Low Molecular Weight Branched PEI Binding to Linear DNA

Abstract

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...

Polyethylenimine-based nanocarriers in co-delivery of drug and gene: a developing horizon

“…They performed the same ITC tests using PEI 25 kDa instead of 600 Da PEI. This means that a large PEI can attach to DNA multivalently, while the small PEI can be linked monovalently [19]. The study of Lungu, C.N., et al in 2016 on linear and branched PEIs and their spatial characteristics showed that LPEIs modify their geometry easily compared to BPEIs and are more adaptable to a specific binding sites.…”

“…Recently, non-viral vectors, which are highly efficient, have been developed by modifying low molecular weight (600 Da) with a variety of functional groups [19]. Because of the high density of the charge and the so-called ‘sponge-proton effect’, PEI has more advantages over other cationic polymers than other non-viral vectors.…”

Development of Tailor-Made Dendrimer Ternary Complexes for Drug/Gene Co-Delivery in Cancer

“…The properties of PEI namely the molecular weight, the architecture of the chains or the degree of branching are fundamental parameters in determining the physicochemical characteristics of nucleic acids-PEI complexes. These properties also strongly influence both the cytotoxicity displayed by the complexes and the efficiency of cellular transfection [ 9 , 46 , 47 ].…”

Multipotent miRNA Sponge-Loaded Magnetic Nanodroplets with Ultrasound/Magnet-Assisted Delivery for Hepatocellular Carcinoma Therapy

“…T2-weighted MRI of SLMNDs-20% showed a strong negative contrast signal as compared with non-magnetic NDs, and SLMNDs-20% could achieve an accumulation to the sidewall of the centrifuge tubes under magnetic force within 20 min (Figure G). Furthermore, SLMNDs-20% were easy to agglomerate with the addition of a small amount of PEI because PEI molecules bound adjacently on the DNA lattice at low concentrations, while they located far from each other on DNA at high concentrations. , When the amount of PEI was above 12 mg [keeping the ratio of nitrogen to phosphorus (N/P) ≥ 3, the agglomeration gradually disappeared (Figure H). Besides, AGE proved that the addition of PEI inhibited the staining of the sponge loaded on pre-SLMNDs-20%.…”

“…During the venous circulation, nucleic acids are easily decomposed by nuclease in the blood. Fortunately, many research studies revealed that polyethylenimine (PEI) can not only enhance delivery efficiency and expand expression duration for both in vitro and in vivo nucleic acid transfection − but also protect combined DNA from enzyme digestion in the serum, which inspired us to use PEI for protection of the genes loaded on NDs. PEI has a high density of positive charges to interact with the negatively charged cell membranes and internalize itself combined with nucleic acid into the cell through endocytosis.…”