Transfection efficiency and uptake process of polyplexes in human lung endothelial cells: a comparative study in non‐polarized and polarized cells

Mennesson, Eric; Erbacher, Patrick; Piller, Véronique; Kiéda, Claudine; Midoux, Patrick; Pichon, Chantal

doi:10.1002/jgm.727

Cited by 22 publications

(16 citation statements)

References 41 publications

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“…As polarized apical cells are generally resistant to lipid-based transfection agents, [42] this successful demonstration of target gene inhibition may be predicated on our use of polyethyleneimine (PEI) to transport siRNA into polarized epithelium. Indeed, the success of this approach is consistent with previous reports demonstrating that PEI-polyplexes are capable of transfecting polarized monolayers, whether of endothelial or epithelial origin [43]. Although speculative, in vivo targeting of asthmatic epithelium may actually be less challenging, given the characteristic loss of epithelial barrier integrity [44] and mucus metaplasia [45] associated with allergic asthma.…”

Section: Discussionsupporting

confidence: 83%

Evaluation of nasal epithelium sampling as a tool in the preclinical development of siRNA‐based therapeutics for asthma

Healey

Evans

Hopkin

et al. 2013

J Cellular Molecular Medi

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The development of siRNA-based asthma therapeutics is currently hampered by a paucity of relevant biomarkers and the need to ascertain tissue-specific gene targeting in the context of active disease. Epithelial STAT6 expression is fundamental to asthma pathogenesis in which inflammatory changes are found throughout the respiratory tract. Therefore, to improve preclinical evaluation, we tested the efficacy of STAT6-targeting siRNA within nasal epithelial cells (NEC's) obtained from asthmatic and non-asthmatic donors. STAT6 expression was invariant in both donor groups and amenable to suppression by siRNA treatment. In addition, STAT6 mRNA was also suppressible by apically delivered siRNA treatment in comparative differentiated nasal epithelial cell-line monolayer cultures. Analysis of donor NEC's showed consistent elevation in CCL26 (eotaxin-3) mRNA within the asthmatic group suggesting potential as a relevant biomarker. Furthermore, targeting of STAT6 with siRNA attenuated IL-13-driven CCL26 expression in these cells, pointing to the utility of this approach in preclinical testing. Finally, siRNA-mediated suppression of STAT6 was independent of donor disease phenotype or epithelial cell differentiation status, signifying therapeutic potential.

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Section: Discussionsupporting

confidence: 83%

Evaluation of nasal epithelium sampling as a tool in the preclinical development of siRNA‐based therapeutics for asthma

Healey

Evans

Hopkin

et al. 2013

J Cellular Molecular Medi

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“…Various studies have been undertaken to investigate the mechanisms of cellular uptake and intracellular trafficking of macromolecular drug carriers with different charged groups. Studies revealed that three specific endocytic pathways - the clathrin-mediated endocytosis, the caveolae-mediated endocytosis and macropinocytosis facilitate internalization of PEI-DNA polyplexes in some cells [44]. DeSimone and coworkers studied the internalization of microfabricated monodisperse cylindrical particles in a variety of cells.…”

Section: Discussionmentioning

confidence: 99%

Endocytic uptake of a large array of HPMA copolymers: Elucidation into the dependence on the physicochemical characteristics

Liu

Bauer

Callahan

et al. 2010

Journal of Controlled Release

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Endocytic uptake and subcellular trafficking of a large array of HPMA (N-(2-hydroxypropyl) methacrylamide) based copolymers possessing positively or negatively charged residues, or hydrophobic groups were evaluated by flow cytometry and living cell confocal microscopy in cultured prostate cancer cells. The degrees of cellular uptake of various copolymer fractions with narrow polydispersities were quantified. The copolymer charge was the predominant physicochemical feature in terms of cellular uptake. Fast and efficient uptake occurred in positively charged copolymers due to non-specific adsorptive endocytosis, whereas slow uptake of negatively charged copolymers was observed. The uptake of copolymers was also molecular weight dependent. The copolymers were internalized into the cells through multiple endocytic pathways: positively charged copolymers robustly engaged clathrin-mediated endocytosis, macropinocytosis and dynamin-dependent endocytosis, while weakly negatively charged copolymers weakly employed these pathways; strongly negatively charged copolymers only mobilized macropinocytosis. HPMA copolymer possessing 4 mol% of moderately hydrophobic functional groups did not show preferential uptake. All copolymers ultimately localized in late endosomes/lysosomes via early endosomes; with varying kinetics among the copolymers. This study indicates that cell entry and subsequent intracellular trafficking of polymeric drug carriers are strongly dependent on the physicochemical characteristics of the nanocarrier, such as charge and molecular weight.

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“…Evidences exist showing that the size of the complexes may help in determining the endocytosis pathway [50]. However there is no clear consensus on the relationship between the complex size and their internalization mechanism since also changes in chemical nature of the compound may affect the pathway of complex entry [51].…”

Section: Large-sized Lipoplexes Promote Efficient Gene Transfer In Vitromentioning

confidence: 99%

Physicochemical Parameters of Non-Viral Vectors that Govern Transfection Efficiency

Barteau¹,

Chèvre²,

Letrou-Bonneval³

et al. 2008

CGT

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Gene therapy is based on the vectorization of nucleic acids to target cells and their subsequent expression. Cationic lipids and polymers are the most widely used vectors for the delivery of DNA into cultured cells. Nowadays, numerous reagents made of these cationic molecules are commercially available and used by researchers from the academic and industrial field. By contrast their evaluations in preclinical programs have revealed that their use for in vivo applications will be more problematic than their massive use in vitro. This is mostly due to the physicochemical properties of cationic vectors/DNA complexes, which are the result of their mode of interaction. Indeed, these cationic vectors interact through electrostatic forces with negatively charged DNA. This results in the formation of highly organized positively charged supramolecular structures where DNA molecules are condensed. Association of DNA with cationic lipids under a micellar or liposomal form leads to lamellar organization with DNA molecules sandwiched between lipid bilayers. Although the lamellar phase is the common described structure, as evidenced by small-angle X-ray scattering and electron microscopy, some cationic lipid combined with a hexagonal forming lipid could also result with DNA in an inverted hexagonal structure. Despite a lot of effort, the precise mechanism of gene transfer with cationic vector is still ill-defined. Here, our objective was to overview the main relationships between the physico chemical properties of cationic lipid/DNA complexes and their transfection efficiency. An overview of a new class of vectors consisting of amphiphilic block copolymers designed for in vivo delivery is also presented and discussed.

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Transfection efficiency and uptake process of polyplexes in human lung endothelial cells: a comparative study in non‐polarized and polarized cells

Abstract: We show that the efficiency of polyplex uptake by HLMEC and their internalization mechanism are polymer-dependent. By contrast with His-polyplexes, the HLMEC polarization has little influence on the uptake process and on the transfection efficiency of PEI-polyplexes.

Cited by 22 publications

References 41 publications

Evaluation of nasal epithelium sampling as a tool in the preclinical development of siRNA‐based therapeutics for asthma

Evaluation of nasal epithelium sampling as a tool in the preclinical development of siRNA‐based therapeutics for asthma

Endocytic uptake of a large array of HPMA copolymers: Elucidation into the dependence on the physicochemical characteristics

Physicochemical Parameters of Non-Viral Vectors that Govern Transfection Efficiency

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