A Scaffold-Assisted 3D Cancer Cell Model for Surface-Enhanced Raman Scattering-Based Real-Time Sensing and Imaging.

Despite recent advances in the development of scaffold-based three-dimensional (3D) cell models, challenges persist in imaging and monitoring cell behavior within these complex structures due to their heterogeneous cell distribution and geometries. Incorporating sensors into 3D scaffolds provides a potential solution for real-time, in situ sensing and imaging of biological processes such as cell growth and disease development. We introduce a 3D printed hydrogel-based scaffold capable of supporting both surface-enhanced Raman scattering (SERS) biosensing and imaging of 3D breast cancer cell models. The scaffold incorporates plasmonic nanoparticles and SERS tags, for sensing and imaging, respectively. We demonstrate the scaffold's adaptability and modularity in supporting breast cancer spheroids, thereby enabling spatial and temporal monitoring of tumor evolution.

Remodeling arteries: studying the mechanical properties of 3D-bioprinted hybrid photoresponsive materials.

3D-printed cell models are currently in the spotlight of medical research. Whilst significant advances have been made, there are still aspects that require attention to achieve more realistic models which faithfully represent the in vivo environment. In this work we describe the production of an artery model with cyclic expansive properties, capable of mimicking the different physical forces and stress factors that cells experience in physiological conditions. The artery wall components are reproduced using 3D printing of thermoresponsive polymers with inorganic nanoparticles (NPs) representing the outer tunica adventitia, smooth muscle cells embedded in extracellular matrix representing the tunica media, and finally a monolayer of endothelial cells as the tunica intima. Cyclic expansion can be induced thanks to the inclusion of photo-responsive plasmonic NPs embedded within the thermoresponsive ink composition, resulting in changes in the thermoresponsive polymer hydration state and hence volume, in a stimulated on-off manner. By changing the thermoresponsive polymer composition, the transition temperature and pulsatility can be efficiently tuned. We show the direct effect of cyclic expansion and contraction on the overlying cell layers by analyzing transcriptional changes in mechanoresponsive mesenchymal genes associated with such microenvironmental physical cues. The technique described herein involving stimuli-responsive 3D printed tissue constructs, also described as four- dimensional (4D) printing, offers a novel approach for the production of dynamic biomodels.

Machine Learning-Assisted High-Throughput SERS Classification of Cell Secretomes.

During the response to different stress conditions, damaged cells react in multiple ways, including the release of a diverse cocktail of metabolites. Moreover, secretomes from dying cells can contribute to the effectiveness of anticancer therapies and can be exploited as predictive biomarkers. The nature of the stress and the resulting intracellular responses are key determinants of the secretome composition, but monitoring such processes remains technically arduous. Hence, there is growing interest in developing tools for noninvasive secretome screening. In this regard, it has been previously shown that the relative concentrations of relevant metabolites can be traced by surface-enhanced Raman scattering (SERS), thereby allowing label-free biofluid interrogation. However, conventional SERS approaches are insufficient to tackle the requirements imposed by high-throughput modalities, namely fast data acquisition and automatized analysis. Therefore, machine learning methods were implemented to identify cell secretome variations while extracting standard features for cell death classification. To this end, ad hoc microfluidic chips were devised, to readily conduct SERS measurements through a prototype relying on capillary pumps made of filter paper, which eventually would function as the SERS substrates. The developed strategy may pave the way toward a faster implementation of SERS into cell secretome classification, which can be extended even to laboratories lacking highly specialized facilities.

Hybrid core-shell nanoparticles for cell-specific magnetic separation and photothermal heating.

Hyperthermia, as the process of heating a malignant site above 42 °C to trigger cell death, has emerged as an effective and selective cancer therapy strategy. Various modalities of hyperthermia have been proposed, among which magnetic and photothermal hyperthermia are known to benefit from the use of nanomaterials. In this context, we introduce herein a hybrid colloidal nanostructure comprising plasmonic gold nanorods (AuNRs) covered by a silica shell, onto which iron oxide nanoparticles (IONPs) are subsequently grown. The resulting hybrid nanostructures are responsive to both external magnetic fields and near-infrared irradiation. As a result, they can be applied for the targeted magnetic separation of selected cell populations - upon targeting by antibody functionalization - as well as for photothermal heating. Through this combined functionality, the therapeutic effect of photothermal heating can be enhanced. We demonstrate both the fabrication of the hybrid system and its application for targeted photothermal hyperthermia of human glioblastoma cells.

Challenges for optical nanothermometry in biological environments.

Temperature monitoring is useful in medical diagnosis, and essential during hyperthermia treatments to avoid undesired cytotoxic effects. Aiming to control heating doses, different temperature monitoring strategies have been developed, largely based on luminescent materials, a.k.a. nanothermometers. However, for such nanothermometers to work, both excitation and emission light beams must travel through tissue, making its optical properties a relevant aspect to be considered during the measurements. In complex tissues, heterogeneity, and real-time alterations as a result of therapeutic treatment may have an effect on light-tissue interaction, hindering accuracy in the thermal reading. In this Tutorial Review we discuss various methods in which nanothermometers can be used for temperature sensing within heterogeneous environments. We discuss recent developments in optical (nano)thermometry, focusing on the incorporation of luminescent nanoparticles into complex in vitro and in vivo models. Methods formulated to avoid thermal misreading are also discussed, considering their respective advantages and drawbacks.

In Vivo Evaluation of Multifunctional Gold Nanorods for Boron Neutron Capture and Photothermal Therapies.

The incidence and mortality of cancer demand more innovative approaches and combination therapies to increase treatment efficacy and decrease off-target side effects. We describe a boron-rich nanoparticle composite with potential applications in both boron neutron capture therapy (BNCT) and photothermal therapy (PTT). Our strategy is based on gold nanorods (AuNRs) stabilized with polyethylene glycol and functionalized with the water-soluble complex cobalt bis(dicarbollide) ([3,3'-Co(1,2-C2B9H11)2]-), commonly known as COSAN. Radiolabeling with the positron emitter copper-64 (64Cu) enabled in vivo tracking using positron emission tomography imaging. 64Cu-labeled multifunctionalized AuNRs proved to be radiochemically stable and capable of being accumulated in the tumor after intravenous administration in a mouse xenograft model of gastrointestinal cancer. The resulting multifunctional AuNRs showed high biocompatibility and the capacity to induce local heating under external stimulation and trigger cell death in heterogeneous cancer spheroids as well as the capacity to decrease cell viability under neutron irradiation in cancer cells. These results position our nanoconjugates as suitable candidates for combined BNCT/PTT therapies.

Nd3+-Doped Lanthanum Oxychloride Nanocrystals as Nanothermometers.

The development of optical nanothermometers operating in the near-infrared (NIR) is of high relevance toward temperature measurements in biological systems. We propose herein the use of Nd3+-doped lanthanum oxychloride nanocrystals as an efficient system with intense photoluminescence under NIR irradiation in the first biological transparency window and emission in the second biological window with excellent emission stability over time under 808 nm excitation, regardless of Nd3+ concentration, which can be considered as a particular strength of our system. Additionally, surface passivation through overgrowth of an inert LaOCl shell around optically active LaOCl/Nd3+ cores was found to further enhance the photoluminescence intensity and also the lifetime of the 1066 nm, 4F3/2 to 4I11/2 transition, without affecting its (ratiometric) sensitivity toward temperature changes. As required for biological applications, we show that the obtained (initially hydrophobic) nanocrystals can be readily transferred into aqueous solvents with high, long-term stability, through either ligand exchange or encapsulation with an amphiphilic polymer.

Development of an accurate method for dispersion and quantification of carbon nanotubes in biological media.

Understanding the biological effects triggered by nanomaterials is crucial, not only in nanomedicine but also in toxicology. The dose-response relation is relevant in biological tests due to its use for determining appropriate dosages for drugs and toxicity limits. Carbon nanotubes can trigger numerous unusual biological effects, many of which could have unique applications in biotechnology and medicine. However, their resuspension in saline solutions and the accurate determination of their concentration after dispersion in biological media are major handicaps to identify the magnitude of the response of organisms as a function of this exposure. This difficulty has led to inconsistent results and misinterpretations of their in vivo behavior, limiting their potential use in nanomedicine. The lack of a suitable protocol that allows comparing different studies of the content of carbon nanotubes and their adequate resuspension in culture cell media gives rise to this study. Here, we describe a methodology to functionalize, resuspend and determine the carbon nanotube concentration in biocompatible media based on UV-Vis spectroscopy. This method allows us to accurately estimate the concentration of these resuspended carbon nanotubes, after removing bundles and micrometric aggregates, which can be used as a calibration standard, for dosage-dependent studies in biological systems. This method can also be extended to any other nanomaterial to properly quantify the actual concentration.

Dye-doped biodegradable nanoparticle SiO2 coating on zinc- and iron-oxide nanoparticles to improve biocompatibility and for in vivo imaging studies.

In vivo imaging and therapy represent one of the most promising areas in nanomedicine. Particularly, the identification and localization of nanomaterials within cells and tissues are key issues to understand their interaction with biological components, namely their cell internalization route, intracellular destination, therapeutic activity and possible cytotoxicity. Here, we show the development of multifunctional nanoparticles (NPs) by providing luminescent functionality to zinc and iron oxide NPs. We describe simple synthesis methods based on modified Stöber procedures to incorporate fluorescent molecules on the surface of oxide NPs. These procedures involve the successful coating of NPs with size-controlled amorphous silica (SiO2) shells incorporating standard chromophores like fluorescein, rhodamine B or rhodamine B isothiocyanate. Specifically, spherical Fe3O4 NPs with an average size of 10 nm and commercial ZnO NPs (ca. 130 nm), both coated with an amorphous SiO2 shell of ca. 15 and 24 nm thickness, respectively, are presented. The magnetic nanoparticles, with a major presence of magnetite, show negligible coercitivity. Hence, interactions (dipolar) are very weak and the cores are in the superparamagnetic regime. Spectroscopic measurements confirm the presence of fluorescent molecules within the SiO2 shell, making these hybrid NPs suitable for bioimaging. Thus, our coating procedures improve NP dispersibility in physiological media and allow the identification and localization of intracellular ZnO and Fe3O4 NPs using confocal microscopy imaging preserving the fluorescence of the NP. We demonstrate how both Fe3O4 and ZnO NPs coated with luminescent SiO2 are internalized and accumulated in the cell cytoplasm after 24 hours. Besides, the SiO2 shell provides a platform for further functionalization that enables the design of targeted therapeutic strategies. Finally, we studied the degradation of the shell in different physiological environments, pointing out that the SiO2 coating is stable enough to reach the target cells maintaining its original structure. Degradation took place only 24 hours after exposure to different media.

High-Pressure Melting Curve of Zintl Sodium Silicide Na4Si4 by In Situ Electrical Measurements.

The inorganic chemistry of the Na-Si system at high pressure is fascinating, with a large number of interesting compounds accessible in the industrial pressure scale, below 10 GPa. In particular, Na4Si4 is stable in this whole pressure range and thus plays an important role in understanding the thermodynamics and kinetics underlying materials synthesis at high pressures and high temperatures. In the present work, the melting curve of the Zintl compound Na4Si4 made of Na+ and Si44- tetrahedral cluster ions is studied at high pressures up to 5 GPa, by using in situ electrical measurements. During melting, the insulating Na4Si4 solid transforms into an ionic conductive liquid that can be probed through the conductance of the whole high-pressure cell, i.e., the system constituted of the sample, the heater, and the high-pressure assembly. Na4Si4 melts congruently in the studied pressure range, and its melting point increases with pressure with a positive slope dTm/dp of 20(4) K/GPa.

Nature of Hexagonal Silicon Forming via High-Pressure Synthesis: Nanostructured Hexagonal 4H Polytype.

Hexagonal Si allotropes are expected to enhance light absorption in the visible range as compared to common cubic Si with diamond structure. Therefore, synthesis of these materials is crucial for the development of Si-based optoelectronics. In this work, we combine in situ high-pressure high-temperature synthesis and vacuum heating to obtain hexagonal Si. High pressure is one of the most promising routes to stabilize these allotropes. It allows one to obtain large-volume nanostructured ingots by a sequence of direct solid-solid transformations, ensuring high-purity samples for detailed characterization. Thanks to our synthesis approach, we provide the first evidence of a polycrystalline bulk sample of hexagonal Si. Exhaustive structural analysis, combining fine-powder X-ray and electron diffraction, afforded resolution of the crystal structure. We demonstrate that hexagonal Si obtained by high-pressure synthesis correspond to Si-4H polytype (ABCB stacking) in contrast with Si-2H (AB stacking) proposed previously. This result agrees with prior calculations that predicted a higher stability of the 4H form over 2H form. Further physical characterization, combining experimental data and ab initio calculations, have shown a good agreement with the established structure. Strong photoluminescence emission was observed in the visible region for which we foresee optimistic perspectives for the use of this material in Si-based photovoltaics.

Nano-ZnO leads to tubulin macrotube assembly and actin bundling, triggering cytoskeletal catastrophe and cell necrosis.

Zinc is a crucial element in biology that plays chief catalytic, structural and protein regulatory roles. Excess cytoplasmic zinc is toxic to cells so there are cell-entry and intracellular buffering mechanisms that control intracellular zinc availability. Tubulin and actin are two zinc-scavenging proteins that are essential components of the cellular cytoskeleton implicated in cell division, migration and cellular architecture maintenance. Here we demonstrate how exposure to different ZnO nanostructures, namely ZnO commercial nanoparticles and custom-made ZnO nanowires, produce acute cytotoxic effects in human keratinocytes (HaCat) and epithelial cells (HeLa) triggering a dose-dependent cell retraction and collapse. We show how engulfed ZnO nanoparticles dissolve intracellularly, triggering actin filament bundling and structural changes in microtubules, transforming these highly dynamic 25 nm diameter polymers into rigid macrotubes of tubulin, severely affecting cell proliferation and survival. Our results demonstrate that nano-ZnO causes acute cytoskeletal collapse that triggers necrosis, followed by a late reactive oxygen species (ROS)-dependent apoptotic process.