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The exact role of malignant ascites in the development of intraperitoneal metastases remains unclear, and the mechanisms by which extracellular vesicles (EVs) promote tumor progression in the pre‐metastatic niche have not been fully discovered. In this study, we characterized ascites from high‐grade epithelial ovarian cancer patients. Small‐EVs (30–150 nm) were isolated from two sources—the bulk ascites and the ascitic fluid‐derived tumor cell cultures—and assessed with a combination of imaging, proteomic profiling, and protein expression analyses. In addition, Gene Ontology and pathway analysis were performed using different databases and bioinformatic tools. The results proved that the small‐EVs derived from the two sources exhibited significantly different stiffness and size distributions. The bulk ascitic fluid‐derived small‐EVs were predominantly involved in the complement and coagulation cascade. Small‐EVs derived from ascites cell cultures contained a robust proteomic profile of extracellular matrix remodeling regulators, and we observed an increase in transforming growth factor‐β‐I (TGFβI), plasminogen activator inhibitor 1 (PAI‐1), and fibronectin expression after neoadjuvant chemotherapy. When measured in the two sources, we demonstrated that fibronectin exhibited opposite expression patterns in small‐EVs in response to chemotherapy. These findings highlight the importance of an ascites cell isolation workflow in investigating the treatment‐induced cancer adaption processes., The mechanisms by which extracellular vesicles (EVs) promote ovarian cancer progression in the pre‐metastatic niche have not been fully discovered. This study showed that sampling ascites before and after chemotherapy allows characterizing small‐EVs released by tumor cells, reflecting their biological status. Small‐EVs derived from ascitic fluid‐derived tumor cell cultures contained a robust proteomic profile of extracellular matrix remodeling regulators.

Lyotropic liquid crystalline nanoparticles with bicontinuous cubic internal nanostructure, known as cubosomes, have been proposed as nanocarriers in various medical applications. However, as these nanoparticles show a certain degree of cytotoxicity, particularly against erythrocytes, their application in systemic administrations is limited to date. Intending to produce a more biocompatible formulation, we prepared cubosomes for the first time stabilized with a biodegradable polyphosphoester-analog of the commonly used Pluronic F127. The ABA-triblock copolymer poly(methyl ethylene phosphate)-block-poly(propylene oxide)-block-poly(methyl ethylene phosphate) (PMEP-b-PPO-b-PMEP) was prepared by organocatalyzed ring-opening polymerization of MEP. The cytotoxic features of the resulting formulation were investigated against two different cell lines (HEK-293 and HUVEC) and human red blood cells. The response of the complement system was also evaluated. Results proved the poly(phosphoester)-based formulation was significantly less toxic than that prepared using Pluronic F127 with respect to all the tested cell lines and, more importantly, hemolysis assay and complement system activation tests demonstrated its very high hemocompatibility. The potentially biodegradable poly(phosphoester)-based cubosomes represent a new and versatile platform for preparation of functional and smart nanocarriers.

Chimerism status evaluation of post-allogeneic hematopoietic stem cell transplantation samples is essential to predict post-transplant relapse. The most commonly used technique capable of detecting small increments of chimerism is quantitative real-time PCR. Although this method is already used in several laboratories, previously described protocols often lack sensitivity and the amount of the DNA required for each chimerism analysis is too high. In the present study, we compared a novel semi-nested allele-specific real-time PCR (sNAS-qPCR) protocol with our in-house standard allele-specific real-time PCR (gAS-qPCR) protocol. We selected two genetic markers and analyzed technical parameters (slope, y-intercept, R2, and standard deviation) useful to determine the performances of the two protocols. The sNAS-qPCR protocol showed better sensitivity and precision. Moreover, the sNAS-qPCR protocol requires, as input, only 10 ng of DNA, which is at least 10-fold less than the gAS-qPCR protocols described in the literature. Finally, the proposed sNAS-qPCR protocol could prove very useful for performing chimerism analysis with a small amount of DNA, as in the case of blood cell subsets.

Lorella Pascolo,1 Barbara Bortot,1 Nuria Benseny-Cases,2 Alessandra Gianoncelli,3 Giovanni Tosi,4 Barbara Ruozi,4 Clara Rizzardi,5 Eleonora De Martino,1 Maria Angela Vandelli,4 Giovanni Maria Severini11Institute for Maternal and Child Health, Istituto di Ricovero e Cura a Carattere Scientifico Burlo Garofolo, Trieste, Italy; 2European Synchrotron Radiation Facility, Polygone Scientifique Louis Néel, Grenoble, France; 3Elettra-Sincrotrone Trieste, Area Science Park, Basovizza, Trieste, Italy; 4Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 5Department of Anatomical Pathology, Department of Pathology and Forensic Medicine, University of Trieste, Trieste, ItalyAbstract: Poly-lactide-co-glycolide (PLGA) is one of the few polymers approved by the US Food and Drug Administration as a carrier for drug administration in humans; therefore, it is one of the most used materials in the formulation of polymeric nanoparticles (NPs) for therapeutic purposes. Because the cellular uptake of polymeric NPs is a hot topic in the nanomedicine field, the development of techniques able to ensure incontrovertible evidence of the presence of NPs in the cells plays a key role in gaining understanding of their therapeutic potential. On the strength of this premise, this article aims to evaluate the application of synchrotron radiation-based Fourier transform infrared spectroscopy (SR-FTIR) spectromicroscopy and SR X-ray fluorescence (SR-XRF) microscopy in the study of the in vitro interaction of PLGA NPs with cells. To reach this goal, we used PLGA NPs, sized around200nm and loaded with superparamagnetic iron oxide NPs (PLGA-IO-NPs; Fe3O4; size,10–15nm). After exposing human mesothelial (MeT5A) cells to PLGA-IO-NPs (0.1mg/mL), the cells were analyzed after fixation both by SR-FTIR spectromicroscopy and SR-XRF microscopy setups. SR-FTIR-SM enabled the detection of PLGA NPs at single-cell level, allowing polymer detection inside the biological matrix by the characteristic band in the1,700–2,000cm-1region. The precise PLGA IR-signature (1,750cm-1centered pick) also was clearly evident within an area of high amide density. SR-XRF microscopy performed on the same cells investigated under SR-FTIR microscopy allowed us to put in evidence the Fe presence in the cells and to emphasize the intracellular localization of the PLGA-IO-NPs. These findings suggest that SR-FTIR and SR-XRF techniques could be two valuable tools to follow the PLGA NPs’ fate in in vitro studies on cell cultures.Keywords: PLGA-NPs, cell targeting, SR-FTIR, SR-XRF, imaging