Exosomes are cell‐derived nanovesicles with a proven intercellular signaling role in inflammation processes and immune response. Due to their natural origin and liposome‐like structure, these nanometer‐scale vesicles have emerged as novel platforms for therapy and diagnosis. In this work, goat milk exosomes are isolated and fully characterized in terms of their physicochemical properties, proteomics, and biochemical profile in healthy mice, and used to detect inflammatory processes by optical imaging. For the in vitro and in vivo experiments, the exosomes are covalently labeled with the commercial fluorophores sulfo‐Cyanine 5 and BODIPY‐FL to create nanoprobes. In vitro studies using confocal imaging, flow cytometry, and colorimetric assays confirm the internalization of the nanoprobes as well their lack of cytotoxicity in macrophage populations RAW 264.7. Optical imaging in the mouse peritoneal region confirms the in vivo ability of one of the nanoprobes to localize inflammatory processes. In vivo imaging shows exosome uptake in the inflamed peritoneal region, and flow‐cytometric analysis of peritonitis exudates confirms the uptake by macrophage and neutrophil populations. These results support the promising use of goat milk exosomes as natural probes in the detection of inflammatory processes.
The vertiginous increase in the use of extracellular vesicles and especially exosomes for therapeutic applications highlights the necessity of advanced techniques for gaining a deeper knowledge of their pharmacological properties. Herein, we report a novel chemical approach for the robust attachment of commercial fluorescent dyes to the exosome surface with covalent binding. The applicability of the methodology was tested on milk and cancer cell-derived exosomes (from U87 and B16F10 cancer cells). We demonstrated that fluorescent labeling did not modify the original physicochemical properties of exosomes. We tested this nanoprobe in cell cultures and healthy mice to validate its use for in vitro and in vivo applications. We confirmed that these fluorescently labeled exosomes could be successfully visualized with optical imaging.
Hollow organs such as the lungs pose a considerable challenge for post-mortem imaging in preclinical research owing to their extremely low contrast and high structural complexity. The aim of our study was to enhance the contrast of tuberculosis lesions for their stratification by 3D x-ray–based virtual slicing. Organ samples were taken from five control and five tuberculosis-infected mice. Micro-Computed Tomography (CT) scans of the subjects were acquired in vivo (without contrast agent) and post-mortem (with contrast agent). The proposed contrast-enhancing technique consists of x-ray contrast agent uptake (silver nitrate and iodine) by immersion. To create the histology ground-truth, the CT scan of the paraffin block guided the sectioning towards specific planes of interest. The digitalized histological slides reveal the presence, extent, and appearance of the contrast agents in lung structures and organized aggregates of immune cells. These findings correlate with the contrast-enhanced micro-CT slice. The abnormal densities in the lungs due to tuberculosis disease are concentrated in the right tail of the lung intensity histograms. The increase in the width of the right tail (~376%) indicates a contrast enhancement of the details of the abnormal densities. Postmortem contrast agents enhance the x-ray attenuation in tuberculosis lesions to allow 3D visualization by polychromatic x-ray CT, providing an advantageous tool for virtual slicing of whole lungs. The proposed contrast-enhancing technique combined with computational methods and the diverse micro-CT modalities will open the doors to the stratification of lesion types associated with infectious diseases.
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