Polyplex-Induced Cytosolic Nuclease Activation Leads to Differential Transgene Expression

Rattan, Rahul; Vaidyanathan, Sriram; Wu, Gordon S.-H.; Shakya, Anisha; Orr, Bradford G.; Holl, Mark M. Banaszak

doi:10.1021/mp400103f

Cited by 22 publications

(60 citation statements)

References 33 publications

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“…For cationic lipid-based and poly-L-lysine polyplexes[36], it is also noted that the PEG changes the internal structure of the polyplex thus having potential impact on both cellular uptake and oligonucleotide release. It has also been noted that PEGylation can reduce acute inflammatory responses, which may help to minimize the impact of nucleases on gene expression efficiency [20, 37]. …”

Section: Resultsmentioning

confidence: 99%

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The role of caveolin-1 and syndecan-4 in the internalization of PEGylated PAMAM dendrimer polyplexes into myoblast and hepatic cells

Shen

Dongen

Han

et al. 2014

European Journal of Pharmaceutics and Biopharmaceutics

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To improve gene delivery efficiency of PEGylated poly(amidoamine) dendrimers in livers and muscles, the roles of syndecan-4 receptor and caveolin-1 protein in the endocytosis of PEGylated generation 5 (G5-PEG) or 7 (G7-PEG) dendrimers and plasmid DNA polyplexes were explored in C2C12 and HepG2 cells. Expression levels of syndecan-4 for both cell lines were downregulated by transfection of the cells with syndecan-4 specific siRNA. Caveolin-1 was upregulated by infecting the cells with adenovirus vector expressed caveolin-1 (Ad-CAV-1). The impact of syndecan-4 and caveolin-1 on endocytosis of G5-PEG/DNA or G7-PEG/DNA polyplexes was then measured by flow cytometry. Our results demonstrate that downregulation of syndecan-4 and upregulation of caveolin-1 significantly improved internalization of PEG-PAMAM dendrimer polyplexes in HepG2 cells; however, in C2C12 cells, downregulation of syndecan-4 decreased the internalization of the polyplexes while upregulation of caveolin-1 had no effect on internalization. Gene expression results for G5-PEG/pGFP on the two cell lines exhibited the same trends for syndecan-4 and caveolin-1 as was observed for endocytosis of the polyplexes. This study gives a clue how to take strategies by up- or down-regulation of the expressions of Syndecan-4 and Caveolin-1 to improve in vivo gene delivery efficiency of the PEG-PAMAM dendrimers in clinical transgenic therapy.

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Section: Resultsmentioning

confidence: 99%

“…G5 and G7 dendrimers were purified before use according to published procedures [20]. Terminal amine groups of the two dendrimers were modified by PEG 5000 at a ratio of 8% (molar ratio of PEG to surface amine per dendrimer) according to a procedure published previously [3].…”

Section: Methodsmentioning

confidence: 99%

The role of caveolin-1 and syndecan-4 in the internalization of PEGylated PAMAM dendrimer polyplexes into myoblast and hepatic cells

Shen

Dongen

Han

et al. 2014

European Journal of Pharmaceutics and Biopharmaceutics

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“…Polyplex solutions were mixed at an N:P of 10:1 and 1:1 at a concentration of 500 nM G5-NH 2 -TAMRA n based on published protocols. 40 …”

Section: Methodsmentioning

confidence: 99%

Fluorophore:Dendrimer Ratio Impacts Cellular Uptake and Intracellular Fluorescence Lifetime

et al. 2015

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G5-NH2-TAMRAn (n = 1–4, 5+, and 1.5avg) were prepared with n = 1–4 as a precise dye:dendrimer ratio, 5+ as a mixture of dendrimers with 5 or more dye per dendrimer, and 1.5avg as a Poisson distribution of dye:dendrimer ratios with a mean of 1.5 dye per dendrimer. The absorption intensity increased sublinearly with n whereas the fluorescence emission and lifetime decreased with an increasing number of dyes per dendrimer. Flow cytometry was employed to quantify uptake into HEK293A cells. Dendrimers with 2–4 dyes were found to have greater uptake than dendrimer with a single dye. Fluorescence lifetime imaging microscopy (FLIM) showed that the different dye:dendrimer ratio alone was sufficient to change the fluorescence lifetime of the material observed inside cells. We also observed that the lifetime of G5-NH2-TAMRA5+ increased when present in the cell as compared to solution. However, cells treated with G5-NH2-TAMRA1.5avg did not exhibit the high lifetime components present in G5-NH2-TAMRA1 and G5-NH2-TAMRA5+. In general, the effects of dye:dendrimer ratio on fluorescence lifetime were of similar magnitude to environmentally induced lifetime shifts.

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“…27 To investigate the mechanism of interaction of polymers and polyplexes with the cell membrane and to understand the role of these membrane interactions in gene transfection, we used the IF-16 and trypan blue assays to test hypotheses 1 and 2 generated from our previous work, 13,15 as well as new hypothesis (3) regarding the relationship of vector charge density to partition constant.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Measurement of Cationic Polymer Vector and Polymer–pDNA Polyplex Intercalation into the Cell Plasma Membrane

et al. 2015

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Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1x to 100x the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30-50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 minutes. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N: P ration of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40- 50 nA) than L-PEI polyplexes (< 20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (< 20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials.

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Polyplex-Induced Cytosolic Nuclease Activation Leads to Differential Transgene Expression

Cited by 22 publications

References 33 publications

The role of caveolin-1 and syndecan-4 in the internalization of PEGylated PAMAM dendrimer polyplexes into myoblast and hepatic cells

The role of caveolin-1 and syndecan-4 in the internalization of PEGylated PAMAM dendrimer polyplexes into myoblast and hepatic cells

Fluorophore:Dendrimer Ratio Impacts Cellular Uptake and Intracellular Fluorescence Lifetime

Quantitative Measurement of Cationic Polymer Vector and Polymer–pDNA Polyplex Intercalation into the Cell Plasma Membrane

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