Lipopolyamine-Mediated Single Nanoparticle Formation of Calf Thymus DNA Analyzed by Fluorescence Correlation Spectroscopy

Purpose The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N⁴,N⁹-dioleoylspermine and N¹-cholesteryl spermine carbamate. Methods Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen (PG) to allow fluorescence intensity fluctuation measurement and analysis. Results N⁴,N⁹-Dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 × 10⁻¹² m²/s and particle number (PN) = 0.7 at ammonium/phosphate (N/P) charge ratio=1.0–1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N¹-Cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5–2.5) with D = 1.3 × 10⁻¹² m²/s and PN value of 5.2. N⁴,N⁹-Dioleoylspermine is a more efficient DNA-condensing agent than N¹-cholesteryl spermine carbamate. Conclusions FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.