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



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 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.


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.


cationic polymers cytotoxicity DNA-complexes gene delivery linear polyamidoamines 



Branched poly(ethyleneimine)


Ethidium bromide


Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone


2′,7′-dichlorodihydrofluorescein diacetate


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


Lactate dehydrogenase




Mitochondrial membrane potential


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






Phosphate buffer saline


Plasmid DNA


Polyethylene glycol






Reactive oxygen species


Polymer repeating unit to nucleotide ratio



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


  1. 1.
    Hunter AC. Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity. Adv Drug Deliv Rev. 2006;58(14):1523–31.CrossRefPubMedGoogle Scholar
  2. 2.
    De Smedt SC, Demeester J, Hennink WE. Cationic polymer based gene delivery systems. Pharm Res. 2000;17(2):113–26.CrossRefPubMedGoogle Scholar
  3. 3.
    Tros de Ilarduya C, Sun Y, Düzgüneş N. Gene delivery by lipoplexes and polyplexes. Eur J Pharm Sci. 2010;40(3):159–70.CrossRefPubMedGoogle Scholar
  4. 4.
    Lv H, Zhang S, Wang B, Cui S, Yan J. Toxicity of cationic lipids and cationic polymers in gene delivery. J Control Release. 2006;114(1):100–9.CrossRefPubMedGoogle Scholar
  5. 5.
    Jones CH, Chen C-K, Ravikrishnan A, Rane S, Pfeifer BA. Overcoming Nonviral Gene Delivery Barriers: Perspective and Future. Mol Pharm. 2013;10(11):4082–98.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hunter AC, Moghimi SM. Cationic carriers of genetic material and cell death: a mitochondrial tale. Biochim Biophys Acta. 2010;1797(6–7):1203–9.CrossRefPubMedGoogle Scholar
  7. 7.
    Grandinetti G, Smith AE, Reineke TM. Membrane and nuclear permeabilization by polymeric pDNA vehicles: efficient method for gene delivery or mechanism of cytotoxicity? Mol Pharm. 2012;9(3):523–38.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Vaidyanathan S, Orr BG, Banaszak Holl MM. Role of Cell Membrane–Vector Interactions in Successful Gene Delivery. Acc Chem Res. 2016;49(8):1486–93.CrossRefPubMedGoogle Scholar
  9. 9.
    van de Wetering P, Cherng J-Y, Talsma H, Hennink WE. Relation between transfection efficiency and cytotoxicity of poly(2-(dimethylamino)ethyl methacrylate)/plasmid complexes. J Control Release. 1997;49(1):59–69.CrossRefGoogle Scholar
  10. 10.
    Ogris M, Brunner S, Schüller S, Kircheis R, Wagner E. PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther. 1999;6(4):595–605.CrossRefPubMedGoogle Scholar
  11. 11.
    Walker GF, Fella C, Pelisek J, Fahrmeir J, Boeckle S, Ogris M, et al. Toward synthetic viruses: endosomal pH-triggered deshielding of targeted polyplexes greatly enhances gene transfer in vitro and in vivo. Mol Ther. 2005;11(3):418–25.CrossRefPubMedGoogle Scholar
  12. 12.
    Sung SJ, Min SH, Cho KY, Lee S, Min YJ, Yeom YI, et al. Effect of polyethylene glycol on gene delivery of polyethylenimine. Biol Pharm Bull. 2003;26(4):492–500.CrossRefPubMedGoogle Scholar
  13. 13.
    Hill IR, Garnett MC, Bignotti F, Davis SS. In vitro cytotoxicity of poly(amidoamine)s: relevance to DNA delivery. Biochim Biophys Acta. 1999;1427(2):161–74.CrossRefPubMedGoogle Scholar
  14. 14.
    Richardson S, Ferruti P, Duncan R. Poly(amidoamine)s as potential endosomolytic polymers: evaluation in vitro and body distribution in normal and tumour-bearing animals. J Drug Target. 1999;6(6):391–404.CrossRefPubMedGoogle Scholar
  15. 15.
    Pettit MW, Griffiths P, Ferruti P, Richardson SC. Poly(amidoamine) polymers: soluble linear amphiphilic drug-delivery systems for genes, proteins and oligonucleotides. Ther Deliv. 2011;2(7):907–17.CrossRefPubMedGoogle Scholar
  16. 16.
    Martello F, Piest M, Engbersen JFJ, Ferruti P. Effects of branched or linear architecture of bioreducible poly(amido amine)s on their in vitro gene delivery properties. J Control Release. 2012;164(3):372–9.CrossRefPubMedGoogle Scholar
  17. 17.
    Jones NA, Hill IR, Stolnik S, Bignotti F, Davis SS, Garnett MC. Polymer chemical structure is a key determinant of physicochemical and colloidal properties of polymer-DNA complexes for gene delivery. Biochim Biophys Acta. 2000;1517(1):1–18.CrossRefPubMedGoogle Scholar
  18. 18.
    Richardson SC, Pattrick NG, Man YK, Ferruti P, Duncan R. Poly(amidoamine)s as potential nonviral vectors: ability to form interpolyelectrolyte complexes and to mediate transfection in vitro. Biomacromolecules. 2001;2(3):1023–8.CrossRefPubMedGoogle Scholar
  19. 19.
    Moghimi SM, Symonds P, Murray JC, Hunter AC, Debska G, Szewczyk A. A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. Mol Ther. 2005;11(6):990–5.CrossRefPubMedGoogle Scholar
  20. 20.
    Ranucci E, Ferruti P, Suardi MA, Manfredi A. Poly(amidoamine)s with 2-Dithiopyridine Side Substituents as Intermediates to Peptide–Polymer Conjugates. Macromol Rapid Commun. 2007;28(11):1243–50.CrossRefGoogle Scholar
  21. 21.
    Ranucci E, Ferruti P, Lattanzio E, Manfredi A, Rossi M, Mussini PR, et al. Acid-base properties of poly(amidoamine)s. J Polym Sci A Polym Chem. 2009;47(24):6977–91.CrossRefGoogle Scholar
  22. 22.
    Rejman J, Oberle V, Zuhorn IS, Hoekstra D. Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J. 2004;377(Pt 1):159–69.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Richardson SC, Pattrick NG, Lavignac N, Ferruti P, Duncan R. Intracellular fate of bioresponsive poly(amidoamine)s in vitro and in vivo. J Control Release. 2010;142(1):78–88.CrossRefPubMedGoogle Scholar
  24. 24.
    Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial ROS-induced ROS release: an update and review. Biochim Biophys Acta. 2006;1757(5–6):509–17.Google Scholar
  25. 25.
    Bowman EJ, Siebers A, Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A. 1988;85(21):7972–6.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Crider BP, Xie XS, Stone DK. Bafilomycin inhibits proton flow through the H+ channel of vacuolar proton pumps. J Biol Chem. 1994;269(26):17379–81.PubMedGoogle Scholar
  27. 27.
    Clague MJ, Urbe S, Aniento F, Gruenberg J. Vacuolar ATPase activity is required for endosomal carrier vesicle formation. J Biol Chem. 1994;269(1):21–4.PubMedGoogle Scholar
  28. 28.
    Borgonovo B, Cocucci E, Racchetti G, Podini P, Bachi A, Meldolesi J. Regulated exocytosis: a novel, widely expressed system. Nat Cell Biol. 2002;4(12):955–62.CrossRefPubMedGoogle Scholar
  29. 29.
    Völkl H, Friedrich F, Häussinger D, Lang F. Effect of cell volume on Acridine Orange fluorescence in hepatocytes. Biochem J. 1993;295(Pt 1):11–4.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Zhao H, Cai Y, Santi S, Lafrenie R, Lee H. Chloroquine-Mediated Radiosensitization Is Due to the Destablization of the Lysosomal Membrane and Subsequent Induction of Cell Death by Necrosis. Radiat Res. 2005;164(3):250–7.CrossRefPubMedGoogle Scholar
  31. 31.
    Solomon VR, Lee H. Chloroquine and its analogs: A new promise of an old drug for effective and safe cancer therapies. Eur J Pharmacol. 2009;625(1):220–33.Google Scholar
  32. 32.
    De Duve C, De Barsy T, Poole B, Trouet A, Tulkens P, Fo VH. Lysosomotropic agents. Biochem Pharmacol. 1974;23(18):2495–531.CrossRefPubMedGoogle Scholar
  33. 33.
    Ohkuma S, Poole B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A. 1978;75(7):3327–31.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Luzio JP, Pryor PR, Bright NA. Lysosomes: fusion and function. Nat Rev Mol Cell Biol. 2007;8(8):622–32.CrossRefPubMedGoogle Scholar
  35. 35.
    Rémy-Kristensen A, Clamme J-P, Vuilleumier C, Kuhry J-G, Mély Y. Role of endocytosis in the transfection of L929 fibroblasts by polyethylenimine/DNA complexes. Biochim Biophys Acta Biomembr. 2001;1514(1):21–32.CrossRefGoogle Scholar
  36. 36.
    Vercauteren D, Deschout H, Remaut K, Engbersen JF, Jones AT, Demeester J, et al. Dynamic colocalization microscopy to characterize intracellular trafficking of nanomedicines. ACS Nano. 2011;5(10):7874–84.CrossRefPubMedGoogle Scholar
  37. 37.
    Cohen S, Coue G, Beno D, Korenstein R, Engbersen JF. Bioreducible poly(amidoamine)s as carriers for intracellular protein delivery to intestinal cells. Biomaterials. 2012;33(2):614–23.CrossRefPubMedGoogle Scholar
  38. 38.
    Benjaminsen RV, Mattebjerg MA, Henriksen JR, Moghimi SM, Andresen TL. The possible "proton sponge" effect of polyethylenimine (PEI) does not include change in lysosomal pH. Mol Ther. 2013;21(1):149–57.CrossRefPubMedGoogle Scholar
  39. 39.
    Barbucci R, Casolaro M, Ferruti P, Barone V, Leli F, Oliva L. Macroinorganics. 7. Property structure relationships for polymeric bases whose monomeric units behave independently toward protonation. Macromolecules. 1981;14(5):1203–9.CrossRefGoogle Scholar
  40. 40.
    Khayat Z, Griffiths PC, Grillo I, Heenan RK, King SM, Duncan R. Characterising the size and shape of polyamidoamines in solution as a function of pH using neutron scattering and pulsed-gradient spin-echo NMR. Int J Pharm. 2006;317(2):175–86.CrossRefPubMedGoogle Scholar
  41. 41.
    Ferruti P, Manzoni S, Richardson SCW, Duncan R, Pattrick NG, Mendichi R, et al. Amphoteric Linear Poly(amido-amine)s as Endosomolytic Polymers: Correlation between Physicochemical and Biological Properties. Macromolecules. 2000;33(21):7793–800.CrossRefGoogle Scholar
  42. 42.
    Griffiths PC, Khayat Z, Tse S, Heenan RK, King SM, Duncan R. Studies on the mechanism of interaction of a bioresponsive endosomolytic polyamidoamine with interfaces. 1. Micelles as model surfaces. Biomacromolecules. 2007;8(3):1004–12.CrossRefPubMedGoogle Scholar
  43. 43.
    Battaglia G, Crea F, Crea P, De Stefano C, Sammartano S. Medium effect on the acid-base properties of branched polyethylenimine in different aqueous electrolyte solutions. J Chem Eng Data. 2009;54(2):502–10.CrossRefGoogle Scholar
  44. 44.
    Grandinetti G, Ingle NP, Reineke TM. Interaction of poly(ethylenimine)-DNA polyplexes with mitochondria: implications for a mechanism of cytotoxicity. Mol Pharm. 2011;8(5):1709–19.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Pattrick NG, Richardson SC, Casolaro M, Ferruti P, Duncan R. Poly(amidoamine)-mediated intracytoplasmic delivery of ricin A-chain and gelonin. J Control Release. 2001;77(3):225–32.CrossRefPubMedGoogle Scholar

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

Personalised recommendations