zeta potential= -58.5 mV). P3DL/PAH/PSSCMA demonstrated a pH-dependent swelling transition; from pH 1 to 7 (102.2% increase). The size increased by 33.0% in reduction response study after incubating with 10 mM glutathione (day 7). HT-29 cells showed high viabilities (86.7%) after treatment with the fabricated nanospheres at 0.8 µg/mL. Cellular internalization was successful with more than 70.0% nanospheres detected in HT-29 cells. Therefore, this fabricated nanospheres may be considered as potential nanocarriers for colon cancer-targeted chemotherapeutic drug delivery.
The work described in this study aimed to express CYP2C8 wild-type and mutant proteins in bacterial expression system and to use the expressed proteins to investigate the structural and functional consequences of a reported allele CYP2C8(*)4 (carrying Ile264Met substitution) on protein activity. Ile264 was replaced by three different amino acids resulting in three mutant constructs, 2C8I264M, 2C8I264R and 2C8I264D. The presence of isoleucine at position 264 in CYP2C8 was found to be important for proper haem insertion and protein folding; whereas bulkier or charged residues were highly disruptive resulting in inactive proteins with minimum spectral and catalytic activities. This was evidenced from the low levels of Soret peak at 450 nm and negligible levels of tolbutamide methylhydroxylase activity. Kinetic study using paclitaxel indicated that all three mutants exhibited only 9.7 to 35.4% of the activity level observed in the wild-type. In addition, the mutants were more sensitive to proteinase K digestion, indicating a possible alteration of conformation. The combined effects of protein instability and compromised catalytic activity resulted in defective CYP2C8 protein which may have clinical implications in carriers of CYP2C8*4, particularly in terms of their capacity to clear potent drugs and their susceptibility to adverse drug reactions.