Nanostructural and nanomechanical alterations of photosensitized lipid membranes due to light induced formation of reactive oxygen species

Abstract

Photosensitization has a wide range of applications in vastly distant fields. Three key components must be present at the same time to trigger the related photodynamic effect: light, the photosensitizer (PS) and oxygen. Irradiating the sensitizer leads to the formation of reactive oxygen species (ROS). Since PSs are accumulated preferably in lipid membranes, the study of photoinduced damage to membrane lipids can greatly increase our understanding of the effect of ROS on membranes in pathological as well as therapeutic conditions. We aimed to characterize the topographical and nanomechanical changes in supported lipid bilayers (SLBs) evoked by light-induced ROS formation. SLBs were prepared on mica surfaces by deposition of liposomes containing unsaturated lipid components. Topographical changes of SLBs were imaged by atomic force microscopy (AFM), and ROS-induced nanomechanical alterations of the membranes were assessed by AFM force measurements. To shed light on chemical alterations of the membrane constituents, infrared spectra were recorded. In the AFM images of porphyrin-containing membranes nanoscopic, bilayer-spanning holes were detected after irradiation. The measured rupture forces increased as a result of irradiation. These phenomena did not occur in membranes lacking unsaturated lipid components, emphasizing their role in ROS-mediated disruption confirmed by infrared spectroscopy results.

Fig. 1

Effect of ROS on SLBs. 5 × 5 μm AFM height-contrast images. The color scale for height is given in the upper right corner. Orange and yellow tones mark the liquid-disordered and liquid-ordered domains, respectively. (a) DPPC-DOPC-MPE SLB before and (b) after irradiation: the membrane is covered in nanoscale holes. The holes are more distinct and numerous in the liquid-ordered domains. The inserted height section profile shows the height along the green line. (c) DPPC-DOPC SLB before and d) after adding H₂O₂ to it. Membrane holes alike to holes in photosensitized membranes were observed.

Fig. 2

Evolution of membrane holes. (a) A chronological sequence of height-contrast AFM images were taken of the same region of DPPC-DOPC-MPE (ca. 2.35 × 1.75 μm) SLB during continuous irradiation. The frame rate is ca. 4 min. Liquid-ordered domains are displayed in yellow the holes appear as dark spots. Appearance, expansion or fusion, reduction or total disappearance of holes is observable (see the different colored circles). The 8th frame shows the image transformed by ImageJ where the “disappeared” holes are marked with yellow crosses. (b) The cumulative distributions of relative hole diameters for the series of frames are presented, fitted with the Gaussian curves. The fit parameters are given in the table. c stands for the center w for width and h stands for height. (c) The total area of the holes for each frame. Curly brackets show the linear proportion of increments in the total area covered by holes.

Fig. 3

Typical force-distance curves for demonstration while piercing through an SLB. (a) In most cases, the rupture of the bilayer consists of a sequential puncture through each of the layer. F₁ and F₂ mark these rupture forces, and x₁ and x₂ mark the layer thickness. (b) Some cases, the rupture of the bilayer can happen in one step, referred to as complete rupture.

Fig. 4

Scatterplot and distribution of (F, x) data of every membrane rupture event for DPPC-DOPC sample. (a) 2D map of the distribution with fitted Gaussian density functions along the two axes blue color means the 1st, red means the 2nd, and black means the complete rupture event. The density function of the summation of respective rupture thicknesses (x₁ + x₂) is also presented with a dashed green curve. (b) Cumulative distribution of F₁, F₂, and F with the same color codes with solid curves before irradiation. (c) Similar distributions are with dashed curves after irradiation. The grey line shows the median of complete rupture forces. n = 1024 for all data sets, but not every point is displayed in the scatterplot to avoid visual clutter.

Fig. 5

Scatterplot and distribution of (F, x) data of the 1st and 2nd rupture events before and after irradiation for DPPC-DOPC-MPE SLB. (a) 2D maps of the distribution with fitted Gaussian density functions along the horizontal (x) axis. 1st rupture events are represented with blue, 2nd rupture events are represented with red, and the cumulative distribution of F1 and F2 are with the same color coding. (b) The same representation for the curves after irradiation the fitted curves are displayed with dashed lines. The arrows present the shift in the median F1 and F2 force due to irradiation.

Fig. 6

Joint cumulative distribution functions of F1 and F2 forces and the individual components as Gaussian density functions for the four different samples before and after the treatment. (a) DPPC-DOPC-MPE as reference, (b) DPPC-MPE, (c) DPPC-DOPC(+ H2O2), d) DPPC-DOPC-MPE(+ GO + C). We used the same representation as before blue color means the F1, red means the F2 and green means F1 and F2 together. The functions displayed with a solid line represent the values before irradiation, the functions displayed with dashed lines represent the values after irradiation. Since the distribution for F1 and F2 overlap each other, a joint cumulative distribution function was produced from all the recorded breakthrough forces. From these curves, density functions were derived after smoothing and derivation. We then fitted the results as a decomposition of Gaussians. The components could be identified as separate F1 and F2 distributions. This figure compares the effects of the treatments on the indicated samples with the DPPC-DOPC-MPE sample.

Fig. 7

FTIR spectra of DOPC-MPE sample. (a) The FTIR spectrum of DOPC-MPE vesicles before (black) and after (red) irradiation. A small blue circle indicates a characteristic lipid peak used for normalization. Lilac and green areas are enlarged below to present the characteristic spectral changes: the increased νas(C = O) peak (b), and the shifted and increased νas(PO2−) peak (c).