Encapsulated Rose Bengal Enhances the Photodynamic Treatment of Triple-Negative Breast Cancer Cells.

Among breast cancer subtypes, triple-negative breast cancer stands out as the most aggressive, with patients facing a 40% mortality rate within the initial five years. The limited treatment options and unfavourable prognosis for triple-negative patients necessitate the development of novel therapeutic strategies. Photodynamic therapy (PDT) is an alternative treatment that can effectively target triple-negative neoplastic cells such as MDA-MB-231. In this in vitro study, we conducted a comparative analysis of the PDT killing rate of unbound Rose Bengal (RB) in solution versus RB-encapsulated chitosan nanoparticles to determine the most effective approach for inducing cytotoxicity at low laser powers (90 mW, 50 mW, 25 mW and 10 mW) and RB concentrations (50 µg/mL, 25 µg/mL, 10 µg/mL and 5 µg/mL). Intracellular singlet oxygen production and cell uptake were also determined for both treatment modalities. Dark toxicity was also assessed for normal breast cells. Despite the low laser power and concentration of nanoparticles (10 mW and 5 µg/mL), MDA-MB-231 cells experienced a substantial reduction in viability (8 ± 1%) compared to those treated with RB solution (38 ± 10%). RB nanoparticles demonstrated higher singlet oxygen production and greater uptake by cancer cells than RB solutions. Moreover, RB nanoparticles display strong cytocompatibility with normal breast cells (MCF-10A). The low activation threshold may be a crucial advantage for specifically targeting malignant cells in deep tissues.

The fate of organic species upon sintering of thiol-stabilised gold nanoparticles under different atmospheric conditions.

Understanding and controlling the sintering behavior of gold nanoparticles is important for applications such as printed electronics, catalysis and sensing that utilise these materials. Here we examine the processes by which thiol-protected gold nanoparticles thermally sinter under a variety of atmospheres. We find that upon sintering, the surface-bound thiyl ligands exclusively form the corresponding disulfide species when released from the gold surface. Experiments conducted using air, hydrogen, nitrogen, or argon atmospheres revealed no significant differences between the temperatures of the sintering event nor on the composition of released organic species. When conducted under high vacuum, the sintering event occurred at lower temperatures compared to ambient pressures in cases where the resulting disulfide had relatively high volatility (dibutyl disulfide). Hexadecylthiol-stabilized particles exhibited no significant differences in the temperatures of the sintering event under ambient pressures compared to high vacuum conditions. We attribute this to the relatively low volatility of the resultant dihexadecyl disulfide product.

Photodynamic Treatment of Human Breast and Prostate Cancer Cells Using Rose Bengal-Encapsulated Nanoparticles.

Cancer, a prominent cause of death, presents treatment challenges, including high dosage requirements, drug resistance, poor tumour penetration and systemic toxicity in traditional chemotherapy. Photodynamic therapy, using photosensitizers like rose bengal (RB) with a green laser, shows promise against breast cancer cells in vitro. However, the hydrophilic RB struggles to efficiently penetrate the tumour site due to the unique clinical microenvironment, aggregating around rather than entering cancer cells. In this study, we have synthesized and characterized RB-encapsulated chitosan nanoparticles with a peak particle size of ~200 nm. These nanoparticles are readily internalized by cells and, in combination with a green laser (λ = 532 nm) killed 94-98% of cultured human breast cancer cells (MCF-7) and prostate cancer cells (PC3) at a low dosage (25 µg/mL RB-nanoparticles, fluence ~126 J/cm2, and irradiance ~0.21 W/cm2). Furthermore, these nanoparticles are not toxic to cultured human normal breast cells (MCF10A), which opens an avenue for translational applications.

Fabrication and characterization of chitosan nanoparticles using the coffee-ring effect for photodynamic therapy.

BACKGROUND AND OBJECTIVES: Biocompatible nanoparticles have been increasingly used in a variety of medical applications, including photodynamic therapy. Although the impact of synthesis parameters and purification methods is reported in previous studies, it is still challenging to produce a reliable protocol for the fabrication, purification, and characterization of nanoparticles in the 200-300 nm range that are highly monodisperse for biomedical applications. STUDY DESIGN/MATERIALS AND METHODS: We investigated the synthesis of chitosan nanoparticles in the 200-300 nm range by evaluating the chitosan to sodium tripolyphosphate (TPP) mass ratio and acetic acid concentration of the chitosan solution. Chitosan nanoparticles were also crosslinked to rose bengal and incubated with human breast cancer cells (MCF-7) to test photodynamic activity using a green laser (λ = 532 nm, power = 90 mW). RESULTS: We established a simple protocol to fabricate and purify biocompatible nanoparticles with the most frequent size occurring between 200 and 300 nm. This was achieved using a chitosan to TPP mass ratio of 5:1 in 1% v/v acetic acid at a pH of 5.5. The protocol involved the formation of nanoparticle coffee rings that showed the particle shape to be spherical in the first approximation. Photodynamic treatment with rose bengal-nanoparticles killed ~98% of cancer cells. CONCLUSION: A simple protocol was established to prepare and purify spherical and biocompatible chitosan nanoparticles with a peak size of ~200 nm. These have remarkable antitumor activity when coupled with photodynamic treatment.

Silicon deposition on guard cells increases stomatal sensitivity as mediated by K+ efflux and consequently reduces stomatal conductance.

Silicon (Si) has been widely reported to improve plant resistance to water stress via various mechanisms including cuticular Si deposition to reduce leaf transpiration. However, there is limited understanding of the effects of Si on stomatal physiology, including the underlying mechanisms and implications for resistance to water stress. We grew tall fescue (Festuca arundinacea Schreb. cv. Fortuna) hydroponically, with or without Si, and treated half of the plants with 20% polyethylene glycol to impose physiological drought (osmotic stress). Scanning electron microscopy in conjunction with X-ray mapping found that Si was deposited on stomatal guard cells and as a sub-cuticular layer in Si-treated plants. Plants grown in Si had a 28% reduction in stomatal conductance and a 23% reduction in cuticular conductance. When abscisic acid was applied exogenously to epidermal leaf peels to promote stomatal closure, Si plants had 19% lower stomatal aperture compared to control plants (i.e. increased stomatal sensitivity) and an increased efflux of guard cell K+ ions. However, the changes in stomatal physiology with Si were not substantial enough to improve water stress resistance, as shown by a lack of significant effect of Si on water potential, growth, photosynthesis and water-use efficiency. Our findings suggest a novel underlying mechanism for reduced stomatal conductance with Si application; specifically, that Si deposition on stomatal guard cells promotes greater stomatal sensitivity as mediated by guard cell K+ efflux.

Semitransparent bandages based on chitosan and extracellular matrix for photochemical tissue bonding.

BACKGROUND: Extracellular matrices (ECMs) are often used in reconstructive surgery to enhance tissue regeneration and remodeling. Sutures and staples are currently used to fix ECMs to tissue although they can be invasive devices. Other sutureless and less invasive techniques, such as photochemical tissue bonding, cannot be coupled to ECMs because of their intrinsic opacity to light. RESULTS: We succeeded in fabricating a biocompatible and adhesive device that is based on ovine forestomach matrix (OFM) and a chitosan adhesive. The natural opacity of the OFM has been overcome by adding the adhesive into the matrix that allows for the light to effectively penetrate through it. The OFM-chitosan device is semitransparent (attenuation length ~ 106 µm) and can be photoactivated by green light to bond to tissue. This device does not require sutures or staples and guarantees a bonding strength of ~ 23 kPa. CONCLUSIONS: A new semitransparent and biocompatible bandage has been successfully fabricated and characterized for sutureless tissue bonding.

Oriented epitaxial TiO2 nanowires for water splitting.

Highly oriented epitaxial rutile titanium dioxide (TiO2) nanowire arrays have been hydrothermally grown on polycrystalline TiO2 templates with their orientation dependent on the underlying TiO2 grain. Both the diameter and areal density of the nanowires were tuned by controlling the precursor concentration, and the template surface energy and roughness. Nanowire tip sharpness was influenced by precursor solubility and diffusivity. A new secondary ion mass spectrometer technique has been developed to install additional nucleation sites in single crystal TiO2 templates and the effect on nanowire growth was probed. Using the acquired TiO2 nanowire synthesis knowhow, an assortment of nanowire arrays were installed upon the surface of undoped TiO2 photo-electrodes and assessed for their photo-electrochemical water splitting performance. The key result obtained was that the presence of short and dispersed nanowire arrays significantly improved the photocurrent when the illumination intensity was increased from 100 to 200 mW cm-2. This is attributed to the alignment of the homoepitaxially grown nanowires to the [001] direction, which provides the fastest charge transport in TiO2 and an improved pathway for photo-holes to find water molecules and undertake oxidation. This result lays a foundation for achieving efficient water splitting under conditions of concentrated solar illumination.

Real-time monitoring of peptide grafting onto chitosan films using capillary electrophoresis.

Chitosan, being antimicrobial and biocompatible, is attractive as a cell growth substrate. To improve cell attachment, arginine-glycine-aspartic acid-serine (RGDS) peptides were covalently grafted to chitosan films, through the widely used coupling agents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC-HCl) and N-hydroxysuccinimide (NHS), via the carboxylic acid function of the RGDS molecule. The grafting reaction was monitored, for the first time, in real time using free-solution capillary electrophoresis (CE). This enabled fast separation and determination of the peptide and all other reactants in one separation with no sample preparation. Covalent RGDS peptide grafting onto the chitosan film surface was demonstrated using solid-state NMR of swollen films. CE indicated that oligomers of RGDS, not simply RGDS, were grafted on the film, with a likely hyperbranched structure. To assess the functional properties of the grafted films, cell growth was compared on control and peptide-grafted chitosan films. Light microscopy and polymerase chain reaction (PCR) analysis demonstrated greatly improved cell attachment to RGDS-grafted chitosan films.

Rose bengal-encapsulated chitosan nanoparticles for the photodynamic treatment of Trichophyton species.

Rose bengal (RB) solutions coupled with a green laser have proven to be efficient in clearing resilient nail infections caused by Trichophyton rubrum in a human pilot study and in extensive in vitro experiments. Nonetheless, the RB solution can become diluted or dispersed over the tissue and prevented from penetrating the nail plate to reach the subungual area where fungal infection proliferates. Nanoparticles carrying RB can mitigate the problem of dilution and are reported to effectively penetrate through the nail. For this reason, we have synthesized RB-encapsulated chitosan nanoparticles with a peak distribution size of ~200 nm and high reactive oxygen species (ROS) production. The RB-encapsulated chitosan nanoparticles aPDT were shown to kill more than 99% of T. rubrum, T. mentagrophytes, and T. interdigitale spores, which are the common clinically relevant pathogens in onychomycosis. These nanoparticles are not cytotoxic against human fibroblasts, which promotes their safe application in clinical translation.

Assessing the quantification of acetylation in konjac glucomannan via ATR-FTIR and solid-state NMR spectroscopy.

Dietary fiber like konjac glucomannan (KGM) is important in maintaining good human health. There is no established method for quantifying the average degree of acetylation DA of this polysaccharide. Polysaccharides are notoriously difficult to dissolve. In this study, KGM could not be fully dissolved in common solvents and was characterized in the solid state. ATR-FTIR spectroscopy enabled a fast qualitative assessment of acetylation, selective to the outer layer of KGM particles, and identifying excipients like magnesium stearate. Average DA was quantified for the first time with solid-state 13C NMR in KGM: semi-quantitative measurements on the same arbitrary scale by cross polarization (1 to 2 days) were calibrated with a few longer single-pulse excitation measurements (approximately 1 week). DA values ranged from 4 to 8% of the hexoses in the backbone, in agreement with previously reported values. This method could be used for quality control and standardization of KGM products.

Synthesis, characterization, and multilayer assembly of pH sensitive graphene-polymer nanocomposites.

pH sensitive graphene-polymer composites have been prepared by the modification of graphene basal planes with pyrene-terminated poly(2-N,N'-(dimethyl amino ethyl acrylate) (PDMAEA) and poly(acrylic acid) (PAA) via pi-pi stacking. The pyrene-terminal PDMAEA and PAA were synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization with a pyrene-functional RAFT agent. The graphene-polymer composites were found to demonstrate phase transfer behavior between aqueous and organic media at different pH values. Atomic force microscopy (AFM) analysis revealed that the thicknesses of the graphene-polymer sheets were approximately 3.0 nm when prepared using PDMAEA (M(n): 6800 and PDI: 1.12). The surface coverage of polymer chains on the graphene basal plane was calculated to be 5.3 x 10(-11) mol cm(-2) for PDMAEA and 1.3 x 10(-10) mol cm(-2) for PAA. The graphene-polymer composites were successfully characterized using X-ray photoelectron spectroscopy (XPS), attenuated total reflection infrared (ATR-IR) spectroscopy, and thermogravimetric analysis (TGA). Self-assembly of the two oppositely charged graphene-polymer composites afforded layer-by-layer (LbL) structures as evidenced by high-resolution scanning electron microscopy (SEM) and quartz crystal microbalance (QCM) measurements.

X-ray mapping and scatter diagram analysis of the discoloring products resulting from the interaction of artist's pigments.

The discoloring interaction between the artist's pigments cadmium yellow and the copper-containing malachite, an interaction that is conjectured to cause black spotting in oil paintings of the 19th and early 20th centuries, was examined using X-ray mapping and scatter diagram analysis. The application of these coupled techniques confirmed that copper sulfide phases were produced during discoloration reaction. Scatter diagram analysis indicated that two copper sulfide stoichiometries (CuS and Cu3S2) were present as reaction products where previously only crystalline CuS (covellite) had been identified by X-ray diffraction. The results demonstrate the potential of X-ray mapping coupled with scatter diagram analysis for the identification of both crystalline and X-ray amorphous phases produced by such complex heterogeneous interactions and their applicability to the investigation of interactions of artists' pigments.

Porous chitosan adhesives with L-DOPA for enhanced photochemical tissue bonding.

L-3,4-dihydroxyphenylalanine (L-DOPA) is a naturally occurring catechol that is known to increase the adhesive strength of various materials used for tissue repair. With the aim of fortifying a porous and erodible chitosan-based adhesive film, L-DOPA was incorporated in its fabrication for stronger photochemical tissue bonding (PTB), a repair technique that uses light and a photosensitiser to promote tissue adhesion. The results showed that L-DOPA did indeed increase the tissue bonding strength of the films when photoactivated by a green LED, with a maximum strength recorded of approximately 30 kPa, 1.4 times higher than in its absence. The addition of L-DOPA also did not appreciably change the swelling, mechanical and erodible properties of the film. This study showed that strong, porous and erodible adhesive films for PTB made from biocompatible materials can be obtained through a simple inclusion of a natural additive such as L-DOPA, which was simply mixed with chitosan without any chemical modifications. In vitro studies using human fibroblasts showed no negative effect on cell proliferation indicating that these films are biocompatible. The films are convenient for various surgical applications as they can provide strong tissue support and a microporous environment for cellular infusion without the use of sutures. STATEMENT OF SIGNIFICANCE: Tissue adhesives are not as strong as sutures on wounds under stress. Our group has previously demonstrated that strong sutureless tissue repair can be realised with chitosan-based adhesive films that photochemically bond to tissue when irradiated with green light. The advantage of this technique is that films are easier to handle than glues and sutures, and their crosslinking reactions can be controlled with light. However, these films are not optimal for high-tension tissue regenerative applications because of their non-porous structure, which cannot facilitate cell and nutrient exchange at the wound site. The present study resolves this issue, as we obtained a strong and porous photoactivated chitosan-based adhesive film, by simply using freeze drying and adding L-DOPA.

Radular stylus of Cryptochiton stelleri: A multifunctional lightweight and flexible fiber-reinforced composite.

Chitons are herbivorous invertebrates that use rows of ultrahard magnetite-based teeth connected to a flexible belt (radula) to rasp away algal deposits growing on and within rocky outcrops along coastlines around the world. Each tooth is attached to the radula by an organic structure (stylus) that provides mechanical support during feeding. However, the underlying structures within the stylus, and their subsequent function within the chiton have yet to be investigated. Here, we investigate the macrostructural architecture, the regional material and elemental distribution and subsequent nano-mechanical properties of the stylus from the Northern Pacific dwelling Cryptochiton stelleri. Using a combination of µ-CT imaging, optical and electron microscopy, as well as elemental analysis, we reveal that the stylus is a highly contoured tube, mainly composed of alpha-chitin fibers, with a complex density distribution. Nanoindentation reveals regiospecific and graded mechanical properties that can be correlated with both the elemental composition and material distribution. Finite element modeling shows that the unique macroscale architecture, material distribution and elemental gradients have been optimized to preserve the structural stability of this flexible, yet robust functionally-graded fiber-reinforced composite tube, providing effective function during rasping. Understanding these complex fiber-based structures offers promising blueprints for lightweight, multifunctional and integrated materials.

Porous Chitosan Films Support Stem Cells and Facilitate Sutureless Tissue Repair.

Photochemical tissue bonding with chitosan-based adhesive films is an experimental surgical technique that avoids the risk of thermal tissue injuries and the use of sutures to maintain strong tissue connection. This technique is advantageous over other tissue repair methods as it is minimally invasive and does not require mixing of multiple components before or during application. To expand the capability of the film to beyond just a tissue bonding device and promote tissue regeneration, in this study, we designed bioadhesive films that could also support stem cells. The films were modified with oligomeric chitosan to tune their erodibility and made porous through freeze-drying for better tissue integration. Of note, porous adhesive films (pore diameter ∼110 µm), with 10% of the chitosan being oligomeric, could retain similar tissue bonding strengths (13-15 kPa) to that of the nonporous chitosan-based adhesives used in previous studies when photoactivated. When tested in vitro, these films exhibited a mass loss of ∼20% after 7 days, swelling ratios of ∼270-300%, a percentage elongation of ∼90%, and both a tensile strength and Young's modulus of ∼1 MPa. The physical properties of the films were suitable for maintaining the viability and multipotency of bone-marrow-derived human mesenchymal stem cells over the duration of culture. Thus, these biocompatible, photoactivated porous, and erodible adhesive films show promise for applications in controlled cell delivery and regenerative medicine.

Optimization of Sample Preparation processes of Bone Material for Raman Spectroscopy.

Raman spectroscopy has recently been investigated for use in the calculation of postmortem interval from skeletal material. The fluorescence generated by samples, which affects the interpretation of Raman data, is a major limitation. This study compares the effectiveness of two sample preparation techniques, chemical bleaching and scraping, in the reduction of fluorescence from bone samples during testing with Raman spectroscopy. Visual assessment of Raman spectra obtained at 1064 nm excitation following the preparation protocols indicates an overall reduction in fluorescence. Results demonstrate that scraping is more effective at resolving fluorescence than chemical bleaching. The scraping of skeletonized remains prior to Raman analysis is a less destructive method and allows for the preservation of a bone sample in a state closest to its original form, which is beneficial in forensic investigations. It is recommended that bone scraping supersedes chemical bleaching as the preferred method for sample preparation prior to Raman spectroscopy.

Ultrasensitive Colorimetric and Ratiometric Detection of Cu2+: Acid-Base Properties, Complexation, and Binding Studies.

Herein, we report the synthesis and characterization of a chemosensor, 5-(diethylamino)-2-(2,3-dihydro-1H-perimidin-2-yl)phenol (HL), synthesized from a condensation between 4-(diethylamino)salicylaldehyde and 1,8-diaminonaphthalene. Upon investigation of the sensing properties of HL, it was found that this sensor may be employed for simple yet efficient detection of Cu2+ in aqueous methanol solutions. The selective and ratiometric response to Cu2+ yielded an outstandingly low limit of detection of 3.7 nM by spectrophotometry and is also useful as a naked-eye sensor from 2.5 µM. The system was studied by spectrophotometric pH titrations to determine Cu2+ binding constants and complex speciation. Binding of Cu2+ to HL occurs in 1:1 stoichiometry, in good agreement with high-resolution electrospray ionization mass spectrometry (ESI-HRMS) results, Cu2+ titrations, and Job's plot experiments, while the coordination geometry was tentatively assigned as square pyramidal by spectroscopic studies.