Patterned Threadlike Micelles and DNA-Tethered Nanoparticles: A Structural Study of PEGylated Cationic Liposome–DNA Assemblies

Abstract

The self-assembly of oppositely charged biomacromolecules has been extensively studied due to its pertinence in the design of functional nanomaterials. Using cryo electronic microscopy (cryo-EM), optical light scattering and fluorescence microscopy, we investigated the structure and phase behavior of PEGylated (PEG: poly(ethylene-glycol)) cationic liposome–DNA nanoparticles (CL–DNA NPs) as a function of DNA length, topology (linear and circular) and ρchg (the molar charge ratio of cationic lipid to anionic DNA). Although all NPs studied showed a lamellar internal nanostructure, NPs formed with short (~ 2 kbps), linear, polydisperse DNA were defect-rich and contained smaller domains. Unexpectedly, we found distinctly different equilibrium structures away from the isoelectric point. At ρchg > 1, in the excess cationic lipid regime, thread-like micelles rich in PEG-lipid were found to coexist with NPs, cationic liposomes and spherical micelles. At high concentrations these PEGylated thread-like micelles formed a well-ordered, patterned morphology with highly uniform inter-micellar spacing. At ρchg < 1, in the excess DNA regime and with no added salt, individual NPs were tethered together via long, linear DNA (48 kbps λ-phage DNA) into a biopolymer-mediated floc. Our results provide insight on what equilibrium nanostructures can form when oppositely charged macromolecules self-assemble in aqueous media. Self-assembled, well-ordered thread-like micelles and tethered nanoparticles may have a broad range of applications in bionanotechnology, including nanoscale lithograpy and the development of lipid-based multi-functional nanoparticle networks.

Cationic liposome–nucleic acid nanoparticle assemblies with applications in gene delivery and gene silencing

“…While Cryo-TEM studies are consistent with the DLS results, they further reveal (figure 3, bottom) differences in the detailed structure and morphology of PEGylated CL-DNA NPs that depend on DNA length and shape [40]. In contrast with polydisperse salmon sperm DNA, NPs formed with λ-DNA or pGL3 can also exhibit hollow cores in addition to the lamellar morphology from core to surface.…”

“…Complexes which contain sufficient PEG-lipids (such that the PEG chains transition from the mushroom to the brush conformation) sterically stabilize CL-DNA complexes against flocculation caused by van der Waals attractions [55][56][57]. Experiments have shown that PEGylated complexes resist aggregation even after extensive centrifugation [40]. Thus, the use of a PEG-lipid improves colloidal stability, and as experiments show PEGylation leads to the spontaneous formation of equilibrium nanoparticles (NPs).…”

Quantitative Intracellular Localization of Cationic Lipid–Nucleic Acid Nanoparticles with Fluorescence Microscopy

“…A recent cryo-EM study has found that the DNA length and topology can also influence the average number of layers found in each particle (38). NPs formed with long, linear DNA (48 kbps, lambda DNA) or circular plasmid (2 kbps, pDNA) results in more layers than NPs formed with polydisperse, linear DNA (2 kbps, salmon DNA).…”

“…Dual fluorescent labeling permits discrimination between CL–DNA NPs and cationic liposomes lacking DNA (which coexist at equilibrium, see Fig. 4B) (24,38). Furthermore, dual-labeled CL–DNA complexes allow direct visualization of vector-cargo disassociation (39,45).…”

“…Thus it is necessary to use computer software to automate the measurements and allow data to be extracted from large numbers of cells. As of today, numerous research groups use quantitative analysis of fluorescent imaging to study gene delivery vectors (37,38,44,46–51). Intracellular localization is an interesting feature that can be measured from fluorescence microscopy.…”