The influence of alkyl pyridinium sponge toxins on membrane properties, cytotoxicity, transfection and protein expression in mammalian cells

Abstract

The ability of two alkyl pyridinium sponge toxin preparations (poly-APS and halitoxin) to form transient pores/lesions in cell membranes and allow transfection of plasmid cDNA have been investigated using HEK 293 cells. Poly-APS and halitoxin preparations caused a collapse in membrane potential, reductions in input resistance and increased Ca2+ permeability. At least partial recovery was observed after poly-APS application but recovery was more rarely seen with halitoxin. The transfection with plasmid cDNAs for an enhanced green fluorescent protein (EGFP) and human tumour necrosis factor receptor 2 (TNFR2) was assessed for both toxin preparations and compared with lipofectamine. Stable transfection was achieved with poly-APS although it was less efficient than lipofectamine. These results show that viable cells transfected with alien cDNA can be obtained using novel transient pore-forming alkyl pyridinium sponge toxins and a simple pre-incubation protocol. This provides the first proof of principle that pore-forming alkyl pyridinium compounds can be used to deliver cDNA to the intracellular environment without permanently compromising the plasma membrane.

Chemical synthesis and biological activities of 3-alkyl pyridinium polymeric analogues of marine toxins

“…Different biological activities were described for poly-APS (1) [26] of which the transient pore-forming capability below its lethal concentration [30] is considered the most promising for developing a new molecular delivery tool to the wide area of biomedical research. In spite of the success of using poly-APS (1) as a delivery tool for large molecules including proteins and cDNA [30], its development was hampered by a lack of knowledge of how structural modifications affected the biological activity and the difficulties in getting a sustainable supply of a single bioactive compound from the natural resource.…”

“…Initially, basic electrophysiological experiments were conducted to determine whether poly-APS (1; 5 μg ml −1 ) applied for ∼20 s caused a reversible collapse of the resting membrane potential and input resistance of undifferentiated mouse ES cells, similar to effects previously described in other cell types [17,20,30]. The mean resting membrane potential was −43±3 mV under control conditions, and at the peak of the response to poly-APS (1; 5 μg ml −1 ) this value was significantly reduced to −13±3 mV (n=6; P< 0.005).…”

“…This allowed the use of both APS12 (9a) and APS12-2 (9b) at a higher concentration (5 μg ml −1 ) than that previously used for poly-APS (1; 0.5 μg ml −1 ) [30] in the transfection experiments. Transfection efficiency was determined by flow cytometry as a percentage of the number of cells expressing EGFP to the total number of cell population.…”

“…Small and macromolecules including cDNA and peptides can diffuse into intracellular compartments through the pores formed. Poly-APS (1) can, thus, act as a transfecting tool that is mechanistically distinct from lipofection systems, where genetic material is introduced into the cell via liposomes [17,20,30]. However, the development of this transfecting agent has been hampered by the lack of knowledge about the structure-activity relationship (SAR) and the difficulties in getting a sustainable and consistent supply of a single bioactive compound from the natural resource.…”

Quaternary Pyridinium Salts: A Review

“…So, these salts are applied for electrodialysis of salt solutions 71) . The properties of the membranes in mammalian cells can be modified by the presence of alkylpyridinium sponge toxin which increases their porosity 72) .The action of laurylpyridinium salts on the membranes of tumor cells have also been reported 73) . Gene delivery Gene therapy is helpful for the treatment of various diseases including cancer, AIDS, arthritis, and many more.…”