Concanavalin A Delivers a Photoactive Protein to the Bacterial Wall.

Modular supramolecular complexes, where different proteins are assembled to gather targeting capability and photofunctional properties within the same structures, are of special interest for bacterial photodynamic inactivation, given their inherent biocompatibility and flexibility. We have recently proposed one such structure, exploiting the tetrameric bacterial protein streptavidin as the main building block, to target S. aureus protein A. To expand the palette of targets, we have linked biotinylated Concanavalin A, a sugar-binding protein, to a methylene blue-labelled streptavidin. By applying a combination of spectroscopy and microscopy, we demonstrate the binding of Concanavalin A to the walls of Gram-positive S. aureus and Gram-negative E. coli. Photoinactivation is observed for both bacterial strains in the low micromolar range, although the moderate affinity for the molecular targets and the low singlet oxygen yields limit the overall efficiency. Finally, we apply a maximum entropy method to the analysis of autocorrelation traces, which proves particularly useful when interpreting signals measured for diffusing systems heterogeneous in size, such as fluorescent species bound to bacteria.

Versatile Supramolecular Complex for Targeted Antimicrobial Photodynamic Inactivation.

We report the development of a supramolecular structure endowed with photosensitizing properties and targeting capability for antimicrobial photodynamic inactivation. Our synthetic strategy uses the tetrameric bacterial protein streptavidin, labeled with the photosensitizer eosin, as the main building block. Biotinylated immunoglobulin G (IgG) from human serum, known to associate with Staphylococcus aureus protein A, was bound to the complex streptavidin-eosin. Fluorescence correlation spectroscopy and fluorescence microscopy demonstrate binding of the complex to S. aureus. Efficient photoinactivation is observed for S. aureus suspensions treated with IgG-streptavidin-eosin at concentrations higher than 0.5 µM and exposed to green light. The proposed strategy offers a flexible platform for targeting a variety of molecules and microbial species.

Salivary Proteome Changes in Response to Acute Psychological Stress Due to an Oral Exam Simulation in University Students: Effect of an Olfactory Stimulus.

The autonomic nervous system (ANS) plays a crucial role both in acute and chronic psychological stress eliciting changes in many local and systemic physiological and biochemical processes. Salivary secretion is also regulated by ANS. In this study, we explored salivary proteome changes produced in thirty-eight University students by a test stress, which simulated an oral exam. Students underwent a relaxation phase followed by the stress test during which an electrocardiogram was recorded. To evaluate the effect of an olfactory stimulus, half of the students were exposed to a pleasant odor diffused in the room throughout the whole session. Saliva samples were collected after the relaxation phase (T0) and the stress test (T1). State anxiety was also evaluated at T0 and T1. Salivary proteins were separated by two-dimensional electrophoresis, and patterns at different times were compared. Spots differentially expressed were trypsin digested and identified by mass spectrometry. Western blot analysis was used to validate proteomic results. Anxiety scores and heart rate changes indicated that the fake exam induced anxiety. Significant changes of α-amylase, polymeric immunoglobulin receptor (PIGR), and immunoglobulin α chain (IGHA) secretion were observed after the stress test was performed in the two conditions. Moreover, the presence of pleasant odor reduced the acute social stress affecting salivary proteome changes. Therefore, saliva proteomic analysis was a useful approach to evaluate the rapid responses associated to an acute stress test also highlighting known biomarkers.

A Photoactive Supramolecular Complex Targeting PD-L1 Reveals a Weak Correlation between Photoactivation Efficiency and Receptor Expression Levels in Non-Small-Cell Lung Cancer Tumor Models.

Photo-immunotherapy uses antibodies conjugated to photosensitizers to produce nanostructured constructs endowed with targeting properties and photo-inactivation capabilities towards tumor cells. The superficial receptor density on cancer cells is considered a determining factor for the efficacy of the photodynamic treatment. In this work, we propose the use of a photoactive conjugate that consists of the clinical grade PD-L1-binding monoclonal antibody Atezolizumab, covalently linked to either the well-known photosensitizer eosin or the fluorescent probe Alexa647. Using single-molecule localization microscopy (direct stochastic optical reconstruction microscopy, dSTORM), and an anti-PD-L1 monoclonal antibody labelled with Alexa647, we quantified the density of PD-L1 receptors exposed on the cell surface in two human non-small-cell lung cancer lines (H322 and A549) expressing PD-L1 to a different level. We then investigated if this value correlates with the effectiveness of the photodynamic treatment. The photodynamic treatment of H322 and A549 with the photo-immunoconjugate demonstrated its potential for PDT treatments, but the efficacy did not correlate with the PD-L1 expression levels. Our results provide additional evidence that receptor density does not determine a priori the level of photo-induced cell death.

Targeted photoimmunotherapy for cancer.

Photodynamic therapy (PDT) is a clinically approved procedure that can exert a curative action against malignant cells. The treatment implies the administration of a photoactive molecular species that, upon absorption of visible or near infrared light, sensitizes the formation of reactive oxygen species. These species are cytotoxic and lead to tumor cell death, damage vasculature, and induce inflammation. Clinical investigations demonstrated that PDT is curative and does not compromise other treatment options. One of the major limitations of the original method was the low selectivity of the photoactive compounds for malignant over healthy tissues. The development of conjugates with antibodies has endowed photosensitizing molecules with targeting capability, so that the compounds are delivered with unprecedented precision to the site of action. Given their fluorescence emission capability, these supramolecular species are intrinsically theranostic agents.

The Interaction of Hypericin with SARS-CoV-2 Reveals a Multimodal Antiviral Activity.

Hypericin is a photosensitizing drug that is active against membrane-enveloped viruses and therefore constitutes a promising candidate for the treatment of SARS-CoV-2 infections. The antiviral efficacy of hypericin is largely determined by its affinity toward viral components and by the number of active molecules loaded on single viruses. Here we use an experimental approach to follow the interaction of hypericin with SARS-CoV-2, and we evaluate its antiviral efficacy, both in the dark and upon photoactivation. Binding to viral particles is directly visualized with fluorescence microscopy, and a strong affinity for the viral particles, most likely for the viral envelope, is measured spectroscopically. The loading of a maximum of approximately 30 molecules per viral particle is estimated, despite with marked heterogeneity among particles. Because of this interaction, nanomolar concentrations of photoactivated hypericin substantially reduce virus infectivity on Vero E6 cells, but a partial effect is also observed in dark conditions, suggesting multiple mechanisms of action for this drug.