Multiscale Analysis of Metal Oxide Nanoparticles in Tissue: Insights into Biodistribution and Biotransformation

Abstract

Metal oxide nanoparticles have emerged as exceptionally potent biomedical sensors and actuators due to their unique physicochemical features. Despite fascinating achievements, the current limited understanding of the molecular interplay between nanoparticles and the surrounding tissue remains a major obstacle in the rationalized development of nanomedicines, which is reflected in their poor clinical approval rate. This work reports on the nanoscopic characterization of inorganic nanoparticles in tissue by the example of complex metal oxide nanoparticle hybrids consisting of crystalline cerium oxide and the biodegradable ceramic bioglass. A validated analytical method based on semiquantitative X-ray fluorescence and inductively coupled plasma spectrometry is used to assess nanoparticle biodistribution following intravenous and topical application. Then, a correlative multiscale analytical cascade based on a combination of microscopy and spectroscopy techniques shows that the topically applied hybrid nanoparticles remain at the initial site and are preferentially taken up into macrophages, form apatite on their surface, and lead to increased accumulation of lipids in their surroundings. Taken together, this work displays how modern analytical techniques can be harnessed to gain unprecedented insights into the biodistribution and biotransformation of complex inorganic nanoparticles. Such nanoscopic characterization is imperative for the rationalized engineering of safe and efficacious nanoparticle-based systems.

Keywords: analytical imaging; biological fate; multiscale; nanosafety; spectral unmixing.

Figure 1

a) BG/ceria hybrid nanoparticles were produced using flame spray pyrolysis. Scanning transmission electron micrograph overlaid with energy‐dispersive x‐ray spectroscopy maps show the elemental distribution within the particles. b) Ceria recovery in the hybrid nanoparticles was quantified using different elemental analysis techniques. The standard digestion method using hydrofluoric acid (HF) fails due to the formation of CeF₃. Digestion using a mixture of H₂O₂ and HNO₃ showed high recovery rates for both bare particles and on particle‐spiked tissues. c) The nanoparticles in suspension were applied to the subcutis in a rat skin flap model. d) Biodistribution of BG/ceria hybrid nanoparticles 7 days after application (intravenous injection into the inferior vena cava or topical application to the subcutis). In the case of the topical application, more than 99.96% stayed at the initial site of application (n = 3).

Figure 2

Reconstructed computer tomographs of a) a large part of a rat skin flap and b) a smaller rat skin biopsy. Green frames indicate the same region. High‐intensity pixels are colored red and represent nanoparticles. The nanoparticles are easily identifiable and are located along the subcutis of the rat.

Figure 3

a) Macrophage‐stained (CD68) histology section of skin rat tissue. b) XRF measuring spots and Ce signal. c) Overlay of (a) and (b). The Ce distribution is limited to the subcutaneous region of the skin and is maximal (green) around the macrophages (brown staining). Blue frame indicates ToF‐SIMS measurement region d) ToF‐SIMS map with C_xH_y (green) as a tissue marker, SiO_x (blue) as a substrate marker, and ceria (Ce_xO_yH_z, red). e) The same region with CN + CNO (green), O + OH (blue) and phosphate (PO₂ + PO₃, red). The co‐location of ceria and phosphate indicates the mineralization of the BG component. f–k) Raman spectroscopy signatures of tissues from nanoparticle‐treated and untreated rats. Non‐negative matrix factorization (NMF) was used to separate different constituents, compare Figure S8 in the Supporting Information. f) Average Raman spectra of the as‐prepared nanoparticles versus treated tissue. Raman intensity of treated tissue is normalized according to the peak intensity of ceria at 465 cm⁻¹. The average spectrum can be decomposed into an inorganic part <600 cm⁻¹ and a biological part >600 cm⁻¹. g) The lipid component spectra of the treated and untreated tissue samples show a higher occurrence of lipid components in the treated sample. The shaded area denotes the standard deviation from bootstrap resampling. h) The lipid and cholesterol components that were extracted match their specific peaks reported in the literature.^{[ 59} , ⁶⁰ , ^{61 ]} i) The ceria component shows the localization of the nanoparticles in the tissue sample. j) There is an increased lipid occurrence around the nanoparticles. k) Additionally, increased amounts of cholesterol are found in the particle region.

Figure 4

a–c) Histological images of rat skin tissue. Same‐color frames indicate approximate region. d) Corresponding ToF‐SIMS map with proline (blue) as a tissue marker, Si (green) as a substrate marker, and Ce (red). e) Raman merged spectral map of the same region showing CeO₂, f) CH/CH₂ (lipids), g) amide I (proteins), and h) PO_x (apatite). i) Overlay of the previous maps with CeO₂ in red, CH/CH₂ in blue, amide I in green, and PO_x in blue. j) Electron micrograph of the same region confirms particle distribution around a blood vessel.

Figure 5

a) Density‐dependent color secondary electron micrograph of nanoparticle‐containing tissue macrophages next to a blood vessel. Nanoparticles show up in yellow (compare EDXS in Figure S8 in the Supporting Information). b) Focused‐ion beam cut trench giving access to cross‐sectional imaging of a c) nanoparticle‐containing macrophage. Red arrows indicate the same region.