Cyclic Stretch-Induced Mechanical Stress Applied at 1 Hz Frequency Can Alter the Metastatic Potential Properties of SAOS-2 Osteosarcoma Cells.

Recently, there has been an increasing focus on cellular morphology and mechanical behavior in order to gain a better understanding of the modulation of cell malignancy. This study used uniaxial-stretching technology to select a mechanical regimen able to elevate SAOS-2 cell migration, which is crucial in osteosarcoma cell pathology. Using confocal and atomic force microscopy, we demonstrated that a 24 h 0.5% cyclic elongation applied at 1 Hz induces morphological changes in cells. Following mechanical stimulation, the cell area enlarged, developing a more elongated shape, which disrupted the initial nuclear-to-cytoplasm ratio. The peripheral cell surface also increased its roughness. Cell-based biochemical assays and real-time PCR quantification showed that these morphologically induced changes are unrelated to the osteoblastic differentiative grade. Interestingly, two essential cell-motility properties in the modulation of the metastatic process changed following the 24 h 1 Hz mechanical stimulation. These were cell adhesion and cell migration, which, in fact, were dampened and enhanced, respectively. Notably, our results showed that the stretch-induced up-regulation of cell motility occurs through a mechanism that does not depend on matrix metalloproteinase (MMP) activity, while the inhibition of ion-stretch channels could counteract it. Overall, our results suggest that further research on mechanobiology could represent an alternative approach for the identification of novel molecular targets of osteosarcoma cell malignancy.

Conjugates of Gold Nanoparticles and Antitumor Gold(III) Complexes as a Tool for Their AFM and SERS Detection in Biological Tissue.

Citrate-capped gold nanoparticles (AuNPs) were functionalized with three distinct antitumor gold(III) complexes, e.g., [Au(N,N)(OH)2][PF6], where (N,N)=2,2'-bipyridine; [Au(C,N)(AcO)2], where (C,N)=deprotonated 6-(1,1-dimethylbenzyl)-pyridine; [Au(C,N,N)(OH)][PF6], where (C,N,N)=deprotonated 6-(1,1-dimethylbenzyl)-2,2'-bipyridine, to assess the chance of tracking their subcellular distribution by atomic force microscopy (AFM), and surface enhanced Raman spectroscopy (SERS) techniques. An extensive physicochemical characterization of the formed conjugates was, thus, carried out by applying a variety of methods (density functional theory-DFT, UV/Vis spectrophotometry, AFM, Raman spectroscopy, and SERS). The resulting gold(III) complexes/AuNPs conjugates turned out to be pretty stable. Interestingly, they exhibited a dramatically increased resonance intensity in the Raman spectra induced by AuNPs. For testing the use of the functionalized AuNPs for biosensing, their distribution in the nuclear, cytosolic, and membrane cell fractions obtained from human lymphocytes was investigated by AFM and SERS. The conjugates were detected in the membrane and nuclear cell fractions but not in the cytosol. The AFM method confirmed that conjugates induced changes in the morphology and nanostructure of the membrane and nuclear fractions. The obtained results point out that the conjugates formed between AuNPs and gold(III) complexes may be used as a tool for tracking metallodrug distribution in the different cell fractions.

Detection and localization of gold nanoshells inside cells: near-field approximation.

The optical properties of metal nanoparticles play a fundamental role for their use in a wide range of applications. In hyperthermia treatment, for example, gold nanoshells (NSs, dielectric core+gold shell) pre-embedded in a cancer cell absorb energy when exposed to appropriate wavelengths of a laser beam and heat up, thereby destroying the cancer cell. In this process, nevertheless, healthy tissues (not targeted by the NSs) along the laser path are not affected; this is because most biological soft tissues have a relatively low light absorption coefficient in the near-infrared (NIR) regions-a characteristic known as the tissue optical window. Over such a window, NIR light transmits through the tissues with scattering-limited attenuation and minimal heating, thereby avoiding damage to healthy tissues. As a consequence, the identification of NSs assumed a fundamental role for the further development of such cancer treatment. Recently, we have demonstrated the possibility to identify 100-150 nm diameter gold NSs inside mouse cells using a scanning near-optical microscope (SNOM). In this paper, we provide a numerical demonstration that the SNOM is able to locate NSs inside the cell with a particle-aperture distance of about 100 nm. This result was obtained by developing an analytical approach based on the calculation of the dyadic Green function in the near-field approximation. The implications of our findings will remarkably affect further investigations on the interaction between NSs and biological systems.

Active stabilization of a pseudoheterodyne scattering scanning near field optical microscope.

Scattering scanning near-field optical microscopes (s-SNOMs) based on pseudoheterodyne detection and operating at ambient conditions typically suffer from instabilities related to the variable optical path length of the interferometer arms. These cause strong oscillations in the measured optical amplitude and phase comparable with those of the signal and, thus, resulting in dramatic artifacts. Besides hampering the comparison between the topography and the optical measurements, such oscillations may lead to misinterpretations of the physical phenomena occurring at the sample surface, especially for nanostructured materials. Here, we propose a stabilizing method based on interferometer phase control, which improves substantially the image quality and allows the correct extraction of optical phase and amplitude for both micro- and nanostructures. This stabilization method expands the measurement capabilities of s-SNOM to any slowly time-dependent phenomena that require long-term stability of the system. We envisage that active stabilization will increase the technological significance of s-SNOMs and will have far-reaching applications in the field of heat transfer and nanoelectronics.

Semitransparent Perovskite Solar Cells with Ultrathin Protective Buffer Layers.

Semitransparent perovskite solar cells (ST-PSCs) are increasingly important in a range of applications, including top cells in tandem devices and see-through photovoltaics. Transparent conductive oxides (TCOs) are commonly used as transparent electrodes, with sputtering being the preferred deposition method. However, this process can damage exposed layers, affecting the electrical performance of the devices. In this study, an indium tin oxide (ITO) deposition process that effectively suppresses sputtering damage was developed using a transition metal oxides (TMOs)-based buffer layer. An ultrathin (<10 nm) layer of evaporated vanadium oxide or molybdenum oxide was found to be effective in protecting against sputtering damage in ST-PSCs for tandem applications, as well as in thin perovskite-based devices for building-integrated photovoltaics. The identification of minimal parasitic absorption, the high work function and the analysis of oxygen vacancies denoted that the TMO layers are suitable for use in ST-PSCs. The highest fill factor (FF) achieved was 76%, and the efficiency (16.4%) was reduced by less than 10% when compared with the efficiency of gold-based PSCs. Moreover, up-scaling to 1 cm2-large area ST-PSCs with the buffer layer was successfully demonstrated with an FF of ∼70% and an efficiency of 15.7%. Comparing the two TMOs, the ST-PSC with an ultrathin V2Ox layer was slightly less efficient than that with MoOx, but its superior transmittance in the near infrared and greater light-soaking stability (a T80 of 600 h for V2Ox compared to a T80 of 12 h for MoOx) make V2Ox a promising buffer layer for preventing ITO sputtering damage in ST-PSCs.

Oxidized Alginate Dopamine Conjugate: A Study to Gain Insight into Cell/Particle Interactions.

Background: We had previously synthetized a macromolecular prodrug consisting of oxidized Alginate and dopamine (AlgOx-Da) for a potential application in Parkinson disease (PD). Methods: In the present work, we aimed at gaining an insight into the interactions occurring between AlgOx-Da and SH-SY5Y neuronal cell lines in view of further studies oriented towards PD treatment. With the scope of ascertaining changes in the external and internal structure of the cells, multiple methodologies were adopted. Firstly, fluorescently labeled AlgOx-Da conjugate was synthetized in the presence of fluorescein 5(6)-isothiocyanate (FITC), providing FITC-AlgOx-Da, which did not alter SH-SY5Y cell viability according to the sulforhodamine B test. Furthermore, the uptake of FITC-AlgOx-Da by the SH-SY5Y cells was studied using scanning near-field optical microscopy and assessments of cell morphology over time were carried out using atomic force microscopy. Results: Notably, the AFM methodology confirmed that no relevant damage occurred to the neuronal cells. Regarding the effects of DA on the intracellular reactive oxygen species (ROS) production, AlgOx-Da reduced them in comparison to free DA, while AlgOx did almost not influence ROS production. Conclusions: these findings seem promising for designing in vivo studies aiming at administering Oxidized Alginate Dopamine Conjugate for PD treatment.

Oxidized Alginate Dopamine Conjugate: In Vitro Characterization for Nose-to-Brain Delivery Application.

BACKGROUND: The blood-brain barrier (BBB) bypass of dopamine (DA) is still a challenge for supplying it to the neurons of Substantia Nigra mainly affected by Parkinson disease. DA prodrugs have been studied to cross the BBB, overcoming the limitations of DA hydrophilicity. Therefore, the aim of this work is the synthesis and preliminary characterization of an oxidized alginate-dopamine (AlgOX-DA) conjugate conceived for DA nose-to-brain delivery. METHODS: A Schiff base was designed to connect oxidized polymeric backbone to DA and both AlgOX and AlgOX-DA were characterized in terms of Raman, XPS, FT-IR, and 1H- NMR spectroscopies, as well as in vitro mucoadhesive and release tests. RESULTS: Data demonstrated that AlgOX-DA was the most mucoadhesive material among the tested ones and it released the neurotransmitter in simulated nasal fluid and in low amounts in phosphate buffer saline. Results also demonstrated the capability of scanning near-field optical microscopy to study the structural and fluorescence properties of AlgOX, fluorescently labeled with fluorescein isothiocyanate microstructures. Interestingly, in SH-SY5Y neuroblastoma cell line up to 100 µg/mL, no toxic effect was derived from AlgOX and AlgOX-DA in 24 h. CONCLUSIONS: Overall, the in vitro performances of AlgOX and AlgOX-DA conjugates seem to encourage further ex vivo and in vivo studies in view of nose-to-brain administration.

Tissue degeneration in ALS affected spinal cord evaluated by Raman spectroscopy.

The Raman spectral features from spinal cord tissue sections of transgenic, ALS model mice and non-transgenic mice were compared using 457 nm excitation line, profiting from the favourable signal intensity obtained in the molecular fingerprint region at this wavelength. Transverse sections from four SOD1G93A mice at 75 days and from two at 90 days after birth were analysed and compared with sections of similarly aged control mice. The spectra acquired within the grey matter of tissue sections from the diseased mice is markedly different from the grey matter signature of healthy mice. In particular, we observe an intensity increase in the spectral windows 450-650 cm-1 and 1050-1200 cm-1, accompanied by an intensity decrease in the lipid contributions at ~1660 cm-1, ~1440 cm-1 and ~1300 cm-1. Axons demyelination, loss of lipid structural order and the proliferation and aggregation of branched proteoglycans are related to the observed spectral modifications. Furthermore, the grey and white matter components of the spinal cord sections could also be spectrally distinguished, based on the relative intensity of characteristic lipid and protein bands. Raman spectra acquired from the white matter regions of the SOD1G93A mice closely resembles those from control mice.

An imaging dataset of cervical cells using scanning near-field optical microscopy coupled to an infrared free electron laser.

Using a scanning near-field optical microscope coupled to an infrared free electron laser (SNOM-IR-FEL) in low-resolution transmission mode, we collected chemical data from whole cervical cells obtained from 5 pre-menopausal, non-pregnant women of reproductive age, and cytologically classified as normal or with different grades of cervical cell dyskaryosis. Imaging data are complemented by demography. All samples were collected before any treatment. Spectra were also collected using attenuated total reflection, Fourier-transform (ATR-FTIR) spectroscopy, to investigate the differences between the two techniques. Results of this pilot study suggests SNOM-IR-FEL may be able to distinguish cervical abnormalities based upon changes in the chemical profiles for each grade of dyskaryosis at designated wavelengths associated with DNA, Amide I/II, and lipids. The novel data sets are the first collected using SNOM-IR-FEL in transmission mode at the ALICE facility (UK), and obtained using whole cells as opposed to tissue sections, thus providing an 'intact' chemical profile. These data sets are suited to complementing future work on image analysis, and/or applying the newly developed algorithm to other datasets collected using the SNOM-IR-FEL approach.

Imaging cervical cytology with scanning near-field optical microscopy (SNOM) coupled with an IR-FEL.

Cervical cancer remains a major cause of morbidity and mortality among women, especially in the developing world. Increased synthesis of proteins, lipids and nucleic acids is a pre-condition for the rapid proliferation of cancer cells. We show that scanning near-field optical microscopy, in combination with an infrared free electron laser (SNOM-IR-FEL), is able to distinguish between normal and squamous low-grade and high-grade dyskaryosis, and between normal and mixed squamous/glandular pre-invasive and adenocarcinoma cervical lesions, at designated wavelengths associated with DNA, Amide I/II and lipids. These findings evidence the promise of the SNOM-IR-FEL technique in obtaining chemical information relevant to the detection of cervical cell abnormalities and cancer diagnosis at spatial resolutions below the diffraction limit (≥0.2 µm). We compare these results with analyses following attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy; although this latter approach has been demonstrated to detect underlying cervical atypia missed by conventional cytology, it is limited by a spatial resolution of ~3 µm to 30 µm due to the optical diffraction limit.

ELF non ionizing radiation changes the distribution of the inner chemical functional groups in human epithelial cell (HaCaT) culture.

Human skin cell culture (HaCaT) that has been exposed to an AC magnetic field undergoes detectable changes in its biochemical properties and shapes. Such changes were observed by infrared wavelength-selective scanning near-field optical microscopy with a resolution of 80-100 nm. We specifically investigated the changes in the distribution of the inner chemical functional groups and in the cell morphology induced by a 24 h exposure to a 1 mT (rms), 50 Hz sinusoidal magnetic field in a temperature regulated solenoid. These results further accentuate the crucial questions, raised by several recent studies, about the impact of low-frequency electromagnetic field on human cells.

Chemically resolved imaging of biological cells and thin films by infrared scanning near-field optical microscopy.

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths (lambda) corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 micro m ( approximately lambda/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research.