Independent versus Cooperative Binding in Polyethylenimine–DNA and Poly(<scp>l</scp>-lysine)–DNA Polyplexes

Ketola, Tiia Maaria; Hanzlíková, Martina; Leppänen, Linda; Raviña, Manuela; Bishop, Corey J.; Green, Jordan J.; Urtti, Arto; Lemmetyinen, Helge; Yliperttula, Marjo; Vuorimaa‐Laukkanen, Elina

doi:10.1021/jp404812a

Cited by 30 publications

(51 citation statements)

References 50 publications

(136 reference statements)

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“…Recently, Ketola et al showed that the equilibrium constant of 25 kDa branched PEI to plasmid DNA (7164 bp) using time-resolved fluorescence spectroscopy was 7.3×10 hydroxyethyl) piperazine-N′-2-ethanesulfonic acid (HEPES) and 75 mM Sodium chloride (NaCl) (pH 7.4). 14) This value is slightly lower than the value obtained from our previous study under similar ionic and pH conditions (1.0-1. ).…”

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confidence: 60%

Low Molecular Weight Branched PEI Binding to Linear DNA

Utsuno

Kono

Tanaka

et al. 2016

CHEMICAL & PHARMACEUTICAL BULLETIN

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Polyethylenimine (PEI) is one of the most versatile non-viral vectors used in gene therapy, especially for delivering plasmid DNA to human cells. However, a good understanding of PEI binding to DNA, the fundamental basis for the functioning of PEI as a vector, has been missing in the literature. In this study, PEI (branched, 600 Da) binding to DNA was examined by isothermal titration calorimetry (ITC), quartz crystal microbalance (QCM) and a complementary set of analysis tools. We demonstrated that a separation between the binding heat and the condensation heat is needed and that the excluded site model should be used for PEI binding stage in the ITC analysis. The equilibrium constant for PEI binding to DNA was determined to be 2.5 10 5 M 1 from the ITC analysis, and as 2.3 10 5 M 1 from the QCM analysis. Additionally, we suggested that the 600 Da branched PEI binds to the major groove of DNA and the rearrangement of PEI on DNA may be difficult to occur because of the small dissociation rate. The binding analysis presented here can be employed to improve our understanding of the functioning of PEI and PEI-like non-viral vectors.Key words DNA condensation; polyethylenimine; isothermal titration calorimetry; quartz crystal microbalance; thermodynamics; kinetics The cationic polyelectrolyte polyethylenimine (PEI) is one of the most versatile polymers used for non-viral gene delivery.1-3) PEI binds to DNA via electrostatic interactions between the positively charged PEI amine groups and the negatively charged nucleic acid phosphate groups in the DNA backbone. PEI, by neutralizing the DNA molecule, facilitates its condensation into particles in the 'nano-meter' range.4) It is believed that PEI's strong transfection ability is due to its effective ability to condense the DNA molecule into a nanoparticle, 5-7) which facilitates its cellular uptake and subsequent intracellular trafficking. PEI is available in a broad range of molecular weights. Higher molecular weight PEIs have high transfection efficiency, but lead to excessive cytotoxicity. Low molecular weight PEIs have demonstrated low toxicity on cells, but they also display low transfection efficiency.5) Recently, highly efficient non-viral vectors have been developed by modifying low molecular weight (600 Da) PEI with a variety of functional groups. [8][9][10][11][12] To better understand PEI interactions with the DNA molecule, we previously determined the thermodynamic parameters of PEI binding to DNA using isothermal titration calorimetry (ITC). 13) In that study, two types of binding modes were found to describe the interactions between PEI and DNA. One type of binding involves PEI binding to the DNA groove, another likely binding mode involves external binding of PEI to the DNA phosphate backbone. Recently, Ketola et al. showed that the equilibrium constant of 25 kDa branched PEI to plasmid DNA (7164 bp) using time-resolved fluorescence spectroscopy was 7.3×10 ). 13)The observed difference may be due to the difference in the mass and architecture of the PEI u...

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confidence: 60%

Low Molecular Weight Branched PEI Binding to Linear DNA

Utsuno

Kono

Tanaka

et al. 2016

CHEMICAL & PHARMACEUTICAL BULLETIN

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“…We observed that DNA loading was maximum when only non-degradable, highly charged polymers were used in the middle layers of the formulation, rather than more weakly charged and biodegradable polymers. This loading difference may be due to the differences in binding affinity between the varying cationic polymers and DNA [24, 28]. …”

Section: Resultsmentioning

confidence: 99%

Degradable polymer-coated gold nanoparticles for co-delivery of DNA and siRNA

2015

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Gold nanoparticles have utility for in vitro, ex vivo, and in vivo imaging applications as well as for serving as a scaffold for therapeutic delivery and theranostic applications. Starting with gold nanoparticles as a core, layer-by-layer degradable polymer coatings enable co-delivery of both DNA and short interfering RNA simultaneously. To engineer release kinetics, polymers which degrade through two different mechanisms can be utilized to construct hybrid inorganic/polymeric particles. During fabrication of the nanoparticles, the zeta potential reverses upon the addition of each oppositely charged polyelectrolyte layer and the final nanoparticle size reaches approximately 200 nm in diameter. When the hybrid gold/polymer/nucleic acid nanoparticles are added to human primary brain cancer cells in vitro, they are internalizable by cells and reach the cytoplasm and nucleus as visualized by transmission electron microscopy and observed through exogenous gene expression. This nanoparticle delivery leads to both exogenous DNA expression and siRNA-mediated knockdown, with the knockdown efficacy superior to that of Lipofectamine® 2000, a commercially available transfection reagent. These gold/polymer/nucleic acid hybrid nanoparticles are an enabling theranostic platform technology capable of delivering combinations of genetic therapies to human cells.

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“…If the binding affinity [31] between the polymer types of interest vary substantially it may also be beneficial to assess the degradation rate even within the same cell type [14] as this could change the proportion of DNA in an unbound state vs. a polymer-bound state, affecting DNA degradation rate. Furthermore, if the Cy3 density conjugated to pDNA varies, re-calibrating the plasmid conversion would also be necessary.…”

Section: Discussionmentioning

confidence: 99%

Quantification of cellular and nuclear uptake rates of polymeric gene delivery nanoparticles and DNA plasmids via flow cytometry

et al. 2016

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Non-viral, biomaterial-mediated gene delivery has the potential to treat many diseases, but is limited by low efficacy. Elucidating the bottlenecks of plasmid mass transfer can enable an improved understanding of biomaterial structure-function relationships, leading to next-generation rationally designed non-viral gene delivery vectors. As proof of principle, we transfected human primary glioblastoma cells using a poly(beta-amino ester) complexed with eGFP plasmid DNA. The polyplexes transfected 70.6 ± 0.6% of the cells with 101 ± 3% viability. The amount of DNA within the cytoplasm, nuclear envelope, and nuclei was assessed at multiple time points using fluorescent dye conjugated plasmid up to 24 hours post-transfection using a quantitative multi-well plate-based flow cytometry assay. Conversion to plasmid counts and degradation kinetics were accounted for via quantitative PCR (plasmid degradation rate constants were determined to be 0.62 hr−1 and 0.084 hr−1 for fast and slow phases respectively). Quantitative cellular uptake, nuclear association, and nuclear uptake rate constants were determined by using a four-compartment first order mass-action model. The rate limiting step for these poly(beta-amino ester)/DNA polyplex nanoparticles was determined to be cellular uptake (7.5×10−4 hr−1) and only 0.1% of the added dose was taken up by the human brain cancer cells, whereas 12% of internalized DNA successfully entered the nucleus (the rate of nuclear internalization of nuclear associated plasmid was 1.1 hr−1). We describe an efficient new method for assessing cellular and nuclear uptake rates of non-viral gene delivery nanoparticles using flow cytometry to improve understanding and design of polymeric gene delivery nanoparticles.

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Independent versus Cooperative Binding in Polyethylenimine–DNA and Poly(l-lysine)–DNA Polyplexes

Cited by 30 publications

References 50 publications

Low Molecular Weight Branched PEI Binding to Linear DNA

Low Molecular Weight Branched PEI Binding to Linear DNA

Degradable polymer-coated gold nanoparticles for co-delivery of DNA and siRNA

Quantification of cellular and nuclear uptake rates of polymeric gene delivery nanoparticles and DNA plasmids via flow cytometry

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