Design and evaluation of new pH-sensitive amphiphilic cationic lipids for siRNA delivery

Abstract

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 72 hours. 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 72 hours 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

Principles in the design of ligand-targeted cancer therapeutics and imaging agents

“…peptides 156,157 , pore-forming and caged fusogenic or cationic lipids 156,[158][159][160] , and photothermally activated complexes 161 have all been investigated for use in promoting drug egress from endosomes 162 . However, in each of these cases, notable heterogeneity and bulk have been added to the intended drug product, rendering the path to regulatory approval more difficult.…”

Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA

“…20 It is well known that the physiological pH in cancer cells is lower compared to that in blood and normal tissues, and it is about 6.0 in early endosomes, and reduces to 5.0 during the progression to late endosomes and lysosomes. 21 pH-sensitive PEG-lipid can be degraded even in weak acid, in favor of endosomal escape, followed by cytoplasmic release of the liposome-incorporated drug. 22 Ketal, 23 vinyl ester, 24 orthoester, 25 hydrazone, 26 and Schiff base 27,28 are several examples of pH-degradable bonds that are hydrolyzed relatively rapidly at pH 6.0 but stable at neutral conditions.…”

Multifunctional Cationic Lipid-Based Nanoparticles Facilitate Endosomal Escape and Reduction-Triggered Cytosolic siRNA Release

“…19,20 Among these carriers, ECO ((1-aminoethyl)iminobis[ N -(oleicylcysteinyl-1-amino-ethyl)propionamide]) emerged as a lead multifunctional carrier for further development because of its effectiveness for mediating potent gene silencing in both cancerous and non-cancerous cells. 19 …”