Plasmid linearization changes shape and efficiency of transfection complexes

Abstract

Abstract:The ability to efficiently transfect plasmid DNA (pDNA) into eukaryotic cells has exerted major impact on scientific research in recent years, and translation to clinical application is ongoing, but challenging. In addi tion to the choice of the delivery vector, the topology of the DNA seems to be a key factor for efficient transfection. The nanostructured DNA/Vector complexes may differ in size, charge, and shape, for example. This study therefore investigated the transfection efficiency of circular versus linearized plasmid DNA using a GFP expressing vector with Lipofectamine2000 and linear 25 kDA polyethylen imine (PEI). Transfection efficiency and cytotoxicity were measured by flow cytometry and fluorescence micro scopy. Shape was determined by transmission electron microscopy. Transfection agent concentrations were cho sen below the toxicity level. We determined the optimal N/P ratio over 48 h by using two different concentrations of plasmid DNA. With the increase of DNA concentra tion and increasing N/P ratio, transfection efficiency also increased. Our results showed a better transfection effi ciency with the circular compared to the linearized DNA, under the same experimental conditions for both Lipo fectamine and PEI. In electron microscopy, there was a notable difference in the shape of the complexes: circu lar DNA had random coil appearance in well compacted, roughly spherical shape, while linearized DNA appeared as wormlike strands, both, when complexed with Lipo fectamine or with polyethyleneimine. This generates the hypothesis that the shape of the transfection particle may be an important factor for successful gene transfer.

Novel Coagulation Factor VIII Gene Therapy in a Mouse Model of Hemophilia A by Lipid-Coated Fe3O4 Nanoparticles

“…DPPC-Fe 3 O 4 -carrying DNA nanoparticles with a small size might completely combine the supercoiled circular plasmid DNA and can achieve a higher transfection efficiency in the targeted cells, possibly through membrane endocytic machinery. In a previous study, Lehner et al [ 38 ] also demonstrated a similar result: a better transfection efficiency with polyethylenimine (PEI)-carrying circular form DNA nanoparticles compared to that of linear form DNA was found. Under electron microscopy observation, the researchers also found a remarkable difference in the shape of the DNA nanoparticle complexes: circular DNA had a smaller size with a well compacted and roughly spherical shape, while PEI-carried linear DNA nanoparticles appeared larger in size with string-like strand confirmation [ 38 ].…”

“…TEM was performed to visualize the confirmations of circular and linearized plasmid DNA/DPPC-Fe 3 O 4 nanoparticles in an aqueous environment as previously described [ 38 ]. Briefly, the plasmid DNA/DPPC-Fe 3 O 4 complex was deposited on the carbon-coated copper grid.…”

Freezing-Assisted Gene Delivery Combined with Polyampholyte Nanocarriers

“…Surprisingly, jetPEI luciferase expression was found to be higher than Lipofectamine 3000 in both the nonfrozen and freeze concentration conditions. Other studies have reported that jetPEI can provide a higher transfection efficiency compared to Lipofectamine. , One possible reason for the reduced transfection efficiency of Lipofectamine 3000 compared to jetPEI is that it can adsorb onto large anionic serum protein aggregates. These large aggregates most likely will not be able to cross the cell membrane and deliver pDNA to the cells; it is possible that pDNA-jetPEI could prevent this aggregation.…”

Polyethyleneimine-Based Lipopolyplexes as Carriers in Anticancer Gene Therapies

“…For example, branched chains with high MW seem to work better with oligonucleotides, as opposed to 25 kDa lPEI, which is considered the gold standard when transfecting cells with plasmid DNA (pDNA). Interestingly, plasmid linearization hampers the complexation process [ 74 , 85 , 86 ]. Moreover, Kwok and Hart observed that pDNA complexes are more stable than those with siRNA, and the branched form of PEI is more effective in the case of RNA [ 87 ].…”

“…Reverse lipopolyplexes, briefly mentioned earlier and illustrated in Figure 1 , exactly fulfill the idea of transporting polyplexes as the external layer of the liposomal carrier. Exposure of polyethyleneimine helps to circumvent some limitations of lipid carriers, namely short circulation times (especially considering lipoplexes), simultaneously increasing endosomal release due to its increased efficiency as a proton sponge [ 86 , 95 ]. The standard procedure utilizes either non-covalent adsorption of polyethyleneimine to negatively charged lipids and surfactants constituting the preformed vesicles [ 108 , 109 ] or covalent modification of PEI with some hydrophobic anchor that enables close interaction with lipid bilayers.…”