Raman microscopy for noninvasive imaging of pharmaceutical nanocarriers: intracellular distribution of cationic liposomes of different composition

Abstract

Nanotechnology is playing an increasing role in targeted drug delivery into pathological tissues. Drug-loaded pharmaceutical nanocarriers can be delivered into diseased sites by passive targeting (spontaneous accumulation of nanocarriers in the areas with affected vasculature) or by active targeting (via site-specific ligands attached to the surface of drug-loaded nanocarriers). Subsequent level of targeting requires cellular internalization of nanocarriers and their specific association with certain individual cell organelles. The control over intracellular distribution of pharmaceutical nanocarriers requires effective and noninvasive methods of their visualization inside cells. In an attempt to enhance cellular internalization of pharmaceutical nanocarriers and their association with mitochondria specifically, we have prepared three types of cationic liposomes and investigated their intracellular distribution. The analysis was performed using Raman microspectroscopy in order to provide morphological information as well as biochemical signatures of the sample. It was demonstrated that Raman microscopy allows evaluation of the extent of mitochondrial association depending on the liposome composition.

Figure 1

Raman spectra of the liposome preparations modified with three distinct cationic moieties: DOTAP, SA, and STPP. Spectra were collected from dried liposomal solutions onto a CaF2 slides.

Figure 2

Raman images of HeLa cells incubated with DOTAP-modified liposomes (top row), SA-modified liposomes (middle row), and STPP-modified liposomes (bottom row), structures of which are shown on the left. Cell cultures were incubated for 1, 3, and 6 hrs, with DOTAP-, SA-, or STPP-modified liposomes. Generated VCA-abundance images. The colors are representative of nuclei and cell body (blue), mitochondria-rich regions (green), and internalized liposomes (red).

Figure 3

Raman image of a HeLa cell incubated with STPP-modified liposomes for 3hrs. Nucleus and cell body (blue), mitochondria-rich regions (green), and internalized liposomes aggregations (red). Color-coded corresponding spectra are shown on the right.

Figure 4

Raman images of HeLa cell incubated for 3 hours STPP-modified liposomes. (A) blue region denotes nuclear and cytoplasmic protein content; red—internalized liposomes; yellow—membrane-rich organelles. (B) is the fluorescence image of the Mitotracker of the cell. (C) is the analyzed image in A overlayed with B fluorescence stain Mitotracker®, pseudo-colored green.