Publications

The interaction of nanoparticles with biological media is a topic of general interest for drug delivery systems and among those for active nanoparticles, also called nanomotors. Herein, we report the use of super resolution microscopy, in particular, stochastic optical reconstruction microscopy (STORM), to characterize the formation of a protein corona around active enzyme-powered nanomotors. First, we characterized the distribution and number of enzymes on nano-sized particles and characterized their motion capabilities. Then, we incubated the nanomotors with fluorescently labelled serum proteins. Interestingly, we observed a significant decrease of protein corona formation (20%) and different composition, which was studied by proteomic analysis. Moreover, motion was not hindered, as nanomotors displayed enhanced diffusion regardless of the protein corona. Elucidating how active particles interact with biological media and maintain their self-propulsion after protein corona formation will pave the way for the use of these systems in complex biological fluids in biomedicine.; The interaction of self-propelled nanomotors with biological media is of outmost relevance when considering their actuation within biological contexts. Here, we explored how protein corona forms around active nanomotors using STORM.

JTD Keywords: Gold, Impact, Nanoparticle uptake, Plasma, Size, Surface-properties

Hydroxyapatite-based materials have been widely used in countless applications, such as bone regeneration, catalysis, air and water purification or protein separation. Recently, much interest has been given to controlling the aspect ratio of hydroxyapatite crystals from bulk samples. The ability to exert control over the aspect ratio may revolutionize the applications of these materials towards new functional materials. Controlling the shape, size and orientation of HA crystals allows obtaining high aspect ratio structures, improving several key properties of HA materials such as molecule adsorption, ion exchange, catalytic reactions, and even overcoming the well-known brittleness of ceramic materials. Regulating the morphogenesis of HA crystals to form elongated oriented fibres has led to flexible inorganic synthetic sponges, aerogels, membranes, papers, among others, with applications in sustainability, energy and catalysis, and especially in the biomedical field.; Hydroxyapatite-based materials have been widely used in countless applications, such as bone regeneration, catalysis, air and water purification or protein separation.

JTD Keywords: Bone, Calcium-phosphate, Doped hydroxyapatite, Fire-resistant, Hydrothermal synthesis, Metal-ions, Nanoparticles, Nanowires, Particle-size, Porous nanocomposite

Nanomedicine presents innovative solutions for cancer treatment, including photothermal therapy (PTT). PTT centers on the design of photoactivatable nanoparticles capable of absorbing non-toxic near-infrared light, generating heat within target cells to induce cell death. The successful transition from benchside to bedside application of PTT critically depends on the core properties of nanoparticles responsible for converting light into heat and the surface properties for precise cell-specific targeting. Precisely targeting the intended cells remains a primary challenge in PTT. In recent years, a groundbreaking approach has emerged to address this challenge by functionalizing nanocarriers and enhancing cell targeting. This strategy involves the creation of biomimetic nanoparticles that combine desired biocompatibility properties with the immune evasion mechanisms of natural materials. This review comprehensively outlines various strategies for designing biomimetic photoactivatable nanocarriers for PTT, with a primary focus on its application in cancer therapy. Additionally, we shed light on the hurdles involved in translating PTT from research to clinical practice, along with an overview of current clinical applications.

JTD Keywords: biomimetic nanoparticles, cancer treatment, diagnosis, drug-delivery, erythrocyte-membrane, facile synthesis, iron-oxide nanoparticles, magnetic nanoparticles, membrane-camouflaged nanoparticles, metastatic breast-cancer, size, stem-cells, Biomimetic nanoparticles, Cancer treatment, Membrane-camouflaged nanoparticles, Photothermal therapy

The formation of a protein corona, where proteins spontaneously adhere to the surface of nanomaterials in biological environments, leads to changes in their physicochemical properties and subsequently affects their intended biomedical functionalities. Most current methods to study protein corona formation are ensemble-averaging and either require fluorescent labeling, washing steps, or are only applicable to specific types of particles. Here we introduce real-time all-optical nanoparticle analysis by scattering microscopy (RONAS) to track the formation of protein corona in full serum, at the single-particle level, without any labeling. RONAS uses optical scattering microscopy and enables real-time and in situ tracking of protein adsorption on metallic and dielectric nanoparticles with different geometries directly in blood serum. We analyzed the adsorbed protein mass, the affinity, and the kinetics of the protein adsorption at the single particle level. While there is a high degree of heterogeneity from particle to particle, the predominant factor in protein adsorption is surface chemistry rather than the underlying nanoparticle material or size. RONAS offers an in-depth understanding of the mechanisms related to protein coronas and, thus, enables the development of strategies to engineer efficient bionanomaterials.

JTD Keywords: Dielectric nanoparticles, Optical microscopy, Plasmonic nanoparticles, Protein corona, Protein corona,plasmonic nanoparticles,dielectricnanoparticles,optical microscopy,single particle, Silica nanoparticles,gold nanoparticles,surface-charge,adsorption,albumin,binding,size,transferrin,mas, Single particles

Alzheimer's disease is characterized by a combination of several neuropathological hallmarks, such as extracellular aggregates of beta amyloid (Aβ). Numerous alternatives have been studied for inhibiting Aβ aggregation but, at this time, there are no effective treatments available. Here, we developed the tri-component nanohybrid system AuNPs@POM@PEG based on gold nanoparticles (AuNPs) covered with polyoxometalates (POMs) and polyethylene glycol (PEG). In this work, AuNPs@POM@PEG demonstrated the inhibition of the formation of amyloid fibrils, showing a 75% decrease in Aβ aggregation in vitro. As it is a potential candidate for the treatment of Alzheimer's disease, we evaluated the cytotoxicity of AuNPs@POM@PEG and its ability to cross the blood-brain barrier (BBB). We achieved a stable nanosystem that is non-cytotoxic below 2.5 nM to human neurovascular cells. The brain permeability of AuNPs@POM@PEG was analyzed in an in vitro microphysiological model of the BBB (BBB-on-a-chip), containing 3D human neurovascular cell co-cultures and microfluidics. The results show that AuNPs@POM@PEG was able to cross the brain endothelial barrier in the chip and demonstrated that POM does not affect the barrier integrity, giving the green light to further studies into this system as a nanotherapeutic.

JTD Keywords: beta-amyloid, blood-brain barrier organ-on-a-chip, cellular uptake, citrate, cytotoxicity, electrocatalytic reduction, gold nanoparticles, hypothesis, nanorods, polyoxometalates, size, stability, surface, Alzheimers-disease, Blood–brain barrier organ-on-a-chip, Gold nanoparticles, Nanovehicle, Polyoxometalates, Β-amyloid

Prior studies demonstrated that encapsulation in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) enhanced the delivery of enzymes used for replacement therapy (ERT) of lysosomal storage disorders (LSDs). This study examined how the copolymer lactide:glycolide ratio impacts encapsulation, physicochemical characteristics, stability, and release under lysosomal conditions. Hyaluronidase, deficient in mucopolysaccharidosis IX, was encapsulated in NPs synthesized using 50:50, 60:40, or 75:25 lactide:glycolide copolymers. All NPs had diameters compatible with cellular transport (≤168 nm) and polydispersity indexes (≤0.16) and ζ-potentials (≤-35 mV) compatible with colloidal stability. Yet, their encapsulation efficiency varied, with 75:25 NPs and 60:40 NPs having the lowest and highest EE, respectively (15% vs. 28%). Under lysosomal conditions, the 50:50 copolymer degraded fastest (41% in 1 week), as expected, and the presence of a targeting antibody coat did not alter this result. Additionally, 60:40 NPs destabilized fastest (<1 week) because of their smaller diameter, and 75:25 NPs did not destabilize in 4 weeks. All formulations presented burst release under lysosomal conditions (56-78% of the original load within 30 min), with 50:50 and 60:40 NPs releasing an additional small fraction after week 1. This provided 4 weeks of sustained catalytic activity, sufficient to fully degrade a substrate. Altogether, the 60:40 NP formulation is preferred given its higher EE, and 50:50 NPs represent a valid alternative, while the highest stability of 75:25 NPs may impair lysosomes. These results can guide future studies aiming to translate PLGA NP-based ERT for this and other LSDs.

JTD Keywords: biodegradation, copolymer ratio, degradation, drug-delivery, emulsification, enzyme release, enzyme replacement therapy, hyaluronidase, mechanisms, microspheres, nanoparticle stability, poly(lactide-co-glycolide) nanoparticles, size, sphingomyelinase, transport, Central-nervous-system, Copolymer ratio, Enzyme release, Enzyme replacement therapy, Hyaluronidase, Lysosomal storage disorder, Nanoparticle stability, Poly(lactide-co-glycolide) nanoparticles

Nanoparticles (NPs) are used to encapsulate therapeutic cargos and deliver them specifically to the target site. The intracellular trafficking of NPs dictates the NP-cargo distribution within different cellular compartments, and thus governs their efficacy and safety. Knowledge in this field is crucial to understand their biological fate and improve their rational design. However, there is a lack of methods that allow precise localization and quantification of individual NPs within distinct cellular compartments simultaneously. Here, we address this issue by proposing a correlative light and electron microscopy (CLEM) method combining direct stochastic optical reconstruction microscopy (dSTORM) and transmission electron microscopy (TEM). We aim at combining the advantages of both techniques to precisely address NP localization in the context of the cell ultrastructure. Individual fluorescently-labelled poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) NPs were directly visualized by dSTORM and assigned to cellular compartments by TEM. We first tracked NPs along the endo-lysosomal pathway at different time points, then demonstrated the effect of chloroquine on their intracellular distribution (i.e. endosomal escape). The proposed protocol can be applied to fluorescently labelled NPs and/or cargo, including those not detectable by TEM alone. Our studies are of great relevance to obtain important information on NP trafficking, and crucial for the design of more complex nanomaterials aimed at cytoplasmic/nucleic drug delivery.

JTD Keywords: chemistry, delivery, endocytosis, endosomal escape, exocytosis, fluorescence, light, size, tomography, Cellular uptake

A bottom-up approach to fabricating monodisperse, two-component polymersomes that possess phase-separated ("patchy") chemical topology is presented. This approach is compared with already-existing top-down preparation methods for patchy polymer vesicles, such as film rehydration. These findings demonstrate a bottom-up, solvent-switch self-assembly approach that produces a high yield of nanoparticles of the target size, morphology, and surface topology for drug delivery applications, in this case patchy polymersomes of a diameter of ≈50 nm. In addition, an image processing algorithm to automatically calculate polymersome size distributions from transmission electron microscope images based on a series of pre-processing steps, image segmentation, and round object identification is presented.© 2023 Wiley-VCH GmbH.

JTD Keywords: assemblies, copolymers, evolution, membranes, micelles, ph, phase separation, polymersomes, rafts, self-assembly, size, vesicles, Cell biology, Drug delivery, Phase separation, Polymersomes, Self-assembly, Vesicles

Sprouting angiogenesis is a core biological process critical to vascular development. Its accurate simulation, relevant to multiple facets of human health, is of broad, interdisciplinary appeal. This study presents an in-silico model replicating a microfluidic assay where endothelial cells sprout into a biomimetic extracellular matrix, specifically, a large-pore, low-concentration fibrin-based porous hydrogel, influenced by chemotactic factors. We introduce a novel approach by incorporating the extracellular matrix and chemotactic factor effects into a unified term using a single parameter, primarily focusing on modelling sprouting dynamics and morphology. This continuous model naturally describes chemotactic-induced sprouting with no need for additional rules. In addition, we extended our base model to account for matrix sensing and degradation, crucial aspects of angiogenesis. We validate our model via a hybrid in-silico experimental method, comparing the model predictions with experimental results derived from the microfluidic setup. Our results underscore the intricate relationship between the extracellular matrix structure and angiogenic sprouting, proposing a promising method for predicting the influence of the extracellular matrix on angiogenesis.Copyright © 2023 Ferre-Torres, Noguera-Monteagudo, Lopez-Canosa, Romero-Arias, Barrio, Castaño and Hernandez-Machado.

JTD Keywords: angiogenesis, biomimmetic, chemotaxis, endothelial cells, filopodia, growth, in silico model, mathematical models, mechanisms, metalloproteinase, migration, morphogenesis, phase field, pore-size, simulation, Angiogenesis, Biomimmetic, Chemotaxis, Endothelial cells, Extracellular matrix, In silico model, Mathematical models, Phase field, Tip cells

JTD Keywords: alpha-tricalcium phosphate, cement, crystallinity, hydrolysis, hydroxyapatite, particle-size, powders, relative humidity, setting reaction, stability, tcp, vapor sorption, Calcium-phosphate, Moisture

Drug and gene delivery systems based on polymeric nanoparticles offer a greater efficacy and a reduced toxicity compared to traditional formulations. Recent studies have evidenced that their internalization, biodistribution and efficacy can be affected, among other factors, by their mechanical properties. Here, we analyze by means of Atomic Force Microscopy force spectroscopy how composition, surface functionalization and loading affect the mechanics of nanoparticles. For this purpose, nanoparticles made of Poly(lactic-co-glycolic) (PLGA) and Ethyl cellulose (EC) with different functionalizations and loading were prepared by nano-emulsion templating using the Phase Inversion Composition method (PIC) to form the nano-emulsions. A multiparametric nanomechanical study involving the determination of the Young's modulus, maximum deformation and breakthrough force was carried out. The obtained results showed that composition, surface functionalization and loading affect the nanomechanical properties in a different way, thus requiring, in general, to consider the overall mechanical properties after the addition of a functionalization or loading. A graphical representation method has been proposed enabling to easily identify mechanically equivalent formulations, which is expected to be useful in the development of soft polymeric nanoparticles for pre-clinical and clinical use.Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.

JTD Keywords: afm, atomic-force microscopy, cell, delivery-systems, drug-delivery, emulsification approach, internalization, mechanics of nanoparticles, nanomedicine, nanoparticle functionalization, particles, protein corona, size, young?s modulus, Afm, Loaded plga nanoparticles, Mechanics of nanoparticles, Nanomedicine, Nanoparticle functionalization, Polymeric nanoparticles, Young’s modulus

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations-dolines-capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli.© 2022. The Author(s).

JTD Keywords: cavin, cell-migration, cholesterol, extracellular-matrix, nanoscale organization, particle-size, polarization, size distribution, tension, Plasma-membrane

Nanoparticles represent a promising class of material for nanomedicine and molecular biosensing. The formation of a protein corona due to nonspecific particle-protein interactions is a determining factor for the biological fate of nanoparticles in vivo and strongly impacts the performance of nanoparticles when used as biosensors. Nonspecific interactions are usually highly heterogeneous, yet little is known about the heterogeneity of the protein corona that may lead to inter- and intraparticle differences in composition and protein distribution. Here, we present a super-resolution microscopic approach to study the protein corona on single silica nanoparticles and subsequent cellular interactions using multicolor stimulated emission depletion (STED) microscopy. We demonstrate that STED resolves structural features of protein corona on single particles including the distribution on the particle surface and the degree of protein internalization in porous particles. Using multicolor measurements of multiple labeled protein species, we determine the composition of the protein corona at the single-particle level. We quantify particle-to-particle differences in the composition and find that the composition is considerably influenced by the particle geometry. In a subsequent cellular uptake measurement, we demonstrate multicolor STED of protein corona on single particles internalized by cells. Our study shows that STED microscopy opens the window toward mechanistic understanding of protein coronas and aids in the rational design of nanoparticles as nanomedicines and biosensors.

JTD Keywords: insights, multicolor microscopy, nanoparticles, protein corona, quantification, size, sted microscopy, Fluorescence, Quantification, Sted microscopy

3DNA holds promise as a carrier for drugs that can be intercalated into its core or linked to surface arms. Coupling 3DNA to an antibody targeting intercellular adhesion molecule 1 (ICAM-1) results in high lung-specific biodistributions in vivo. While the role of individual parameters on ICAM-1 targeting has been studied for other nanocarriers, it has never been examined for 3DNA or in a manner capable of revealing the hierarchic interplay among said parameters. In this study, we used 2-layer vs. 4-layer anti-ICAM 3DNA and radiotracing to examine biodistribution in mice. We found that, below saturating conditions and within the ranges tested, the density of targeting antibodies on 3DNA is the most relevant parameter driving lung targeting over liver clearance, compared to the number of antibodies per carrier, total antibody dose, 3DNA dose, 3DNA size, or the administered concentration, which influenced the dose in organs but not the lung specific-over-liver clearance ratio. Data predicts that lung-specific delivery of intercalating (core loaded) drugs can be tuned using this biodistribution pattern, while that of arm-linked (surface loaded) drugs requires a careful parametric balance because increasing anti-ICAM density reduces the number of 3DNA arms available for drug loading.

JTD Keywords: 3dna nanocarrier, acid sphingomyelinase, antibody, carrier design parameters, carriers, dna nanostructures, doxorubicin, drug type, icam-1, inflammation, lung targeting, multiparametric hierarchy, nanoparticles, size, 3dna nanocarrier, Intracellular delivery, Multiparametric hierarchy

Polypropylene (PP) surgical meshes, with different knitted architectures, were chemically functionalized with gold nanoparticles (AuNPs) and 4-mercaptothiazole (4-MB) to transform their fibers into a surface enhanced Raman scattering (SERS) detectable plastic material. The application of a thin layer of poly[N-isopropylacrylamide-co-N,N'-methylene bis(acrylamide)] (PNIPAAm-co-MBA) graft copolymer, covalently polymerized to the mesh-gold substrate, caused the conversion of the inert plastic into a thermoresponsive material, resulting in the first PP implantable mesh with both SERS and temperature stimulus responses. AuNPs were homogeneously distributed over the PP yarns, offering a clear SERS recognition together with higher PNIPAAm lower critical solution temperature (LCST ∼ 37 °C) than without the metallic particles (LCST ∼ 32 °C). An infrared thermographic camera was used to observe the polymer-hydrogel folding-unfolding process and to identify the new value of the LCST, connected with the heat generation by plasmonic-resonance gold NPs. The development of SERS PP prosthesis will be relevant for the bioimaging and biomarker detection of the implant by using the plasmonic effect and Raman vibrational spectroscopy for minimally invasive interventions (such as laparoscopy), to prevent patient inflammatory processes. Furthermore, Raman sources have been proved to not damage the cells, like happens with near-infrared irradiation, representing another advantage of moving to SERS approaches. The findings reported here offer unprecedented application possibilities in the biomedical field by extrapolating the material functionalization to other nonabsorbable polymer made devices (e.g., surgical sutures, grapes, wound dressings, among others).

JTD Keywords: gold nanoparticles, poly(n-isopropylacrylamide), polymers, polypropylene, raman-spectroscopy, reduction, resonance, sers spectroscopy, size, surface functionalization, Gold nanoparticles, Polypropylene, Surface functionalization

Thermal scanning probe lithography (tSPL) is a nanofabrication method for the chemical and physical nanopatterning of a large variety of materials and polymer resists with a lateral resolution of 10 nm and a depth resolution of 1 nm. In this Primer, we describe the working principles of tSPL and highlight the characteristics that make it a powerful tool to locally and directly modify material properties in ambient conditions. We introduce the main features of tSPL, which can pattern surfaces by locally delivering heat using nanosized thermal probes. We define the most critical patterning parameters in tSPL and describe post-patterning analysis of the obtained results. The main sources of reproducibility issues related to the probe and the sample as well as the limitations of the tSPL technique are discussed together with mitigation strategies. The applications of tSPL covered in this Primer include those in biomedicine, nanomagnetism and nanoelectronics; specifically, we cover the fabrication of chemical gradients, tissue-mimetic surfaces, spin wave devices and field-effect transistors based on two-dimensional materials. Finally, we provide an outlook on new strategies that can improve tSPL for future research and the fabrication of next-generation devices.

JTD Keywords: Beam lithography, Design, Feature size, Force microscope cantilevers, Mos2, Polymer, Silicon, Speed, Thermochemical nanolithography, Tip

Antibody-functionalized nanoparticles (NPs) are commonly used to increase the targeting selectivity toward cells of interest. At a molecular level, the number of functional antibodies on the NP surface and the density of receptors on the target cell determine the targeting interaction. To rationally develop selective NPs, the single-molecule quantitation of both parameters is highly desirable. However, techniques able to count molecules with a nanometric resolution are scarce. Here, we developed a labeling approach to quantify the number of functional cetuximabs conjugated to NPs and the expression of epidermal growth factor receptors (EGFRs) in breast cancer cells using direct stochastic optical reconstruction microscopy (dSTORM). The single-molecule resolution of dSTORM allows quantifying molecules at the nanoscale, giving a detailed insight into the distributions of individual NP ligands and cell receptors. Additionally, we predicted the fraction of accessible antibody-conjugated NPs using a geometrical model, showing that the total number exceeds the accessible number of antibodies. Finally, we correlated the NP functionality, cell receptor density, and NP uptake to identify the highest cell uptake selectivity regimes. We conclude that single-molecule functionality mapping using dSTORM provides a molecular understanding of NP targeting, aiding the rational design of selective nanomedicines.

JTD Keywords: active targeting, active targeting dstorm, antibodies, dstorm, heterogeneity, multivalency, nanomedicine, nanoparticle functionality, size, super-resolution microscopy, surface, Active targeting, Antibodies, Cell membranes, Cell receptors, Cytology, Direct stochastic optical reconstruction microscopy, Dstorm, Heterogeneity, Ligands, Medical nanotechnology, Molecules, Nanomedicine, Nanoparticle functionality, Nanoparticle targeting, Nanoparticles, Optical reconstruction, Single molecule, Stochastic systems, Stochastics, Super-resolution microscopy, Superresolution microscopy

The characterization of newly synthesized materials is a cornerstone of all chemistry and nanotechnology laboratories. For this purpose, a wide array of analytical techniques have been standardized and are used routinely by laboratories across the globe. With these methods we can understand the structure, dynamics and function of novel molecular architectures and their relations with the desired performance, guiding the development of the next generation of materials. Moreover, one of the challenges in materials chemistry is the lack of reproducibility due to improper publishing of the sample preparation protocol. In this context, the recent adoption of the reporting standard MIRIBEL (Minimum Information Reporting in Bio–Nano Experimental Literature) for material characterization and details of experimental protocols aims to provide complete, reproducible and reliable sample preparation for the scientific community. Thus, MIRIBEL should be immediately adopted in publications by scientific journals to overcome this challenge. Besides current standard spectroscopy and microscopy techniques, there is a constant development of novel technologies that aim to help chemists unveil the structure of complex materials. Among them super-resolution microscopy (SRM), an optical technique that bypasses the diffraction limit of light, has facilitated the study of synthetic materials with multicolor ability and minimal invasiveness at nanometric resolution. Although still in its infancy, the potential of SRM to unveil the structure, dynamics and function of complex synthetic architectures has been highlighted in pioneering reports during the last few years. Currently, SRM is a sophisticated technique with many challenges in sample preparation, data analysis, environmental control and automation, and moreover the instrumentation is still expensive. Therefore, SRM is currently limited to expert users and is not implemented in characterization routines. This perspective discusses the potential of SRM to transition from a niche technique to a standard routine method for material characterization. We propose a roadmap for the necessary developments required for this purpose based on a collaborative effort from scientists and engineers across disciplines.

JTD Keywords: blinking, fluorophore, intramolecular spirocyclization, localization, nanoparticles, resolution limit, reveals, single-molecule fluorescence, stimulated-emission, Characterization techniques, Diffraction, Distributed computer systems, Environmental management, Information reporting, Material chemistry, Materials characterization, Minimum information, Optical reconstruction microscopy, Optical resolving power, Sample preparation, Structure dynamics, Structure functions, Super-resolution microscopy, Synthesized materials

Underpowered trials risk inaccurate results. Recruitment to stroke rehabilitation randomised controlled trials (RCTs) is often a challenge. Statistical simulations offer an important opportunity to explore the adequacy of sample sizes in the context of specific outcome measures. We aimed to examine and compare the adequacy of stroke rehabilitation RCT sample sizes using the Barthel Index (BI) or modified Rankin Scale (mRS) as primary outcomes.We conducted computer simulations using typical experimental event rates (EER) and control event rates (CER) based on individual participant data (IPD) from stroke rehabilitation RCTs. Event rates are the proportion of participants who experienced clinically relevant improvements in the RCT experimental and control groups. We examined minimum sample size requirements and estimated the number of participants required to achieve a number needed to treat within clinically acceptable boundaries for the BI and mRS.We secured 2350 IPD (18 RCTs). For a 90% chance of statistical accuracy on the BI a rehabilitation RCT would require 273 participants per randomised group. Accurate interpretation of effect sizes would require 1000s of participants per group. Simulations for the mRS were not possible as a clinically relevant improvement was not detected when using this outcome measure.Stroke rehabilitation RCTs with large sample sizes are required for accurate interpretation of effect sizes based on the BI. The mRS lacked sensitivity to detect change and thus may be unsuitable as a primary outcome in stroke rehabilitation trials.Copyright © 2021 Elsevier Inc. All rights reserved.

JTD Keywords:  , barthel index, design, increasing value, modified rankin scale, randomised controlled trials, recruitment, reducing waste, reliability, sample size calculations, simulations, stroke rehabilitation, Adult, Article, Barthel index, Calculation, Computer simulation, Controlled study, Effect size, Female, Human, Human experiment, Major clinical study, Male, Modified rankin scale, Numbers needed to treat, Outcome assessment, Randomised controlled trials, Randomized controlled trial, Randomized controlled-trials, Rankin scale, Recruitment, Rehabilitation, Sample size, Sample size calculations, Simulations, Stroke rehabilitation

Quantum dots (QDs) are semiconductor nanoparticles with unique optical and electronic properties, whose interest as potential nano-theranostic platforms for imaging and sensing is increasing. The design and use of QDs requires the understanding of cell-nanoparticle interactions at a microscopic and nanoscale level. Model systems such as supported lipid bilayers (SLBs) are useful, less complex platforms mimicking physico-chemical properties of cell membranes. In this work, we investigated the effect of topographical homogeneity of SLBs bearing different surface charge in the adsorption of hydrophilic QDs. Using quartz-crystal microbalance, a label-free surface sensitive technique, we show significant differences in the interactions of QDs onto homogeneous and inhomogeneous SLBs formed following different strategies. Within short time scales, QDs adsorb onto topographically homogeneous, defect-free SLBs is driven by electrostatic interactions, leading to no layer disruption. After prolonged QD exposure, the nanomechanical stability of the SLB decreases suggesting nanoparticle insertion. In the case of inhomogeneous, defect containing layers, QDs target preferentially membrane defects, driven by a subtle interplay of electrostatic and entropic effects, inducing local vesicle rupture and QD insertion at membrane edges. © 2021

JTD Keywords: adsorption, atomic force microscopy, bilayer formation, gold nanoparticles, hydrophilic quantum dots, lipid membrane defects, model, nanomechanics, quartz crystal microbalance with dissipation, size, supported lipid bilayers, surfaces, Atomic force microscopy, Atomic-force-microscopy, Cytology, Defect-free, Electronic properties, Electrostatics, Hydrophilic quantum dot, Hydrophilic quantum dots, Hydrophilicity, Hydrophilics, Lipid bilayers, Lipid membrane defect, Lipid membrane defects, Lipid membranes, Lipids, Nanocrystals, Nanomechanics, Optical and electronic properties, Quartz, Quartz crystal microbalance with dissipation, Quartz crystal microbalances, Quartz-crystal microbalance, Semiconductor nanoparticles, Semiconductor quantum dots, Supported lipid bilayers

Background: In recent decades, gold nanoparticle (Au NP)-based cancer therapy has been heavily debated. The physico-chemical properties of AuNPs can be exploited in photothermal therapy, making them a powerful tool for selectively killing cancer cells. However, the synthetic side products and capping agents often induce a strong activation of the inflammatory pathways of macrophages, thus limiting their further applications in vivo. Methods: Here, we described a green method to obtain stable polyphenol-capped AuNPs (Au NPs@polyphenols), as polyphenols are known for their anti-inflammatory and anticancer properties. These NPs were used in human macrophages to test key inflammation-related markers, such as NF-κB, TNF-α, and interleukins-6 and 8. The results were compared with similar NPs obtained by a traditional chemical route (without the polyphenol coating), proving the potential of Au NPs@polyphenols to strongly promote the shutdown of inflammation. This was useful in developing them for use as heat-synergized tools in the thermal treatment of two types of cancer cells, namely, breast cancer (MCF-7) and neuroblastoma (SH-SY5Y) cells. The cell viability, calcium release, oxidative stress, HSP-70 expression, mitochondrial, and DNA damage, as well as cytoskeleton alteration, were evaluated. Results: Our results clearly demonstrate that the combined strategy markedly exerts anticancer effects against the tested cancer cell, while neither of the single treatments (only heat or only NPs) induced significant changes. Conclusions: Au NP@polyphenols may be powerful agents in cancer treatment.

JTD Keywords: antioxidant, aunps, biocompatibility, biology, calcium, cancer, green synthesis, inflammation response, inhibition, interleukin-6, mechanisms, natural polyphenols, physico-chemical properties, polyphenols, size, thermal treatment, Aunps, Cancer, Green synthesis, Inflammation response, Nobilis l. leaves, Physico-chemical properties, Polyphenols, Thermal treatment

In vitro research for the study of type 2 diabetes (T2D) is frequently limited by the availability of a functional model for islets of Langerhans. To overcome the limitations of obtaining pancreatic islets from different sources, such as animal models or human donors, immortalized cell lines as the insulin-producing INS1E beta-cells have appeared as a valid alternative to model insulin-related diseases. However, immortalized cell lines are mainly used in flat surfaces or monolayer distributions, not resembling the spheroid-like architecture of the pancreatic islets. To generate islet-like structures, the use of scaffolds appeared as a valid tool to promote cell aggregations. Traditionally-used hydrogel encapsulation methods do not accomplish all the requisites for pancreatic tissue engineering, as its poor nutrient and oxygen diffusion induces cell death. Here, we use cryogelation technology to develop a more resemblance scaffold with the mechanical and physical properties needed to engineer pancreatic tissue. This study shows that carboxymethyl cellulose (CMC) cryogels prompted cells to generate beta-cell clusters in comparison to gelatin-based scaffolds, that did not induce this cell organization. Moreover, the high porosity achieved with CMC cryogels allowed us to create specific range pseudoislets. Pseudoislets formed within CMC-scaffolds showed cell viability for up to 7 d and a better response to glucose over conventional monolayer cultures. Overall, our results demonstrate that CMC-scaffolds can be used to control the organization and function of insulin-producing beta-cells, representing a suitable technique to generate beta-cell clusters to study pancreatic islet function.

JTD Keywords: biomaterial, cryogel, pancreatic islets, scaffold, tissue engineering, ?-cell, Architecture, Beta-cell, Beta-cell heterogeneity, Biomaterial, Carboxymethyl cellulose, Cell culture, Cell death, Cell engineering, Cell organization, Cells, Cellulose, Cryogel, Cryogels, Cytoarchitecture, Delivery, Encapsulation methods, Gelation, Gene-expression, Immortalized cells, Insulin, Insulin secretory responses, Islets of langerhans, Mechanical and physical properties, Monolayer culture, Monolayers, Pancreatic islets, Pancreatic tissue, Pancreatic-islets, Proliferation, Scaffold, Scaffolds, Scaffolds (biology), Size, Tissue, Tissue engineering, Β-cell

The functionalization of nanoparticles with functional moieties is a key strategy to achieve cell targeting in nanomedicine. The interplay between size and ligand number is crucial for the formulation performance and needs to be properly characterized to understand nanoparticle structure-activity relations. However, there is a lack of methods able to measure both size and ligand number at the same time and at the single particle level. Here, we address this issue by introducing a correlative light and electron microscopy (CLEM) method combining super-resolution microscopy (SRM) and transmission electron microscopy (TEM) imaging. We apply our super-resCLEM method to characterize the relationship between size and ligand number and density in PLGA-PEG nanoparticles. We highlight how heterogeneity found in size can impact ligand distribution and how a significant part of the nanoparticle population goes completely undetected in the single-technique analysis. Super-resCLEM holds great promise for the multiparametric analysis of other parameters and nanomaterials.

JTD Keywords: cellular uptake, correlative light and electron microscopy (clem), density, electron microscopy (em), functionalization, heterogeneity, nanomedicine, nanoparticles, pegylation, plga, progress, quantification, size, Correlative light and electron microscopy (clem), Electron microscopy (em), Heterogeneity, Nanomedicine, Nanoparticles, Physicochemical characterization, Super-resolution microscopy (srm)

A dual approach employing peptidic biofunctionalization and laser micro-patterns on dental zirconia was explored, with the aim of providing a flexible tool to improve tissue integration of restorations. Direct laser interference patterning with a femtosecond Ti:Sapphire laser was employed, and two periodic grooved patterns were produced with a periodicity of 3 and 10 μm. A platform containing the cell-adhesive RGD and the osteogenic DWIVA peptides was used to functionalize the grooved surfaces. Topography and surface damage were characterized by confocal laser scanning (CLSM), scanning electron and scanning transmission electron microscopy techniques. The surface patterns exhibited a high homogeneity and subsurface damage was found in the form of nano-cracks and nano-pores, at the bottom of the valleys. Accelerated tests in water steam were carried out to assess hydrothermal degradation resistance, which slightly decreased after the laser treatment. Interestingly, the detrimental effects of the laser modification were reverted by a post-laser thermal treatment. The attachment of the molecule was verified trough fluorescence CLSM and X-ray photoelectron spectroscopy. Finally, the biological properties of the surfaces were studied in human mesenchymal stem cells. Cell adhesion, morphology, migration and differentiation were investigated. Cells on grooved surfaces displayed an elongated morphology and aligned along the patterns. On these surfaces, migration was greatly enhanced along the grooves, but also highly restricted in the perpendicular direction as compared to flat specimens. After biofunctionalization, cell number and cell area increased and well-developed cell cytoskeletons were observed. However, no effects on cell migration were found for the peptidic platform. Although some osteogenic potential was found in specimens grooved with a periodicity of 10 μm, the largest effects were observed from the biomolecule, which favored upregulation of several genes related to osteoblastic differentiation in all the surfaces.

JTD Keywords: alumina toughened zirconia, cell alignment, grain-size, implants, interference, laser patterning, osteogenic differentiation, osteointegration, peptides, surface functionalization, surface-topography, tissue, titanium surface, Laser patterning, Low-temperature degradation, Osteointegration, Peptides, Surface functionalization, Zirconia

Enzyme-powered nanomotors are an exciting technology for biomedical applications due to their ability to navigate within biological environments using endogenous fuels. However, limited studies into their collective behavior and demonstrations of tracking enzyme nanomotors in vivo have hindered progress toward their clinical translation. Here, we report the swarming behavior of urease-powered nanomotors and its tracking using positron emission tomography (PET), both in vitro and in vivo. For that, mesoporous silica nanoparticles containing urease enzymes and gold nanoparticles were used as nanomotors. To image them, nanomotors were radiolabeled with either I on gold nanoparticles or F-labeled prosthetic group to urease. In vitro experiments showed enhanced fluid mixing and collective migration of nanomotors, demonstrating higher capability to swim across complex paths inside microfabricated phantoms, compared with inactive nanomotors. In vivo intravenous administration in mice confirmed their biocompatibility at the administered dose and the suitability of PET to quantitatively track nanomotors in vivo. Furthermore, nanomotors were administered directly into the bladder of mice by intravesical injection. When injected with the fuel, urea, a homogeneous distribution was observed even after the entrance of fresh urine. By contrast, control experiments using nonmotile nanomotors (i.e., without fuel or without urease) resulted in sustained phase separation, indicating that the nanomotors’ self-propulsion promotes convection and mixing in living reservoirs. Active collective dynamics, together with the medical imaging tracking, constitute a key milestone and a step forward in the field of biomedical nanorobotics, paving the way toward their use in theranostic applications. 124 18

JTD Keywords: cell, reversal, silica nanoparticles, size, step, transport, Propelled micromotors

© 2020 American Chemical Society. The synthesis of hydroxyapatite (HAp) with different shapes and sizes has attracted increasing attention because the applicability of this ceramic material depends on structure-properties relationships (i.e., the dimensions and morphology of HAp crystals determine properties such as the bioactivity and mechanical strength). Although different synthetic routes based on the addition of surfactants, organic modifiers, or dispersants have been proposed to control the growth of HAp crystals, many efforts are being devoted to simplify the whole process using simple parameters such as pH. However, the control of the morphology is still poor and shows low reproducibility. In this work, a new additive-free synthetic route, which is based on the hydrothermal method and the utilization of nonaqueous solvents, is proposed. The influence of the synthesis parameters such as pH, concentration of starting solutions, and the solvent on relevant features such as phase purity, crystallinity, crystallite size, and morphology has been examined using spectroscopic techniques, X-ray diffraction, and scanning electron microscopy. As a consequence, this work presents an easy and robust method based only on the use of organic solvent and the control of the pH that produces pure and crystalline HAp with a controlled shape and size. This method has been used to elucidate some of the key aspects of the crystal growth mechanism and to synthesize HAp crystals with different and well-defined shapes (e.g., belts, rods, flakes needle-like, or polymorph) and sizes, in a reproducible way.

JTD Keywords: biomaterial, bone, crystals, ethanol, size, solubility, Morphology

Self-assembly of fluorenylmethoxycarbonyl-protected diphenylalanine (FmocFF) in water is widely known to produce hydrogels. Typically, confocal microscopy is used to visualize such hydrogels under wet conditions, that is, without freezing or drying. However, key aspects of hydrogels like fiber diameter, network morphology and mesh size are sub-diffraction limited features and cannot be visualized effectively using this approach. In this work, we show that it is possible to image FmocFF hydrogels by Points Accumulation for Imaging in Nanoscale Topography (PAINT) in native conditions and without direct gel labelling. We demonstrate that the fiber network can be visualized with improved resolution (≈50 nm) both in 2D and 3D. Quantitative information is extracted such as mesh size and fiber diameter. This method can complement the existing characterization tools for hydrogels and provide useful information supporting the design of new materials.

JTD Keywords: FmocFF, Hydrogels, Mesh size, PAINT, Super-resolution

A numerical analysis of the polarization force between a sharp conducting probe and a dielectric film of finite lateral dimensions on a metallic substrate is presented with the double objective of (i) determining the conditions under which the film can be approximated by a laterally infinite film and (ii) proposing an analytical model valid in this limit. We show that, for a given dielectric film, the critical diameter above which the film can be modeled as laterally infinite depends not only on the probe geometry, as expected, but mainly on the film thickness. In particular, for films with intermediate to large thicknesses (>100 nm), the critical diameter is nearly independent from the probe geometry and essentially depends on the film thickness and dielectric constant following a relatively simple phenomenological expression. For films that can be considered as laterally infinite, we propose a generalized analytical model valid in the thin-ultrathin limit (<20-50 nm) that reproduces the numerical calculations and the experimental data. Present results provide a general framework under which accurate quantification of electrostatic force microscopy measurements on dielectric films on metallic substrates can be achieved.

JTD Keywords: Dielectric constant, Dielectric films, Electrostatic force microscopy, Quantification, Analytical models, Electric force microscopy, Electrostatic force, Film thickness, Permittivity, Probes, Substrates, Ultrathin films, Accurate quantifications, Electrostatic force microscopy, Finite size effect, Lateral dimension, Metallic substrate, Numerical calculation, Polarization forces, Quantification, Dielectric films

Background: All living organisms are made of individual and identifiable cells, whose number, together with their size and type, ultimately defines the structure and functions of an organism. While the total cell number of lower organisms is often known, it has not yet been defined in higher organisms. In particular, the reported total cell number of a human being ranges between 1012 and 1016 and it is widely mentioned without a proper reference. Aim: To study and discuss the theoretical issue of the total number of cells that compose the standard human adult organism. Subjects and methods: A systematic calculation of the total cell number of the whole human body and of the single organs was carried out using bibliographical and/or mathematical approaches. Results: A current estimation of human total cell number calculated for a variety of organs and cell types is presented. These partial data correspond to a total number of 3.72×1013. Conclusions: Knowing the total cell number of the human body as well as of individual organs is important from a cultural, biological, medical and comparative modelling point of view. The presented cell count could be a starting point for a common effort to complete the total calculation.

JTD Keywords: Cell size, Human cell number, Organ, Theoretical issue, Total cell count

A technique for producing micrometer-scale structures over large, nonplanar chitosan surfaces is described. The technique makes use of the rheological characteristics (deformability) of the chitosan to create freestanding, three-dimensional scaffolds with controlled shapes, incorporating defined microtopography. The results of an investigation into the technical limits of molding different combinations of shapes and microtopographies are presented, highlighting the versatility of the technique when used irrespectively with inorganic or delicate organic moulds. The final, replicated scaffolds presented here are patterned with arrays of one-micrometer-tall microstructures over large areas. Structural integrity is characterized by the measurement of structural degradation. Human umbilical vein endothelial cells cultured on a tubular scaffold show that early cell growth is conditioned by the microtopography and indicate possible uses for the structures in biomedical applications. For those applications requiring improved chemical and mechanical resistance, the structures can be replicated in poly(dimethyl siloxane).

JTD Keywords: Biocompatible Materials/ chemistry, Cell Adhesion, Cell Culture Techniques/ methods, Cell Proliferation, Cells, Cultured, Chitosan/ chemistry, Crystallization/methods, Endothelial Cells/ cytology/ physiology, Humans, Materials Testing, Nanostructures/ chemistry/ ultrastructure, Nanotechnology/methods, Particle Size, Surface Properties, Tissue Engineering/methods

Two Cys-containing analogues of the anticancer drug Kahalalide F are synthesized and conjugated to 20 and 40 nm gold nanoparticles (GNPs). The resulting complexes are characterized by different analytical techniques to confirm the attachment of peptide to the GNPs. The self-assembly capacity of a peptide dramatically influences the final ratio number of molecules per nanoparticle, saturating the nanoparticle surface and prompting multilayered capping on the surface. In such way, the nanoparticle could act as a concentrator for the delivery of drugs, thereby increasing bioactivity. The GNP sizes and the conjugation have influence on the biological activities. Kahalalide F analogues conjugated with GNPs are located subcellularly at lysosome-like bodies, which may be related to the action mechanism of Kahalalide F. The results suggest that the selective delivery and activity of Kahalalide F analogues can be improved by conjugating the peptides to GNPs.

JTD Keywords: Electrical detection, Cellular uptake, Drug-delivery, Cancer-cells, Peptide, Size, Surface, Absorption, Scattering, Therapy

Shape-dependent local differentials in cell proliferation are considered to be a major driving mechanism of structuring processes in vivo, such as embryogenesis, wound healing, and angiogenesis. However, the specific biophysical signaling by which changes in cell shape contribute to cell cycle regulation remains poorly understood. Here, we describe our study of the roles of nuclear volume and cytoskeletal mechanics in mediating shape control of proliferation in single endothelial cells. Micropatterned adhesive islands were used to independently control cell spreading and elongation. We show that, irrespective of elongation, nuclear volume and apparent chromatin decondensation of cells in G1 systematically increased with cell spreading and highly correlated with DNA synthesis (percent of cells in the S phase). In contrast, cell elongation dramatically affected the organization of the actin cytoskeleton, markedly reduced both cytoskeletal stiffness (measured dorsally with atomic force microscopy) and contractility (measured ventrally with traction microscopy), and increased mechanical anisotropy, without affecting either DNA synthesis or nuclear volume. Our results reveal that the nuclear volume in G1 is predictive of the proliferative status of single endothelial cells within a population, whereas cell stiffness and contractility are not. These findings show that the effects of cell mechanics in shape control of proliferation are far more complex than a linear or straightforward relationship. Our data are consistent with a mechanism by which spreading of cells in G1 partially enhances proliferation by inducing nuclear swelling and decreasing chromatin condensation, thereby rendering DNA more accessible to the replication machinery.

JTD Keywords: Cell Line, Cell Nucleus/ physiology, Cell Proliferation, Cell Size, Computer Simulation, Endothelial Cells/ cytology/ physiology, G1 Phase/ physiology, Humans, Mechanotransduction, Cellular/ physiology, Models, Biological, Statistics as Topic

In a previous work, we studied the interaction of β-amyloid fibrils (Aβ) with gold nanoparticles (AuNP) conjugated with the peptide CLPFFD-NH2. Here, we studied the effect of changing the residue sequence of the peptide CLPFFD-NH2 on the efficiency of conjugation to AuNP, the stability of the conjugates, and the affinity of the conjugates to the Aβ fibrils. We conjugated the AuNP with CLPFFD-NH2 isomeric peptides (CDLPFF-NH2 and CLPDFF-NH2) and characterized the resulting conjugates with different techniques including UV−Vis, TEM, EELS, XPS, analysis of amino acids, agarose gel electrophoresis, and CD. In addition, we determined the proportion of AuNP bonded to the Aβ fibrils by ICP-MS. AuNP-CLPFFD-NH2 was the most stable of the conjugates and presented more affinity for Aβ fibrils with respect to the other conjugates and bare AuNP. These findings help to better understand the way peptide sequences affect conjugation and stability of AuNP and their interaction with Aβ fibrils. The peptide sequence, the steric effects, and the charge and disposition of hydrophilic and hydrophobic residues are crucial parameters when considering the design of AuNP peptide conjugates for biomedical applications.

JTD Keywords: Self-assembled monolayers, Aggregation, Dispersions, Adsorption, Particles, Design, Size