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Pharmaceutical Research

, 35:86 | Cite as

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

  • Ammar A. Y. Almulathanon
  • Elisabetta Ranucci
  • Paolo Ferruti
  • Martin C. Garnett
  • Cynthia Bosquillon
Research Paper

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.

KEY WORDS

cationic polymers cytotoxicity DNA-complexes gene delivery linear polyamidoamines 

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

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Division of Molecular Therapeutics and Formulation, School of PharmacyUniversity of NottinghamNottinghamUK
  2. 2.Pharmacy College,University of Mosul,MosulIraq
  3. 3.Dipartimento di Chimica,Università degli Studi di MilanoMilanItaly

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