Identification of GB3 as a Novel Biomarker of Tumor-Derived Vasculature in Neuroblastoma Using a Stiffness-Based Model.

Neuroblastoma (NB) is a childhood cancer in sympathetic nervous system cells. NB exhibits cellular heterogeneity, with adrenergic and mesenchymal states displaying distinct tumorigenic potentials. NB is highly vascularized, and blood vessels can form through various mechanisms, including endothelial transdifferentiation, leading to the development of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs. Therefore, identifying new TEC biomarkers is vital for effective NB therapies. A stiffness-based platform simulating human arterial and venous stiffness was developed to study NB TECs in vitro. Adrenergic cells cultured on arterial-like stiffness transdifferentiated into TECs, while mesenchymal state cells did not. The TECs derived from adrenergic cells served as a model to explore new biomarkers, with a particular focus on GB3, a glycosphingolipid receptor implicated in angiogenesis, metastasis, and drug resistance. Notably, the TECs unequivocally expressed GB3, validating its novelty as a marker. To explore targeted therapeutic interventions, nanoparticles functionalized with the non-toxic subunit B of the Shiga toxin were generated, because they demonstrated a robust affinity for GB3-positive cells. Our results demonstrate the value of the stiffness-based platform as a predictive tool for assessing NB aggressiveness, the discovery of new biomarkers, and the evaluation of the effectiveness of targeted therapeutic strategies.

Tuneable hydrogel patterns in pillarless microfluidic devices.

Organ-on-chip (OOC) technology has recently emerged as a powerful tool to mimic physiological or pathophysiological conditions through cell culture in microfluidic devices. One of its main goals is bypassing animal testing and encouraging more personalized medicine. The recent incorporation of hydrogels as 3D scaffolds into microfluidic devices has changed biomedical research since they provide a biomimetic extracellular matrix to recreate tissue architectures. However, this technology presents some drawbacks such as the necessity for physical structures as pillars to confine these hydrogels, as well as the difficulty in reaching different shapes and patterns to create convoluted gradients or more realistic biological structures. In addition, pillars can also interfere with the fluid flow, altering the local shear forces and, therefore, modifying the mechanical environment in the OOC model. In this work, we present a methodology based on a plasma surface treatment that allows building cell culture chambers with abutment-free patterns capable of producing precise shear stress distributions. Therefore, pillarless devices with arbitrary geometries are needed to obtain more versatile, reliable, and biomimetic experimental models. Through computational simulation studies, these shear stress changes are demonstrated in different designed and fabricated geometries. To prove the versatility of this new technique, a blood-brain barrier model has been recreated, achieving an uninterrupted endothelial barrier that emulates part of the neurovascular network of the brain. Finally, we developed a new technology that could avoid the limitations mentioned above, allowing the development of biomimetic OOC models with complex and adaptable geometries, with cell-to-cell contact if required, and where fluid flow and shear stress conditions could be controlled.

A New Microfluidic Device to Facilitate Functional Precision Medicine Assays.

Functional precision medicine (FPM) has emerged as a new approach to improve cancer treatment. Despite its potential, FPM assays present important limitations such as the number of cells and trained personnel required. To overcome these impediments, here we describe a novel microfluidic platform that can be used to perform FPM assays, optimizing the use of primary cancer cells and simplifying the process by using microfluidics to automatize the process.

Engineering Biomimetic Nanoparticles through Extracellular Vesicle Coating in Cancer Tissue Models.

Using nanoparticles (NPs) in drug delivery has exhibited promising therapeutic potential in various cancer types. Nevertheless, several challenges must be addressed, including the formation of the protein corona, reduced targeting efficiency and specificity, potential immune responses, and issues related to NP penetration and distribution within 3-dimensional tissues. To tackle these challenges, we have successfully integrated iron oxide nanoparticles into neuroblastoma-derived extracellular vesicles (EVs) using the parental labeling method. We first developed a tissue-engineered (TE) neuroblastoma model, confirming the viability and proliferation of neuroblastoma cells for at least 12 days, supporting its utility for EV isolation. Importantly, EVs from long-term cultures exhibited no differences compared to short-term cultures. Concurrently, we designed Rhodamine (Rh) and Polyacrylic acid (PAA)-functionalized magnetite nanoparticles (Fe3O4@PAA-Rh) with high crystallinity, purity, and superparamagnetic properties (average size: 9.2 ± 2.5 nm). We then investigated the internalization of Fe3O4@PAA-Rh nanoparticles within neuroblastoma cells within the TE model. Maximum accumulation was observed overnight while ensuring robust cell viability. However, nanoparticle internalization was low. Taking advantage of the enhanced glucose metabolism exhibited by cancer cells, glucose (Glc)-functionalized nanoparticles (Fe3O4@PAA-Rh-Glc) were synthesized, showing superior cell uptake within the 3D model without inducing toxicity. These glucose-modified nanoparticles were selected for parental labeling of the TE models, showing effective NP encapsulation into EVs. Our research introduces innovative approaches to advance NP delivery, by partially addressing the challenges associated with 3D systems, optimizing internalization, and enhancing NP stability and specificity through EV-based carriers. Also, our findings hold the promise of more precise and effective cancer therapies while minimizing potential side effects.

Advances in current in vitro models on neurodegenerative diseases.

Many neurodegenerative diseases are identified but their causes and cure are far from being well-known. The problem resides in the complexity of the neural tissue and its location which hinders its easy evaluation. Although necessary in the drug discovery process, in vivo animal models need to be reduced and show relevant differences with the human tissues that guide scientists to inquire about other possible options which lead to in vitro models being explored. From organoids to organ-on-a-chips, 3D models are considered the cutting-edge technology in cell culture. Cell choice is a big parameter to take into consideration when planning an in vitro model and cells capable of mimicking both healthy and diseased tissue, such as induced pluripotent stem cells (iPSC), are recognized as good candidates. Hence, we present a critical review of the latest models used to study neurodegenerative disease, how these models have evolved introducing microfluidics platforms, 3D cell cultures, and the use of induced pluripotent cells to better mimic the neural tissue environment in pathological conditions.

Polyoxometalate-Decorated Gold Nanoparticles Inhibit ß-Amyloid Aggregation and Cross the Blood-Brain Barrier in a µphysiological Model.

Alzheimer's disease is characterized by a combination of several neuropathological hallmarks, such as extracellular aggregates of beta amyloid (Aß). Numerous alternatives have been studied for inhibiting Aß 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 Aß 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.

Ferulic acid-loaded polymeric nanoparticles prepared from nano-emulsion templates facilitate internalisation across the blood-brain barrier in model membranes.

A hydroxycinnamic acid derivative, namely ferulic acid (FA) has been successfully encapsulated in polymeric nanoparticles (NPs) based on poly(lactic-co-glycolic acid) (PLGA). FA-loaded polymeric NPs were prepared from O/W nano-emulsion templates using the phase inversion composition (PIC) low-energy emulsification method. The obtained PLGA NPs exhibited high colloidal stability, good drug-loading capacity, and particle hydrodynamic diameters in the range of 74 to 117 nm, depending on the FA concentration used. In vitro drug release studies confirmed a diffusion-controlled mechanism through which the amount of released FA reached a plateau at 60% after 6 hours-incubation. Five kinetic models were used to fit the FA release data as a function of time. The Weibull distribution and Korsmeyer-Peppas equation models provided the best fit to our experimental data and suggested quasi-Fickian diffusion behaviour. Moderate dose-response antioxidant and radical scavenging activities of FA-loaded PLGA NPs were demonstrated using the DPPH˙ assay achieving inhibition activities close to 60 and 40%, respectively. Cell culture studies confirmed that FA-loaded NPs were not toxic according to the MTT colorimetric assay, were able to internalise efficiently SH-SY5Y neuronal cells and supressed the intracellular ROS-level induced by H2O2 leading to 52% and 24.7% of cellular viability at 0.082 and 0.041 mg mL-1, respectively. The permeability of the NPs through the blood brain barrier was tested with an in vitro organ-on-a-chip model to evaluate the ability of the FA-loaded PLGA and non-loaded PLGA NPs to penetrate to the brain. NPs were able to penetrate the barrier, but permeability decreased when FA was loaded. These results are promising for the use of loaded PLGA NPs for the management of neurological diseases.

3D bioprinted functional skeletal muscle models have potential applications for studies of muscle wasting in cancer cachexia.

Acquired muscle diseases such as cancer cachexia are responsible for the poor prognosis of many patients suffering from cancer. In vitro models are needed to study the underlying mechanisms of those pathologies. Extrusion bioprinting is an emerging tool to emulate the aligned architecture of fibers while implementing additive manufacturing techniques in tissue engineering. However, designing bioinks that reconcile the rheological needs of bioprinting and the biological requirements of muscle tissue is a challenging matter. Here we formulate a biomaterial with dual crosslinking to modulate the physical properties of bioprinted models. We design 3D bioprinted muscle models that resemble the mechanical properties of native tissue and show improved proliferation and high maturation of differentiated myotubes suggesting that the GelMA-AlgMA-Fibrin biomaterial possesses myogenic properties. The electrical stimulation of the 3D model confirmed the contractile capability of the tissue and enhanced the formation of sarcomeres. Regarding the functionality of the models, they served as platforms to recapitulate skeletal muscle diseases such as muscle wasting produced by cancer cachexia. The genetic expression of 3D models demonstrated a better resemblance to the muscular biopsies of cachectic mouse models. Altogether, this biomaterial is aimed to fabricate manipulable skeletal muscle in vitro models in a non-costly, fast and feasible manner.

Hyaluronic acid-based bioink improves the differentiation and network formation of neural progenitor cells.

Introduction: Three-dimensional (3D) bioprinting is a promising technique for the development of neuronal in vitro models because it controls the deposition of materials and cells. Finding a biomaterial that supports neural differentiation in vitro while ensuring compatibility with the technique of 3D bioprinting of a self-standing construct is a challenge. Methods: In this study, gelatin methacryloyl (GelMA), methacrylated alginate (AlgMA), and hyaluronic acid (HA) were examined by exploiting their biocompatibility and tunable mechanical properties to resemble the extracellular matrix (ECM) and to create a suitable material for printing neural progenitor cells (NPCs), supporting their long-term differentiation. NPCs were printed and differentiated for up to 15 days, and cell viability and neuronal differentiation markers were assessed throughout the culture. Results and Discussion: This composite biomaterial presented the desired physical properties to mimic the ECM of the brain with high water intake, low stiffness, and slow degradation while allowing the printing of defined structures. The viability rates were maintained at approximately 80% at all time points. However, the levels of ß-III tubulin marker increased over time, demonstrating the compatibility of this biomaterial with neuronal cell culture and differentiation. Furthermore, these cells showed increased maturation with corresponding functional properties, which was also demonstrated by the formation of a neuronal network that was observed by recording spontaneous activity via Ca2+ imaging.

BBB-on-a-chip with integrated micro-TEER for permeability evaluation of multi-functionalized gold nanorods against Alzheimer's disease.

BACKGROUND: The lack of predictive models that mimic the blood-brain barrier (BBB) hinders the development of effective drugs for neurodegenerative diseases. Animal models behave differently from humans, are expensive and have ethical constraints. Organ-on-a-chip (OoC) platforms offer several advantages to resembling physiological and pathological conditions in a versatile, reproducible, and animal-free manner. In addition, OoC give us the possibility to incorporate sensors to determine cell culture features such as trans-endothelial electrical resistance (TEER). Here, we developed a BBB-on-a-chip (BBB-oC) platform with a TEER measurement system in close distance to the barrier used for the first time for the evaluation of the permeability performance of targeted gold nanorods for theranostics of Alzheimer's disease. GNR-PEG-Ang2/D1 is a therapeutic nanosystem previously developed by us consisting of gold nanorods (GNR) functionalized with polyethylene glycol (PEG), angiopep-2 peptide (Ang2) to overcome the BBB and the D1 peptide as beta amyloid fibrillation inhibitor, finally obtaining GNR-PEG-Ang2/D1 which showed to be useful for disaggregation of the amyloid in in vitro and in vivo models. In this work, we evaluated its cytotoxicity, permeability, and some indications of its impact on the brain endothelium by employing an animal-free device based on neurovascular human cells. RESULTS: In this work, we fabricated a BBB-oC with human astrocytes, pericytes and endothelial cells and a TEER measuring system (TEER-BBB-oC) integrated at a micrometric distance of the endothelial barrier. The characterization displayed a neurovascular network and the expression of tight junctions in the endothelium. We produced GNR-PEG-Ang2/D1 and determined its non-cytotoxic range (0.05-0.4 nM) for plated cells included in the BBB-oC and confirmed its harmless effect at the highest concentration (0.4 nM) in the microfluidic device. The permeability assays revealed that GNR-PEG-Ang2/D1 cross the BBB and this entry is facilitated by Ang2 peptide. Parallel to the permeability analysis of GNR-PEG-Ang2/D1, an interesting behavior of the TJs expression was observed after its administration probably related to the ligands on the nanoparticle surface. CONCLUSIONS: BBB-oC with a novel TEER integrated setup which allow a correct read-out and cell imaging monitoring was proven as a functional and throughput platform to evaluate the brain permeability performance of nanotherapeutics in a physiological environment with human cells, putting forward a viable alternative to animal experimentation.

Preservation of critical quality attributes of mesenchymal stromal cells in 3D bioprinted structures by using natural hydrogel scaffolds.

Three dimensional (3D) bioprinting is an emerging technology that enables complex spatial modeling of cell-based tissue engineering products, whose therapeutic potential in regenerative medicine is enormous. However, its success largely depends on the definition of a bioprintable zone, which is specific for each combination of cell-loaded hydrogels (or bioinks) and scaffolds, matching the mechanical and biological characteristics of the target tissue to be repaired. Therefore proper adjustment of the bioink formulation requires a compromise between: (i) the maintenance of cellular critical quality attributes (CQA) within a defined range of specifications to cell component, and (ii) the mechanical characteristics of the printed tissue to biofabricate. Herein, we investigated the advantages of using natural hydrogel-based bioinks to preserve the most relevant CQA in bone tissue regeneration applications, particularly focusing on cell viability and osteogenic potential of multipotent mesenchymal stromal cells (MSCs) displaying tripotency in vitro, and a phenotypic profile of 99.9% CD105+ /CD45,- 10.3% HLA-DR,+ 100.0% CD90,+ and 99.2% CD73+ /CD31- expression. Remarkably, hyaluronic acid, fibrin, and gelatin allowed for optimal recovery of viable cells, while preserving MSC's proliferation capacity and osteogenic potency in vitro. This was achieved by providing a 3D structure with a compression module below 8.8 ± 0.5 kPa, given that higher values resulted in cell loss by mechanical stress. Beyond the biocompatibility of naturally occurring polymers, our results highlight the enhanced protection on CQA exerted by bioinks of natural origin (preferably HA, gelatin, and fibrin) on MSC, bone marrow during the 3D bioprinting process, reducing shear stress and offering structural support for proliferation and osteogenic differentiation.

Quantitative Image Analysis of Fibrillar Collagens Reveals Novel Diagnostic and Prognostic Biomarkers and Histotype-Dependent Aberrant Mechanobiology in Lung Cancer.

Fibrillar collagens are the most abundant extracellular matrix components in non-small cell lung cancer (NSCLC). However, the potential of collagen fiber descriptors as a source of clinically relevant biomarkers in NSCLC is largely unknown. Similarly, our understanding of the aberrant collagen organization and associated tumor-promoting effects is very scarce. To address these limitations, we identified a digital pathology approach that can be easily implemented in pathology units based on CT-FIRE software (version 2; https://loci.wisc.edu/software/ctfire) analysis of Picrosirius red (PSR) stains of fibrillar collagens imaged with polarized light (PL). CT-FIRE settings were pre-optimized to assess a panel of collagen fiber descriptors in PSR-PL images of tissue microarrays from surgical NSCLC patients (106 adenocarcinomas [ADC] and 89 squamous cell carcinomas [SCC]). Using this approach, we identified straightness as the single high-accuracy diagnostic collagen fiber descriptor (average area under the curve = 0.92) and fiber density as the single descriptor consistently associated with a poor prognosis in both ADC and SCC independently of the gold standard based on the TNM staging (hazard ratio, 2.69; 95% CI, 1.55-4.66; P < .001). Moreover, we found that collagen fibers were markedly straighter, longer, and more aligned in tumor samples compared to paired samples from uninvolved pulmonary tissue, particularly in ADC, which is indicative of increased tumor stiffening. Consistently, we observed an increase in a panel of stiffness-associated processes in the high collagen fiber density patient group selectively in ADC, including venous/lymphatic invasion, fibroblast activation (α-smooth muscle actin), and immune evasion (programmed death-ligand 1). Similarly, a transcriptional correlation analysis supported the potential involvement of the major YAP/TAZ pathway in ADC. Our results provide a proof-of-principle to use CT-FIRE analysis of PSR-PL images to assess new collagen fiber-based diagnostic and prognostic biomarkers in pathology units, which may improve the clinical management of patients with surgical NSCLC. Our findings also unveil an aberrant stiff microenvironment in lung ADC that may foster immune evasion and dissemination, encouraging future work to identify therapeutic opportunities.

Microfluidic-based dynamic BH3 profiling predicts anticancer treatment efficacy.

Precision medicine is starting to incorporate functional assays to evaluate anticancer agents on patient-isolated tissues or cells to select for the most effective. Among these new technologies, dynamic BH3 profiling (DBP) has emerged and extensively been used to predict treatment efficacy in different types of cancer. DBP uses synthetic BH3 peptides to measure early apoptotic events ('priming') and anticipate therapy-induced cell death leading to tumor elimination. This predictive functional assay presents multiple advantages but a critical limitation: the cell number requirement, that limits drug screening on patient samples, especially in solid tumors. To solve this problem, we developed an innovative microfluidic-based DBP (µDBP) device that overcomes tissue limitations on primary samples. We used microfluidic chips to generate a gradient of BIM BH3 peptide, compared it with the standard flow cytometry based DBP, and tested different anticancer treatments. We first examined this new technology's predictive capacity using gastrointestinal stromal tumor (GIST) cell lines, by comparing imatinib sensitive and resistant cells, and we could detect differences in apoptotic priming and anticipate cytotoxicity. We then validated µDBP on a refractory GIST patient sample and identified that the combination of dactolisib and venetoclax increased apoptotic priming. In summary, this new technology could represent an important advance for precision medicine by providing a fast, easy-to-use and scalable microfluidic device to perform DBP in situ as a routine assay to identify the best treatment for cancer patients.

In vitro modelling of human proprioceptive sensory neurons in the neuromuscular system.

Proprioceptive sensory neurons (pSN) are an essential and undervalued part of the neuromuscular circuit. A protocol to differentiate healthy and amyotrophic lateral sclerosis (ALS) human neural stem cells (hNSC) into pSN, and their comparison with the motor neuron (MN) differentiation process from the same hNSC sources, facilitated the development of in vitro co-culture platforms. The obtained pSN spheroids cultured interact with human skeletal myocytes showing the formation of annulospiral wrapping-like structures between TrkC + neurons and a multinucleated muscle fibre, presenting synaptic bouton-like structures in the contact point. The comparative analysis of the genetic profile performed in healthy and sporadic ALS hNSC differentiated to pSN suggested that basal levels of ETV1, critical for motor feedback from pSN, were much lower for ALS samples and that the differences between healthy and ALS samples, suggest the involvement of pSN in ALS pathology development and progression.

Phosphorylation disrupts long-distance electron transport in cytochrome c.

It has been recently shown that electron transfer between mitochondrial cytochrome c and the cytochrome c1 subunit of the cytochrome bc1 can proceed at long-distance through the aqueous solution. Cytochrome c is thought to adjust its activity by changing the affinity for its partners via Tyr48 phosphorylation, but it is unknown how it impacts the nanoscopic environment, interaction forces, and long-range electron transfer. Here, we constrain the orientation and separation between cytochrome c1 and cytochrome c or the phosphomimetic Y48pCMF cytochrome c, and deploy an array of single-molecule, bulk, and computational methods to investigate the molecular mechanism of electron transfer regulation by cytochrome c phosphorylation. We demonstrate that phosphorylation impairs long-range electron transfer, shortens the long-distance charge conduit between the partners, strengthens their interaction, and departs it from equilibrium. These results unveil a nanoscopic view of the interaction between redox protein partners in electron transport chains and its mechanisms of regulation.

Determination of the nanoscale electrical properties of olfactory receptor hOR1A1 and their dependence on ligand binding: Towards the development of capacitance-operated odorant biosensors.

The transduction of odorant binding into cellular signaling by olfactory receptors (ORs) is not understood and knowing its mechanism would enable developing new pharmacology and biohybrid electronic detectors of volatile organic compounds bearing high sensitivity and selectivity. The electrical characterization of ORs in bulk experiments is subject to microscopic models and assumptions. We have directly determined the nanoscale electrical properties of ORs immobilized in a fixed orientation, and their change upon odorant binding, using electrochemical scanning tunneling microscopy (EC-STM) in near-physiological conditions. Recordings of current versus time, distance, and electrochemical potential allows determining the OR impedance parameters and their dependence with odorant binding. Our results allow validating OR structural-electrostatic models and their functional activation processes, and anticipating a novel macroscopic biosensor based on ORs.

Substrate adhesion determines migration during mesenchymal cell condensation in chondrogenesis.

Mesenchymal condensation is a prevalent morphogenetic transition that is essential in chondrogenesis. However, the current understanding of condensation mechanisms is limited. In vivo, progenitor cells directionally migrate from the surrounding loose mesenchyme towards regions of increasing matrix adherence (the condensation centers), which is accompanied by the upregulation of fibronectin. Here, we focused on the mechanisms of cell migration during mesenchymal cell condensation and the effects of matrix adherence. Dendrimer-based nanopatterns of the cell-adhesive peptide arginine-glycine-aspartic acid (RGD), which is present in fibronectin, were used to regulate substrate adhesion. We recorded collective and single-cell migration of mesenchymal stem cells, under chondrogenic induction, using live-cell imaging. Our results show that the cell migration mode of single cells depends on substrate adhesiveness, and that cell directionality controls cell condensation and the fusion of condensates. Inhibition experiments revealed that cell-cell interactions mediated by N-cadherin (also known as CDH2) are also pivotal for directional migration of cell condensates by maintaining cell-cell cohesion, thus suggesting a fine interplay between cell-matrix and cell-cell adhesions. Our results shed light on the role of cell interactions with a fibronectin-depositing matrix during chondrogenesis in vitro, with possible applications in regenerative medicine. This article has an associated First Person interview with the first author of the paper.

Recent advances in engineering nanotopographic substrates for cell studies.

Cells sense their environment through the cell membrane receptors. Interaction with extracellular ligands induces receptor clustering at the nanoscale, assembly of the signaling complexes in the cytosol and activation of downstream signaling pathways, regulating cell response. Nanoclusters of receptors can be further organized hierarchically in the cell membrane at the meso- and micro-levels to exert different biological functions. To study and guide cell response, cell culture substrates have been engineered with features that can interact with the cells at different scales, eliciting controlled cell responses. In particular, nanoscale features of 1-100 nm in size allow direct interaction between the material and single cell receptors and their nanoclusters. Since the first "contact guidance" experiments on parallel microstructures, many other studies followed with increasing feature resolution and biological complexity. Here we present an overview of the advances in the field summarizing the biological scenario, substrate fabrication techniques and applications, highlighting the most recent developments.

Multicolor Super-Resolution Microscopy of Protein Corona on Single Nanoparticles.

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.

Development of a Custom-Made 3D Printing Protocol with Commercial Resins for Manufacturing Microfluidic Devices.

The combination of microfluidics and photo-polymerization techniques such as stereolithography (SLA) has emerged as a new field which has a lot of potential to influence in such important areas as biological analysis, and chemical detection among others. However, the integration between them is still at an early stage of development. In this article, after analyzing the resolution of a custom SLA 3D printer with commercial resins, microfluidic devices were manufactured using three different approaches. First, printing a mold with the objective of creating a Polydimethylsiloxane (PDMS) replica with the microfluidic channels; secondly, open channels have been printed and then assembled with a flat cover of the same resin material. Finally, a closed microfluidic device has also been produced in a single process of printing. Important results for 3D printing with commercial resins have been achieved by only printing one layer on top of the channel. All microfluidic devices have been tested successfully for pressure-driven fluid flow.