In Resistive Pulse Sensing, nanoparticles dispersed in solution are individually detected and characterized during their translocation through a narrow pore or channel. Electrophoretic force and fluid flow can be precisely adjusted to direct nanoparticles toward the sensing zone. The impact of various factors on nanoparticle translocation dynamics, including solution ionic strength, pH, applied potential difference, and pipette tip geometry, has been extensively investigated. In this work, we focus on the role of pipette filling height, an experimental parameter often overlooked despite its significant impact on the overall pressure gradient and the resulting flow through the pipette tip. We used a solution of NaCl 150 mM plus 0.1 % v/v Triton X-100 at pH 7.2, a pipette with radius of approximately 200 nm and a voltage of +/- 200 mV. Our findings reveal that the pipette filling height emerges as the critical factor dictating the translocation direction of negatively charged 160 nm PMMA particles and surpassing the combined effect of electrokinetic forces. Ultimately, our results indicate that considering the pipette filling level could enhance the accurate interpretation of experimental results, offering an additional parameter for fine-tuning nanoparticles dynamics, thus providing a valuable tool to researchers in this field.

Over the past few decades, finding more efficient and selective administration routes has gained significant attention due to its crucial role in the bioavailability, absorption rate and pharmacokinetics of therapeutic substances. The pulmonary delivery of drugs has become an attractive target of scientific and biomedical interest in the health care research area, as the lung, thanks to its high permeability and large absorptive surface area and good blood supply, is capable of absorbing pharmaceuticals either for local deposition or for systemic delivery. Nevertheless, the pulmonary drug delivery is relatively complex, and strategies to mitigate the effects of mechanical, chemical and immunological barriers are required. Herein, engineered erythrocytes, the Erythro–Magneto–Hemagglutinin (HA)–virosomes (EMHVs), are used as a novel strategy for efficiently delivering drugs to the lungs. EMHV bio-based carriers exploit the physical properties of magnetic nanoparticles to achieve effective targeting after their intravenous injection thanks to an external magnetic field. In addition, the presence of hemagglutinin fusion proteins on EMHVs’ membrane allows the DDS to anchor and fuse with the target tissue and locally release the therapeutic compound. Our results on the biomechanical and biophysical properties of EMHVs, such as the membrane robustness and deformability and the high magnetic susceptibility, as well as their in vivo biodistribution, highlight that this bio-inspired DDS is a promising platform for the controlled and lung-targeting delivery of drugs, and represents a valuable alternative to inhalation therapy to fulfill unmet clinical needs.

The plant-derived natural alkaloid berberine displays therapeutic potential to treat several pathological conditions, including dyslipidemias, diabetes and cardiovascular disorders. However, data on berberine effects during embryonic development are scarce and in part controversial. In this study, using zebrafish embryos as vertebrate experimental model, we address the effects of berberine treatment on cardiovascular system development and functionality. Starting from the observation that berberine induces developmental toxicity and pericardial edema in a time- and concentration-dependent manner, we found that treated embryos display cardiac looping defects and, at later stages, present an abnormal heart characterized by a stretched morphology and atrial endocardial/myocardial detachment. Furthermore, berberine affected cardiac functionality of the embryos, promoting bradycardia and reducing the cardiac output, the atrial shortening fraction percentage and the atrial stroke volume. We also found that, during development, berberine interferes with the angiogenic process, without altering vascular permeability. These alterations are associated with increased levels of vascular endothelial growth factor aa (vegfaa) mRNA, suggesting an important role for Vegfaa as mediator of berberine-induced cardiovascular defects. Altogether, these data indicate that berberine treatment during vertebrate development leads to an impairment of cardiovascular system morphogenesis and functionality, suggesting a note of caution in its use during pregnancy and lactation.

In this work biocompatible polymethylmethacrylate nanoparticles (PMMA-NPs) were used as carrier of a molecular beacon (MB) for sensing survivin mRNA in cancer cells. MBs are oligonucleotide sequences generating a fluorescent signal when they hybridize with their target. They constitute potential theranostic agents as they can act at the same time as sensors, able to detect endogenous nucleic acids, and as drug, by silencing the target mRNA. NPs offer numerous advantages over conventional drug delivery approaches, such as the possibility of multiple functionalization for improving the imaging, diagnosis and targeted therapy. In particular, PMMA-NPs used in this study consist of a hydrophobic PMMA core covalently functionalized with fluorescein and an external hydrophilic shell decorated with primary amine groups and quaternary ammonium salts. The aim of the work was to evaluate by confocal microscopy and fluorescence measurements: a) the ability of PMMA-NPs to promote, in human A549 cancer cells, the internalization of a MB specific for survivin mRNA; b) the involvement of endocytosis in the NP uptake; c) the NP fate at different times of cell incubation to verify their localization in lysosomes; d) the MB localization on the Endoplasmic Reticulum (ER) where the target mRNA is located. The results obtained demonstrated: a) PMMA-NPs efficiently promote the MB internalization generating a specific fluorescent signal in the presence of survivin mRNA expression; b) the involvement of endocytosis in the NP uptake; c) the NP localization in lysosomes at different times of cell incubation and their subsequent release in the cell culture medium; d) the MB fluorescence localization in proximity of the ER where the target mRNA is presumably located.

One of the main goals in nanomedicine is the development of effective drug delivery systems. Polymeric nanoparticles (NPs) have been exploited as nanocarriers for their stability, handiness and biocompatibility. In this context, NPs have been coupled to molecular beacons (MBs), theranostic agents that conjugate the sensing of specific mRNA with a silencing activity. The aim of this work was to evaluate by confocal microscopy and fluorescence measurements: a) the involvement of endocytosis in the NP uptake; b) the NP fate at different times of cell incubation to verify their localization in lysosomes; c) the MB localization on the Endoplasmic Reticulum (ER) where the target mRNA is located. Results: a) PMMA-NPs promote endocytosis; b) strong co-localization of NP and lysosome tracker fluorescence after 2- 48 hours of incubation; c) co-localization of the MB fluorescence with the ER-marker signal after 90' of incubation. The data highlight the ability of the PMMA-NPs to promote the survivin-MB internalization by endocytosis and demonstrate that the PMMA-NPs are an appropriate delivery system capable of being eliminated by cells; these evidences also contribute to consider the MB as an effective tool for the intracellular sensing.

To dissect the TBX5 regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type, CRE, Tbx5lox/+and Tbx5del/+ mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt–Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between TBX5, arrhythmia and heart development.

Background: TBX5 mutations cause Holt-Oram syndrome (HOS) characterized by upper limb and cardiac malformations, but can also contribute to early-onset of atrial fibrillation. Focusing on miRNAs involved in TBX5 regulatory circuits with a cardiac relevant role, we identified miR-182-5p, belonging to miR-183 cluster, found upregulated in Tbx5-depleted hearts of mouse and zebrafish embryos. Methods: To functionally analyse the miR-182-5p role in developing heart, miR-182-5p was dysregulated in zebrafish zygotes of Tg(Myl7:EGFP) and Tg(myl7:gCaMP) transgenic lines. To stably deregulate miR-182-5p in zebrafish heart we exploited the Gal4/UAS system to restrict the miR-182 expression into cardiac context. For physiological analyses we performed the mechanogram of cardiac contraction and electrocardiogram recording. To understand miR-182-5p downstream regulation, in silico analyses, followed by ddPCR/real-time quantifications on dissected zebrafish hearts and rescue experiments both in transient and stable miR-182-5p overexpressing zebrafish embryos were performed. Results: Depletion of Tbx5 from cardiomyocytes increased the expression of miR-182 cluster family that is controlled by Kruppel-like factor 4 (KLF4), a transcription factor repressed by Tbx5. Both transient and stable upregulation of miR-182 in zebrafish affect heart morphology, calcium handling and the onset of arrhythmia while its cardiac-specific downregulation decreases cardiac defects in zebrafish HOS hearts. Expression analyses on selected miR-182-5p putative targets revealed that several calcium channel proteins resulted downregulated in miR-182-5p overexpressing hearts. Transgenic zebrafish line stably overexpressing miR-182-5p in the heart manifested arrhythmia overtime with or without cardiac structural defects. Conclusion: We identified miR-182-5p as a potential suitable target to interfere in the circuit between upstream genetic abnormalities and downstream effectors leading to arrhythmia occurrence.

Despite the accepted notion that erythrocytes are uniquely deformable cells, the apparent Young's modulus values reported in the literature do not differ so much from those of other cells. We devised to measure the local deformability of living immobilized human erythrocytes at a low force, in contact-free mode, using an application of Scanning Ion Conductance Microscopy (SICM) previously developed in our laboratory. Reversible indentations were induced by forces of up to few hundreds pN. The indentation did not grow linearly with the force. The apparent Young's modulus varied from 0.2 to 1.5 kPa applying forces from 20 to 500 pN on a cell surface area of about 0.2 mu m(2), exhibiting a progressive stiffening at increasing force. Control measurements showed that A549 cells exhibit a constant value of the apparent Young's modulus (about 2 kPa) for forces up to about 800 pN. These findings show that SICM is a suitable tool to investigate cell mechanical properties, when forces in the range of tens of pN are required, in the absence of mechanical contact between probe and sample. The nonlinear deformation of the erythrocyte has to be taken into account in modeling the complex regulation mechanism of the microvascular beds.

Polymethylmethacrylate core-shell fluorescent nanoparticles promote, in human lung A549 cancer cells, the internalization of a molecular beacon (MB) specific for survivin mRNA, an anti-apoptotic protein overexpressed in cancer cells.AIMS:To design an effective drug delivery system, the knowledge of the uptake mechanism and of the nanoparticles (NPs) and MB fate is required.MATERIALS AND METHODS AND KEY FINDINGS:Experiments with dextran as marker for endocytosis showed that in the presence of NPs the number of endocytic vesicles per cell doubled and their mean size significantly (p < 0.001) increased with respect to controls in absence of NPs, indicating an involvement of NPs in the endocytotic process. By using LysoTracker™ Deep Red, as marker of lysosomes, we found that nanoparticles co-localize with lysosomes. Moreover, a cellular release of nanoparticles detected in the culture medium, suggested a role of lysosomal exocytosis in nanoparticle elimination. The MB fluorescence in proximity of the labeled Endoplasmic Reticulum was indicative that the opening of the MB occurs in proximity of its target mRNA.SIGNIFICANCE:The results show the involvement of endocytotic pathway in the uptake of NPs, which are an appropriate delivery system capable of being eliminated by cells. Furthermore the data confirm that the MB can be considered an effective tool for the intracellular sensing.

Wireless neuronal stimulation, mediated by ultrasounds and piezoelectric nanoparticles, represents an unprecedented approach aimed at cell activation. Recently, we demonstrated that barium titanate nanoparticles behave as excellent nanotransducers, by eliciting specific cell response following treatment with ultrasounds. In this chapter, we describe in detail the techniques exploited to investigate the nanoparticle/cell interactions and the activation of the neuronal-like cultures in terms of sodium and calcium fluxes.

Modeling biological systems in vitro has contributed to clarification of complex mechanisms in simplified and controlled experimental conditions. Mouse embryonic stem (mES) cells can be successfully differentiated toward specific neuronal cell fates, thus representing an attractive tool to dissect, in vitro, mechanisms that underlie complex neuronal features. In this study, we generated and characterized a reporter mES cell line, called Tph2(GFP), in which the vital reporter GFP replaces the tryptophan hydroxylase 2 (Tph2) gene. Tph2GFP mES cells selectively express GFP upon in vitro differentiation toward the serotonergic fate, they synthesize serotonin, possess excitable membranes, and show the typical morphological, morphometrical, and molecular features of in vivo serotonergic neurons. Thanks to the vital reporter GFP, we highlighted by time-lapse video microscopy several dynamic processes such as cell migration and axonal outgrowth in living cultures. Finally, we demonstrated that predifferentiated Tph2GFP cells are able to terminally differentiate, integrate, and innervate the host brain when grafted in vivo. On the whole, the present study introduces the Tph2GFP mES cell line as a useful tool allowing accurate developmental and dynamic studies and representing a reliable platform for the study of serotonergic neurons in health and disease.

One of the main goals of nanomedicine in cancer is the development of effective drug delivery systems, primarily nanoparticles. Survivin, an overexpressed anti-apoptotic protein in cancer, represents a pharmacological target for therapy and a Molecular Beacon (MB) specific for survivin mRNA is available. In this study, the ability of polymethylmethacrylate nanoparticles (PMMA-NPs) to promote survivin MB uptake in human A549 cells was investigated. Fluorescent and positively charged core PMMA-NPs of nearly 60nm, obtained through an emulsion co-polymerization reaction, and the MB alone were evaluated in solution, for their analytical characterization; then, the MB specificity and functionality were verified after adsorption onto the PMMA-NPs. The carrier ability of PMMA-NPs in A549 was examined by confocal microscopy. With the optimized protocol, a hardly detectable fluorescent signal was obtained after incubation of the cells with the MB alone (fluorescent spots per cell of 1.90±0.40 with a mean area of 1.04±0.20µm2), while bright fluorescent spots inside the cells were evident by using the MB loaded onto the PMMA-NPs. (27.50±2.30 fluorescent spots per cell with a mean area of 2.35±0.16µm2). These results demonstrate the ability of the PMMA-NPs to promote the survivin-MB internalization, suggesting that this complex might represent a promising strategy for intracellular sensing and for the reduction of cancer cell proliferation.

BACKGROUND:The Zolfino bean is a variety of Phaseolus vulgaris, which is cultivated in a limited area of Tuscany, Italy, and is widely appreciated for its flavor and culinary uses. OBJECTIVES:A yellow Zolfino landrace cultivated in the Leccio-Reggello area was characterized and compared with three other varieties of Phaseolus vulgaris (i.e. the Borlotto, Cannellino, and Corona beans) in terms of its general features and potential as an antioxidant/anti-inflammatory agent. DESIGN:The length, width, thickness, equatorial section surface, weight, volume, and seed coat section were measured in all the beans. The seed surface area was also estimated by an original empirical method. The ability of the different beans to interfere with the enzymes of the polyol pathway (that is, aldose reductase (AR) and sorbitol dehydrogenase) was tested using the supernatant after soaking the beans at room temperature and after thermal treatment, which simulated the bean-cooking process in a controlled fashion. RESULTS:Concerning the general features, Zolfino was comparable with other beans, except Corona, in terms of surface-volume ratio, which possesses the lowest tegument thickness. Moreover, Zolfino appears the most effective in inhibiting AR activity. The inhibitory ability is unaffected by thermal treatment and appears to be associated with compound(s) present in the coat of the bean. CONCLUSIONS:The ability of Zolfino to inhibit AR, thus reducing the flux of glucose through the polyol pathway, highlights the features of Zolfino as a functional food, potentially useful in treating the dysfunctions linked to the hyperactivity of AR, such as diabetic complications or inflammatory responses.

Scanning ion-conductance microscopy (SICM) belongs to the family of scanning-probe microscopies. The spatial resolution of these techniques is limited by the size of the probe. In SICM the probe is a pipette, obtained by heating and pulling a glass capillary tubing. The size of the pipette tip is therefore an important parameter in SICM experiments. However, the characterization of the tip is not a consolidated routine in SICM experimental practice. In addition, potential and limitations of the different methods available for this characterization may not be known to all users. We present an overview of different methods for characterizing size and geometry of the pipette tip, with the aim of collecting and facilitating the use of several pieces of information appeared in the literature in a wide interval of time under different disciplines. In fact, several methods that have been developed for pipettes used in cell physiology can be also fruitfully employed in the characterization of the SICM probes. The overview includes imaging techniques, such as scanning electron microscopy and atomic Force microscopy, and indirect methods, which measure some physical parameter related to the size of the pipette. Examples of these parameters are the electrical resistance of the pipette filled with a saline solution and the surface tension at the pipette tip. We discuss advantages and drawbacks of the methods, which may be helpful in answering a wide range of experimental questions.

Background: Boron nitride nanotubes (BNNTs) represent a new opportunity for drug delivery and clinical therapy. The present work has the objective to investigate pectin-coated BNNTs (P-BNNTs) for their biocompatibility on macrophage cultures, since these cells are among the first components of the immune system to interact with administered nanoparticles.Methods: As first step, the potential toxicity of P-BNNTs is verified in terms of proliferation, oxidative stress induction and apoptosis/necrosis phenomena. Thereafter, the modulation of immune cell response following P-BNNT exposure is evaluated at gene and protein level, in particular focusing on cytokine release. Finally, P-BNNT internalization is assessed through transmission electron microscopy and confocal microscopy.Results: The results proved that P-BNNTs are not toxic for macrophages up to 50 mu g/ml after 24 h of incubation. The cytokine expression is not affected by P-BNNT administration both at gene and protein level. Moreover, PBNNTs are internalized by macrophages without impairments of the cell structures.Conclusions: Collected data suggest that P-BNNTs cause neither adverse effects nor inflammation processes in macrophages.General significance: These findings represent the first and fundamental step in immune compatibility evaluation of BNNTs, mandatory before any further pre-clinical testing. (C) 2016 Elsevier B.V. All rights reserved.

Human cutaneous melanoma is an aggressive and chemotherapy-resistant type of cancer. AM251 is a cannabinoid type 1 (CB1) receptor antagonist/inverse agonist with off-target antitumor activity against pancreatic and colon cancer cells. The current study aimed to characterize the in-vitro antimelanoma activity of AM251. The BRAF V600E mutant melanoma cell line, A375, was used as an in-vitro model system. Characterization tools included a cell viability assay, nuclear morphology assessment, gene expression, western blot, flow cytometry with Annexin V-FITC/7-AAD double staining, cell cycle analyses, and measurements of changes in intracellular cAMP and calcium concentrations. AM251 exerted a marked cytotoxic effect against A375 human melanoma cells with potency comparable with that observed for cisplatin without significant changes in the human dermal fibroblasts viability. AM251, at a concentration that approximates the IC50, downregulated genes encoding antiapoptotic proteins (BCL2 and survivin) and increased transcription levels of proapoptotic BAX, induced alteration of Annexin V reactivity, DNA fragmentation, chromatin condensation in the cell nuclei, and G2/M phase arrest.AM251 also induced a 40% increase in the basal cAMP levels, but it did not affect intracellular calcium concentrations. The involvement of GPR55, TRPA1, and COX-2 in the AM251 mechanism of action was excluded. The combination of AM251 with celecoxib produced a synergistic antitumor activity, although the mechanism underlying this effect remains to be elucidated. This study provides the first evidence of a proapoptotic effect and G2/M cell cycle arrest of AM251 on A375 cells. This compound may be a potential prototype for the development of promising diarylpyrazole derivatives to be evaluated in human cutaneous melanoma.

Tetragonal barium titanate nanoparticles (BTNPs) have been exploited as nanotransducers owing to their piezoelectric properties, in order to provide indirect electrical stimulation to SH-SY5Y neuron-like cells. Following application of ultrasounds to cells treated with BTNPs, fluorescence imaging of ion dynamics revealed that the synergic stimulation is able to elicit a significant cellular response in terms of calcium and sodium fluxes; moreover, tests with appropriate blockers demonstrated that voltage-gated membrane channels are activated. The hypothesis of piezoelectric stimulation of neuron-like cells was supported by lack of cellular response in the presence of cubic nonpiezoelectric BTNPs, and further corroborated by a simple electroelastic model of a BTNP subjected to ultrasounds, according to which the generated voltage is compatible with the values required for the activation of voltage-sensitive channels.

The use of antisense oligonucleotide molecular beacons, able to generate a fluorescent signal when they hybridize with their target mRNA, represents an innovative strategy in cancer. This approach is able to conjugate the ability of sensing specific mRNA with the pharmacological silencing activity, preventing the overexpression of proteins associated to cancer development. In this context, this strategy minimizes the non-specific toxicity addressing the therapy mainly towards the tumor cells by using effective delivery systems. The aim of this work was to investigate the ability of polymethylmethacrylate nanoparticles (PMMA-NPs) to act as vehicle of an oligonucleotide molecular beacon (MB) targeting survivin mRNA in A549 human lung adenocarcinoma epithelial cells. Furthermore, this paper focuses the attention on the need for having an appropriate healthy control in in-vitro experiments. In particular, the survivin-MB was firstly characterized in solution in order to verify its functionality and then the PMMA-NPs ability to promote the MB internalization was verified in A549 cells by confocal microscopy. Confluent Human Dermal Fibroblasts from adult (HDFa) were used as healthy control. The results showed that PMMA-NPs promote the survivin-MB cellular up-take and that the use of 10 μg/mL PMMA-NPs as carrier for survivin-MB for 1h 30 min might be a promising strategy to reduce cancer cell proliferation avoiding detectable consequences on the healthy cells.

In cancer research, the use of antisense oligonucleotide molecular beacons, able to generate a fluorescent signal when they hybridize with their target mRNA, may represent an innovative strategy that conjugates the ability of sensing specific mRNA with the pharmacological silencing activity, preventing the overexpression of proteins associated to cancer development. In cancer context, this approach minimizes the non-specific toxicity and addresses the therapy mainly towards the tumor cells by using effective delivery systems. The aim of this work was to investigate the ability of polymethylmethacrylate nanoparticles (PMMA-NPs) to act as carrier of a theranostic agent, an oligonucleotide molecular beacon (MB) targeting survivin mRNA in A549 human lung adenocarcinoma epithelial cells. Moreover, this paper highlights the need for having an appropriate healthy control in in-vitro experiments. This is a problem widely discussed and felt by the scientific community and often represents a limit recognized in many experimental approaches. In particular, the survivin-MB was firstly characterized in solution in order to verify its functionality and then the PMMA-NPs ability to promote the MB internalization was verified in A549 cells by confocal microscopy. Confluent Human Dermal Fibroblasts from adult (HDFa) were used as healthy control. The results obtained allow us to assess that PMMA-NPs promote the survivin-MB cellular up-take and that the use of 10 μg/mL PMMA-NPs as carrier for survivin-MB for 1h 30 mins might be a promising strategy to reduce cancer cell proliferation avoiding detectable consequences on the healthy cells.