Removal of cells from body fluids by magnetic separation in batch and continuous mode: influence of bead size, concentration, and contact time

The magnetic separation of pathogenic compounds from body fluids is an appealing therapeutic concept. Recently, removal of a diverse array of pathogens has been demonstrated using extracorporeal dialysis-type devices. The contact time between the fluid and the magnetic beads in such devices is limited to a few minutes. This poses challenges, particularly if large compounds such as bacteria or cells need to be removed. Here, we report on the feasibility to remove cells from body fluids in a continuous dialysis type of setting. We assessed tumor cell removal efficiencies from physiological fluids with or without white blood cells using a range of different magnetic bead sizes (50–4000 nm), concentrations, and contact times. We show that tumor cells can be quantitatively removed from body fluids within acceptable times (1– 2 min) and bead concentrations (0.2 mg per mL). We further present a mathematical model to describe the minimal bead number concentration needed to remove a certain number of cells, in the presence of competing nonspecific uptake. The present study paves the way for investigational studies to assess the therapeutic potential of cell removal by magnetic blood purification in a dialysis-like setting

Correlative cathodoluminescence electron microscopy bioimaging: towards single protein labelling with ultrastructural context

The understanding of living systems and their building blocks relies heavily on the assessment of structure–function relationships at the nanoscale. Ever since the development of the first optical microscope, the reliance of scientists across disciplines on microscopy has increased. The development of the first electron microscope and with it the access to information at the nanoscale has prompted numerous disruptive discoveries. While fluorescence imaging allows identification of specific entities based on the labelling with fluorophores, the unlabelled constituents of the samples remain invisible. In electron microscopy on the other hand, structures can be comprehensively visualized based on their distinct electron density and geometry. Although electron microscopy is a powerful tool, it does not implicitly provide information on the location and activity of specific organic molecules. While correlative light and electron microscopy techniques have attempted to unify the two modalities, the resolution mismatch between the two data sets poses major challenges. Recent developments in optical super resolution microscopy enable high resolution correlative light and electron microscopy, however, with considerable constraints due to sample preparation requirements. Labelling of specific structures directly for electron microscopy using small gold nanoparticles (i.e. immunogold) has been used extensively. However, identification of specific entities solely based on electron contrast, and the differentiation from endogenous dense granules, remains challenging. Recently, the use of correlative cathodoluminescence electron microscopy (CCLEM) imaging based on luminescent inorganic nanocrystals has been proposed. While nanometric resolution can be reached for both the electron and the optical signal, high energy electron beams are potentially damaging to the sample. In this review, we discuss the opportunities of (volumetric) multi-color single protein labelling based on correlative cathodoluminescence electron microscopy, and its prospective impact on biomedical research in general. We elaborate on the potential challenges of correlative cathodoluminescence electron microscopy-based bioimaging and benchmark CCLEM against alternative high-resolution correlative imaging techniques.ISSN:2040-3364ISSN:2040-337

Uptake, distribution and radio-enhancement effects of gold nanoparticles in tumor microtissues

Radiotherapy is an integral and highly effective part of cancer therapy, applicable in over 50% of patients affected by cancer. Due to the low specificity of the X-ray irradiation, the maximal radiation dose is greatly limited in order to avoid damage to surrounding healthy tissue. The limitations in applicable dose oftentimes result in the survival of a subpopulation of radio-resistant cells that then cause cancer reoccurence. Approaches based on tumor-targeted high atomic number inorganic nanoparticles have been proposed to locally increase the photoelectric absorption cross-section of tumors relative to healthy tissue. However, the complex interplay between the nanoparticle radio-enhancers and the tumor tissue has led to poor translation of in vitro findings to (pre)clinics. Here, we report the development of a tumor microtissue model along with analytical imaging for the quantitative assessment of nanoparticle-based radio-enhancement as a function of nanoparticle size, uptake and intratissural distribution. The advanced in vitro model exhibits key features of cancerous tissues, including diminished susceptibility to drugs and attenuated response to nanoparticle treatment compared to corresponding conventional 2D cell cultures. Whereas radio-enhancement effects between 2D and 3D cell cultures were comparable for 5 nm gold particles, the limited penetration of 50 nm gold nanoparticles into 3D microtissues led to a significantly reduced radio-enhancement effect in 3D compared to 2D. Taken together, tumor microtissues, which in stark contrast to 2D cell culture exhibit tissue-like features, may provide a valuable high-throughput intermediate pre-selection step in the preclinical translation of nanoparticle-based radio-enhancement therapy designs.ISSN:2516-023

Inorganic nanohybrids combat antibiotic-resistant bacteria hiding within human macrophages

Bacterial infections are one of the main health concerns humanity faces today and bacterial resistances and protection mechanisms are set to aggravate the issue in the coming years. An increasing number of bacterial strains evades antibiotic treatment by hiding inside cells. Conventional antimicrobial agents are unable to penetrate or be retained in the infected mammalian cells. Recent approaches to overcome these limitations have focused on load-carrier systems, requiring a triggered discharge leading to complex release kinetics. The unison of potent antimicrobial activity with high mammalian cell compatibility is a prerequisite for intracellular activity, which is not well-met by otherwise well-established inorganic systems, such as silver-based nanoparticles. In this work, load and carrier are combined into one functional inorganic nanoparticle system, which unites antimicrobial activity with mammalian cell compatibility. These multicomponent nanohybrids based on cerium oxide are produced in one step, yet unite complex materials. The nanoparticles form suprastructures of similar size and surface charge as bacteria, therefore facilitating the uptake into the same subcellular compartments, where they unleash their antibacterial effect. Such intrinsically antibacterial nanohybrids significantly reduce bacterial survival inside macrophages without harming the latter. Furthermore, blocking of nanoparticle endocytosis and subcellular electron microscopy elucidate the mechanism of action. Taken together, this work presents the first demonstration of antibacterial activity of ceria-based nanoparticles inside of mammalian cells and offers a route to straightforward and robust intracellular antibacterial agents that do not depend on payload delivery or biological constituents.ISSN:2040-3364ISSN:2040-337

Bi2O3 boosts brightness, biocompatibility and stability of Mn-doped Ba3(VO4)2 as NIR-II contrast agent

Deep-tissue fluorescence imaging remains a major challenge as there is limited availability of bright biocompatible materials with high photo- and chemical stability. Contrast agents with emission wavelengths above 1000 nm are most favorable for deep tissue imaging, offering deeper penetration and less scattering than those operating at shorter wavelengths. Organic fluorophores suffer from low stability while inorganic nanomaterials (e.g. quantum dots) are based typically on heavy metals raising toxicity concerns. Here, we report scalable flame aerosol synthesis of water-dispersible Ba3(VO4)2 nanoparticles doped with Mn5+ which exhibit a narrow emission band at 1180 nm upon near-infrared excitation. Their co-synthesis with Bi2O3 results in even higher absorption and ten-fold increased emission intensity. The addition of Bi2O3 also improved both chemical stability and cytocompatibility by an order of magnitude enabling imaging deep within tissue. Taken together, these bright particles offer excellent photo-, chemical and colloidal stability in various media with cytocompatibility to HeLa cells superior to existing commercial contrast agents.ISSN:2050-7518ISSN:2050-750