Abstract
This study aimed to further explore the mechanisms behind the ability of certain linear polyamidoamines (PAAs) to transfect cells with minimal cytotoxicity. The transfection efficiency of DNA complexed with a PAA of a molecular weight over 10kDa or 25kDa branched polyethyleneimine (BPEI) was compared in A549 cells using a luciferase reporter gene assay. The impact of endo/lysosomal escape on transgene expression was investigated by transfecting cells in presence of bafilomycin A1 or chloroquine. Cytotoxicity caused by the vectors was evaluated by measuring cell metabolic activity, lactate dehydrogenase release, formation of reactive oxygen species and changes in mitochondrial membrane potential. The luciferase activity was ~3-fold lower after transfection with PAA polyplexes than with BPEI complexes at the optimal polymer to nucleotide ratio (RU:Nt). However, in contrast to BPEI vectors, PAA polyplexes caused negligible cytotoxic effects. The transfection efficiency of PAA polyplexes was significantly reduced in presence of bafilomycin A1 while chloroquine enhanced or decreased transgene expression depending on the RU:Nt. PAA polyplexes displayed a pH-dependent endo/lysosomal escape which was not associated with cytotoxic events, unlike observed with BPEI polyplexes. This is likely due to their greater interactions with biological membranes at acidic than neutral pH.
Highlights
One of the greatest challenges in gene therapy remains the development of safe and effective vehicles to deliver fragile exogenous nucleic acids into host cells
The transfection efficiency of PAA polyplexes was significantly reduced in presence of bafilomycin A1 while chloroquine enhanced or decreased transgene expression depending on the RU:Nt
Bafilomycin A1 was purchased from Alfa Aesar (Fisher Scientific, Loughborough, UK). 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), Carbonyl cyanide 4(trifluoromethoxy) phenylhydrazone (FCCP), branched polyethyleneimine (BPEI) (25 kDa), ethidium bromide (EtBr, 1mg/ml solution), Coomassie Brilliant Blue R250, agarose powder and all other chemicals or cell culture reagents were purchased from Sigma-Aldrich (Gillingham, UK)
Summary
One of the greatest challenges in gene therapy remains the development of safe and effective vehicles to deliver fragile exogenous nucleic acids into host cells. Linear polyamidoamines (PAAs) are a group of biodegradable cationic polymers that have more recently been explored as gene delivery systems Some of those polymers exhibit a transfection efficiency similar to that obtained with PEI but with a low cytotoxicity compared to other polycationic vectors [13,14,15,16]. The present work explored cellular events induced by DNA polyplexes prepared using a linear PAA with a methylenebisacrylamide/dimethylethylenediamine (MBA-DMEDA) backbone of a molecular weight over 10kDa. Amongst a series of structurally diverse PAAs, MBADMEDA based polymers showed the greatest ability to complex DNA as well as to promote transgene expression [17], with a similar transfection ability as the ISA group of PAAs described elsewhere [18]. Polyplexes made with a commercially available branched PEI (BPEI) of 25kDa known to be highly efficient as a gene delivery vehicle and highly toxic [19] were tested alongside for comparison
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