Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer

Boeckle, Sabine; Gersdorff, Katharina von; Piepen, Silke van der; Culmsee, Carsten; Wagner, Ernst; Ogris, Manfred

doi:10.1002/jgm.598

Cited by 416 publications

(426 citation statements)

References 24 publications

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“…To enable rational and focused design of siRNA delivery systems, detailed knowledge of the interaction between the vector polymers and the siRNA as well as between the polyplexes and the cells is necessary. PEI has been extensively characterized for DNA delivery [30][31][32]40]. For instance, it has been shown that high efficiency requires N/P ratios > 8 [30,40], while full condensation of the DNA is reached at N/P3-4 [30,31,41].…”

Section: Discussionmentioning

confidence: 99%

“…PEI has been extensively characterized for DNA delivery [30][31][32]40]. For instance, it has been shown that high efficiency requires N/P ratios > 8 [30,40], while full condensation of the DNA is reached at N/P3-4 [30,31,41]. This indicates that a fraction of the vector has a role unrelated to condensation of DNA, and this fraction has been speculated to be free in solution or at least only weakly associated to the nucleic acid-containing polyplex.…”

Section: Discussionmentioning

confidence: 99%

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Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

et al. 2016

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Future improvements of non-viral vectors for siRNA delivery require better understanding of intracellular processing and vector interactions with target cells. Here, we have compared the siRNA delivery properties of a lipid derivative of bPEI 1.8kDa (DOPE-PEI) with branched polyethyleneimine (bPEI) with average molecular weights of 1.8kDa (bPEI 1.8kDa) and 25 kDa (bPEI 25kDa). We find mechanistic differences between the DOPE-PEI conjugate and bPEI regarding siRNA condensation and intracellular processing. bPEI 1.8kDa and bPEI 25kDa have similar properties with respect to condensation capability, but are very different regarding siRNA decondensation, cellular internalization and induction of reporter gene knockdown. Lipid conjugation of bPEI 1.8kDa improves the siRNA delivery properties, but with markedly different 2 formulation requirements and mechanisms of action compared to conventional PEIs. Interestingly, strong knockdown using bPEI 25kDa is dependent on the presence of a free vector fraction which does not increase siRNA uptake. Finally, we have investigated the effect on lysosomal pH induced by these vectors to elucidate the differences in the proton sponge effect between lipid conjugated PEI and conventional PEI: Neither DOPE-PEI nor bPEI 25kDa affected lysosomal pH as a function of time, underlining that the possible proton sponge effect is not associated with changes in lysosomal pH.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

et al. 2016

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“…43 Complexes had a final DNA concentration of 10 mg ml À1 . The presented data (Supplementary Table S3; Supplementary information) are means of three measurements, with each measurement averaging the data of 10 sub-runs.…”

Section: Polyplex Size and B-potential Measurementmentioning

confidence: 99%

“…Transfection efficiency was evaluated 24 h after treatment by measuring luciferase reporter gene expression using a luminometer (Lumat LB 9507; Berthold Technologies, Bad Wildbad, Germany) as previously described. 43 Two nanogram of recombinant luciferase (Promega, Mannheim, Germany) correspond to 10 7 light units. Protein concentration was determined using a BCA assay (Pierce, Rockford, IL, USA) with bovine serum albumin as a standard.…”

Section: In Vitro Transfection and Metabolic Activitymentioning

confidence: 99%

Novel degradable oligoethylenimine acrylate ester-based pseudodendrimers for in vitro and in vivo gene transfer

et al. 2007

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“…The transfection complex of linearized pCR3.1-CYP2B1 plasmid with PEI was generated at an N/P ratio (nitrogen in PEI/phosphate in DNA) of 6 in HBS (HEPES-buffered saline: 20 mM HEPES pH 7.1, 150 mM NaCl) at a final DNA concentration of 20 mg/ml. 13 Forty-eight hours after transfection, cells were selected with 0.5 mg geneticin per ml culture medium. To obtain subclones, the surviving cells were re-seeded in a 96-well plate at 1 cell/well after 2 weeks of geneticin selection.…”

Section: Cell Culturementioning

confidence: 99%

Effects of hypoxia and limited diffusion in tumor cell microenvironment on bystander effect of P450 prodrug therapy

et al. 2006

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Cytochrome P450 (CYP) enzyme 2B1 metabolizes the anticancer prodrug cyclophosphamide (CPA) to 4-hydroxy-CPA, which decomposes to the cytotoxic metabolites acrolein and phosphoramide mustard. We have evaluated the bystander cytotoxicity of CPA in combination with CYP2B1 gene-directed enzyme prodrug therapy using a cell culture-based agarose overlay technique. This method mimics the tumor microenvironment by limiting the diffusion of metabolites and by reducing the oxygen concentration to levels similar to those found in solid tumors. Under these conditions, the CYP activity of CYP2B1-expressing tumor cells was decreased by 80% compared to standard aerobic conditions. Despite this decrease in metabolic activity, a potent bystander effect was observed, resulting in up to 90% killing by CPA of a tumor cell population comprised of only B20% CYP-expressing tumor cells. Similarly, transient transfection of a small fraction (B14%) of a human hepatoma Huh7 cell population with a CYP2B1 expression plasmid followed by short-term treatment with CPA (5 h) led to an eradication of 95% of the cells. No such bystander effect was observed without the agarose overlay. These findings suggest that the agarose overlay technique is very useful as an in vitro test system for investigation of the bystander effect of CYP/CPA and other enzyme/prodrug combinations under conditions that mimic the hypoxic conditions present in solid tumors in vivo.

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Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer

Cited by 416 publications

References 24 publications

Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

Novel degradable oligoethylenimine acrylate ester-based pseudodendrimers for in vitro and in vivo gene transfer

Effects of hypoxia and limited diffusion in tumor cell microenvironment on bystander effect of P450 prodrug therapy

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