Development of Small, Homogeneous pDNA Particles Condensed with Mono-cationic Detergents and Encapsulated in a Multifunctional Envelope-type Nano Device

Abstract

Non-viral DNA vectors are promising gene delivery systems and a variety of non-viral DNA vectors have been developed to date. Recently, we developed a novel non-viral gene delivery system-multifunctional envelope-type nano device (MEND). The MEND system has high transfection activity, similar to that of adenovirus vector, which is a potent viral vector. However, conventional MEND is relatively large and heterogeneous (approximately 300 nm), probably because they contain relatively large-and heterogeneous-pDNA particles condensed with polycations, such as poly-L-lysine. Small particle size is important for in vivo delivery, because large particles are rapidly eliminated from systemic circulation. Moreover, heterogeneous size of drug carriers is difficult to apply to clinical applications. Here, we describe construction of small homogeneous MEND. First, we screened mono-cationic detergents (MCD(s)) to obtain optimal pDNA condensed particles. We determined that benzyldimethylhexadecylammonium chloride (BDHAC) and thonzonium bromide (TB) were optimal pDNA condensers. Next, we packaged the condensed pDNA particles into a lipid bi-layer. The resulting lipid-encapsulated pDNA particles were then equipped with octaarginine to facilitate cell-uptake (R8-MEND (MCD)). The carrier showed high transfection activity in cultured HeLa cells. Furthermore, the R8-MEND (MCD) were small and homogeneous compared with conventional MEND. These results indicate that R8-MEND (MCD) has potential as a novel non-viral delivery system for clinical application.

Investigation of siRNA Nanoparticle Formation Using Mono-Cationic Detergents and Its Use in Gene Silencing in Human HeLa Cells

“…We considered that Type-E MCDs, which have an L-chain and an aromatic group, may form siRNA/MCD nanoparticles more effectively than Type-C and -D MCDs, due to the greater hydrophobicity of Type-E MCDs. A similar tendency was also observed when pDNA was used [20]. In the case of MCDs containing a dodecyl group L-chain (E-1), aggregation was observed at high N/P ratios, although negatively charged nanoparticles were formed at low N/P ratios.…”

“…In this experiment, we prepared the R8-D-MEND containing an siRNA nanoparticle complexed with the E-3 MCD, which showed high transfection activity as a DNA condenser [20]. Gene silencing efficiencies after 12, 24, 48 and 72 h of transfection are shown in Figure 2A.…”

“…We previously reported on an optimal mono-cationic detergent (MCD), a quaternary ammonium salt detergent, for use as a pDNA condensation agent that results in efficient release, and showed a high transfection activity in cultured HeLa cells [20]. Moreover, the results of MCD screening showed that the hydrophobic nature of the MCD is important for the condensation of pDNA.…”

Strategies for optimizing targeting and delivery of mucosal HIV vaccines

“…poly‐ L ‐lysine) interact with pDNA molecules through a highly cooperative process, leading to condensation of multiple molecules of pDNA. This process has been found to be quasi‐irreversible 112, 113 and leads to formation of relatively large particles containing multiple pDNA molecules per particle. Unlike polycations, interaction of pDNA with oligo‐ and mono‐cationic detergents is reversible and condenses pDNA into discrete particles that might contain only one pDNA molecule.…”

“…The solubility and unfolding of DNA in mono‐cationic detergent condensed pDNA formulations requires additional stabilization for effective transfection of cells in vivo . Stabilized monomolecular plasmid DNA nano‐particles (∼60 nm) packaged into a lipid bilayer and loaded with the cell‐penetrating peptide octaarginine generated particles of ∼100 nm diameter with low toxicity and transfection efficiencies similar to Ad vector and the larger poly‐ L ‐lysine membrane particles 112, 113. A recent study showed that poly‐propylene sulfide polymer 20 nm particles could enter lymphatic vessels and targeted 50% of resident lymph node DC and macrophages without the need for specific targeting molecules 108.…”