Pore forming polyalkylpyridinium salts from marine sponges versus synthetic lipofection systems: distinct tools for intracellular delivery of cDNA and siRNA

Debra Mclaggan,Roderick H Scott,Marcel Jaspars,David J Macewan,Kristina Sepčić,Noppadon Adjimatera,Ian S Blagbrough

doi:10.1186/1472-6750-6-6

Abstract

BackgroundHaplosclerid marine sponges produce pore forming polyalkylpyridinium salts (poly-APS), which can be used to deliver macromolecules into cells. The aim of this study was to investigate the delivery of DNA, siRNA and lucifer yellow into cells mediated by poly-APS and its potential mechanisms as compared with other lipofection systems (lipofectamine and N4,N9-dioleoylspermine (LipoGen)). DNA condensation was evaluated and HEK 293 and HtTA HeLa cells were used to investigate pore formation and intracellular delivery of cDNA, siRNA and lucifer yellow.ResultsPoly-APS and LipoGen were both found to be highly efficient DNA condensing agents. Fura-2 calcium imaging was used to measure calcium transients indicative of cell membrane pore forming activity. Calcium transients were evoked by poly-APS but not LipoGen and lipofectamine. The increases in intracellular calcium produced by poly-APS showed temperature sensitivity with greater responses being observed at 12°C compared to 21°C. Similarly, delivery of lucifer yellow into cells with poly-APS was enhanced at lower temperatures. Transfection with cDNA encoding for the expression enhanced green fluorescent protein was also evaluated at 12°C with poly-APS, lipofectamine and LipoGen. Intracellular delivery of siRNA was achieved with knockdown in beta-actin expression when lipofectamine and LipoGen were used as transfection reagents. However, intracellular delivery of siRNA was not achieved with poly-APS.ConclusionPoly-APS mediated pore formation is critical to its activity as a transfection reagent, but lipofection systems utilise distinct mechanisms to enable delivery of DNA and siRNA into cells.

Highlights

Haplosclerid marine sponges produce pore forming polyalkylpyridinium salts, which can be used to deliver macromolecules into cells
DNA condensation study of poly-APS and LipoGen The DNA condensation experiments were run with a plasmid encoding for enhanced green fluorescent protein or luciferase, and with calf thymus DNA
Many compounds that bind to DNA, including DNA condensing agents used in non-viral gene therapy (NVGT), can displace ethidium bromide (EthBr) from EthBr-DNA complexes

Summary

Introduction

Haplosclerid marine sponges produce pore forming polyalkylpyridinium salts (polyAPS), which can be used to deliver macromolecules into cells. Poly-APS is a marine toxin preparation extracted from the sponge Reniera sarai, which can form reversible pores or lesions in cell membranes [1] This toxin preparation primarily contains a cocktail of two polymeric 1,3-octylpyridinium salts of 5.5 and 19 kDa sizes [4]. There has to date been no reported study on the interaction of this polycationic toxin with DNA and further information is required on the mechanism of transfection and the versatility of macromolecule (DNA or siRNA) delivery This is important given that our data suggest membrane poration is required for polyAPS-mediated transfection because low molecular weight pyridinium surfactant monomers and dimers can be efficient non-toxic agents for gene delivery [8]. In contrast, pyridinium surfactant monomers are not efficient pore formers and appear to package DNA for delivery by fusion and/or endocytosis

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