Synthesis and Preliminary Investigations of the siRNA Delivery Potential of Novel, Single-Chain Rigid Cationic Carotenoid Lipids

Michael D Pungente,Philip L Leopold,Christer L Øpstad,Vassilia Partali,Emile Jubeli,Tarek Ibrahim,Nada Abdul Khalique,Nour Barakat,Liji Raju,Mais Al-Kawaz,Rachel Jones,Hans-Richard Sliwka

doi:10.3390/molecules17033484

Abstract

The success of nucleic acid delivery requires the development of safe and efficient delivery vectors that overcome cellular barriers for effective transport. Herein we describe the synthesis of a series of novel, single-chain rigid cationic carotenoid lipids and a study of their preliminary in vitro siRNA delivery effectiveness and cellular toxicity. The efficiency of siRNA delivery by the single-chain lipid series was compared with that of known cationic lipid vectors, 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol) and 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine (EPC) as positive controls. All cationic lipids (controls and single-chain lipids) were co-formulated into liposomes with the neutral co-lipid, 1,2-dioleolyl-sn-glycerol-3-phosphoethanolamine (DOPE). Cationic lipid-siRNA complexes of varying (+/−) molar charge ratios were formulated for delivery into HR5-CL11 cells. Of the five single-chain carotenoid lipids investigated, lipids 1, 2, 3 and 5 displayed significant knockdown efficiency with HR5-CL11 cells. In addition, lipid 1 exhibited the lowest levels of cytotoxicity with cell viability greater than 80% at all (+/−) molar charge ratios studied. This novel, single-chain rigid carotenoid-based cationic lipid represents a new class of transfection vector with excellent cell tolerance, accompanied with encouraging siRNA delivery efficiency.

Highlights

Cationic lipids are promising non-viral vector systems for use in small interfering RNAand DNA delivery [1,2,3,4] to effect the introduction of exogenous sequences of DNA into cells to correct defective genes [5,6,7] or to selectively silence gene expression, referred to as RNA interference, or RNAi, through siRNA delivery [8,9,10,11]
Current commercially available cationic lipids used for siRNA delivery have a common structural motif that includes a hydrophilic headgroup linked typically to two non-rigid hydrophobic tails
Five novel lipids, 1–5 having a common rigid C30-carotenoid hydrophobic domain, while differing in the nature of the amphiphilic headgroups, were synthesized and evaluated for liposome and lipoplex formation. These cationic carotenoid lipids were evaluated for their effectiveness to deliver a specific GL2 siRNA to effect knockdown of luciferase activity in the

Summary

Introduction

Cationic lipids are promising non-viral vector systems for use in small interfering RNA (siRNA)and DNA delivery [1,2,3,4] to effect the introduction of exogenous sequences of DNA into cells to correct defective genes [5,6,7] or to selectively silence gene expression, referred to as RNA interference, or RNAi, through siRNA delivery [8,9,10,11]. Cationic lipids are promising non-viral vector systems for use in small interfering RNA (siRNA). Over the past several years, novel cationic lipid vectors have been synthesized and combined with nucleic acids for these purposes [12,13]. In order for the lipid-nucleic acid complex (or lipoplex) to cross the cell membrane, the complex should be charge-neutral or have an excess positive charge overall. The use of cationic lipids facilitates lipoplex formation by developing a charge-neutral complex with the negatively charged nucleic acid (DNA or siRNA). The lipoplex size is important for active endocytosis [14]. Larger particles may have better initial contact with the cells [15], and increased phagocytic activity accompanied by endosomal escape [16]. It should be mentioned that large particles on the order of the size of a cell are inefficient for in vivo administration as they become trapped in the capillary regions

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Conclusion