AimTo carry out a systematic study on the effect of different storage conditions on the number as well as the physical and functional properties of antibacterial extracellular vesicles (EVs) derived from human neutrophilic granulocytes.MethodsProduction of EVs with antibacterial properties was initiated by opsonized Zymosan A particles. The number of released fluorescent EVs was determined by flow cytometry following careful calibration. Physical properties and size of EVs were investigated by flow cytometry, dynamic light scattering and electron microscopy. Functional properties of EVs were tested by bacterial survival assay.ResultsStorage at +20°C or +4°C resulted in a significant decrease of EV number and antibacterial effect after 1 day. Storage at −20°C did not influence the EV number up to 28 days, but induced a shift in EV size and almost complete loss of antibacterial function by 28 days. Storage at −80°C had no significant effect either on EV number or size and allowed partial preservation of the antibacterial function up to 28 days. Snap-freezing did not improve the results, whereas the widely used cryoprotectants induced EV lysis.ConclusionStorage significantly alters both the physical and functional properties of EVs even if the number of EVs stays constant. If storage is needed, EVs should be kept at −80°C, preferably not longer than 7 days. For functional tests, freshly prepared EVs are recommended.

Translational research is changing the practice of modern medicine and the way in which health problems are approached and solved. The use of small-animal models in basic and preclinical sciences is a major keystone for these kinds of research and development strategies, representing a bridge between discoveries at the molecular level and clinical implementation in diagnostics and/or therapeutics. The development of high-resolution in vivo imaging technologies provides a unique opportunity for studying disease in real time, in a quantitative way, at the molecular level, along with the ability to repeatedly and non-invasively monitor disease progression or response to treatment. The greatest advantages of preclinical imaging techniques include the reduction of biological variability and the opportunity to acquire, in continuity, an impressive amount of unique information (without interfering with the biological process under study) in distinct forms, repeated or modulated as needed, along with the substantial reduction in the number of animals required for a particular study, fully complying with 3R (Replacement, Reduction and Refinement) policies. The most suitable modalities for small-animal in vivo imaging applications are based on nuclear medicine techniques (essentially, positron emission tomography [PET] and single photon emission computed tomography [SPECT]), optical imaging (OI), computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance spectroscopy imaging (MRSI), and ultrasound. Each modality has intrinsic advantages and limitations. More recently, aiming to overcome the inherent limitations of each imaging modality, multimodality devices designed to provide complementary information upon the pathophysiological process under study have gained popularity. The combination of high-resolution modalities, like micro-CT or micro-MRI, with highly sensitive techniques providing functional information, such as micro-PET or micro-SPECT, will continue to broaden the horizons of research in such key areas as infection, oncology, cardiology, and neurology, contributing not only to the understanding of the underlying mechanisms of disease, but also providing efficient and unique tools for evaluating new chemical entities and candidate drugs. The added value of small-animal imaging techniques has driven their increasing use by pharmaceutical companies, contract research organizations, and research institutions.

Multimodal nanoparticulate materials are described, offering magnetic, radionuclide, and fluorescent imaging capabilities to exploit the complementary advantages of magnetic resonance imaging (MRI), positron emission tomography/single-photon emission commuted tomography (PET/SPECT), and optical imaging. They comprise Fe3O4@NaYF4 core/shell nanoparticles (NPs) with different cation dopants in the shell or core, including Co0.16Fe2.84O4@NaYF4(Yb, Er) and Fe3O4@NaYF4(Yb, Tm). These NPs are stabilized by bisphosphonate polyethylene glycol conjugates (BP-PEG), and then show a high transverse relaxivity (r2) up to 326 mM–1 s–1 at 3T, a high affinity to [18F]-fluoride or radiometal-bisphosphonate conjugates (e.g., 64Cu and 99mTc), and fluorescent emissions from 500 to 800 nm under excitation at 980 nm. The biodistribution of intravenously administered particles determined by PET/MR imaging suggests that negatively charged Co0.16Fe2.84O4@NaYF4(Yb, Er)-BP-PEG (10K) NPs cleared from the blood pool more slowly than positively charged NPs Fe3O4@NaYF4(Yb, Tm)-BP-PEG (2K). Preliminary results in sentinel lymph node imaging in mice indicate the advantages of multimodal imaging.

Extracellular vesicles (EVs) are important elements of intercellular communication. A plethora of different, occasionally even opposite, physiologic and pathologic effects have been attributed to these vesicles in the last decade. A direct comparison of individual observations is however hampered by the significant differences in the way of elicitation, collection, handling, and storage of the investigated vesicles. In the current work, we carried out a careful comparative study on 3, previously characterized types of EVs produced by neutrophilic granulocytes. We investigated in parallel the modulation of multiple blood-related cells and functions by medium-sized vesicles. We show that EVs released from resting neutrophils exert anti-inflammatory action by reducing production of reactive oxygen species (ROS) and cytokine release from neutrophils. In contrast, vesicles generated upon encounter of neutrophils with opsonized particles rather promote proinflammatory processes as they increase production of ROS and cytokine secretion from neutrophils and activate endothelial cells. EVs released from apoptosing cells were mainly active in promoting coagulation. We thus propose that EVs are "custom made," acquiring selective capacities depending on environmental factors prevailing at the time of their biogenesis.