Palmitic acid (PA) is a saturated long-chain fatty acid with a 16-carbon backbone. PA is the most common saturated fatty acid found in the human body and can be provided in the diet or synthesized endogenously from other fatty acids, carbohydrates, and amino acids. PA represents 20-30% of total fatty acids (FA) in membrane phospholipids (PL), and adipose triacylglycerols (TAG).
The binding of siRNA to lipid-like moieties such as cholesterol, α-tocopherol, and PA increases the hydrophobicity of the siRNA molecule, thereby improving stability and enhancing cellular uptake of siRNA. In addition, when delivering through polymeric delivery carriers, binding to hydrophobic molecules such as cholesterol or PA can make siRNA more efficient.
Fig.1 A) Synthesis of siPA and product confirmation via MALDI-TOF. B) Synthesis of 50B and GPC analysis. C) Schematic of core-loaded siPA-NP and si-NP polyplexes. (Sarett, 2016)
The performance of PEG-b-p(DMAEMA-co-BMA) "50B" can be improved through pairing with siPA hydrophobized siRNA. The hydrophobicity of both the polymer and siPA molecule was essential to improved polyplex stability, which can be attributed to increased Van der Waals interactions between carrier and cargo. These interactions facilitated more efficient siPA loading into NPs and siPA-NP polyplexes were also more resilient to heparan sulfate-induced destabilization. The enhanced pharmacokinetics of siPA-NPs translated to increased bioactivity of siRNA, as assessed by target gene silencing of the model gene luciferase within orthotopic triple negative breast cancer (MDA-MB-231) tumors.
Conjugation of PA to siRNA has shown particular promise as a modification strategy to improve siRNA stability, cellular penetration, and gene silencing. PA is an endogenous post-transcriptional modification commonly found on membrane-associated signaling proteins; while PA is involved in a wide variety of cellular functions, it is especially believed to influence protein-membrane interactions as well as protein uptake and intracellular trafficking. Motivated by the inherent effects of PA on membrane binding and translocation, PA-modified siRNA has been recently tested as an approach to enhance gene silencing in comparison to unmodified siRNA or siRNA conjugated to cholesterol in cancer cell lines in vitro.
Fig.2 The siRNA-PA conjugate efficiently loads into NPs at a lower N:P ratio than unmodified siRNA. (Sarett, 2015)
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