Pioglitazone Phases and Metabolic Effects in Nanoparticle-Treated Cells Analyzed via Rapid Visualization of FLIM Images.

Fluorescence lifetime imaging microscopy (FLIM) has proven to be a useful method for analyzing various aspects of material science and biology, like the supramolecular organization of (slightly) fluorescent compounds or the metabolic activity in non-labeled cells; in particular, FLIM phasor analysis (phasor-FLIM) has the potential for an intuitive representation of complex fluorescence decays and therefore of the analyzed properties. Here we present and make available tools to fully exploit this potential, in particular by coding via hue, saturation, and intensity the phasor positions and their weights both in the phasor plot and in the microscope image. We apply these tools to analyze FLIM data acquired via two-photon microscopy to visualize: (i) different phases of the drug pioglitazone (PGZ) in solutions and/or crystals, (ii) the position in the phasor plot of non-labelled poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), and (iii) the effect of PGZ or PGZ-containing NPs on the metabolism of insulinoma (INS-1 E) model cells. PGZ is recognized for its efficacy in addressing insulin resistance and hyperglycemia in type 2 diabetes mellitus, and polymeric nanoparticles offer versatile platforms for drug delivery due to their biocompatibility and controlled release kinetics. This study lays the foundation for a better understanding via phasor-FLIM of the organization and effects of drugs, in particular, PGZ, within NPs, aiming at better control of encapsulation and pharmacokinetics, and potentially at novel anti-diabetics theragnostic nanotools.

Impact of temporal resolution in single particle tracking analysis.

Temporal resolution is a key parameter in the observation of dynamic processes, as in the case of single molecules motions visualized in real time in two-dimensions by wide field (fluorescence) microscopy, but a systematic investigation of its effects in all the single particle tracking analysis steps is still lacking. Here we present tools to quantify its impact on the estimation of diffusivity and of its distribution using one of the most popular tracking software for biological applications on simulated data and movies. We found important shifts and different widths for diffusivity distributions, depending on the interplay of temporal sampling conditions with various parameters, such as simulated diffusivity, density of spots, signal-to-noise ratio, lengths of trajectories, and kind of boundaries in the simulation. We examined conditions starting from the ones of experiments on the fluorescently labelled receptor p75NTR, a relatively fast-diffusing membrane receptor (diffusivity around 0.5-1 µm2/s), visualized by TIRF microscopy on the basal membrane of living cells. From the analysis of the simulations, we identified the best conditions in cases similar to these ones; considering also the experiments, we could confirm a range of values of temporal resolution suitable for obtaining reliable diffusivity results. The procedure we present can be exploited in different single particle/molecule tracking applications to find an optimal temporal resolution.

Aptamer-based gold nanoparticle aggregates for ultrasensitive amplification-free detection of PSMA.

Early diagnosis is one of the most important factors in determining the prognosis in cancer. Sensitive detection and quantification of tumour-specific biomarkers have the potential to improve significantly our diagnostic capability. Here, we introduce a triggerable aptamer-based nanostructure based on an oligonucleotide/gold nanoparticle architecture that selectively disassembles in the presence of the biomarker of interest; its optimization is based also on in-silico determination of the aptamer nucleotides interactions with the protein of interest. We demonstrate this scheme for the case of Prostate Specific Membrane Antigen (PSMA) and PSMA derived from PSMA-positive exosomes. We tested the disassembly of the system by diameter and count rate measurements in dynamic light scattering, and by inspection of its plasmon resonance shift, upon addition of PSMA, finding appreciable differences down to the sub-picomolar range; this points towards the possibility that this approach may lead to sensors competitive with diagnostic biochemical assays that require enzymatic amplification. More generally, this scheme has the potential to be applied to a broad range of pathologies with specific identified biomarkers.

Quantitative determination of fluorescence labeling implemented in cell cultures.

BACKGROUND: Labeling efficiency is a crucial parameter in fluorescence applications, especially when studying biomolecular interactions. Current approaches for estimating the yield of fluorescent labeling have critical drawbacks that usually lead them to be inaccurate or not quantitative. RESULTS: We present a method to quantify fluorescent-labeling efficiency that addresses the critical issues marring existing approaches. The method operates in the same conditions of the target experiments by exploiting a ratiometric evaluation with two fluorophores used in sequential reactions. We show the ability of the protocol to extract reliable quantification for different fluorescent probes, reagents concentrations, and reaction timing and to optimize labeling performance. As paradigm, we consider the labeling of the membrane-receptor TrkA through 4'-phosphopantetheinyl transferase Sfp in living cells, visualizing the results by TIRF microscopy. This investigation allows us to find conditions for demanding single and multi-color single-molecule studies requiring high degrees of labeling. CONCLUSIONS: The developed method allows the quantitative determination and the optimization of staining efficiency in any labeling strategy based on stable reactions.

Setting up multicolour TIRF microscopy down to the single molecule level.

Investigating biological mechanisms in ever greater detail requires continuous advances in microscopy techniques and setups. Total internal reflection fluorescence (TIRF) microscopy is a well-established technique for visualizing processes on the cell membrane. TIRF allows studies down to the single molecule level, mainly in single-colour applications. Instead, multicolour setups are still limited. Here, we describe our strategies for implementing a multi-channel TIRF microscopy system capable of simultaneous two-channel excitation and detection, starting from a single-colour commercial setup. First, we report some applications at high molecule density and then focus on the challenges we faced for achieving the single molecule level simultaneously in different channels, showing that rigorous optimizations on the setup are needed to increase its sensitivity up to this point, from camera setting to background minimization. We also discuss our strategies regarding crucial points of fluorescent labelling for this type of experiment: labelling strategy, kind of probe, efficiency, and orthogonality of the reaction, all of which are aspects that can influence the achievable results. This work may provide useful guidelines for setting up advanced single-molecule multi-channel TIRF experiments to obtain insights into interaction mechanisms on the cell membrane of living cells.

Targeting Peptides: The New Generation of Targeted Drug Delivery Systems.

Peptides can act as targeting molecules, analogously to oligonucleotide aptamers and antibodies. They are particularly efficient in terms of production and stability in physiological environments; in recent years, they have been increasingly studied as targeting agents for several diseases, from tumors to central nervous system disorders, also thanks to the ability of some of them to cross the blood-brain barrier. In this review, we will describe the techniques employed for their experimental and in silico design, as well as their possible applications. We will also discuss advancements in their formulation and chemical modifications that make them even more stable and effective. Finally, we will discuss how their use could effectively help to overcome various physiological problems and improve existing treatments.

Axonal plasticity in response to active forces generated through magnetic nano-pulling.

Mechanical force is crucial in guiding axon outgrowth before and after synapse formation. This process is referred to as "stretch growth." However, how neurons transduce mechanical input into signaling pathways remains poorly understood. Another open question is how stretch growth is coupled in time with the intercalated addition of new mass along the entire axon. Here, we demonstrate that active mechanical force generated by magnetic nano-pulling induces remodeling of the axonal cytoskeleton. Specifically, the increase in the axonal density of microtubules induced by nano-pulling leads to an accumulation of organelles and signaling vesicles, which, in turn, promotes local translation by increasing the probability of assembly of the "translation factories." Modulation of axonal transport and local translation sustains enhanced axon outgrowth and synapse maturation.

Choosing the Probe for Single-Molecule Fluorescence Microscopy.

Probe choice in single-molecule microscopy requires deeper evaluations than those adopted for less sensitive fluorescence microscopy studies. Indeed, fluorophore characteristics can alter or hide subtle phenomena observable at the single-molecule level, wasting the potential of the sophisticated instrumentation and algorithms developed for advanced single-molecule applications. There are different reasons for this, linked, e.g., to fluorophore aspecific interactions, brightness, photostability, blinking, and emission and excitation spectra. In particular, these spectra and the excitation source are interdependent, and the latter affects the autofluorescence of sample substrate, medium, and/or biological specimen. Here, we review these and other critical points for fluorophore selection in single-molecule microscopy. We also describe the possible kinds of fluorophores and the microscopy techniques based on single-molecule fluorescence. We explain the importance and impact of the various issues in fluorophore choice, and discuss how this can become more effective and decisive for increasingly demanding experiments in single- and multiple-color applications.

Is Raman the best strategy towards the development of non-invasive continuous glucose monitoring devices for diabetes management?

Diabetes has no well-established cure; thus, its management is critical for avoiding severe health complications involving multiple organs. This requires frequent glycaemia monitoring, and the gold standards for this are fingerstick tests. During the last decades, several blood-withdrawal-free platforms have been being studied to replace this test and to improve significantly the quality of life of people with diabetes (PWD). Devices estimating glycaemia level targeting blood or biofluids such as tears, saliva, breath and sweat, are gaining attention; however, most are not reliable, user-friendly and/or cheap. Given the complexity of the topic and the rise of diabetes, a careful analysis is essential to track scientific and industrial progresses in developing diabetes management systems. Here, we summarize the emerging blood glucose level (BGL) measurement methods and report some examples of devices which have been under development in the last decades, discussing the reasons for them not reaching the market or not being really non-invasive and continuous. After discussing more in depth the history of Raman spectroscopy-based researches and devices for BGL measurements, we will examine if this technique could have the potential for the development of a user-friendly, miniaturized, non-invasive and continuous blood glucose-monitoring device, which can operate reliably, without inter-patient variability, over sustained periods.

Fluorescence lifetime microscopy unveils the supramolecular organization of liposomal Doxorubicin.

The supramolecular organization of Doxorubicin (DOX) within the standard Doxoves® liposomal formulation (DOX®) is investigated using visible light and phasor approach to fluorescence lifetime imaging (phasor-FLIM). First, the phasor-FLIM signature of DOX® is resolved into the contribution of three co-existing fluorescent species, each with its characteristic mono-exponential lifetime, namely: crystallized DOX (DOXc, 0.2 ns), free DOX (DOXf, 1.0 ns), and DOX bound to the liposomal membrane (DOXb, 4.5 ns). Then, the exact molar fractions of the three species are determined by combining phasor-FLIM with quantitative absorption/fluorescence spectroscopy on DOXc, DOXf, and DOXb pure standards. The final picture on DOX® comprises most of the drug in the crystallized form (∼98%), with the remaining fractions divided between free (∼1.4%) and membrane-bound drug (∼0.7%). Finally, phasor-FLIM in the presence of a DOX dynamic quencher allows us to suggest that DOXf is both encapsulated and non-encapsulated, and that DOXb is present on both liposome-membrane leaflets. We argue that the present experimental protocol can be applied to the investigation of the supramolecular organization of encapsulated luminescent drugs/molecules all the way from the production phase to their state within living matter.

Pioglitazone-Loaded PLGA Nanoparticles: Towards the Most Reliable Synthesis Method.

Recent findings have proved the benefits of Pioglitazone (PGZ) against atherosclerosis and type 2 diabetes. Since the systematic and controllable release of this drug is of significant importance, encapsulation of this drug in nanoparticles (NPs) can minimize uncontrolled issues. In this context, drug delivery approaches based on several poly(lactic-co-glycolic acid) (PLGA) nanoparticles have been rising in popularity due to their promising capabilities. However, a fully reliable and reproducible synthetic methodology is still lacking. In this work, we present a rational optimization of the most critical formulation parameters for the production of PGZ-loaded PLGA NPs by the single emulsification-solvent evaporation or nanoprecipitation methods. We examined the influence of several variables (e.g., component concentrations, phases ratio, injection flux rate) on the synthesis of the PGZ-NPs. In addition, a comparison of these synthetic methodologies in terms of nanoparticle size, polydispersity index (PDI), zeta potential (ζp), drug loading (DL%), entrapment efficiency (EE%), and stability is offered. According to the higher entrapment efficiency content, enhanced storage time and suitable particle size, the nanoprecipitation approach appears to be the simplest, most rapid and most reliable synthetic pathway for these drug nanocarriers, and we demonstrated a very slow drug release in PBS for the best formulation obtained by this synthesis.

Fluorolabeling of the PPTase-Related Chemical Tags: Comparative Study of Different Membrane Receptors and Different Fluorophores in the Labeling Reactions.

The set-up of an advanced imaging experiment requires a careful selection of suitable labeling strategies and fluorophores for the tagging of the molecules of interest. Here we provide an experimental workflow to allow evaluation of fluorolabeling performance of the chemical tags target of phosphopantetheinyl transferase enzymes (PPTases), once inserted in the sequence of different proteins of interest. First, S6 peptide tag was fused to three different single-pass transmembrane proteins (the tyrosine receptor kinases TrkA and VEGFR2 and the tumor necrosis factor receptor p75NTR), providing evidence that all of them can be conveniently albeit differently labeled. Moreover, we chose the S6-tagged TrkA construct to test eight different organic fluorophores for the PPTase labeling of membrane receptors in living cells. We systematically compared their non-specific internalization when added to a S6-tag negative cell culture, the percentage of S6-TrkA expressing cells effectively labeled and the relative mean fluorescence intensity, their photostability upon conjugation, and ratio of specific (cellular) versus background (glass-adhered) signal. This allowed to identify which fluorophores are actually recommended for these labeling reactions. Finally, we compared the PPTase labeling of a purified, YBBR-tagged Nerve Growth Factor with two differently charged organic dyes. We detected some batch-to-batch variability in the labeling yield, regardless of the fluorophore used. However, upon purification of the fluorescent species and incubation with living primary DRG neurons, no significant difference could be appreciated in both internalization and axonal transport of the labeled neurotrophins.

Lysosome Dynamic Properties during Neuronal Stem Cell Differentiation Studied by Spatiotemporal Fluctuation Spectroscopy and Organelle Tracking.

We investigated lysosome dynamics during neuronal stem cell (NSC) differentiation by two quantitative and complementary biophysical methods based on fluorescence: imaging-derived mean square displacement (iMSD) and single-particle tracking (SPT). The former extracts the average dynamics and size of the whole population of moving lysosomes directly from imaging, with no need to calculate single trajectories; the latter resolves the finest heterogeneities and dynamic features at the single-lysosome level, which are lost in the iMSD analysis. In brief, iMSD analysis reveals that, from a structural point of view, lysosomes decrement in size during NSC differentiation, from 1 µm average diameter in the embryonic cells to approximately 500 nm diameter in the fully differentiated cells. Concomitantly, iMSD analysis highlights modification of key dynamic parameters, such as the average local organelle diffusivity and anomalous coefficient, which may parallel cytoskeleton remodeling during the differentiation process. From average to local, SPT allows mapping heterogeneous dynamic responses of single lysosomes in different districts of the cells. For instance, a dramatic decrease of lysosomal transport in the soma is followed by a rapid increase of transport in the projections at specific time points during neuronal differentiation, an observation compatible with the hypothesis that lysosomal active mobilization shifts from the soma to the newborn projections. Our combined results provide new insight into the lysosome size and dynamics regulation throughout NSC differentiation, supporting new functions proposed for this organelle.

Effects of fixatives on myelin molecular order probed with RP-CARS microscopy.

When live imaging is not feasible, sample fixation allows preserving the ultrastructure of biological samples for subsequent microscopy analysis. This process could be performed with various methods, each one affecting differently the biological structure of the sample. While these alterations were well-characterized using traditional microscopy, little information is available about the effects of the fixatives on the spatial molecular orientation of the biological tissue. We tackled this issue by employing rotating-polarization coherent anti-Stokes Raman scattering (RP-CARS) microscopy to study the effects of different fixatives on the myelin sub-micrometric molecular order and micrometric morphology. RP-CARS is a novel technique derived from CARS microscopy that allows probing spatial orientation of molecular bonds while maintaining the intrinsic chemical selectivity of CARS microscopy. By characterizing the effects of the fixation procedures, the present work represents a useful guide for the choice of the best fixation technique(s), in particular for polarization-resolved CARS microscopy. Finally, we show that the combination of paraformaldehyde and glutaraldehyde can be effectively employed as a fixative for RP-CARS microscopy, as long as the effects on the molecular spatial distribution, here characterized, are taken into account.

Graphene Promotes Axon Elongation through Local Stall of Nerve Growth Factor Signaling Endosomes.

Several works reported increased differentiation of neuronal cells grown on graphene; however, the molecular mechanism driving axon elongation on this material has remained elusive. Here, we study the axonal transport of nerve growth factor (NGF), the neurotrophin supporting development of peripheral neurons, as a key player in the time course of axonal elongation of dorsal root ganglion neurons on graphene. We find that graphene drastically reduces the number of retrogradely transported NGF vesicles in favor of a stalled population in the first 2 days of culture, in which the boost of axon elongation is observed. This correlates with a mutual charge redistribution, observed via Raman spectroscopy and electrophysiological recordings. Furthermore, ultrastructural analysis indicates a reduced microtubule distance and an elongated axonal topology. Thus, both electrophysiological and structural effects can account for graphene action on neuron development. Unraveling the molecular players underneath this interplay may open new avenues for axon regeneration applications.

Molecular insight on the altered membrane trafficking of TrkA kinase dead mutants.

We address the contribution of kinase domain structure and catalytic activity to membrane trafficking of TrkA receptor tyrosine kinase. We conduct a systematic comparison between TrkA-wt, an ATP-binding defective mutant (TrkA-K544N) and other mutants displaying separate functional impairments of phosphorylation, ubiquitination, or recruitment of intracellular partners. We find that only K544N mutation endows TrkA with restricted membrane mobility and a substantial increase of cell surface pool already in the absence of ligand stimulation. This mutation is predicted to drive a structural destabilization of the αC helix in the N-lobe by molecular dynamics simulations, and enhances interactions with elements of the actin cytoskeleton. On the other hand, a different TrkA membrane immobilization is selectively observed after NGF stimulation, requires both phosphorylation and ubiquitination to occur, and is most probably related to the signaling abilities displayed by the wt but not mutated receptors. In conclusion, our results allow to distinguish two different TrkA membrane immobilization modes and demonstrate that not all kinase-inactive mutants display identical membrane trafficking.

Fast-diffusing p75NTR monomers support apoptosis and growth cone collapse by neurotrophin ligands.

The p75 neurotrophin (NT) receptor (p75NTR) plays a crucial role in balancing survival-versus-death decisions in the nervous system. Yet, despite 2 decades of structural and biochemical studies, a comprehensive, accepted model for p75NTR activation by NT ligands is still missing. Here, we present a single-molecule study of membrane p75NTR in living cells, demonstrating that the vast majority of receptors are monomers before and after NT activation. Interestingly, the stoichiometry and diffusion properties of the wild-type (wt) p75NTR are almost identical to those of a receptor mutant lacking residues previously believed to induce oligomerization. The wt p75NTR and mutated (mut) p75NTR differ in their partitioning in cholesterol-rich membrane regions upon nerve growth factor (NGF) stimulation: We argue that this is the origin of the ability of wt p75NTR , but not of mut p75NTR, to mediate immature NT (proNT)-induced apoptosis. Both p75NTR forms support proNT-induced growth cone retraction: We show that receptor surface accumulation is the driving force for cone collapse. Overall, our data unveil the multifaceted activity of the p75NTR monomer and let us provide a coherent interpretative frame of existing conflicting data in the literature.

Silver-nanoparticles as plasmon-resonant enhancers for eumelanin's photoacoustic signal in a self-structured hybrid nanoprobe.

Developing safe and high efficiency contrast tools is an urgent need to allow in vivo applications of photoacoustics (PA), an emerging biomolecular imaging methodology, with poor invasiveness, deep penetration, high spatial resolution and excellent endogenous contrast. Eumelanins hold huge promise as biocompatible, endogenous photoacoustic contrast agents. However, their huge potential is still unexplored due to the difficulty to achieve at the same time poor aggregation in physiologic environment and high PA contrast. This study addresses both issues through the design of a biocompatible photoacoustic nanoprobe, named MelaSil_Ag-NPs, relying on silica-templated eumelanin formation as well as eumelanins redox and metal chelating properties to reduce Ag+ ions and control the growth of generated metal nanoparticles. This strategy allowed self-structuring of the system into a core-shell architecture, where the Ag core was found to boost PA signal, despite the poor eumelanin content. Obtained hybrid nanoplatforms, showed stable photoacoustic properties even under long irradiation. Furthermore, conjugation with rhodamine isothiocyanate allowed particles detection through fluorescent imaging proving their multifunctional potentialities. In addition, they were stable towards aggregation and efficiently endocytosed by human pancreatic cancer cells (BxPC3 and Panc-1) displaying no significant cytotoxicity. Such numerous features prove huge potential of those nanoparticles as a multifunctional platform for biomedical applications.

Targeting Inflammation With Nanosized Drug Delivery Platforms in Cardiovascular Diseases: Immune Cell Modulation in Atherosclerosis.

Atherosclerosis (AS) is a disorder of large and medium-sized arteries; it consists in the formation of lipid-rich plaques in the intima and inner media, whose pathophysiology is mostly driven by inflammation. Currently available interventions and therapies for treating atherosclerosis are not always completely effective; side effects associated with treatments, mainly caused by immunodepression for anti-inflammatory molecules, limit the systemic administration of these and other drugs. Given the high degree of freedom in the design of nanoconstructs, in the last decades researchers have put high effort in the development of nanoparticles (NPs) formulations specifically designed for either drug delivery, visualization of atherosclerotic plaques, or possibly the combination of both these and other functionalities. Here we will present the state of the art of these subjects, the knowledge of which is necessary to rationally address the use of NPs for prevention, diagnosis, and/or treatment of AS. We will analyse the work that has been done on: (a) understanding the role of the immune system and inflammation in cardiovascular diseases, (b) the pathological and biochemical principles in atherosclerotic plaque formation, (c) the latest advances in the use of NPs for the recognition and treatment of cardiovascular diseases, (d) the cellular and animal models useful to study the interactions of NPs with the immune system cells.

Peripheral Neuron Survival and Outgrowth on Graphene.

Graphene displays properties that make it appealing for neuroregenerative medicine, yet its interaction with peripheral neurons has been scarcely investigated. Here, we culture on graphene two established models for peripheral neurons: PC12 cells and DRG primary neurons. We perform a nano-resolved analysis of polymeric coatings on graphene and combine optical microscopy and viability assays to assess the material cytocompatibility and influence on differentiation. We find that differentiated PC12 cells display a remarkably increased neurite length on graphene (up to 27%) with respect to controls. Notably, DRG primary neurons survive both on bare and coated graphene. They present dense axonal networks on coated graphene, while they form cell islets characterized by dense axonal bundles on uncoated graphene. These findings indicate that graphene holds potential for nerve tissue regeneration and might pave the road to novel concepts of active nerve conduits.