Gold Nanocluster Extracellular Vesicle Supraparticles: Self-Assembled Nanostructures for Three-Dimensional Uptake Visualization.

Extracellular vesicles (EVs) are secreted by the vast majority of cells and are being intensively studied due to their emerging involvement in a variety of cellular communication processes. However, the study of their cellular uptake and fate has been hampered by difficulty in imaging EVs against the cellular background. Here, we show that EVs combined with hydrophobic gold nanoclusters (AuNCs) can self-assemble into supraparticles, offering an excellent labeling strategy for high-resolution electron microscopic imaging in vitro. We have tracked and visualized the reuptake of breast cancer cell-derived EV AuNC supraparticles into their parent cells, from early endocytosis to lysosomal degradation, using focused ion beam-scanning electron microscopy (FIB-SEM). The presence of gold within the EVs and lysosomes was confirmed via DF-STEM EDX analysis of lift-out sections. The demonstrated formation of AuNC EV supraparticles will facilitate future applications in EV imaging as well as the EV-assisted cellular delivery of AuNCs.

Prognostic implications of post-stress ejection fraction decrease detected by gated SPECT in the absence of stress-induced perfusion abnormalities.

PURPOSE: The prognostic meaning of a post-stress ejection fraction (EF) decrease detected by perfusion gated SPECT is still unclear.We therefore followed up patients with post-stress EF decrease in the absence of stress-induced perfusion abnormalities. METHODS: We prospectively enrolled 57 consecutive patients with post-stress EF drop ≥ 5 EF units and summed difference score (SDS) ≤ 1. They were followed up for more than 1 year and their outcome was compared with a group of sex- and age-matched controls with the same SDS but without EF decrease. RESULTS: During follow-up there were 13 events (1 cardiac death, 1 non-fatal myocardial infarction, 1 congestive heart failure and 10 late revascularizations). In the control group we registered six events. There was a significant difference (p<0.0001) between the event-free survival curves of the two groups. CONCLUSION: The event rate of patients with post-stress EF decrease ≥ 5 EF units is relatively high and is significantly worse than that of a control group of patients with similarly normal SDS but without EF changes. Therefore, a post-stress EF decrease without stress-induced perfusion abnormalities should be cautiously interpreted.

Tuneable peptide cross-linked nanogels for enzyme-triggered protein delivery.

Many diseases are associated with the dysregulated activity of enzymes, such as matrix metalloproteinases (MMPs). This dysregulation can be leveraged in drug delivery to achieve disease- or site-specific cargo release. Self-assembled polymeric nanoparticles are versatile drug carrier materials due to the accessible diversity of polymer chemistry. However, efficient loading of sensitive cargo, such as proteins, and introducing functional enzyme-responsive behaviour remain challenging. Herein, peptide-crosslinked, temperature-sensitive nanogels for protein delivery were designed to respond to MMP-7, which is overexpressed in many pathologies including cancer and inflammatory diseases. The incorporation of N-cyclopropylacrylamide (NCPAM) into N-isopropylacrylamide (NIPAM)-based copolymers enabled us to tune the polymer lower critical solution temperature from 33 to 44 °C, allowing the encapsulation of protein cargo and nanogel-crosslinking at slightly elevated temperatures. This approach resulted in nanogels that were held together by MMP-sensitive peptides for enzyme-specific protein delivery. We employed a combination of cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), small angle neutron scattering (SANS), and fluorescence correlation spectroscopy (FCS) to precisely decipher the morphology, self-assembly mechanism, enzyme-responsiveness, and model protein loading/release properties of our nanogel platform. Simple variation of the peptide linker sequence and combining multiple different crosslinkers will enable us to adjust our platform to target specific diseases in the future.

Controlled Dendrimersome Nanoreactor System for Localized Hypochlorite-Induced Killing of Bacteria.

Antibiotic resistance is a serious global health problem necessitating new bactericidal approaches such as nanomedicines. Dendrimersomes (DSs) have recently become a valuable alternative nanocarrier to polymersomes and liposomes due to their molecular definition and synthetic versatility. Despite this, their biomedical application is still in its infancy. Inspired by the localized antimicrobial function of neutrophil phagosomes and the versatility of DSs, a simple three-component DS-based nanoreactor with broad-spectrum bactericidal activity is presented. This was achieved by encapsulation of glucose oxidase (GOX) and myeloperoxidase (MPO) within DSs (GOX-MPO-DSs), self-assembled from an amphiphilic Janus dendrimer, that possesses a semipermeable membrane. By external addition of glucose to GOX-MPO-DS, the production of hypochlorite (-OCl), a highly potent antimicrobial, by the enzymatic cascade was demonstrated. This cascade nanoreactor yielded a potent bactericidal effect against two important multidrug resistant pathogens, Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), not observed for H2O2 producing nanoreactors, GOX-DS. The production of highly reactive species such as -OCl represents a harsh bactericidal approach that could also be cytotoxic to mammalian cells. This necessitates the development of strategies for activating -OCl production in a localized manner in response to a bacterial stimulus. One option of locally releasing sufficient amounts of substrate using a bacterial trigger (released toxins) was demonstrated with lipidic glucose-loaded giant unilamellar vesicles (GUVs), envisioning, e.g., implant surface modification with nanoreactors and GUVs for localized production of bactericidal agents in the presence of bacterial growth.

Molecular imaging of extracellular vesicles in vitro via Raman metabolic labelling.

Extracellular vesicles (EVs) are biologically-derived nanovectors important for intercellular communication and trafficking. As such, EVs show great promise as disease biomarkers and therapeutic drug delivery vehicles. However, despite the rapidly growing interest in EVs, understanding of the biological mechanisms that govern their biogenesis, secretion, and uptake remains poor. Advances in this field have been hampered by both the complex biological origins of EVs, which make them difficult to isolate and identify, and a lack of suitable imaging techniques to properly study their diverse biological roles. Here, we present a new strategy for simultaneous quantitative in vitro imaging and molecular characterisation of EVs in 2D and 3D based on Raman spectroscopy and metabolic labelling. Deuterium, in the form of deuterium oxide (D2O), deuterated choline chloride (d-Chol), or deuterated d-glucose (d-Gluc), is metabolically incorporated into EVs through the growth of parent cells on medium containing one of these compounds. Isolated EVs are thus labelled with deuterium, which acts as a bio-orthogonal Raman-active tag for direct Raman identification of EVs when introduced to unlabelled cell cultures. Metabolic deuterium incorporation demonstrates no apparent adverse effects on EV secretion, marker expression, morphology, or global composition, indicating its capacity for minimally obstructive EV labelling. As such, our metabolic labelling strategy could provide integral insights into EV biocomposition and trafficking. This approach has the potential to enable a deeper understanding of many of the biological mechanisms underpinning EVs, with profound implications for the design of EVs as therapeutic delivery vectors and applications as disease biomarkers.

Remote Magnetic Nanoparticle Manipulation Enables the Dynamic Patterning of Cardiac Tissues.

The ability to manipulate cellular organization within soft materials has important potential in biomedicine and regenerative medicine; however, it often requires complex fabrication procedures. Here, a simple, cost-effective, and one-step approach that enables the control of cell orientation within 3D collagen hydrogels is developed to dynamically create various tailored microstructures of cardiac tissues. This is achieved by incorporating iron oxide nanoparticles into human cardiomyocytes and applying a short-term external magnetic field to orient the cells along the applied field to impart different shapes without any mechanical support. The patterned constructs are viable and functional, can be detected by T2 *-weighted magnetic resonance imaging, and induce no alteration to normal cardiac function after grafting onto rat hearts. This strategy paves the way to creating customized, macroscale, 3D tissue constructs with various cell-types for therapeutic and bioengineering applications, as well as providing powerful models for investigating tissue behavior.

The prognostic role of end of treatment FDG-PET-CT in patients with diffuse large B cell lymphoma can be improved by considering it with absolute monocyte count at diagnosis.

It is well established that some patients with diffuse large B-cell lymphoma (DLBCL) and the negative end of treatment PET-CT (EOT-PET-CT) will relapse, while a proportion with positive uptake can still obtain long-term EFS. We reviewed data of 200 consecutive, previously untreated patients with DLBCL recorded in Italy and Israel between 2007 and 2015. We found that patients with negative EOT-PET-CT with AMC > 630/mmc have a 3-years EFS of 72%, compared to those with AMC ≤ 630/mmc that have an EFS of 84%. Furthermore, considering patients with positive EOT-PET-CT, those with AMC > 630/mmc have a 3-years EFS of 8%, while those with AMC ≤ 630/mmc have an EFS of 38%. Thus, it appears that combining the gold standard for response evaluation EOT-PET-CT with a simple and inexpensive parameter like AMC at diagnosis, further improves prognostication in DLBCL. Applying this simple method can be useful for all doctors working in lymphoma clinical practice.

Enantioselective Synthesis of Spiro[indolizidine-1,3'-oxindoles].

A three-step procedure for the enantioselective synthesis of spiro[indolizidine-1,3'-oxindoles], consisting of a stereoselective cyclocondensation reaction between (S)-tryptophanol and a prochiral or racemic δ-oxoester, bromination of the resulting oxazolopiperidone lactam, and a final stereoselective spirocyclization, is reported.

SBP ratio in exercise stress testing: validation by perfusion imaging.

AIMS: The 3-min SBP ratio (SBPR) was proposed to detect significant coronary artery disease (CAD), but its relationship with abnormalities detected by myocardial perfusion-gated single-photon emission computed tomography (SPECT) was never examined. METHODS: In 156 consecutive patients submitted to resting and exercise gated SPECT for suspect CAD, perfusion scores, ejection fraction and transient ischemic dilatation (TID) were evaluated and compared with SBPR. RESULTS: There were weak correlations between SBPR and summed stress score (ρ = 0.232, P < 0.004), and summed difference score (SDS) (ρ = 0.228, P < 0.004). According to receiver operating characteristic analysis, SBPR was poorly effective for identifying patients with inducible ischemia (SDS ≥ 2): area under curve = 0.64, (P < 0.002), sensitivity = 82%, specificity = 40%. No relationship was registered between SBPR and postexercise ejection fraction drop, and the relationship with TID was poor (ρ = 0.263, P < 0.001). CONCLUSION: In patients submitted to gated SPECT for suspect CAD, SBPR appears poorly effective for the detection of significant CAD, and does not show any valuable relationship with exercise-induced functional abnormalities.

Integrated FDG-PET/CT imaging is useful in the approach to carcinoid tumors of the lung.

BACKGROUND: Carcinoids enter the differential diagnosis of the solitary pulmonary nodule. Bronchial carcinoids have been traditionally considered as FDG-PET negative but recent studies have found an higher sensitivity of integrated FDG-PET/CT for the detection of these neoplasms. The purpose of this study was to investigate the value of integrated FDG-PET/CT for the evaluation of SPN suspected to be carcinoids. METHODS: All patients with pathologically proven bronchial carcinoids who had FDG-PET/CT scans between 2006 and 2012 have been retrospectively reviewed. PET/CT was performed with the same scanner and the same technique for all patients. The following data were retrieved: age, sex CT findings (side, location, size, shape, margins), SUVmax, type of operation, pathological findings (size and number of mitoses). Regarding PET findings, only SUVmax was considered, whereas the visual assessment was not undertaken. Carcinoids were defined as typical and atypical and as central and peripheral. The long-term follow-up was also recorded. The SUVmax was compared with the other clinical, radiological and pathological variables to find any significant difference or correlation. RESULTS: Twenty-five patients were retrieved, 24 typical and one atypical carcinoid, 21 peripheral and 4 central lesions. The mean diameter on CT-scan was 25.3 mm and the clinical size correlated well with the pathological size. Sixty percent of the tumors were ovoid and 68% had smooth margins. The mean SUVmax was 3.6 (range 1.4-12.9). All the lesions were completely resected. The regression analysis showed a direct correlation between the SUVmax and the tumor size (p = 0.004). No further correlations were found between the SUVmax and the other variables. None of the patients had recurrent disease or died during the follow-up. CONCLUSIONS: Our study showed that FDG-PET/CT might be a useful tool in the evaluation of SPNs suspected to be bronchial carcinoids. When a solitary pulmonary nodule shows an ovoid/round shape and smooth margins on the CT scan and demonstrates an FDG uptake higher than that of the normal lung and with a SUVmax value >1-1.5, a carcinoid should be suspected. If benign lesions can be presumably excluded, surgical resection or at least a biopsy of the lesion is recommended.