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Targeted delivery of functionalized PLGA nanoparticles to macrophages by complexation with the yeast Saccharomyces cerevisiae
Nanoparticles (NPs) are able to deliver a variety of substances into eukaryotic cells. However, their usage is often hampered by a lack of specificity, leading to the undesired uptake of NPs by virtually all cell types. In contrast to this, yeast is known to be specifically taken up into immune cells after entering the body. Therefore, we investigated the interaction of biodegradable surface-modified poly(lactic-co-glycolic acid) (PLGA) particles with yeast cells to overcome the unspecificity of the particulate carriers. Cells of different Saccharomyces cerevisiae strains were characterized regarding their interaction with PLGA-NPs under isotonic and hypotonic conditions. The particles were shown to efficiently interact with yeast cells leading to stable NP/yeast-complexes allowing to associate or even internalize compounds. Notably, applying those complexes to a coculture model of HeLa cells and macrophages, the macrophages were specifically targeted. This novel nano-in-micro carrier system suggests itself as a promising tool for the delivery of biologically active agents into phagocytic cells combining specificity and efficiency.
PDA Indolylmaleimides Induce Anti-Tumor Effects in Prostate Carcinoma Cell Lines Through Mitotic Death
Castrate resistant prostate cancer in men shares several characteristics with canine prostate cancer (PCa). Due to current insufficient therapies, evaluating novel therapeutic agents for late-stage PCa is of considerable interest for both species. PDA indolylmaleimides showed anticancer effects in several neoplastic cell lines. Herein, a comparative characterization of PDA-66 and PDA-377 mediated effects was performed in human and canine PCa cell lines, which is also the first detailed characterization of these agents on cells derived from solid tumors in general. While PDA-377 showed only weak growth inhibition on human PCa cell lines, PDA-66 inhibited proliferation and induced apoptosis in human and canine cell lines with concentrations in the low micromolar range. Morphological characterization and whole transcriptome sequencing revealed that PDA-66 induces mitotic death through its microtubule-depolymerizing ability. PDA-66 appears to be a worthwhile anti-mitotic agent for further evaluation. The similarities in cellular and molecular response observed in the cell lines of both origins form a solid basis for the use of canine PCa in vivo models to gain valuable interchangeable data to the advantage of both species.
Object detection networks and augmented reality for cellular detection in fluorescence microscopy
Object detection networks are high-performance algorithms famously applied to the task of identifying and localizing objects in photography images. We demonstrate their application for the classification and localization of cells in fluorescence microscopy by benchmarking four leading object detection algorithms across multiple challenging 2D microscopy datasets. Furthermore we develop and demonstrate an algorithm that can localize and image cells in 3D, in close to real time, at the microscope using widely available and inexpensive hardware. Furthermore, we exploit the fast processing of these networks and develop a simple and effective augmented reality (AR) system for fluorescence microscopy systems using a display screen and back-projection onto the eyepiece. We show that it is possible to achieve very high classification accuracy using datasets with as few as 26 images present. Using our approach, it is possible for relatively nonskilled users to automate detection of cell classes with a variety of appearances and enable new avenues for automation of fluorescence microscopy acquisition pipelines. © 2020 Waithe et al.
Exogenous supply of Hsp47 triggers fibrillar collagen deposition in skin cell cultures in vitro
Background: Collagen is a structural protein that provides mechanical stability and defined architectures to skin. In collagen-based skin disorders this stability is lost, either due to mutations in collagens or in the chaperones involved in collagen assembly. This leads to chronic wounds, skin fragility, and blistering. Existing approaches to treat such conditions rely on administration of small molecules to simulate collagen production, like 4-phenylbutyrate (4-PBA) or growth factors like TGF-β. However, these molecules are not specific for collagen synthesis, and result in unsolicited side effects. Hsp47 is a collagen-specific chaperone with a major role in collagen biosynthesis. Expression levels of Hsp47 correlate with collagen deposition. This article explores the stimulation of collagen deposition by exogenously supplied Hsp47 (collagen specific chaperone) to skin cells, including specific collagen subtypes quantification. Results: Here we quantify the collagen deposition level and the types of deposited collagens after Hsp47 stimulation in different in vitro cultures of cells from human skin tissue (fibroblasts NHDF, keratinocytes HaCat and endothelial cells HDMEC) and mouse fibroblasts (L929 and MEF). We find upregulated deposition of fibrillar collagen subtypes I, III and V after Hsp47 delivery. Network collagen IV deposition was enhanced in HaCat and HDMECs, while fibril-associated collagen XII was not affected by the increased intracellular Hsp47 levels. The deposition levels of fibrillar collagen were cell-dependent i.e. Hsp47-stimulated fibroblasts deposited significantly higher amount of fibrillar collagen than Hsp47-stimulated HaCat and HDMECs. Conclusions: A 3-fold enhancement of collagen deposition was observed in fibroblasts upon repeated dosage of Hsp47 within the first 6 days of culture. Our results provide fundamental understanding towards the idea of using Hsp47 as therapeutic protein to treat collagen disorders.