Synthetic
small interfering RNA (
siRNA) has become the basis of a new generation of gene-silencing
cancer therapeutics. However, successful implementation of this novel
therapy relies on the ability to effectively deliver
siRNA into target cells and to prevent degradation of
siRNA in lysosomes after endocytosis. In this study, our goal was to design and optimize new amphiphilic cationic
lipid carriers that exhibit selective pH-sensitive endosomal membrane disruptive capabilities to allow for the efficient release of their
siRNA payload into the cytosol. The pH sensitive
siRNA carriers consist of three domains (cationic head, hydrophobic tail,
amino acid-based linker). A library of eight
lipid carriers were synthesized using solid phase chemistry, and then studied to determine the role of (1) the number of protonable
amines and overall pKa of the cationic head group, (2) the degree of unsaturation of the hydrophobic tail, and (3) the presence of
histidine residues in the
amino acid linker for transfection and silencing efficacy. In vitro screening evaluation of the new carriers demonstrated at least 80% knockdown of a GFP reporter in CHO cells after 72h. The carriers ECO and ECLn performed the best in a
luciferase knockdown study in HT29 human
colon cancer cells, which were found to be more difficult to transfect. They significantly reduced expression of this reporter to 22.7±3.31% and 23.5±5.11% after 72h post-transfection, better than
Lipofectamine RNAiMax. Both ECO and ECLn carriers caused minimal cytotoxicity, preserving relative cell viabilities at 87.3±2.72% and 88.9±6.84%, respectively. A series of
hemolysis assays at various pHs revealed that increasing the number of
amines in the protonable head group, and removing the
histidine residue from the linker, both resulted in improved membrane disruptive activity at the endosomal pH of 6.5. Meanwhile, the cellular uptake into HT29
cancer cells was improved, not only by increasing the
amines of the head group, but also by increasing the degree of unsaturation in the
lipid tails. Due to flexibility of the synthetic procedure, the delivery system could be modified further for different applications. The success of ECO and ECLn for in vitro
siRNA delivery potentially makes them promising candidates for future in vivo studies.