Interaction of Poly(ethylenimine)-DNA Polyplexes with Mitochondria: Implications for a Mechanism of Cytotoxicity

Department of Chemistry & Macromolecules & Interfaces Institute, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States.
Molecular Pharmaceutics (Impact Factor: 4.38). 06/2011; 8(5):1709-19. DOI: 10.1021/mp200078n
Source: PubMed


Poly(ethylenimine) (PEI) and PEI-based systems have been widely studied for use as nucleic acid delivery vehicles. However, many of these vehicles display high cytotoxicity, rendering them unfit for therapeutic use. By exploring the mechanisms that cause cytotoxicity, and through understanding structure-function relationships between polymers and intracellular interactions, nucleic acid delivery vehicles with precise intracellular properties can be tailored for specific function. Previous research has shown that PEI is able to depolarize mitochondria, but the exact mechanism as to how depolarization is induced remains elusive and therefore is the focus of the current study. Potential mechanisms for mitochondrial depolarization include direct mitochondrial membrane permeabilization by PEI or PEI polyplexes, activation of the mitochondrial permeability transition pore, and interference with mitochondrial membrane proton pumps, specifically Complex I of the electron transport chain and F(0)F(1)-ATPase. Herein, confocal microscopy and live cell imaging showed that PEI polyplexes do colocalize to some degree with mitochondria early in transfection, and the degree of colocalization increases over time. Cyclosporin a was used to prevent activation of the mitochondrial membrane permeability transition pore, and it was found that early in transfection cyclosporin a was unable to prevent the loss of mitochondrial membrane potential. Further studies done using rotenone and oligomycin to inhibit Complex I of the electron transport chain and F(0)F(1)-ATPase, respectively, indicate that both of these mitochondrial proton pumps are functioning during PEI transfection. Overall, we conclude that direct interaction between polyplexes and mitochondria may be the reason why mitochondrial function is impaired during PEI transfection.

Download full-text


Available from: Giovanna Grandinetti, Apr 30, 2015
  • Source
    • "The most commonly used is polyethylenimine. However, as explained in the introduction, a number of reports have indicated that PEI can induce cytotoxicity and could cause mitochondrial damages293031. Recently, it was shown, using several polycations (poly-arginine, poly-lysine, poly-histidine and chitosan), that poly-arginine (PolyR) appears to be a good candidate for the formation of a ternary complex with Li28 and a short peptide called OVA made up of eight amino acids (SIINFEKL)[34]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The superparamagnetic iron oxide nanoparticles (SPIONs) have great potential in therapeutic and diagnostic applications. Due to their superparamagnetic behavior, they are used clinically as a Magnetic Resonance Imaging (MRI) contrast agent. Iron oxide nanoparticles are also recognized todays as smart drug-delivery systems. However, to increase their specificity, it is essential to functionalize them with a molecule that effectively targets a specific area of the body. Among the molecules that can fulfill this role, peptides are excellent candidates. Oligonucleotides are recognized as potential drugs for various diseases but suffer from poor uptake and intracellular degradation. In this work, we explore four different strategies, based on the electrostatic interactions between the different partners, to functionalize the surface of SPIONs with a phosphorothioate oligonucleotide (ODN) and a cationic peptide labeled with a fluorophore. The internalization of the nanoparticles has been evaluated in vitro on RAW 264.7 cells. Among these strategies, the “«one-step assembly»”, i.e., the direct complexation of oligonucleotides and peptides on iron oxide nanoparticles, provides the best way of coating for the internalization of the nanocomplexes.
    Preview · Article · Sep 2015 · Nanomaterials
  • Source
    • "The release of these proteins, such as cytochrome c, from the mitochondrial intermembrane space leads to caspase activation and the biochemical execution of cells, characterized by morphological changes and nuclear condensation (Moldoveanu et al., 2013;Parsons & Green, 2010;Tait & Green, 2012). Recent studies have shown that PEI-mediated cytotoxicity is generally characterized by cell death through a mixture of apoptotic and necrotic pathways, interconnected with autophagy responses and mitochondrial dysfunction (Gao et al., 2011;Grandinetti, Ingle, & Reineke, 2011;Hall et al., 2013;Larsen et al., 2012;Lin et al., 2012;Moghimi et al., 2005;Symonds et al., 2005). Polycationic vectors, such as PEI, have previously been shown to induce the release of cytochrome c from mitochondria (Moghimi et al., 2005). "
    [Show abstract] [Hide abstract]
    ABSTRACT: One of the major challenges in the field of nucleic acid delivery is the design of delivery vehicles with attributes that render them safe as well as efficient in transfection. To this end, polycationic vectors have been intensely investigated with native polyethylenimines (PEIs) being the gold standard. PEIs are highly efficient transfectants, but depending on their architecture and size they induce cytotoxicity through different modes of cell death pathways. Here, we briefly review dynamic and integrated cell death processes and pathways, and discuss considerations in cell death assay design and their interpretation in relation to PEIs and PEI-based engineered vectors, which are also translatable for the design and studying the safety of other transfectants.
    Full-text · Article · Nov 2014 · Advances in genetics
    • "This is well illustrated by the finding that DNA polyplexes formed with branched PEI are more stable than linear PEI polyplexes, but linear PEI polyplexes were found to be more effective in gene transfer; apparently intracellular release (Itaka et al., 2004) and also nuclear entry of DNA (Brunner, Furtbauer, Sauer, Kursa, & Wagner, 2002) can be better managed by the lessstable linear PEI polyplexes. Despite many favorable properties, PEI displays also drawbacks: it is nondegradable and significantly toxic (Grandinetti, Ingle, & Reineke, 2011;Moghimi et al., 2005) in a molecular weight-dependent manner. Cytotoxicity includes cell surface and organelle membrane (mitochondria , nucleus) defects, triggering apoptosis, necrosis, and also block of ATP synthesis (Hall et al., 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: For the last five decades cationic polymers have been used for nucleic acids transfection. Our understanding of polymer-nucleic acid interactions and their rational use in delivery has continuously increased. The great improvements in macromolecular chemistry and the recognition of distinct biological extra- and intracellular delivery hurdles triggered several breakthrough developments, including the discovery of natural and synthetic polycations for compaction of nucleic acids into stable nanoparticles termed polyplexes; the incorporation of targeting ligands and surface-shielding of polyplexes to enable receptor-mediated gene delivery into defined target tissues; and strongly improved intracellular transfer efficacy by better endosomal escape of vesicle-trapped polyplexes into the cytosol. These experiences triggered the development of second-generation polymers with more dynamic properties, such as endosomal pH-responsive release mechanisms, or biodegradable units for improved biocompatibility and intracellular release of the nucleic acid pay load. Despite a better biological understanding, significant challenges such as efficient nuclear delivery and persistence of gene expression persist. The therapeutic perspectives widened from pDNA-based gene therapy to application of novel therapeutic nucleic acids including mRNA, siRNA, and microRNA. The finding that different therapeutic pay loads require different tailor-made carriers complicates preclinical developments. Convincing evidence of medical efficacy still remains to be demonstrated. Bioinspired multifunctional polyplexes resembling "synthetic viruses" appear as attractive opportunity, but provide additional challenges: how to identify optimum combinations of functional delivery units, and how to prepare such polyplexes reproducibly in precise form? Design of sequence-defined polymers, screening of combinatorial polymer and polyplex libraries are tools for further chemical evolution of polyplexes.
    No preview · Article · Nov 2014 · Advances in genetics
Show more