Structure and Interaction of Ceramide-Containing Liposomes with Gold Nanoparticles as Characterized by SERS and Cryo-EM

Abstract

Due to the great potential of surface-enhanced Raman scattering (SERS) as local vibrational probe of lipid-nanostructure interaction in lipid bilayers, it is important to characterize these interactions in detail. The interpretation of SERS data of lipids in living cells requires an understanding of how the molecules interact with gold nanostructures and how intermolecular interactions influence the proximity and contact between lipids and nanoparticles. Ceramide, a sphingolipid that acts as important structural component and regulator of biological function, therefore of interest to probing, lacks a phosphocholine head group that is common to many lipids used in liposome models. SERS spectra of liposomes of a mixture of ceramide, phosphatidic acid, and phosphatidylcholine, as well as of pure ceramide and of the phospholipid mixture are reported. Distinct groups of SERS spectra represent varied contributions of the choline, sphingosine, and phosphate head groups and the structures of the acyl chains. Spectral bands related to the state of order of the membrane and moreover to the amide function of the sphingosine head groups indicate that the gold nanoparticles interact with molecules involved in different intermolecular relations. While cryogenic electron microscopy shows the formation of bilayer liposomes in all preparations, pure ceramide was found to also form supramolecular, concentric stacked and densely packed lamellar, nonliposomal structures. That the formation of such supramolecular assemblies supports the intermolecular interactions of ceramide is indicated by the SERS data. The unique spectral features that are assigned to the ceramide-containing lipid model systems here enable an identification of these molecules in biological systems and allow us to obtain information on their structure and interaction by SERS.

Figure 1

Molecular structures of (A) ceramide, (B) phosphatidic acid, and (C) phosphatidylcholine used in the liposome preparations.

Figure 2

Representative SERS spectra of gold nanoparticles (c_AuNP = 6 × 10^–10 M) and liposomes composed of PA/PC, CER, and PA/PC/CER: total lipid concentration, 2 mM; excitation wavelength, 785 nm; intensity, 3.5 × 10⁵ W cm^–2; acquisition time, 1 s; scale bars, 20 cps.

Figure 3

After a hierarchical cluster analysis of the PA/PC/CER liposome data set consisting of 98 spectra, (A) average spectra of four spectral classes are obtained and (B) relative band occurrences of the individual spectra of the four classes C1, C2, C3, and C4 were determined. Liposome composition: 10 mol % CER, 10 mol % PA, and 80 mol % PC. Total lipid concentration: 2 mM. Excitation wavelength: 785 nm. Intensity: 3.5 × 10⁵ W cm^–2. Acquisition time: 1 s.

Figure 4

Cryo-EM micrographs of liposomes composed of (A) PA/PC/CER, (B) CER, and (C) PA/PC prepared with AuNPs. Total lipid concentration: 2 mM. Scale bars: 200 nm.

Figure 5

Cryo-EM micrographs of ceramide preparation: (A) intact liposomes, (B) concentric, stacked lamellar structures, (C) compact, condensed lamellae, and (D) lipid aggregate with many AuNPs. Total lipid concentration: 2 mM. Scale bars: 50 nm.