An Image Processing Algorithm for Facile and Reproducible Quantification of Vomocytosis.

Vomocytosis is a process that occurs when internalized fungal pathogens escape from phagocytes without compromising the viability of the pathogen and the host cell. Manual quantification of time-lapse microscopy videos is currently used as the standard to study pathogen behavior and vomocytosis incidence. However, human-driven quantification of vomocytosis (and the closely related phenomenon, exocytosis) is incredibly burdensome, especially when a large volume of cells and interactions needs to be analyzed. In this study, we designed a MATLAB algorithm that measures the extent of colocalization between the phagocyte and fungal cell (Cryptococcus neoformans; CN) and rapidly reports the occurrence of vomocytosis in a high throughput manner. Our code processes multichannel, time-lapse microscopy videos of cocultured CN and immune cells that have each been fluorescently stained with unique dyes and provides quantitative readouts of the spatiotemporally dynamic process that is vomocytosis. This study also explored metrics, such as the rate of change of pathogen colocalization with the host cell, that could potentially be used to predict vomocytosis occurrence based on the quantitative data collected. Ultimately, the algorithm quantifies vomocytosis events and reduces the time for video analysis from over 1 h to just 10 min, a reduction in labor of 83%, while simultaneously minimizing human error. This tool significantly minimizes the vomocytosis analysis pipeline, accelerates our ability to elucidate unstudied aspects of this phenomenon, and expedites our ability to characterize CN strains for the study of their epidemiology and virulence.

Vomocytosis of Cryptococcus neoformans cells from murine, bone marrow-derived dendritic cells.

Cryptococcus neoformans (CN) cells survive within the acidic phagolysosome of macrophages (MΦ) for extended times, then escape without impacting the viability of the host cell via a phenomenon that has been coined 'vomocytosis'. Through this mechanism, CN disseminate throughout the body, sometimes resulting in a potentially fatal condition-Cryptococcal Meningitis (CM). Justifiably, vomocytosis studies have focused primarily on MΦ, as alveolar MΦ within the lung act as first responders that ultimately expel this fungal pathogen. Herein, we hypothesize that dendritic cells (DCs), an innate immune cell with attributes that include phagocytosis and antigen presentation, can also act as 'vomocytes'. Presciently, this report shows that vomocytosis of CN indeed occurs from murine, bone marrow-derived DCs. Primarily through time-lapse microscopy imaging, we show that rates of vomocytosis events from DCs are comparable to those seen from MΦ and further, are independent of the presence of the CN capsule and infection ratios. Moreover, the phagosome-altering drug bafilomycin A inhibits this phenomenon from DCs. Although DC immunophenotype does not affect the total number of vomocytic events, we observed differences in the numbers of CN per phagosome and expulsion times. Interestingly, these observations were similar in murine, bone marrow-derived MΦ. This work not only demonstrates the vomocytic ability of DCs, but also investigates the complexity of vomocytosis regulation in this cell type and MΦ under multiple modulatory conditions. Understanding the vomocytic behavior of different phagocytes and their phenotypic subtypes is needed to help elucidate the full picture of the dynamic interplay between CN and the immune system. Critically, deeper insight into vomocytosis could reveal novel approaches to treat CM, as well as other immune-related conditions.

The roadmap to micro: Generation of micron-sized polymeric particles using a commercial microfluidic system.

Microfluidic-assisted particle fabrication provides a route to circumvent the disadvantages associated with traditional methods of polymeric particle generation, such as low drug loading efficiency, challenges in controlling encapsulated drug release rates, batch-to-batch variability in particle physical properties and formulation instability. However, this approach primarily produces particles with nanometer size dimensions, which limits drug delivery modalities. Herein, we systematically studied parameters for the generation of micron-sized poly(lactic-co-glycolic) acid (PLGA) particles using a microfluidic system, the NanoAssemblr benchtop. Initially, we used two organic solvents that have been reported suitable for the fabrication of PLGA nanoparticles - acetone and acetonitrile. Subsequently, we methodically manipulated polymer concentration, organic: aqueous flow rate ratios, total flow rate, organic phase composition, and surfactant concentration to develop a route for the fabrication of micron-sized PLGA particles. Further, we incorporated hydroxychloroquine (HCQ), a clinically approved drug for malaria and lymphoma, and measured how its incorporation impacted particle physicochemical properties. Briefly, altering the organic phase composition by including ethyl acetate (less polar solvent), resulted in micron-scale particles, as well as increased polydispersity indexes (PDIs). Adjusting the surfactant concentration of poly vinyl alcohol (PVA) after the addition of these solvent mixtures rendered large particles with lower PDI variability. Moreover, encapsulation of HCQ influenced particle hydrodynamic diameter and PDI in a PVA concentration dependent manner. Finally, we demonstrated that unloaded and HCQ-loaded microparticles did not affect the viability of RAW 264.7 macrophages. This study provides an itinerary for fabricating biocompatible, drug-loaded, micron-sized polymeric particles, particularly when the drug of interest is not readily soluble in conventional organic solvents.

Vomocytosis: Too Much Booze, Base, or Calcium?

Macrophages are well known for their phagocytic activity and their role in innate immune responses. Macrophages eat non-self particles, via a variety of mechanisms, and typically break down internalized cargo into small macromolecules. However, some pathogenic agents have the ability to evade this endosomal degradation through a nonlytic exocytosis process termed vomocytosis. This phenomenon has been most often studied for Cryptococcus neoformans, a yeast that causes roughly 180,000 deaths per year, primarily in immunocompromised (e.g., human immunodeficiency virus [HIV]) patients. Existing dogma purports that vomocytosis involves distinctive cellular pathways and intracellular physicochemical cues in the host cell during phagosomal maturation. Moreover, it has been observed that the immunological state of the individual and macrophage phenotype affect vomocytosis outcomes. Here we compile the current knowledge on the factors (with respect to the phagocytic cell) that promote vomocytosis of C. neoformans from macrophages.

Magnetic nanoparticles loaded with functional RNA nanoparticles.

RNA is now widely acknowledged not only as a multifunctional biopolymer but also as a dynamic material for constructing nanostructures with various biological functions. Programmable RNA nanoparticles (NPs) allow precise control over their formulation and activation of multiple functionalities, with the potential to self-assemble in biological systems. These attributes make them attractive for drug delivery and therapeutic applications. In the present study, we demonstrate the ability of iron oxide magnetic nanoparticles (MNPs) to deliver different types of RNA NPs functionalized with dicer substrate RNAs inside human cells. Our results show that use of functionalized RNA NPs result in statistically higher transfection efficiency compared to the use of RNA duplexes. Furthermore, we show that the nucleic acids in the MNP/RNA NP complexes are protected from nuclease degradation and that they can achieve knockdown of target protein expression, which is amplified by magnetic stimulus. The current work represents the very first report indicating that iron oxide nanoparticles may efficiently protect and deliver programmable RNA NPs to human cells.

Alternating current (AC) susceptibility as a particle-focused probe of coating and clustering behaviour in magnetic nanoparticle suspensions.

HYPOTHESIS: The functionality of magnetic nanoparticles (MNPs) relies heavily on their surface coating, which in turn affects the interactions between MNPs, and the formation of single-core particles or multi-core clusters. In this study we assessed the use of AC susceptibility (ACS) as a magnetic probe of the kinetics of coating and agglomeration of functionalised nanoparticles. We demonstrate the precision and sensitivity of ACS measurements to small changes in MNP coating using arginine-glycine-aspartic acid (RGD) tripeptide binding, and subsequently discuss how ACS can be used to optimise the preparation of polyethyleneimine (PEI) functionalised MNPs aimed at nanomagnetic transfection applications. EXPERIMENTS: We varied the PEI loading of suspensions of MNPs exhibiting a combination of Brownian and Néel relaxation, and used dialysis to study the movement of excess PEI during the coating process. Numerical ACS simulations were employed to determine particle cluster sizes and polydispersity and the results compared with conventional dynamic light scattering (DLS) size measurements. FINDINGS: ACS provided information on the MNP coating and agglomeration process that was not accessible through DLS due to the additional presence of non-magnetic polymer particulates in the suspensions. We consequently derived a simple method to obtain dense, uniform PEI coatings affording high-stability suspensions without excessive quantities of unbound PEI.