Micrometer-sized iron oxide particles (MPIOs) attract increasing interest as contrast agents for cellular tracking by clinical Magnetic Resonance Imaging (MRI). Despite the great potential of MPIOs for in vivo imaging of labeled cells, little is known on the intracellular localization of these particles following uptake due to the lack of techniques with the ability to monitor the particle uptake in vivo at single-cell level. Here, we show that coherent anti-Stokes Raman scattering (CARS) microscopy enables non-invasive, label-free imaging of MPIOs in living cells with sub-micron resolution in three dimensions. CARS allows simultaneous visualization of the cell framework and the MPIOs, where the particles can be readily distinguished from other cellular components of comparable dimensions, and localized inside the cell.

Cellular therapies require methods for noninvasive visualization of transplanted cells. Micron-sized iron oxide particles (MPIOs) generate a strong contrast in magnetic resonance imaging (MRI) and are therefore ideally suited as an intracellular contrast agent to image cells under clinical conditions. However, MPIOs were previously not applicable for clinical use. Here, we present the development and evaluation of silica-based micron-sized iron oxide particles (sMPIOs) with a functionalizable particle surface. Particles with magnetite content of >40% were composed using the sol-gel process. The particle surfaces were covered with COOH groups. Fluorescein, poly-llysine (PLL), and streptavidin (SA) were covalently attached. Monodisperse sMPIOs had an average size of 1.18 µm and an iron content of about 1.0 pg Fe/particle. Particle uptake, toxicity, and imaging studies were performed using HuH7 cells and human and rat hepatocytes. sMPIOs enabled rapid cellular labeling within 4 h of incubation; PLL-modified particles had the highest uptake. In T2*-weighted 3.0 T MRI, the detection threshold in agarose was 1,000 labeled cells, whereas in T1-weighted LAVA sequences, at least 10,000 cells were necessary to induce sufficient contrast. Labeling was stable and had no adverse effects on labeled cells. Silica is a biocompatible material that has been approved for clinical use. sMPIOs could therefore be suitable for future clinical applications in cellular MRI, especially in settings that require strong cellular contrast. Moreover, the particle surface provides the opportunity to create multifunctional particles for targeted delivery and diagnostics.

Adrenocortical carcinoma (ACC) is a heterogeneous malignancy related to poor prognosis and limited treatment options. The orphan drug mitotane (MT) is still a cornerstone in ACC therapy, however, its application is characterized by low aqueous solubility, poor bioavailability, and unfavorable pharmacokinetics, often resulting in below-target plasma concentrations or toxic side effects. Throughout the last decades, nanoparticulate formulations have become attractive carriers to improve anticancer therapy. In this study, injectable MT liposomes (DOPC-MT) and albumin-stabilized MT nanoparticles (BSA-MT) were investigated in depth with respect to their physicochemical properties, and their colloidal and therapeutical stability upon storage. Furthermore, in vitro cytotoxicity was evaluated using the ACC model cell line NCI-H295R for preparing multicellular tumor spheroids, and was compared to non-malignant human dermal fibroblasts. Our results clearly demonstrate that BSA-MT, unlike DOPC-MT, represents a stable and storable MT formulation with a high drug concentration in an aqueous medium. Dual centrifugation was established as a reproducible method for nanoparticle preparation. Although an efficient cytotoxic effect on ACC tumor spheroids was demonstrated, concomitant low toxicity to fibroblasts suggests that higher drug concentrations may be tolerated in vivo. Consequently, BSA-MT is a novel and promising therapeutical approach to address key challenges in MT treatment.

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a b s t r a c tGlass fogging is a phenomenon occurring in lyophilized drug products and can be described as a thin product layer deposited on the inner surface of the glass container, in the area not covered by the lyo cake itself. It is often considered a cosmetic defect; however, the loss of container closure integrity is a potential consequence of the fogging's expansion to the vial neck region, making this a potential critical defect. Thus, a method for predicting the extent of vial fogging before the actual freeze-drying is of particular interest for the pharmaceutical industry. For that reason, we evaluated a simple method ("simulated fogging") applicable to drug product formulations in a specific container closure system. Two different vial types with different surface hydrophilicity were tested using 3 model protein formulations, comparing the simulated fogging test and the degree of fogging after actual lyophilization. The simulated fogging method could predict fogging and showed a correlation to fogging in lyophilized drug product glass vials. We observed that all formulations showed fogging in the hydrophilic vials. By contrast, hydrophobic vials prevented fogging, however, interestingly with remaining defects of so-called droplet formation. Other than extent of fogging, no additional differences of lyophilized cake properties or other product quality attributes were observed between products using the different glass vial types tested.