Fluorescence cross‐correlation spectroscopy as a valuable tool to characterize cationic liposome‐DNA nanoparticle assembly

Abstract

The development of nonviral gene delivery vehicles for therapeutic applications requires methods capable of quantifying the association between the genes and their carrier counterparts. Here we investigate the potential of fluorescence cross-correlation spectroscopy (FCCS) to characterize and optimize the assembly of nonviral cationic liposome (CL)-DNA complexes based on a CL formulation consisting of the cationic lipid DOTAP and zwitterionic lipid DOPC. We use a DNA plasmid for lipoplex loading encoding the Oct4 gene, critically involved in reprogramming somatic cells into induced pluripotent stem cells. We demonstrate that FCCS is able to quantitatively determine the extent of the association between DNA and the liposomes and assess its loading capacity. We also establish that the cationic lipid fraction, being proportional to the liposome membrane charge density, as well as charge ratio between the CLs and anionic DNA play an important role in the degree of interaction between the liposomes and DNA.

Lipid-Nucleic Acid Complexes: Physicochemical Aspects and Prospects for Cancer Treatment

“…The X-ray data further reveals that the DNA macromolecules between the lipid bilayers are organized in a 2D smectic phase [79,88,102], in which the average distance between neighboring DNA chains is tunable by the lipid membrane charge density σ M (Equation (2)) and ρ chg . This is confirmed by fluorescence cross-correlation spectroscopy (FCCS) measurements, which show that for constant ρ chg , higher σ M leads to a larger number of DNA plasmids per particle, while for fixed σ M , a higher ρ chg leads to a smaller number of DNA plasmids per particle since there are more cationic particles on where to distribute the DNA [103]. The existence of divalent cations in solution can also lead to further condensation of DNA [104].…”

In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic Liposome–DNA Complexes

“…Their cationic charge mediates strong electrostatic interactions with the negative charges of nucleic acids, giving rise to the formation of CL-NA complexes (often called lipoplexes) [ 23 , 25 , 26 , 27 ]. These complexes adopt internal nanostructures of lamellar, hexagonal or cubic bicontinuous symmetry, with lipid membranes embedding the nucleic acids [ 24 , 25 , 28 , 29 , 30 , 31 ]. By manipulation of the cationic-to-anionic charge ratio, CR (+/−), between liposomes and nucleic acids, as well as adjusting the lipid membrane charge density [ 32 , 33 ], level of PEGylation [ 34 , 35 ], and inclusion of stimuli-responsive or targeting functionalization [ 36 , 37 , 38 , 39 , 40 , 41 , 42 ], these particles can be made highly efficient.…”

Stability Criterion for the Assembly of Hybrid Lipid-Polymer-Nucleic Acid Nanoparticles

“…For a more quantitative analysis, the auto- and cross-correlation curves (resulting from time traces) can be examined 43,45,50 (Figures 2e-g). The green and red auto-correlation curves contain information on the fluctuation dynamics of species carrying the green and red labels, respectively.…”

“…FCCS has been successfully used to characterize interactions between biologic species such as proteins, nucleic acids and membranes, including in living cells 43,44,[50][51][52] . In the context of nanoparticle characterization, FCCS has been employed to monitor the release of nucleic acids from lipoplexes 53 , and to assess the formation of polymeric 54 and silica nanoparticles 55,56 , lipid bilayer-coated metal organic frameworks 57 , and cationic liposome-DNA complexes 45,58 .…”

“…Here we propose an approach based on dual colour Fluorescence Cross-Correlation Spectroscopy (FCCS) to quantitatively assess the extent of association between polyplexes and liposomes in the formation of LPNPs. By analysing the extent of correlation between the fluorescence intensity fluctuations of two labelled species as they diffuse in-and-out of a confocal volume (Figure 2a), the colocalization/association between them can be quantified [43][44][45] , providing a way to obtain crucial insights about the assembly of LPNPs. Overall, we show that the formation of colloidally-stable nanoparticles containing the three components (polylysine, DNA and liposomes) can be achieved in a regime where polyplexes are initially negative and their charge is inverted to positive by addition of cationic liposomes.…”