Comparison of Gene Transfection and Cytotoxicity Mechanisms of Linear Poly(amidoamine) and Branched Poly(ethyleneimine) Polyplexes

Abstract

Purpose

This study aimed to further explore the mechanisms behind the ability of certain linear polyamidoamines (PAAs) to transfect cells with minimal cytotoxicity.

Methods

The transfection efficiency of DNA complexed with a PAA of a molecular weight over 10 kDa or 25 kDa 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.

Results

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.

Conclusions

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.

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Abbreviations

BPEI:

Branched poly(ethyleneimine)

EtBr:

Ethidium bromide

FCCP:

Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone

H2DCFH-DA:

2′,7′-dichlorodihydrofluorescein diacetate

JC-1:

5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide

LDH:

Lactate dehydrogenase

MBA-DMEDA:

Methylenebisacrylamide/dimethylethylenediamine

MMP:

Mitochondrial membrane potential

MTT:

3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide

PAA:

Polyamidoamine

PB:

PrestoBlue

PBS:

Phosphate buffer saline

pDNA:

Plasmid DNA

PEG:

Polyethylene glycol

PEI:

Poly(ethyleneimine)

PLL:

Poly-L-lysine

ROS:

Reactive oxygen species

RU:Nt:

Polymer repeating unit to nucleotide ratio

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ACKNOWLEDGMENTS AND DISCLOSURES

A.A.Y. Almulathanon was funded by the Ministry of Higher Education and Scientific Research (MOHESR) in Iraq.

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Correspondence to Cynthia Bosquillon.

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Almulathanon, A.A.Y., Ranucci, E., Ferruti, P. et al. Comparison of Gene Transfection and Cytotoxicity Mechanisms of Linear Poly(amidoamine) and Branched Poly(ethyleneimine) Polyplexes. Pharm Res 35, 86 (2018). https://doi.org/10.1007/s11095-017-2328-7

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KEY WORDS

  • cationic polymers
  • cytotoxicity
  • DNA-complexes
  • gene delivery
  • linear polyamidoamines