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An Acid Sensitive Ketal-Based Polyethylene Glycol-Oligoethylenimine Copolymer Mediates Improved Transfection Efficiency at Reduced Toxicity

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Abstract

Purpose

Dynamic PEG-polycation copolymers that release PEG and degrade into small fragments after cell entry might present efficient and biocompatible gene carriers.

Methods

PEG-OEI-MK was synthesized by copolymerization of 5 kDa polyethyleneglycol (PEG) and 800 Da oligoethylenimines through acid-degradable acetone-bis-(N-maleimidoethyl)ketal linkers (MK). To evaluate any benefit of the reversible over stable linkage, also the corresponding pH-stable analog, PEG-OEI-BM, was synthesized via ether linkages. Luciferase and GFP expression plasmids were used for transfections, in vivo biocompatibility was evaluated by intravenous application of polymers in Balb/c mice.

Results

PEG-OEI-MK showed efficient DNA binding as analyzed by ethidium bromide exclusion, resulting in formation of polyplexes with sizes around 100 nm and surface charges of below 5 mV zeta potential. This surface shielding of PEG-OEI-MK polyplexes remained stable at neutral pH 7.4, while polyplexes deshielded and aggregated at pH 5 within 15–30 min. Cell culture experiments demonstrated reduced polymer toxicity compared to the non-PEGylated OEI-MK. Transfection experiments demonstrated reduced gene expression of PEG-OEI-BM compared with the non-PEGylated analog OEI-BM, whereas the pH-reversible polymer PEG-OEI-MK mediated a significant increased transfection efficiency over the non-PEGylated OEI-MK.

Conclusions

PEG-OEI-MK mediated the highest gene transfer at lowest cytotoxicity levels and also best in vivo biocompatibility.

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Abbreviations

AP:

alkaline phosphatase

AST:

aspartate aminotransferase

BM:

1,8-bis-maleimidodiethyleneglycol

DCM:

dichloro methane

EtBr:

ethidium bromide

FCS:

fetal calf serum

HBG:

Hepes buffered glucose (5% (w/v) glucose, 20 mM Hepes, pH 7.4)

0.5× HBS:

Hepes buffered salt (2.5% glucose (w/v), 75 mM NaCl, 20 mM Hepes, pH 7.4)

Hepes:

N-2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid

MFI:

mean fluorescence intensity

MK:

2,2-bis(N-maleimidoethyloxy) propane

m.w.:

molecular weight

Mw:

molecular weight by weight

NaOAc:

sodium acetate

OEI800:

oligoethylenimine with an average molecular weight of 800 Da

PEG5K-SH:

methoxy poly(ethylene glycol) thiol, average molecular weight 5 kDa

PEI25K:

branched polyethylenimine with an average molecular weight of 25 kDa

PEI22K:

linear polyethylenimine with an average molecular weight of 22 kDa

RLU:

relative light units

s.d.:

standard deviation

U/l (37°C):

units per liter plasma, measured at 37°C

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Acknowledgments

We thank Olga Brück for skillful assistance in preparing the manuscript, as well as Wolfgang Roedl and Anna Kulinyak for competent technical assistance. This work was funded by the LMU university, by the DFG projects SFB 486 ‘Nanoman’ and SPP1230, the Nanosystems Initiative Munich (NIM), and the European Community FP6 network project ‘GIANT’.

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  1. Pharmaceutical Biology-Biotechnology, Center for Drug Research and Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, Munich, Germany

    Veronika Knorr, Manfred Ogris & Ernst Wagner

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  1. Veronika Knorr
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  2. Manfred Ogris
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  3. Ernst Wagner
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Correspondence to Ernst Wagner.

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Knorr, V., Ogris, M. & Wagner, E. An Acid Sensitive Ketal-Based Polyethylene Glycol-Oligoethylenimine Copolymer Mediates Improved Transfection Efficiency at Reduced Toxicity. Pharm Res 25, 2937–2945 (2008). https://doi.org/10.1007/s11095-008-9700-6

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