Nanopore Blockade Sensors for Quantitative Analysis Using an Optical Nanopore Assay.

Nanopore sensing is a popular biosensing strategy that is being explored for the quantitative analysis of biomarkers. With low concentrations of analytes, nanopore sensors face challenges related to slow response times and selectivity. Here, we demonstrate an approach to rapidly detect species at ultralow concentrations using an optical nanopore blockade sensor for quantitative detection of the protein vascular endothelial growth factor (VEGF). This sensor relies on monitoring fluorescent polystyrene nanoparticles blocking nanopores in a nanopore array of 676 nanopores. The fluorescent signal is read out using a wide-field fluorescence microscope. Nonspecific blockade events are then distinguished from specific blockade events based on the ability to pull the particles out of the pore using an applied electric field. This allows the detection of VEGF at sub-picomolar concentration in less than 15 min.

Biocompatibility and proteomic profiling of DMSA-coated iron nanocubes in a human glioblastoma cell line.

Background: Superparamagnetic iron core iron oxide shell nanocubes have previously shown superior performance in magnetic resonance imaging T2 contrast enhancement compared with spherical nanoparticles. Methods: Iron core iron oxide shell nanocubes were synthesized, stabilized with dimercaptosuccinic acid (DMSA-NC) and physicochemically characterized. MRI contrast enhancement and biocompatibility were assessed in vitro. Results: DMSA-NC showed a transverse relaxivity of 122.59 mM-1·s-1 Fe. Treatment with DMSA-NC did not induce cytotoxicity or oxidative stress in U-251 cells, and electron microscopy demonstrated DMSA-NC localization within endosomes and lysosomes in cells following internalization. Global proteomics revealed dysregulation of iron storage, transport, transcription and mRNA processing proteins. Conclusion: DMSA-NC is a promising T2 MRI contrast agent which, in this preliminary investigation, demonstrates favorable biocompatibility with an astrocyte cell model.

MRI is a powerful tool used in the diagnosis of cancer, strokes and other injuries. An MRI scan can be improved with the use of iron oxide nanoparticles, which enhance the contrast of the image. In this study we have developed cube-shaped iron nanoparticles (nanocubes), which have been previously shown to be more effective at inducing contrast. We demonstrated that iron-based nanocubes do not damage or induce stress in cells and work effectively as an MRI contrast agent. We further analyzed how the nanocubes may affect cell functioning by investigating changes to protein levels in the cells. The results of this study are promising steps towards using iron-based nanocubes as a tool to improve the clarity of MRI scans for medical imaging and diagnosis. Future work must determine whether these nanocubes work effectively and safely in an animal model, which is a critical step in progressing to their use in clinical settings.

3D Bioprintable Hydrogel with Tunable Stiffness for Exploring Cells Encapsulated in Matrices of Differing Stiffnesses.

In vitro cell models have undergone a shift from 2D models on glass slides to 3D models that better reflect the native 3D microenvironment. 3D bioprinting promises to progress the field by allowing the high-throughput production of reproducible cell-laden structures with high fidelity. The current stiffness range of printable matrices surrounding the cells that mimic the extracellular matrix environment remains limited. The work presented herein aims to expand the range of stiffnesses by utilizing a four-armed polyethylene glycol with maleimide-functionalized arms. The complementary cross-linkers comprised a matrix metalloprotease-degradable peptide and a four-armed thiolated polymer which were adjusted in ratio to tune the stiffness. The modularity of this system allows for a simple method of controlling stiffness and the addition of biological motifs. The application of this system in drop-on-demand printing is validated using MCF-7 cells, which were monitored for viability and proliferation. This study shows the potential of this system for the high-throughput investigation of the effects of stiffness and biological motif compositions in relation to cell behaviors.

Hierarchical assembly of tryptophan zipper peptides into stress-relaxing bioactive hydrogels.

Soft materials in nature are formed through reversible supramolecular assembly of biological polymers into dynamic hierarchical networks. Rational design has led to self-assembling peptides with structural similarities to natural materials. However, recreating the dynamic functional properties inherent to natural systems remains challenging. Here we report the discovery of a short peptide based on the tryptophan zipper (trpzip) motif, that shows multiscale hierarchical ordering that leads to emergent dynamic properties. Trpzip hydrogels are antimicrobial and self-healing, with tunable viscoelasticity and unique yield-stress properties that allow immediate harvest of embedded cells through a flick of the wrist. This characteristic makes Trpzip hydrogels amenable to syringe extrusion, which we demonstrate with examples of cell delivery and bioprinting. Trpzip hydrogels display innate bioactivity, allowing propagation of human intestinal organoids with apical-basal polarization. Considering these extensive attributes, we anticipate the Trpzip motif will prove a versatile building block for supramolecular assembly of soft materials for biotechnology and medicine.

Evaluation of Dimercaptosuccinic Acid-Coated Iron Nanoparticles Immunotargeted to Amyloid Beta as MRI Contrast Agents for the Diagnosis of Alzheimer's Disease.

Nanoparticle-based magnetic contrast agents have opened the potential for magnetic resonance imaging (MRI) to be used for early non-invasive diagnosis of Alzheimer's disease (AD). Accumulation of amyloid pathology in the brain has shown association with cognitive decline and tauopathy; hence, it is an effective biomarker for the early detection of AD. The aim of this study was to develop a biocompatible magnetic nanoparticle targeted to amyloid beta (Aß) plaques to increase the sensitivity of T2-weighted MRI for imaging of amyloid pathology in AD. We presented novel iron core-iron oxide nanoparticles stabilized with a dimercaptosuccinic acid coating and functionalized with an anti-Aß antibody. Nanoparticle biocompatibility and cellular internalization were evaluated in vitro in U-251 glioblastoma cells using cellular assays, proteomics, and transmission electron microscopy. Iron nanoparticles demonstrated no significant in vitro cytotoxicity, and electron microscopy results showed their movement through the endocytic cycle within the cell over a 24 h period. In addition, immunostaining and bio-layer interferometry confirmed the targeted nanoparticle's binding affinity to amyloid species. The iron nanoparticles demonstrated favourable MRI contrast enhancement; however, the addition of the antibody resulted in a reduction in the relaxivity of the particles. The present work shows promising preliminary results in the development of a targeted non-invasive method of early AD diagnosis using contrast-enhanced MRI.

Tuning the Mechanical Properties of Multiarm RAFT-Based Block Copolyelectrolyte Hydrogels via Ionic Cross-Linking for 3D Cell Cultures.

Hydrogels that serve as native extracellular matrix (ECM) mimics are typically naturally derived hydrogels that are physically cross-linked via ionic interactions. This means rapid gelation of synthetic polymers, which give control over the chemical and physical cues in hydrogel formation. Herein, we combine the best of both systems by developing a synthetic hydrogel with ionic cross-linking of block copolyelectrolytes to rapidly create hydrogels. Reversible addition-fragmentation chain-transfer (RAFT) polymerization was used to synthesize oppositely charged polyelectrolyte molecules and, in turn, modulate the mechanical property of stiffness. The mechanical stiffness of a range of 900-3500 Pa was tuned by varying the number of charged ionic groups, the length of the polymer arms, and the polymer concentration. We demonstrate the synthetic polyelectrolyte hydrogel as an ECM mimic for three-dimensional (3D) in vitro cell models using MCF-7 breast cancer cells.

Upconversion Nanoparticle-Based Cell Membrane-Coated cRGD Peptide Bioorthogonally Labeled Nanoplatform for Glioblastoma Treatment.

Glioblastoma is hard to be eradicated partly because of the obstructive blood-brain barrier (BBB) and the dynamic autophagy activities of glioblastoma. Here, hydroxychloroquine (HDX)-loaded yolk-shell upconversion nanoparticle (UCNP)@Zn0.5Cd0.5S nanoparticle coating with the cyclic Arg-Gly-Asp (cRGD)-grafted glioblastoma cell membrane for near-infrared (NIR)-triggered treatment of glioblastoma is prepared for the first time. UCNPs@Zn0.5Cd0.5S (abbreviated as YSN, yolk-shell nanoparticle) under NIR radiation will generate reactive oxygen species for imposing cytotoxicity. HDX, the only available autophagy inhibitor in clinical studies, can enhance cytotoxicity by preventing damaged organelles from being recycled. The cRGD-decorated cell membrane allowed the HDX-loaded nanoparticles to efficiently bypass the BBB and specifically target glioblastoma cells. Exceptional treatment efficacy of the NIR-triggered chemotherapy and photodynamic therapy was achieved in U87 cells and in the mouse glioblastoma model as well. Our results provided proof-of-concept evidence that HDX@YSN@CCM@cRGD could overcome the delivery barriers and achieve targeted treatment of glioblastoma.

How can we use the endocytosis pathways to design nanoparticle drug-delivery vehicles to target cancer cells over healthy cells?

Targeted drug delivery in cancer typically focuses on maximising the endocytosis of drugs into the diseased cells. However, there has been less focus on exploiting the differences in the endocytosis pathways of cancer cells versus non-cancer cells. An understanding of the endocytosis pathways in both cancer and non-cancer cells allows for the design of nanoparticles to deliver drugs to cancer cells whilst restricting healthy cells from taking up anticancer drugs, thus efficiently killing the cancer cells. Herein we compare the differences in the endocytosis pathways of cancer and healthy cells. Second, we highlight the importance of the physicochemical properties of nanoparticles (size, shape, stiffness, and surface chemistry) on cellular uptake and how they can be adjusted to selectively target the dominated endocytosis pathway of cancer cells over healthy cells and to deliver anticancer drug to the target cells. The review generates new thought in the design of cancer-selective nanoparticles based on the endocytosis pathways.

Introducing Stacking Faults into Three-Dimensional Branched Nickel Nanoparticles for Improved Catalytic Activity.

Creating high surface area nanocatalysts that contain stacking faults is a promising strategy to improve catalytic activity. Stacking faults can tune the reactivity of the active sites, leading to improved catalytic performance. The formation of branched metal nanoparticles with control of the stacking fault density is synthetically challenging. In this work, we demonstrate that varying the branch width by altering the size of the seed that the branch grows off is an effective method to precisely tune the stacking fault density in branched Ni nanoparticles. A high density of stacking faults across the Ni branches was found to lower the energy barrier for Ni2+/Ni3+ oxidation and result in enhanced activity for electrocatalytic oxidation of 5-hydroxylmethylfurfural. These results show the ability to synthetically control the stacking fault density in branched nanoparticles as a basis for enhanced catalytic activity.

A Transparent Semiconducting Surface for Capturing and Releasing Single Cells from a Complex Cell Mixture.

Selective isolation of individual target cells from a heterogeneous population is technically challenging; however, the ability to retrieve single cells can have high significance in various aspects of biological research. Here, we present a new photoelectrochemical surface based on a transparent electrode that is compatible with high-resolution fluorescence microscopy for isolating individual rare cells from complex biological samples. This is underpinned by two important factors: (i) careful design of the electrode by patterning discrete Au disks of micron dimension on amorphous silicon-indium tin oxide films and (ii) orthogonal surface chemistry, which modifies the patterned electrode with self-assembly layers of different functionalities, to selectively capture target cells on the Au disks and resist cell binding to the amorphous silicon surface. The co-stimulation of the surface using light from a microscope and an electric potential triggers the reductive desorption of the alkanethiol monolayer from the Au disks to release the single cells of interest from the illuminated regions only. Using circulating tumor cells as a model, we demonstrate the capture of cancer cells on an antibody-coated surface and selective release of single cancer cells with low expression of epithelial cell adhesion molecules.

Rapid and ultrasensitive electrochemical detection of DNA methylation for ovarian cancer diagnosis.

Alterations in DNA methylation, a stable epigenetic marker, are important components in the development of cancer. It is vital to develop diagnostic systems with the ability to rapidly quantify DNA methylation with high sensitivity and selectivity. However, the analysis of DNA methylation must address two main challenges: (i) ultralow abundance and (ii) differentiating methylated cytosine from normal cytosine on target DNA sequence in the presence of an overwhelming background of circulating cell-free DNA. Here we report the development of an ultrasensitive and highly-selective electrochemical biosensor for the rapid detection of DNA methylation in blood. The sensing of DNA methylation involves the hybridization on a network of probe DNA modified gold-coated magnetic nanoparticles (DNA-Au@MNPs) complementary to target DNA, and subsequently enzymatic cleavage to differentiate methylated DNA strands from corresponding unmethylated DNA strands. The biosensor presents a dynamic range from 2 aM to 20 nM for 110 nucleotide DNA sequences containing a single-site methylation with the lowest detected concentration of 2 aM. This DNA-Au@MNPs based sensor provides a promising method to achieve 35 min response time and minimally invasive diagnosis of ovarian cancer.

Zero-valent iron core-iron oxide shell nanoparticles coated with silica and gold with high saturation magnetization.

A new type of gold-coated magnetic nanoparticle with strongly magnetic zero-valent iron core-iron oxide shell were synthesized. The small size of the magnetic cores and the zero-valent iron ensured superparamagnetic behaviour and high saturation magnetization of the overall nanoparticles. The nanoparticles showed stability against magnetic aggregation and good colloidal stability, which is important for many biomedical applications.

Albendazole Release from Silica-Chitosan Nanospheres. In Vitro Study on Cervix Cancer Cell Lines.

In this work, a pH-responsive drug-carrier based on chitosan-silica nanospheres was developed as a carrier for Albendazole (ABZ), a poorly water-soluble anthelmintic drug. Spherical silica nanoparticles were obtained by Stöber method and further etched to obtain mesoporous particles with sizes ranging from 350 to 400 nm. The specific BET area of nanoparticles increased from 15 m2/g to 150 m2/g for etched silica, which also exhibited a uniform pore size distribution. X-ray powder diffraction showed the presence of amorphous phase of silica and a low-intensity peak attributed to ABZ for the drug-loaded nanoparticles. A uniform layer of chitosan was obtained ranging from 10 to 15 nm in thickness due to the small concentration of chitosan used (0.45 mg of chitosan/mg of SiO2). The in vitro evaluation of hybrid nanoparticles was performed using four cervical cancer cell lines CaSki, HeLa, SiHa and C33A, showing a significant reduction in cell proliferation (>85%) after 72 h. Therefore, we confirmed the encapsulation and bioavailability of the drug, which was released in a controlled way, and the presence of chitosan delayed the release, which could be of interest for the development of prolonged release drug delivery systems.

Rapid and ultrasensitive electrochemical detection of circulating tumor DNA by hybridization on the network of gold-coated magnetic nanoparticles.

An accurate and robust method for quantifying the levels of circulating tumor DNA (ctDNA) is vital if this potential biomarker is to be used for the early diagnosis of cancer. The analysis of ctDNA presents unique challenges because of its short half-life and ultralow abundance in early stage cancers. Here we develop an ultrasensitive electrochemical biosensor for rapid detection of ctDNA in whole blood. The sensing of ctDNA is based on hybridization on a network of probe DNA modified gold-coated magnetic nanoparticles (DNA-Au@MNPs). This DNA-Au@MNPs biosensor can selectively detect short- and long-strand DNA targets. It has a broad dynamic range (2 aM to 20 nM) for 22 nucleotide DNA target with an ultralow detection limit of 3.3 aM. For 101 nucleotide ctDNA target, a dynamic range from 200 aM to 20 nM was achieved with a detection limit of 5 fM. This DNA-Au@MNPs based sensor provides a promising method to achieve 20 min response time and minimally invasive cancer early diagnosis.

Formation of Si-Rich Interfaces by Radiation-Induced Diffusion and Microsegregation in CrN/ZrN Nanolayer Coating.

A combination of coating deposition and consequent ion implantation could be beneficial in wear-resistant antifriction surface design and modification. In the present paper, the effects of low-energy 60 keV Si-ion implantation on multinanolayered CrN/ZrN grown on a stainless-steel substrate have been investigated. Complementary experimental (X-ray diffraction, high-resolution transmission electron microscopy, energy-dispersive spectroscopy, secondary ion mass spectrometry) and theoretical (first-principles) methods have been employed to investigate the structure, phase, and composition under a 1 × 10-17 cm-2 irradiation dose. This study has revealed a moderate radiation-tolerance of the CrN/ZrN system, with a 26 nm bilayer period, where the effective ion range after irradiation was below 110 nm. Within the ion range, a decrease in composition homogeneity and structure crystallinity has been found. Si negative ions have been distributed asymmetrically with peak concentrations (10 and 6%) occupying the interfaces between the CrN and ZrN layers. First-principles investigations of the CrN/ZrN(001) heterostructures were carried out to validate the experimental results, which showed that the alignment of Si-rich interfaces closer to chromium layers is a consequence of the lower substitution energy of CrN rather than ZrN. Thus, strong Si-Cr bindings and difference in displacement energies of ZrN and CrN have been attributed as the main factors in Si-rich interface formation. The pin-on-ball tribological test results have exposed the enhancement in wear resistance and the friction coefficient of nanoscale coating via amorphous Si particles descending from interfacial areas and acting as a third-body.

Ultrasensitive detection of programmed death-ligand 1 (PD-L1) in whole blood using dispersible electrodes.

The direct quantification of programmed death-ligand 1 (PD-L1) as a biomarker for cancer diagnosis, prognosis and treatment efficacy is an unmet clinical need. Herein, we demonstrate the first report of rapid, ultrasensitive and selective electrochemical detection of PD-L1 directly in undiluted whole blood using modified gold-coated magnetic nanoparticles as "dispersible electrodes" with an ultralow detection limit of 15 attomolar and a response time of only 15 minutes.

Investigating Spatial Heterogeneity of Nanoparticles Movement in Live Cells with Pair-Correlation Microscopy and Phasor Analysis.

How nanoparticles distribute in living cells and overcome cellular barriers are important criteria in the design of drug carriers. Pair-correlation microscopy is a correlation analysis of fluctuation in the fluorescence intensity obtained by a confocal line scan that can quantify the dynamic properties of nanoparticle diffusion including the number of mobile nanoparticles, diffusion coefficient, and transit time across a spatial distance. Due to the potential heterogeneities in nanoparticle properties and the complexity within the cellular environment, quantification of averaged auto- and pair-correlation profiles may obscure important insights into the ability of nanoparticles to deliver drugs. To overcome this issue, we used phasor analysis to develop a data standardizing method, which can segment the scanned line into several subregions according to diffusion and address the spatial heterogeneity of nanoparticles moving inside cells. The phasor analysis is a fit-free method that represents autocorrelation profiles for each pixel relative to free diffusion on the so-called phasor plots. Phasor plots can then be used to select subpopulations for which the auto- and pair-correlation analysis can be performed separately. We demonstrate the phasor analysis for pair-correlation microscopy for investigating 16 nm, Cy5-labeled silica nanoparticles diffusing across the plasma membrane and green fluorescent proteins (GFP) diffusing across nuclear envelope in MCF-7 cells.

Combining Nanoconfinement in Ag Core/Porous Cu Shell Nanoparticles with Gas Diffusion Electrodes for Improved Electrocatalytic Carbon Dioxide Reduction.

Bimetallic silver-copper electrocatalysts are promising materials for electrochemical CO2 reduction reaction (CO2RR) to fuels and multi-carbon molecules. Here, we combine Ag core/porous Cu shell particles, which entrap reaction intermediates and thus facilitate the formation of C2+ products at low overpotentials, with gas diffusion electrodes (GDE). Mass transport plays a crucial role in the product selectivity in CO2RR. Conventional H-cell configurations suffer from limited CO2 diffusion to the reaction zone, thus decreasing the rate of the CO2RR. In contrast, in the case of GDE-based cells, the CO2RR takes place under enhanced mass transport conditions. Hence, investigation of the Ag core/porous Cu shell particles at the same potentials under different mass transport regimes reveals: (i) a variation of product distribution including C3 products, and (ii) a significant change in the local OH- activity under operation.

How to exploit different endocytosis pathways to allow selective delivery of anticancer drugs to cancer cells over healthy cells.

It was recently shown that it is possible to exploit the nanoparticle shape to selectively target endocytosis pathways found in cancer and not healthy cells. It is important to understand and compare the endocytosis pathways of nanoparticles in both cancer and healthy cells to restrict the healthy cells from taking up anticancer drugs to help reduce the side effects for patients. Here, the clathrin-mediated endocytosis inhibitor, hydroxychloroquine, and the anticancer drug, doxorubicin, are loaded into the same mesoporous silica nanorods. The use of nanorods was found to restrict the uptake by healthy cells but allowed cancer cells to take up the nanorods via the macropinocytosis pathway. Furthermore, it is shown that the nanorods can selectively deliver doxorubicin to the nucleus of breast cancer cells and to the cytoplasm of pancreatic cancer cells. The dual-drug-loaded nanorods were able to selectively kill the breast cancer cells in the presence of healthy breast cells. This study opens exciting possibilities of targeting cancer cells based on the material shape rather than targeting antibodies.

Faceted Branched Nickel Nanoparticles with Tunable Branch Length for High-Activity Electrocatalytic Oxidation of Biomass.

Controlling the formation of nanosized branched nanoparticles with high uniformity is one of the major challenges in synthesizing nanocatalysts with improved activity and stability. Using a cubic-core hexagonal-branch mechanism to form highly monodisperse branched nanoparticles, we vary the length of the nickel branches. Lengthening the nickel branches, with their high coverage of active facets, is shown to improve activity for electrocatalytic oxidation of 5-hydroxymethylfurfural (HMF), as an example for biomass conversion.