Nanoparticles of Cs0.33WO3 as Antibiofilm Agents and Photothermal Treatment to Inhibit Biofilm Formation.

Metal oxide nanoparticles with photothermal properties have attracted considerable research attention for their use in biomedical applications. Cesium tungsten oxide (Cs0.33WO3) nanoparticles (NPs) exhibit strong absorption in the NIR region due to localized surface plasmon resonance, through which they convert light to heat; hence, they can be applied to photothermal treatment for bacteria and biofilm ablation. Herein, Cs0.33WO3 NPs were synthesized through solid-phase synthesis, and their physical properties were characterized through Zetasizer, energy dispersive X-ray spectroscopy, Fourier transform infrared spectrometer, and scanning and transmission electron microscopy (SEM and TEM, respectively). Burkholderia cenocepacia isolates were cultured in tryptic soy broth supplemented with glucose, and the biofilm inhibition and antibiofilm effects of the NPs were determined using a crystal violet assay and the Cell Counting Kit-8 (CCK-8) assay. The biofilm morphology and viability of NP-treated cultures after NIR irradiation were evaluated through SEM and confocal microscopy, respectively. The cytotoxicity of NPs to human macrophages was also assessed using the CCK-8 assay. The NPs effectively inhibited biofilm formation, with a formation rate of <10% and a viability rate of <50% at the concentration of ≥200 µg/mL. The confocal analysis revealed that NIR irradiation markedly enhanced biofilm cytotoxicity after treatment with the NPs. The assay of cytotoxicity to human macrophages demonstrated the biocompatibility of the NPs and NIR irradiation. In sum, the Cs0.33WO3 NPs displayed effective biofilm inhibition and antibiofilm activity at 200 µg/mL treatment concentration; they exhibited an enhancement effect under the NIR irradiation, suggesting Cs0.33WO3 NPs are a potential candidate agent for NIR-irradiated photothermal treatment in bacterial biofilm inhibition and antibiofilm.

Evolution of Detecting Early Onset of Alzheimer's Disease: From Neuroimaging to Optical Immunoassays.

Alzheimer's disease (AD) is a pathological disorder defined by the symptoms of memory loss and deterioration of cognitive abilities over time. Although the etiology is complex, it is mainly associated with the accumulation of toxic amyloid-ß peptide (Aß) aggregates and tau protein-induced neurofibrillary tangles (NFTs). Even now, creating non-invasive, sensitive, specific, and cost-effective diagnostic methods for AD remains challenging. Over the past few decades, polymers, and nanomaterials (e.g., nanodiamonds, nanogold, quantum dots) have become attractive and practical tools in nanomedicine for diagnosis and treatment. This review focuses on current developments in sensing methods such as enzyme-linked immunosorbent assay (ELISA) and surface-enhanced Raman scattering (SERS) to boost the sensitivity in detecting related biomarkers for AD. In addition, optical analysis platforms such as ELISA and SERS have found increasing popularity among researchers due to their excellent sensitivity and specificity, which may go as low as the femtomolar range. While ELISA offers easy technological usage and high throughput, SERS has the advantages of improved mobility, simple electrical equipment integration, and lower cost. Both portable optical sensing techniques are highly superior in terms of sensitivity, specificity, human application, and practicality, enabling the early identification of AD biomarkers.

Phototoxicity effects of NIR-irradiated cesium tungsten oxide (Cs0.33WO3) nanoparticles on zebrafish embryos: A direct immersion study.

Nanoparticles (NPs) with feature sizes ranging between 1 nm and 100 nm have increasingly gained momentum for their versatile functionality as the pharmaceutical agents in many branches of biomedical research and clinical experiments. However, NPs' inherent material toxicity and the concomitant adverse effects of their function, such as photo-physical properties, often remain a major concern over the issues of environmental safety and human health, and require a thorough assessment before a wide-spread usage can be complied. This research herein investigates the intrinsic and photothermal toxicity of Cs0.33WO3 NPs solution in zebrafish larvae through a direct immersion method. Experimentally, the survival, hatching and malformation rates of zebrafish embryo/larvae as functions of the NP feature sizes, concentration and duration of photothermal dose were examined and analyzed. This study verified that the Cs0.33WO3 NPs has an intrinsic toxicity on a scale of a fraction of 1 mg/ml, and the phototoxicity effect of the NIR-irradiated NPs, when irradiated for 30 min, can affect the embryogenesis of zebrafish larvae and causes 60% and 50% in the survival and delayed hatching rates, respectively, as well as a severe malformation.

Monitoring adaptation of skin tissue oxygenation during cycling ergometer exercise by frequency-domain diffuse optical spectroscopy.

In addition to supplying oxygen molecule O2 for metabolic functions during the adaptation to exercise, blood also plays a critical role in heat dissipation for core temperature stabilization. This study investigates the status of hemodynamic oxygenation in the forearm's skin tissue of three participants during a complete ergometer exercise from the resting to exercising, and to recovering conditions using a three-wavelength frequency-domain diffuse reflectance spectroscopy (FD DRS) alongside the monitoring of heartbeat rate and skin temperature. The FD DRS system was synchronized with radiofrequency (RF)-modulated input photon sources and the respective output to extract time-course absorption and scattering coefficients of the skin tissue, which, through the fitting of lambert's law of absorbance, can be used to determine the concentration of oxygenated/deoxygenated hemoglobin molecules, and consequentially, the oxygen saturation of skin tissue and total hemoglobin (THb) concentration. Expressly, a sudden jump in heartbeat rate at the beginning of the exercise, a temporal lag of the rising edge of skin temperature behind that of the THb concentration in the procession of step-wise incremental working intensity, and the uprising of THb in the exhaustion zone in responses to the physiological adaptation to exercise were identified. Finally, conclusive remarks were drawn that the FD DRS system is useful in extracting the hemodynamic properties of forearm skin which is often being neglected in previous exercise physiology studies by DRS-related techniques. The detailed variation of hemodynamic and optical scattering parameters of forearm skin elucidated in the studies can be applied for the analysis of athletes' physiological status, and may be a potential reference for the design of future wearable devices.

Dependence of the Nanoscale Composite Morphology of Fe3O4 Nanoparticle-Infused Lysozyme Amyloid Fibrils on Timing of Infusion: A Combined SAXS and AFM Study.

Understanding the formation process and the spatial distribution of nanoparticle (NP) clusters on amyloid fibrils is an essential step for the development of NP-based methods to inhibit aggregation of amyloidal proteins or reverse the assembling trend of the proto-fibrillary complexes that prompts pathogenesis of neuro degeneration. For this, a detailed structural determination of the diverse hybrid assemblies that are forming is needed, which can be achieved by advanced X-ray scattering techniques. Using a combined solution small angle X-ray scattering (SAXS) and atomic force microscopy (AFM) approach, this study investigates the intrinsic trends of the interaction between lysozyme amyloid fibrils (LAFs) and Fe3O4 NPs before and after fibrillization at nanometer resolution. AFM images reveal that the number of NP clusters interacting with the lysozyme fibers does not increase significantly with NP volume concentration, suggesting a saturation in NP aggregation on the fibrillary surface. The data indicate that the number of non-adsorbed Fe3O4 NPs is highly dependent on the timing of NP infusion within the synthesis process. SAXS data yield access to the spatial distribution, aggregation manner and density of NP clusters on the fibrillary surfaces. Employing modern data analysis approaches, the shape and internal structural morphology of the so formed nanocomposites are revealed. The combined experimental approach suggests that while Fe3O4 NPs infusion does not prevent the fibril-formation, the variation of NP concentration and size at different stages of the fibrillization process can impose a pronounced impact on the superficial and internal structural morphologies of these nanocomposites. These findings may be applicable in devising advanced therapeutic treatments for neurodegenerative diseases and designing novel bio-inorganic magnetic devices. Our results further demonstrate that modern X-ray methods give access to the structure of-and insight into the formation process of-biological-inorganic hybrid structures in solution.

Devising Hyperthermia Dose of NIR-Irradiated Cs0.33WO3 Nanoparticles for HepG2 Hepatic Cancer Cells.

Hyperthermia is one of the most patient-friendly methods to cure cancer diseases owing to its noninvasiveness, minimally induced side-effects and toxicity, and easy implementation, prompting the development of novel therapeutic methods like photothermally triggering dose system. This research herein interrogates the variables of photothermal effects of Cs0.33WO3 nanoparticles (NPs), the duration of irradiation, optical power density and NP concentration, upon HepG2 liver cancer cell line in vitro, leading to the formulation of a near-infrared (NIR)-irradiated thermal dose. Expressly, the NPs with particulate feature sizes of 120 nm were synthesized through a series of oxidation-reduction (REDOX) reaction, thermal annealing and wet-grinding processes, and the subsequent characterization of physical, compositional, optical, photothermal properties were examined using dynamic light scattering (DLS), energy-dispersive X-ray spectroscopy (EDS), scanning and tunneling electron microscopies (SEM and TEM), X-ray diffraction (XRD) and visible-near-infrared (VIS-NIR) photospectroscopy. Cytotoxicity of the NPs and its irradiation parameters were obtained for the HepG2 cells. By incubating the cells with the NPs, the state of endocytosis was verified, and the dependence of cellular survival rate on the variable parameters of photothermal dose was determined while maintaining the medium temperature of the cell-containing culture dish at human body temperature around 36.5 °C.

Hyperthermia Induced by Near-Infrared Laser-Irradiated CsWO3 Nanoparticles Disintegrates Preformed Lysozyme Amyloid Fibrils.

This research study attempts to prove the concept of the applicability of hyperthermia to treating the lysozyme amyloid fibrils (LAF)'s self-assembled fibrillary aggregates by a feedback-modulated temperature controller ranging from 26 °C to 80 °C, and separately, by near-infrared (NIR) laser-irradiated cesium tungstate (CsWO3) nanoparticle (NPs). The dependence of the final morphology of the amyloidal assembly on external heating and the photothermal effect of the NPs on treating the fibrillary assembly were investigated and analyzed. Experimentally, atomic force microscopy (AFM), optical stereoscopy, and scanning electron microscopy (SEM) were used primarily to ensure mutual interaction between LAF and NPs, optically elucidate the surface contour and final fibrillary assembly upon the influence of thermal treatment, and further reveal fine-details of the optical samples. Finally, conclusive remarks are drawn that the fibrillary structures doped with the NPs exhibit an increasing degree of unique orthogonality. As the temperature rises, utter deformation of the dendritic structures of fibrillary assemblies at 70 °C was found, and NIR laser-irradiated CsWO3 NPs have been demonstrated to be useful in topically destructing pre-assembled LAFs, which may be conducive to the future development of neurodegenerative therapeutic techniques.

Effect of the concentration of protein and nanoparticles on the structure of biohybrid nanocomposites.

We present colloidal nanocomposites formed by incorporating magnetite Fe3 O4 nanoparticles (MNPs) with lysozyme amyloid fibrils (LAFs). Preparation of two types of solutions, with and without addition of salt, was carried out to elucidate the structure of MNPs-incorporated fibrillary nanocomposites and to study the effect of the presence of salt on the stability of the nanocomposites. The structural morphology of the LAFs and their interaction with MNPs were analyzed by atomic force microscopy and small-angle x-ray scattering measurements. The results indicate that conformational properties of the fibrils are dependent on the concentration of protein, and the precise ratio of the concentration of the protein and MNPs is crucially important for the stability of the fibrillary nanocomposites. Our results confirm that despite the change in fibrillary morphology induced by the varying concentration of the protein, the adsorption of MNPs on the surface of LAF is morphologically independent. Moreover, most importantly, the samples containing salt have excellent stability for up to 1 year of shelf-life.

Dual Size-Dependent Effect of Fe3O4 Magnetic Nanoparticles Upon Interaction with Lysozyme Amyloid Fibrils: Disintegration and Adsorption.

Nanomedicine compounds containing nanoparticles, such as iron oxides and gold, have been demonstrated to be effective in promoting different magnitudes of interaction with amyloid ß fibrils, of which disintegrating or inhibiting effects are of great importance to treating fibrillary aggregation-induced neurological disorders such as Alzheimer's disease. This research herein studies the interaction between lysozyme amyloid fibrils, a type of fibers derived from hen egg white lysozyme, and Fe3O4 magnetic nanoparticles (MNPs) of an assorted diameter sizes of 5 nm, 10 nm and 20 nm, using atomic force microscopy (AFM). Specifically, the effects of the sizes of negatively charged MNPs on the resultant amyloid fibrillary mixture was investigated. Our results of AFM images indicated that the interaction between MNPs and the fibrils commences immediately after adding MNPs to the fibril solution, and the actions of such MNPs-doped fibrillary interplay, either integration or segmentation, is strongly dependent on the size and volume concentration of MNPs. In the cases of 5 nm and 20 nm particles of equivalent volume concentration, the adsorption and agglomeration of MNPs onto the fibrillary surfaces was observed, whereas, interestingly, MNPs with diameter size of 10 nm enables segmentation of the slender fibrils into debris when a proper implemented volume concentration was found, which signifies utter destruction of the amyloid fibrillary structure.

Dynamic morphogenesis of dendritic structures formation in hen egg white lysozyme fibrils doped with magnetic nanoparticles.

In this research, the dynamic process of aggregation that forms microflower morphology in solution of lysozyme amyloid fibrils doped with spherical or spindle-like magnetic nanoparticles during the process of drying as well as their final microstructures were investigated. The prepared lysozyme amyloid fibrils as well as their mixtures with in-lab synthesized magnetic particles, which were prepared by adding the nanoparticles to the fibrils solution after the process of fibrillation was done, were characterized using brightfield trans-illumination-mode optical microscope, atomic force microscopy (AFM) and scanning electron microscope (SEM). Brightfield optical imaging bases upon photoabsorptive property of the fibrils-nanoparticle composites clearly reveals the morphological features in microscale, and additionally, for the in vivo, live action of the time-dependent process of self-assembly of such composites composed of fibrillary structure incorporated with magnetic particles was optically elucidated at ambient temperature. Moreover, while results of AFM reveal delicate and peculiar association of fibrils with magnetic nanoparticles of different shapes, SEM images illustrate a stark difference in fine detailed final morphology of microstructures associated with spherical and spindle-like nanoparticles. Our results indicated that the interaction between fibrils solution and the nanoparticles commence right after mixing, the dynamic process of forming dendritic structure resembling microflower morphology is on the order of minutes, and its final structure is highly dependent on the shape of magnetic nanoparticles.

Zno Micro/Nanostructures Grown on Sapphire Substrates Using Low-Temperature Vapor-Trapped Thermal Chemical Vapor Deposition: Structural and Optical Properties.

In this research, the Zn(C5H7O2)2·xH2O-based growth of ZnO micro/nanostructures in a low temperature, vapor-trapped chemical vapor deposition system was attempted to optimize structural and optical properties for potential biomedical applications. By trapping in-flow gas molecules and Zinc vapor inside a chamber tube by partially obstructing a chamber outlet, a high pressure condition can be achieved, and this experimental setup has the advantages of ease of synthesis, being a low temperature process, and cost effectiveness. Empirically, the growth process proceeded under a chamber condition of an atmospheric pressure of 730 torr, a controlled volume flow rate of input gas, N2/O2, of 500/500 Standard Cubic Centimeters per Minute (SCCM), and a designated oven temperature of 500 °C. Specifically, the dependence of structural and optical properties of the structures on growth duration and spatially dependent temperature were investigated utilizing scanning electron microscopy, X-ray diffraction (XRD), photoluminescence (PL), and ultraviolet-visible transmission spectroscopy. The experimental results indicate that the grown thin film observed with hexagonal structures and higher structural uniformity enables more prominent structural and optical signatures. XRD spectra present the dominant peaks along crystal planes of (002) and (101) as the main direction of crystallization. In addition, while the structures excited with laser wavelength of 325 nm emit a signature radiation around 380 nm, an ultraviolet lamp with a wavelength of 254 nm revealed distinctive photoluminescence peaks at 363.96 nm and 403.52 nm, elucidating different degrees of structural correlation as functions of growth duration and the spatial gradient of temperature. Transmittance spectra of the structures illustrate typical variation in the wavelength range of 200 nm to 400 nm, and its structural correlation is less significant when compared with PL.

Characterizing the morphologic changes in collagen crosslinked-treated corneas by Fourier transform-second harmonic generation imaging.

PURPOSE: To evaluate the efficacy of using forward second harmonic generation (SHG) and 2-dimensional fast Fourier-transform (2D-FFT) analysis for the label-free characterization and quantification of morphologic changes in the corneal stroma after collagen crosslinking (CXL). SETTING: Department of Physics, National Taiwan University, Taipei, Taiwan. DESIGN: Experimental study. METHODS: En face forward SHG imaging and 2D-FFT analysis were performed on ex vivo porcine corneas at the depths of 100, 200, 400, and 800 µm. Morphologic changes in stromal collagen fiber in control, ultraviolet-A (UVA), riboflavin, and riboflavin-UVA treated porcine corneas were assessed. Hematoxylin-eosin staining and Sirius red staining were performed for comparison. RESULTS: Corneas after CXL treatment tended to have collagen fibers that were wavy compared with the linear pattern in normal corneas. Quantitative 2D-FFT analysis of forward SHG images also showed an increase in the standard deviations of the distribution of stromal collagen fiber orientations, which is indicative of the changed pattern of crosslinked stromal collagen fibers. CONCLUSIONS: Second harmonic generation imaging showed the morphologic changes in stromal collagen after CXL treatment. The linear collagen fibers in normal corneal stroma became wavy after treatment. With the introduction of 2D-FFT analysis, the morphologic changes can be quantified.

Elucidation of the mechanisms of optical clearing in collagen tissue with multiphoton imaging.

Optical clearing (OC) is a promising method to overcome limitations in biomedical depth-resolved optical studies. Mechanisms of OC in purified bovine Achilles tendon, chicken skin, and chicken tendon were studied using time-lapsed, three-dimensional second harmonic generation (SHG) and two-photon fluorescence microscopic imaging. Quantified nonlinear optical measurements allowed temporal separation of two processes in collagen OC with glycerol. The first one is a fast process of tissue dehydration accompanied with collagen shrinkage and the second relatively slow process is glycerol penetration into the interfibrillar space of collagen alongside with CF swelling. The use of 50% glycerol induced less-expressed OC via partial substitution of water molecules with glycerol molecules. We also found that phosphate-buffered saline- and glycerol-treatments were reversible, and fiber morphology and SHG signal intensity were recovered after the removal of immersion agents. It was shown that tissue OC was a dynamic process and elucidation of its physical mechanisms may help choose optimal diagnostic, treatment, and modification regimes for collagen-based as well as other types of biomaterials.

Imaging of biological tissues with pixel-level analysis of second-order susceptibility.

We discuss the recent advances in the development and applications of second-order susceptibility as a contrast mechanism in optical microscopy for biological tissues. We review nonlinear optical methods and approaches for differentiation of tissue structures and discrimination of normal and pathological skin tissues, which have been demonstrated for the potential use in clinical diagnosis. In addition, the potential of second-order susceptibility imaging, encompassing applications in differentiating various types of collagen molecules for clinical diagnosis, is demonstrated. Finally, we discuss future development and application of this technique.

Quantitative analysis of multiphoton excitation autofluorescence and second harmonic generation imaging for medical diagnosis.

In recent years, two-photon excitation fluorescence and second harmonic generation microscopy has become an important tool in biomedical research. The ability of two-photon microscopy to achieve optical sectioning with minimal invasiveness is particularly advantageous for biomedical diagnosis. Advances in the miniaturization of the imaging system have increased its clinical potential, together with the development of quantitative technique for the analysis of data acquired using these imaging modalities. We present a review of the quantitative analysis techniques that have been used successfully with two-photon excitation fluorescence and SHG imaging. Specifically, quantification techniques using ratiometric, morphological, and structural differences to analyze two-photon images will be discussed, and their effectiveness at evaluating dermal and corneal pathologies and cancerous tumor growth will be described.

Spatial orientation mapping of fibers using polarization-sensitive second harmonic generation microscopy.

In this work, we present a non-invasive approach to determine azimuth and elevation angles of collagen fibers capable of generating second harmonic signal. The azimuth angle was determined using the minimum of second harmonic generation (SHG) signal while rotating the plane of polarization of excitation light. The elevation angle was estimated from the ratio of the minimal SHG intensity to the intensity when laser polarization and fiber directions were parallel to each other using experimentally determined calibration curve. Pixel-resolution images of collagen fiber spatial orientation in tendon from bovine leg, chicken leg, and chicken skin were acquired using our approach of SHG polarization-resolved microscopy.

Spatial and temporal analysis of skin glycation by the use of multiphoton microscopy and spectroscopy.

BACKGROUND: Tissue glycation, the main cause of many diabetes-related complications, results in the accumulation of advanced glycation endproducts (AGE). OBJECTIVES: These AGEs are endogenous fluorophores that can serve as a viable pathological indicator for disease diagnostics. Here we explore the capabilities of multiphoton microscopy to non-invasively localize and quantify the skin glycation. METHODS: In our study, multiphoton microscopy and spectroscopy were used to investigate glycation events-induced changes in the intensities of autofluorescence and second harmonic generation on ex vivo human skin. RESULTS: Temporal and spatial dependence of degrees of glycation of the epidermis, collagen and elastin fibers of dermis were evaluated for their relevance to the changes in amplitudes of autofluorescence signals. We found that glycation drastically and linearly increases multiphoton autofluorescence intensity of epidermis and dermal collagen whereas changes in dermal elastin are moderate. We also found decrease in the level of second harmonic generation signal. CONCLUSION: Our study suggests that due to intrinsically weak autofluorescence the dermal collagen is the most sensitive skin tissue to be used for detecting changes in tissue glycation.