A Correlative Imaging Study of in vivo and ex vivo Biodistribution of Solid Lipid Nanoparticles

Abstract

Purpose: Solid lipid nanoparticles are largely used in biomedical research and are characterized by high stability and biocompatibility and are also able to improve the stability of various loaded molecules. In vitro studies demonstrated that these nanoparticles are low cytotoxic, while in vivo studies proved their efficiency as nanocarriers for molecules characterized by a low bioavailability. However, to our knowledge, no data on the systemic biodistribution and organ accumulation of solid lipid nanoparticles in itself are presently available.

Methods: In this view, we investigated the solid lipid nanoparticles biodistribution by a multimodal imaging approach correlating in vivo and ex vivo analyses. We loaded solid lipid nanoparticles with two different fluorophores (cardiogreen and rhodamine) to observe them with an optical imager in the whole organism and in the excised organs, and with fluorescence microscopy in tissue sections. Light and transmission electron microscopy analyses were also performed to evaluate possible structural modification or damage due to nanoparticle administration.

Results: Solid lipid nanoparticles loaded with the two fluorochromes showed good optic characteristics and stable polydispersity. After in vivo administration, they were clearly detectable in the organism. Four hours after the injection, the fluorescent signal occurred in anatomical districts corresponding to the liver and this was confirmed by the ex vivo acquisitions of excised organs. Brightfield, fluorescence and transmission electron microscopy confirmed solid lipid nanoparticles accumulation in hepatocytes without structural damage.

Conclusion: Our results support the systemic biocompatibility of solid lipid nanoparticles and demonstrate their detailed biodistribution from the whole organism to organs until the cells.

Keywords: light microscopy; lipid-based nanoparticles; optical imaging; systemic biodistribution; tissue accumulation; transmission electron microscopy.

Figure 1

Absorption spectra of the fluorophore RH and CG ethanol solution. Emission spectra in ethanol solution of RH (red line, λ_ex= 540 nm) and CG (black line, λ_ex= 690 nm) and as 1:1 (w/w) mixture RH+CG measured at λ_ex= 540 nm (blue line) and λ_ex= 690 nm (green line).

Figure 2

Emission spectra of SLN-RH (A), SLN-CG (B), mixture of SLN-RH and SLN-CG (C) and co-loaded SLN-RH/CG (D). Emission spectra are obtained at different excitation wavelength λex= 540 nm (dotted line) and λex= 690 nm (full line). RH spectra: red line; CG spectra: green line.

Figure 3

Macroscopic appearance of SLN-CG (A), SLN-RH (B), SLN-CG/RH (C) and cryo-TEM photomicrograph of SLN-CG/RH (D).

Figure 4

Plot of the kinetic uptake in the abdominal region (A, B) up to 4 h after injection; SLN biodistribution in living animals 3 h after ip injection (C, D), and fluorescent emission in excised and perfused liver (E, F). (A, C and E) refer to SLN-CG; (B, D and F) refer to SLN-CG/RH.

Figure 5

Light microscopy images of liver sections from control (A–D) and SLN-CG/RH-treated (E–H) mice. (A, E) brightfield images; (B, F) red signal from RH contained in SLN; (C, G) blue signal from DNA stained with Hoechst; (D, H) merge of brightfield image, red and blue fluorescence. Note the red fluorescence signal inside the hepatocytes of the treated mouse. V: centrilobular vein. Bars 50 µm.

Figure 6

Light microscopy images of liver sections from control (A) and SLN-treated (B) mice; note the large amounts of lipid droplets in B (Oil Red O staining). V: centrilobular vein. Bars, 100 µm.

Figure 7

TEM images of liver from control (A) and SLN-treated (B–D) mice. Note the high number of lipid droplets (L) in the hepatocytes of SLN-treated mouse. Some lipid droplets showing a finely granular electron-dense border are extruded from the cell (arrow in (B); high magnification in (C). In addition, the hepatocyte in (B) shows a loosened appearance, with euchromatic nucleus (N), dispersed glycogen clusters (asterisks) and rough endoplasmic reticulum cisternae (ER) arranged in a less orderly pattern (D) in comparison to control (A). Bars 2500 nm (A, B); 1000 nm (C, D).

Figure 8

Light (A–F) and transmission electron (G–I) micrographs of 3T3 cells. 3T3 cells after 1 h (A), 4 h (B) and 24 h (C) treatment with SLN: lipid droplets (stained with Oil Red (O) progressively increase in number and size. 3T3 control cells after 1 h (D), 4 h (E) and 24 h (F) in medium without nanoparticles: only a few small lipid droplets are visible. 3T3 cells after 1 h treatment with SLN (G, H): many lipid droplets (L) are distributed in the cytoplasm, sometimes showing a finely granular border (red arrows). Three SLN (arrows) occur free in the cytoplasm, two of them very close to lipid droplets. After 4 h (I) treatment with SLN, some lipid droplets (L) were extruded from the cells. Note that cytoplasmic organelles such as mitochondria (M) and endoplasmic reticulum (ER), and cell nuclei (N) are well preserved after SLN treatment. Bars 50 µm (A–F); 500 nm (G–I).