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We have efficiently produced collagen-rich microstructures in fibroblast multicellular spheroids (MCSs) as a three-dimensional in vitro tissue analog to investigate silver (Ag) nanoparticle (NP) penetration. The MCS production was examined by changing the seeding cell number (500 to 40,000 cells) and the growth period (1 to 10 days). MCSs were incubated with Ag NP suspensions with a concentration of 5 μg/mL for 24 h. For this study, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to visualize Ag NP localization quantitatively. Thin sections of MCSs were analyzed by LA-ICP-MS with a laser spot size of 8 μm to image distributions of 109Ag, 31P, 63Cu, 66Zn, and 79Br. A calibration using a NP suspension was applied to convert the measured Ag intensity into the number of NPs present. The determined numbers of NPs ranged from 30 to 7200 particles in an outer rim of MCS. The particle distribution was clearly correlated with the presence of 31P and 66Zn and was localized in the outer rim of proliferating cells with a width that was equal to about twice the diameter of single cells. Moreover, abundant collagens were found in the outer rim of MCSs. For only the highest seeding cell number, NPs were completely captured at the outer rim, in a natural barrier reducing particle transport, whereas Eosin (79Br) used as a probe of small molecules penetrated into the core of MCSs already after 1 min of exposure.
We applied high resolution laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with cellular spatial resolution for bioimaging of nanoparticles uptaken by fibroblast multicellular spheroids (MCS). This was used to quantitatively investigate interactions of silver nanoparticles (Ag NPs) and the distributions of intrinsic minerals and biologically relevant elements within thin sections of a fibroblast MCS as a three-dimensional in vitro tissue model. We designed matrix-matched calibration standards for this purpose and printed them using a noncontact piezo-driven array spotter with a Ag NP suspension and multielement standards. The limits of detection for Ag, Mg, P, K, Mn, Fe, Co, Cu, and Zn were at the femtogram (fg) level, which is sufficient to investigate intrinsic minerals in thin MCS sections (20 μm thick). After incubation for 48 h, Ag NPs were enriched in the outer rim of the MCS but not detected in the core. The localization of Ag NPs was inhomogeneous in the outer rim, and they were colocalized with a single-cell-like structure visualized by Fe distribution (pixel size of elemental images: 5 × 0.5 μm). The quantitative value for the total mass of Ag NPs in a thin section by the present method agreed with that obtained by ICP-sector field (SF)-MS with a liquid mode after acid digestion.
We investigated the penetration of silver nanoparticles (Ag NPs) into a three-dimensional in vitro tissue analog using NPs with various sizes and surface coatings, and with different incubation times. A high-Resolution laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) time-of-flight (TOF) instrument was applied for imaging the distributions of elements in thin sample sections (20 μm thick). A fibroblast multicellular spheroid (MCS) was selected as the model system and cultured for more than 8 days to produce a natural barrier formed by the extracellular matrix containing collagen. The MCS was then exposed for up to 48 h to one of four types of Ag NPs (∅ 5 nm citrate coated, ∅ 20 nm citrate coated, ∅ 20 nm polyvinylpyrrolidone coated, and ∅ 50 nm citrate coated). Imaging showed that the penetration pathway was strongly related to steric networks formed by collagen fibrils, and Ag NPs with a hydrodynamic diameter of more than 41 nm were completely trapped in an outer rim of the MCSs even after incubation for 48 h. In addition, we examined the impact of these NPs on essential elements (P, Fe, Cu, and Zn) in
areas of Ag NP accumulation. We observed a linear increase at the sub-femtogram level in the total concentration of Cu (fg per pixel) in samples treated with small or large Ag NPs (∅ 5 nm or ∅ 50 nm) for 48 h.
Directing nanoparticles to the nucleus by attachment of nuclear localization sequences (NLS) is an aim in many applications. Gold nanoparticles modified with two different NLS were studied while crossing barriers of intact cells, including uptake, endosomal escape, and nuclear translocation. By imaging of the nanoparticles and by characterization of their molecular interactions with surface-enhanced Raman scattering (SERS), it is shown that nuclear translocation strongly depends on the particular incubation conditions. After an 1 h of incubation followed by a 24 h chase time, 14 nm gold particles carrying an adenoviral NLS are localized in endosomes, in the cytoplasm, and in the nucleus of fibroblast cells. In contrast, the cells display no nanoparticles in the cytoplasm or nucleus when continuously incubated with the nanoparticles for 24 h. The ultrastructural and spectroscopic data indicate different processing of NLS-functionalized particles in endosomes compared to unmodified particles. NLS functionalized nanoparticles form larger intraendosomal aggregates than unmodified gold nanoparticles. SERS spectra of cells with NLS-functionalized gold nanoparticles contain bands assigned to DNA and were clearly different from those with unmodified gold nanoparticles. The different processing in the presence of an NLS is influenced by a continuous exposure of the cells to nanoparticles and an ongoing nanoparticle uptake. This is supported by mass-spectrometry-based quantification that indicates enhanced uptake of NLS-functionalized nanoparticles compared to unmodified particles under the same conditions. The results contribute to the optimization of nanoparticle analysis in cells in a variety of applications, e.g., in theranostics, biotechnology, and bioanalytics.
This work demonstrates the sub-cellular resolution bio-imaging capabilities of the NWRimage system with Dual Concentric Injector (DCI) technology in a practical application. 3T3 fibroblast cells incubated with gold nanoparticles were imaged, demonstrating the sub-cellular imaging capabilities. A laser ablation system specifically designed for rapid, high resolution imaging was employed. The system´s optical layout is optimized to minimize crater diameters. Sensitivity and speed were achieved using the DCI technology for ultra-fast washout times.
The traceability and availability of nanoparticles enables their use to enhance a variety of nano-biological and nano-medicinal applications. The particular size and shape of nanoparticles determine the uptake rate and pathway into the cell, and therefore impact specific cell components and processes. Selecting specific particle types allows researchers to target the process or structure of interest, with minimal additional impact. This can be used for drug or DNA delivery, and is being explored for use in oncology. Understanding the different uptake mechanisms and impacted processes requires sub-cellular Imaging resolution to determine, for example, whether or not the nanoparticles are reaching the nucleus.
Sub-cellular imaging has traditionally been challenging to achieve with laser ablation ICP-MS due to a lack of sensitivity at small spots. Bioimaging using LA-ICP-MS is a well-established technique, but usually applied on the tissue scale, which depends on larger spot areas where sensitivity is less problematic. The improved sensitivity and washout from the NWRimage has allowed faster imaging of smaller spots. The NWRimage also provides the possibility of true sub-micron spot sizes. This work compares the capabilities of standard laser Ablation (NWR213 system) with results from the NWRimage platform, which has been optimized for imaging applications.
Inflammatory bowel diseases (IBD) comprise mainly ulcerative colitis (UC) and Crohn´s disease (CD). Both forms present with a chronic inflammation of the (gastro) intestinal tract, which induces excessive changes in the composition of the associated extracellular matrix (ECM). In UC, the inflammation is limited to the colon, whereas it can occur throughout the entire gastrointestinal tract in CD. Tools for early diagnosis of IBD are still very limited and highly invasive and measures for standardized evaluation of structural changes are scarce. To investigate an efficient non-invasive way of diagnosing intestinal inflammation and early changes of the ECM, very small superparamagnetic iron oxide nanoparticles (VSOPs) in magnetic resonance imaging (MRI) were applied in two mouse models of experimental colitis: the dextran sulfate sodium (DSS)-induced colitis and the transfer model of colitis. For further validation of ECM changes and inflammation, tissue sections were analyzed by immunohistochemistry. For in depth ex-vivo investigation of VSOPs localization within the tissue, Europium-doped VSOPs served to visualize the contrast agent by imaging mass cytometry (IMC). VSOPs accumulation in the inflamed colon wall of DSS-induced colitis mice was visualized in T2* weighted MRI scans. Components of the ECM, especially the hyaluronic acid content, were found to influence VSOPs binding. Using IMC, colocalization of VSOPs with macrophages and endothelial cells in colon tissue was shown. In contrast to the DSS model, colonic inflammation could not be visualized with VSOP-enhanced MRI in transfer colitis. VSOPs present a potential contrast agent for contrast-enhanced MRI to detect intestinal inflammation in mice at an early stage and in a less invasive manner depending on hyaluronic acid content.
Prostate cancer (PCa) is one of the most common cancers in men. For detection and diagnosis of PCa, non-invasive methods, including magnetic resonance imaging (MRI), can reduce the risk potential of surgical intervention. To explore the molecular characteristics of the tumor, we investigated the applicability of ferumoxytol in PCa in a xenograft mouse model in two different tumor volumes, 500 mm3 and 1000 mm3. Macrophages play a key role in tumor progression, and they are able to internalize iron-oxide particles, such as ferumoxytol. When evaluating T2*-weighted sequences on MRI, a significant decrease of signal intensity between pre- and post-contrast images for each tumor volume (n = 14; p < 0.001) was measured. We, furthermore, observed a higher signal loss for a tumor volume of 500 mm3 than for 1000 mm3. These findings were confirmed by histological examinations and laser ablation inductively coupled plasma-mass spectrometry. The 500 mm3 tumors had 1.5% iron content (n = 14; sigma = 1.1), while the 1000 mm3 tumors contained only 0.4% iron (n = 14; sigma = 0.2). In vivo MRI data demonstrated a correlation with the ex vivo data (R2 = 0.75). The results of elemental analysis by inductively coupled plasma-mass spectrometry correlated strongly with the MRI data (R2 = 0.83) (n = 4). Due to its long retention time in the blood, biodegradability, and low toxicity to patients, ferumoxytol has great potential as a contrast agent for visualization PCa.
The analytical potential of a nanosecond laser ablation inductively coupled plasma mass spectrometer (ns-LA-ICP-SFMS) system, equipped with an ultra-fast wash-out ablation chamber, is critically investigated for fast and highly spatially resolved (∼μm) qualitative elemental distribution within single cells. Initially, a low surface roughness (< 10 nm) thin In–SnO2 layer (total coating thickness ∼200 nm) deposited on glass is employed to investigate the size, morphology and overlapping of laser-induced craters obtained at different laser repetition rates, making use of Atomic Force Microscopy (AFM). Conical craters with a surface diameter of about 2 µm and depths of about 100 nm were measured after a single laser shot. Furthermore, the influence of the sampling distance (i.e. distance between the sample surface and the inner sniffer of the ablation chamber) on the LA-ICP-MS ion signal wash-out time is evaluated. A significant decrease of the transient 120Sn+ ion signal is noticed after slight variations (±200 μm) around the optimum sampling position. Ultra-fast wash-outs (< 10 ms) are achieved reducing the aerosol mixing from consecutive laser shots even when operating the laser at high repetition rates (25 – 100 Hz). Fast and highly spatially resolved images of elemental distribution within mouse embryonic fibroblast cells (NIH/3T3 fibroblast cells) and human cervical carcinoma cells (HeLa cells), incubated with gold nanoparticles (Au NPs) and Cd-based quantum dots (QDs), respectively, are determined at the optimized operating conditions. Elemental distribution of Au and Cd in single cells is achieved using a high scanning speed (50 µm/s) and high repetition rate (100 Hz). The results obtained for the distribution of fluorescent Cd-based QDs within the HeLa cells are in good agreement with those obtained by confocal microscopy. The size, morphology and overlapping of laser-induced craters in the fixed cells are also investigated using AFM, observing conical craters with a surface diameter of about 2.5 µm and depths of about 800 nm after a single laser shot.
Nanoparticles (NPs) have potential applications in medical diagnostics, imaging, drug delivery and other kinds of therapy. Furthermore, studies concerning nanoparticle uptake by cells are important for risk assessment. Size, shape and surface modification of the NPs determine the uptake rate and pathway into the cells, and therefore impact specific cell components and processes. Understanding the different uptake mechanisms and involved processes require sub-cellular resolution to determine, for example, whether the nanoparticles are reaching the nucleus. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is an established quantitative multi-elemental analysis and mapping technique. However, sub-cellular imaging has traditionally been challenging to achieve due to a lack of sensitivity at small laser spots. But now novel laser ablation systems with improved sensitivity and washout time allow imaging at high lateral resolution with spot sizes down to 1 µm.
Here LA-ICP-MS was applied for the imaging of individual fibroblast cells to study the uptake and intracellular processing of metal-containing NPs. To indicate cell morphology the local distribution of naturally occurring elements in cells like P and Zn was measured, too. Our results show that LA-ICP-MS can be used to localise nanoparticle aggregates within cellular compartments. The studied NPs accumulate in the perinuclear region in the course of intracellular processing, but do not enter the cell nucleus. The uptake efficiency depends strongly on the physico-chemical properties of the nanostructures as well as on the incubation conditions like concentration and incubation time. The potential of LA-ICP-MS for analysis at single cell level will be demonstrated.