While few publications have documented the uptake of nanoparticles in plants, this is the first study describing uptake and distribution of the ultra-small anatase TiO 2 in the plant model system Arabidopsis. We modified the nanoparticle surface with Alizarin red S and sucrose, and demonstrated that nanoconjugates traversed cell walls, entered into plant cells, and accumulated in specific subcellular locations. Optical and X-ray fluorescence microscopy co-registered the nanoconjugates in cell vacuoles and nuclei. KeywordsAnatase TiO 2 nanoparticles; TiO 2 nanoconjugates; Arabidopsis thaliana; X-ray fluorescence microscopy (XFM)The application of nanotechnology to plant systems has lagged behind nanomedicine and nanopharmacology in spite of its potential to generate new tools for the delivery of fertilizers, herbicides and insecticides 1 , new ways to manipulate plant genomes 2 and new methods to capture and isolate plant natural products. Compared to the thousands of studies describing the uptake and trafficking of nanoparticles (NPs) in biological systems other than plants, less than twenty reports discussed NP uptake by plant species. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] These studies involved different plant species and different types of NPs which were delivered to intact plants, dissected plant organs or protoplasts using a wide range of application methods. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] Despite the absence of systematic analyses, it has been determined that plants can take up NPs from the environment and transport them through the vascular system to various shoot organs. 4,9,15 However, little is known about the uptake mechanisms involved or the subcellular localization and distribution of the internalized NPs. 16 Uptake efficiency has also Figure S1), and show additional data describing NC distribution and localization in roots, hypotocyls and cotyledons (Figures S2, S3 and S4). This material is available free of charge via the Internet at http://pubs.acs.org. Here, we report on the uptake and localization of anatase titanium dioxide (TiO 2 ) NPs smaller than 5 nm in the plant model system Arabidopsis thaliana. We chose to study the Col-0 accession because this is the most commonly used ecotype within the Arabidopsis research community. 22 The numerous resources developed for this genetic background 23,24 will not only facilitate future analyses of the molecular mechanisms of uptake, intracellular localization and trafficking of NPs, but will also provide opportunities for NP-mediated manipulations of the Arabidopsis genome. In addition, the well-characterized Arabidopsis null mutants and overexpression lines for enzymes of various biochemical pathways offer the possibility for the targeted in planta chemical modification of NP surface with pathway intermediates. TiO 2 NPs with average diameters of 2.8 ± 1.4 nm and NP dispersity of 43% (see Supporting information) were synthesized by a low-temperature alkaline hydrolysis route as described previously 2...
Several recent efforts in radiation biology community worldwide have amassed records and archival tissues from animals exposed to different radionuclides and external beam irradiation. In most cases, these samples come from life-long studies on large animal populations conducted in national laboratories and equivalent institutions throughout Europe, North America, and Japan. While many of these tissues were used for histopathological analyses, much more information may still be obtained from these samples. A new technique suitable for imaging of these tissues is X-Ray Fluorescence Microscopy (XFM). Following development of third generation synchrotrons, XFM has emerged as an ideal technique for study of metal content, speciation, and localization in cells, tissues and organs. Here we review some of the recent XFM literature pertinent to tissue sample studies and present examples of XFM data obtained from tissue sections of beagle dog samples which show that the quality of archival tissues allows XFM investigation.
Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is biologically distinct from HPV-negative HNSCC. Outside of HPV-status, few tumor-intrinsic variables have been identified that correlate to improved survival. As part of exploratory analysis into the trace elemental composition of oropharyngeal squamous cell carcinoma (OPSCC), we performed elemental quanitification by X-ray fluorescence microscopy (XFM) on a small cohort (n = 32) of patients with HPV-positive and -negative OPSCC and identified in HPV-positive cases increased zinc (Zn) concentrations in tumor tissue relative to normal tissue. Subsequent immunohistochemistry of six Zn-binding proteins—zinc-α2-glycoprotein (AZGP1), Lipocalin-1, Albumin, S100A7, S100A8 and S100A9—revealed that only AZGP1 expression significantly correlated to HPV-status (p < 0.001) and was also increased in tumor relative to normal tissue from HPV-positive OPSCC tumor samples. AZGP1 protein expression in our cohort significantly correlated to a prolonged recurrence-free survival (p = 0.029), similar to HNSCC cases from the TCGA (n = 499), where highest AZGP1 mRNA levels correlated to improved overall survival (p = 0.023). By showing for the first time that HPV-positive OPSCC patients have increased intratumoral Zn levels and AZGP1 expression, we identify possible positive prognostic biomarkers in HNSCC as well as possible mechanisms of increased sensitivity to chemoradiation in HPV-positive OPSCC.
This work compares intravenous (IV) versus fluoroscopy-guided transarterial intra-catheter (IC) delivery of iron oxide core-titanium dioxide shell nanoparticles (NPs) in vivo in VX2 model of liver cancer in rabbits. NPs coated with glucose and decorated with a peptide sequence from cortactin were administered to animals with developed VX2 liver cancer. Two hours after NPs delivery tumors, normal liver, kidney, lung and spleen tissues were harvested and used for a series on histological and elemental analysis tests. Quantification of NPs in tissues was done both by bulk inductively coupled plasma mass spectrometry (ICP-MS) analysis and by hard X-ray fluorescence microscopy. Both IV and IC NPs injection are feasible modalities for delivering NPs to VX2 liver tumors with comparable tumor accumulation. It is possible that this is an outcome of the fact that VX2 tumors are highly vascularized and hemorrhagic, and therefore enhanced permeability and retention (EPR) plays the most significant role in accumulation of nanoparticles in tumor tissue. It is, however, interesting to note that IV delivery led to increased sequestration of NPs by spleen and normal liver tissue, while IC delivery lead to more NP positive Kupffer cells. This difference is most likely a direct outcome of blood flow dynamics. Armed with this knowledge about nanoparticle delivery, we plan to test them as radiosensitizers in the future.
Research in cancer nanotechnology is entering its third decade, and the need to study interactions between nanomaterials and cells remains urgent. Heterogeneity of nanoparticle uptake by different cells and subcellular compartments represent the greatest obstacles to a full understanding of the entire spectrum of nanomaterials’ effects. In this work, we used flow cytometry to evaluate changes in cell cycle associated with non-targeted nanocomposite uptake by individual cells and cell populations. Analogous single cell and cell population changes in nanocomposite uptake were explored by X-ray fluorescence microscopy (XFM). Very few nanoparticles are visible by optical imaging without labeling, but labeling increases nanoparticle complexity and the risk of modified cellular uptake. XFM can be used to evaluate heterogeneity of nanocomposite uptake by directly imaging the metal atoms present in the metal-oxide nanocomposites under investigation. While XFM mapping has been performed iteratively in 2D with the same sample at different resolutions, this study is the first example of serial tomographic imaging at two different resolutions. A cluster of cells exposed to non-targeted nanocomposites was imaged with a micron-sized beam in 3D. Next, the sample was sectioned for immunohistochemistry as well as a high resolution “zoomed in” X-ray fluorescence (XRF) tomography with 80 nm beam spot size. Multiscale XRF tomography will revolutionize our ability to explore cell-to-cell differences in nanomaterial uptake.
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