Features of electron-microscopic visualization of soft phospholipid nanoparicles

Transmission electron microscopy (EM) is the way to control structure of lipid nanoparticles. Our work was aimed at study of EM imaging of nanoparticles based on mixtures of dioleoylphosphatidylcholine (DOPE) and phosphatidyl acid (PA), and phosphatidylcholine (PC) and cholesterol (Chol), using contrasting with uranyl acetate (UA), and phospho-tungstic acid (PTA). Suspensions of nanoparticles DOPE/PA (9:2) and PC/Chol (1:1) were adsorbed on formvar film, 1 min, and contrasted with UA, or PTA (pH 0.5), or PTA (pH 7.0), 5-10 sec, then studied in EM Jem-1400 (Jeol, Japan). Spectra of 31P-NMR of samples were recorded on spectrometer AV-300 (Bruker, Germany). Nanoparticles DOPE/PA, UA and PTA (pH 0.5) contrasting: rounded particles, formed by filaments (2-3 nm) of low electron density (ED). Filament morphology corresponds to structures of inverted hexagonal phase (IHP). PTA (pH 7.0) contrasting leads to filament structuring and appearance of membrane-like structures, morphologically corresponding to lamellar phase (LP). However, 31Р-NMR analysis revealed in sample spectrum single signal on 0,189 m.d. (IHP). Nanoparticles PC/Chol, UA and PTA (pH 0.5) contrasting: irregular particles, formed by filaments (2 nm), low ED, corresponding to IHP. The PTA revealed stacks of lipid bilayers in preparation, corresponding to LP. Presence of lipid different phases is confirmed by 31Р-NMR: analysis of preparation spectra, showed LP (signal -0,482 M. D.), IHP and isotropic phase (signals on -0,235, -0,362 M. D.). Thus, EM allows to identify lipid phase state; “traditional” contrasting agents differently identify components in same preparation, suggesting their active interaction with lipid molecules, and indicates necessity of using different contrasting for visualization of lipid nanostructures.