A radio-frequency coupling network for heating of citrate-coated gold nanoparticles for cancer therapy: design and analysis

Gold nanoparticles (GNPs) are nontoxic, can be functionalized with ligands, and preferentially accumulate in tumors. We have developed a 13.56-MHz RF-electromagnetic field (RF-EM) delivery system capable of generating high E-field strengths required for noninvasive, noncontact heating of GNPs. The bulk heating and specific heating rates were measured as a function of NP size and concentration. It was found that heating is both size and concentration dependent, with 5 nm particles producing a 50.6 ± 0.2 °C temperature rise in 30 s for 25 μg/mL gold (125 W input). The specific heating rate was also size and concentration dependent, with 5 nm particles producing a specific heating rate of 356 ± 78 kW/g gold at 16 μg/mL (125 W input). Furthermore, we demonstrate that cancer cells incubated with GNPs are killed when exposed to 13.56 MHz RF-EM fields. Compared to cells that were not incubated with GNPs, three out of four RF-treated groups showed a significant enhancement of cell death with GNPs (p<0.05). GNP-enhanced cell killing appears to require temperatures above 50 °C for the experimental parameters used in this study. Transmission electron micrographs show extensive vacuolization with the combination of GNPs and RF treatment.     

A Universal Platform for Macromolecular Deliveryinto Cells Using Gold Nanoparticle Layers via the Photoporation Effect

Although promising, it is challenging to develop a simple and universal method for the high‐efficiency delivery of biomacromolecules into diverse cells. Here, a universal delivery platform based on gold nanoparticle layer (GNPL) surfaces is proposed. Upon laser irradiation, GNPL surfaces show such good photothermal properties that absorption of the laser energy causes a rapid increase in surface temperature, leading to enhanced membrane permeability of the cultured cells and the diffusion of macromolecules into the cytosol from the surrounding medium. The high‐efficiency delivery of different macromolecules such as dextran and plasmid DNA into different cell types is achieved, including hard‐to‐transfect mouse embryonic fibroblasts (mEFs) and human umbilical vein endothelial cells (HUVECs), while cell viability is well maintained under optimized irradiation conditions. The platform vastly outperforms the leading commercial reagent, Lipofectamine 2000, especially in transfecting hard‐to‐transfect cell lines (plasmid transfection efficiency: ≈53% vs ≈19% for mEFs and ≈44% vs ≈8% for HUVECs). Importantly, as the gold nanoparticles (GNPs) constituting the GNPL are firmly immobilized together, the potential cytotoxicity caused by endocytosis of GNPs is effectively avoided. This platform is reliable, efficient, and cost‐effective with high‐throughput and broad applicability across different cell types, opening up an innovative avenue for high‐efficiency intracellular delivery.     

Maximizing Transfection Efficiency of Vertically Aligned Silicon Nanowire Arrays

Vertically aligned silicon nanowire (VA‐SiNW) arrays are emerging as a powerful new tool for gene delivery by means of mechanical transfection. In order to utilize this tool efficiently, uncertainties around the required design parameters need to be removed. Here, a combination of nanosphere lithography and templated metal‐assisted wet chemical etching is used to fabricate VA‐SiNW arrays with a range of diameters, heights, and densities. This fabrication strategy allows identification of critical parameters of surface topography and consequently the design of SiNW arrays that deliver plasmid with high transfection efficiency into a diverse range of human cells whilst maintaining high cell viability. These results illuminate the cell‐materials interactions that mediate VA‐SiNW transfection and have the potential to transform gene therapy and underpin future treatment modalities.     

Templateless Synthesis of Polygonal Gold Nanoparticles: An Unsupported and Reusable Catalyst with Superior Activity

We report the synthesis of polygonal gold nanoparticles (GNPs) by an in situ reduction technique using ferric ammonium citrate as reducing agent in absence of any surfactant or polymeric template. Transmission electron microscopic analysis and selected area electron diffraction patterns confirmed the formation of well‐crystalline polygonal GNPs grown preferentially along the (111) direction, which is consistent with the results of X‐ray diffractometry analysis. The results of control experiments of HAuCl₄ with tri‐ammonium citrate in presence of different externally added metal ions like Fe³⁺, Ni²⁺, Cu²⁺, Zn²⁺, and Al³⁺ suggested the ion‐induced growth mechanism in the formation of polygonal GNPs. The purified polygonal GNPs were then successfully used as catalyst in the borohydride reduction of three isomeric nitrophenols and also in the aerobic oxidation of different D ‐hexoses (e.g., D ‐glucose, D ‐mannose, D ‐fructose). The catalytic activity of these polygonal GNPs is higher by a factor of 300–1000, depending on the GNP's sample type, in nitrophenol reduction compared to that of spherical GNPs. Similar activity enhancement was also observed in the aerobic oxidation of different D ‐hexoses. These polygonal GNPs catalyst are very stable and could be reused several times in the borohydride reduction of nitrophenols without much losing in their virgin catalytic activity.     

A Combined Photolithographic and Molecular‐Assembly Approach to Produce Functional Micropatterns for Applications in the Biosciences

Chemical patterns have attracted substantial interest for applications in the field of biosensors, fundamental cell–surface interaction studies, tissue engineering, and biomaterials. A novel micropatterning technique is proposed here that combines a top–down approach based on photolithography and a bottom–up strategy through self‐organization of multifunctional molecules. The development of the molecular‐assembly patterning by lift‐off (MAPL) has been driven by the need to economically produce patches incorporating a controlled surface density of bioligands while inhibiting non‐specific adsorption. In the MAPL process, a photoresist pattern is transferred into the desired biochemical pattern by means of spontaneous adsorption of biologically relevant species and photoresist lift‐off. The surface between the interactive patches is subsequently rendered non‐fouling through immobilization of a polycationic poly(ethylene glycol) (PEG)‐graft polymer. We demonstrate that surface density of biotin molecules inside adhesive islands can be tailored quantitatively and that cells grow selectively on cell‐adhesive peptide patterns. MAPL is considered to be a valuable addition to the toolbox of soft‐lithography techniques for life‐science applications combining simplicity (no clean‐room equipment needed), cost‐effectiveness, reproducibility on the scale of whole wafer surfaces, and flexibility in terms of pattern geometry, chemistry, and substrate choice.     

Multifaceted and Nanobored Particle Arrays Sculpted Using Colloidal Lithography

A novel method of fabricating multifaceted and nanobored particle arrays via colloidal lithography using colloidal‐crystal layers as masks for anisotropic reactive‐ion etching (RIE) is reported. The shape of the sculpted particles is dependent on the crystal orientation relative to the etchant flow, the number of colloidal layers, the RIE conditions, and the matrix (or mask) structure in colloidal lithography. Arrays of non‐spherical particles with sculpted shapes, which to date could not otherwise be produced, are fabricated using a tilted anisotropic RIE process and the layer‐by‐layer growth of a colloidal mask. These non‐spherical particles and their ordered arrays can be used for antireflection surfaces, biosensors, and nanopatterning masks, as well as non‐spherical building blocks for novel colloidal crystals. In addition, polymeric particles with patterned holes of controlled depths obtained by the present method can be applied to the fabrication of functional composite particles.     

Simultaneous Culturing of Cell Monolayers and Spheroids on a Single Microfluidic Device for Bridging the Gap between 2D and 3D Cell Assays in Drug Research

Two‐dimensional (2D) cell cultures have been the primary screening tools to predict drug impacts in vitro for decades. However, owing to the lack of tissue‐specific architecture of 2D cultures, secondary screening using three‐dimensional (3D) cell culture models is often necessary. A microfluidic approach that facilitates side‐by‐side 2D and 3D cell culturing in a single microchannel and thus combines the benefits of both set‐ups in drug screening; that is, the uniform spatiotemporal distributions of oxygen, nutrients, and metabolic wastes in 2D, and the tissue‐like architecture, cell–cell, and cell–extracellular matrix interactions only achieved in 3D. The microfluidic platform is made from an organically modified ceramic material, which is inherently biocompatible and supports cell adhesion (2D culture) and metal adhesion (for integration of impedance electrodes to monitor cell proliferation). To induce 3D spheroid formation on another area, a single‐step lithography process is used to fabricate concave microwells, which are made cell‐repellant by nanofunctionalization (i.e., plasma porosification and hydrophobic coating). Thanks to the concave shape of the microwells, the spheroids produced on‐chip can also be released, with the help of microfluidic flow, for further off‐chip characterization after culturing. In this study, the methodology is evaluated for drug cytotoxicity assessment on human hepatocytes.     

Large‐Scale Nano Piezo Force Position Arrays as Ultrahigh‐Resolution Micro‐ and Nanoparticle Tracker

Defining the position of an object on a planar substrate by force sensors is a common technology nowadays. Many products are commercialized worldwide, which make use of force sensors, especially, for instance, touchpads. Here advanced lithography processes together with piezoelectric materials are demonstrated to fabricate an extremely high resolution force sensor. The approach combines a large array of nanoscale piezoelectric lines fabricated on Si wafer by phase‐shift lithography and atomic‐layer‐deposition‐based spacer lithography techniques. These key lithography methods are utilized to fabricate ultralong (cm range) nanolines on the wafer scale. ZnO and P(VDF‐TrFE) are selected here as materials for piezoelectric signal generators. The detection mechanisms are explained and simulations combined with experimental data are demonstrated to prove the concept. The signal generated when an object approaches one single line is in the nanoampere range. The result enables a new and simple path for a device fabrication, which defines the position with micro‐ and nanometer resolution and can be used, for example, as micro‐ and nanoparticle trackers.     

Self‐assembly of Protein Nanoarrays on Block Copolymer Templates

There is considerable interest in developing functional protein arrays on the nanoscale for high‐throughput protein‐based array technology, and for the study of biomolecular and cell interactions at the physical scale of the biomolecules. To these ends, self‐assembly based techniques may be desirable for the nanopatterning of proteins on large sample areas without the use of lithography equipment. We present a fast, general approach for patterning proteins (and potentially other biomolecules) on the nanoscale, which takes advantage of the ability of block copolymers to self‐assemble into ordered surface nanopatterns with defined chemical heterogeneity. We demonstrate nanoarrays of immunoglobulin and bovine serum albumin on polystyrene‐block‐poly(methyl methacrylate) templates, and illustrate the applicability of our technique through immunoassays and DNA sensing performed on the protein nanoarrays. Furthermore, we show that the pattern formation mechanism is a nanoscale effect originating from a combination of fluid flow forces and geometric restrictions templated by an underlying nanopattern with a difference in protein adsorption behavior on adjacent, chemically distinct surfaces. This understanding may provide a framework for extending the patterning approach to other proteins and material systems.     

Capillary Force Lithography: A Versatile Tool for Structured Biomaterials Interface Towards Cell and Tissue Engineering

This Feature Article aims to provide an in‐depth overview of the recently developed molding technologies termed capillary force lithography (CFL) that can be used to control the cellular microenvironment towards cell and tissue engineering. Patterned polymer films provide a fertile ground for controlling various aspects of the cellular microenvironment such as cell–substrate and cell–cell interactions at the micro‐ and nanoscale. Patterning thin polymer films by molding typically involves several physical forces such as capillary, hydrostatic, and dispersion forces. If these forces are precisely controlled, the polymer films can be molded into the features of a polymeric mold with high pattern fidelity and physical integrity. The patterns can be made either with the substrate surface clearly exposed or unexposed depending on the pattern size and material properties used in the patterning. The former (exposed substrate) can be used to adhere proteins or cells on pre‐defined locations of a substrate or within a microfluidic channel using an adhesion‐repelling polymer such as poly(ethylene glycol) (PEG)‐based polymer and hyaluronic acid (HA). Also, the patterns can be used to co‐culture different cells types with molding‐assisted layer‐by‐layer deposition. In comparison, the latter (unexposed substrate) can be used to control the biophysical surrounding of a cell with tailored mechanical properties of the material. The surface micropatterns can be used to engineer cellular and multi‐cellular architecture, resulting in changes of the cell shape and the cytoskeletal structures. Also, the nanoscale patterns can be used to affect various aspects of the cellular behavior, such as adhesion, proliferation, migration, and differentiation.     

Nickel Silicide Nanowire Arrays for Anti‐Reflective Electrodes in Photovoltaics

Conductive nanowires (NWs) provide several advantages as a template and electrode material for solar cells due to their favorable light scattering properties. While the majority of NW solar cell architectures studied are based on semiconductor materials, metallic NWs could provide equivalent anti‐reflection properties, while acting as a low‐resistance back contact for charge transport, and facilitate light scattering in thin layers of semiconductors coated on the surface. However, fabrication of single‐crystalline highly anti‐reflective NWs on low‐cost, flexible substrates remains a challenge to drive down the cost of NW solar cells. In this study, metallic Ni_xSi NW arrays are fabricated by a simple, bottom‐up, and low‐cost method based on the thermal decomposition of silane on the surface of flexible Ni foil substrates without the need for lithography, etching or catalysts. The optical properties of these NW arrays demonstrate broadband suppression of reflection to levels below 1% from 350 nm to 1100 nm, which is among the highest values reported for NWs. A simple route to control the diameter and density of the NWs is introduced based on variations in a carrier gas flow rate. A high‐resolution TEM, XRD and TEM‐EDS study of the NWs reveals that they are single crystalline, with the phase and composition varying between Ni₂Si and NiSi. The nanowire resistivity is measured to be 10^?4 Ω‐cm, suggesting their use as an efficient back electrode material for nanostructured solar cells with favorable light scattering properties.     

Direct Patterning of Metal Chalcogenide Semiconductor Materials

Lithography is one of the most widely used methods for cutting‐edge research and industrial applications, mainly owing to its ability to draw patterns in the micro and even nanoscale. However, the fabrication of semiconductor micro/nanostructures via conventional electron or optical lithography technologies often requires a time‐consuming multistep process and the use of expensive facilities. Herein, a low‐cost, high‐resolution, facile, and versatile direct patterning method based on metal–organic molecular precursors is reported. The ink‐based metal–organic precursors are found to operate as negative resists, with the material exposed by different methods (electron‐beam/laser/heat/ultraviolet (UV)) to render them insoluble in the development process. This technical process can deliver metal chalcogenide semiconductors with arbitrary 2D/3D patterns with sub‐50 nm resolution. Electron beam lithography, two‐photon absorption lithography, thermal scanning probe lithography, and UV photolithography are demonstrated for the direct patterning process. Different metal chalcogenide semiconductor nanodevices, such as photoconductive selenium‐doped Sb₂S₃ nanoribbons, p‐type PbS single‐nanowire field‐effect transistors, and p‐n junction CdS/Cu₂S nanowire solar cells, are fabricated by this method. This direct patterning technique is a versatile and simple micro/nanolithography technology with considerable potential for “lab‐on‐a‐chip” preparation of semiconductor devices.     

Selective Immobilization of Protein Clusters on Polymeric Nanocraters

A method for fabricating chemically nanopatterned surfaces based on a combination of colloidal lithography and plasma‐ enhanced chemical vapor deposition (PECVD) is presented. This method can be applied for the creation of different nanopatterns, and it is in principle not limited in patterning resolution. Nanocraters of poly(acrylic acid) (carboxylic moieties) surrounded by a matrix of poly(ethylene glycol) are fabricated. Chemical force microscopy demonstrates that the process is able to produce the expected surface chemical contrast. Finally, the carboxylic groups of the craters are activated in order to induce the covalent binding of fluorescent‐labeled proteins. Fluorescence investigation using scanning confocal microscopy shows that the proteins are preferentially attached inside the functional craters.     

Fabrication of Biofunctionalized Quasi‐Three‐Dimensional Microstructures of a Nonfouling Comb Polymer Using Soft Lithography

This paper describes a simple set of patterning methods that are applicable to diverse substrates and allow the routine and rapid fabrication of protein patterns embedded within a background that consists of quasi‐three‐dimensional microstructures of a cell‐resistant polymer. The ensemble of methods reported here utilizes three components to create topographically nonfouling polymeric structures that present cell‐adhesive protein patterns in the regions between the microstructures: the first component is an amphiphilic comb polymer that is comprised of a methyl methacrylate backbone and pendant oligo(ethylene glycol) moieties along the side chain, physically deposited films of which are protein‐ and cell‐resistant. The second component of the fabrication methodology involves the use of different variants of soft lithography, such as microcontact printing to create nonfouling topographical features of the comb polymer that demarcate cell‐adhesive regions of the third component: a cell‐adhesive extracellular protein or peptide. The ensemble of methods reported in this paper was used to fabricate quasi‐three‐dimensional patterns that present topographical and biochemical cues on a variety of substrates, and was shown to successfully maintain cellular patterns for up to two months in serum‐containing medium. We believe that this, and other such methods under development that allow independent and systematic control of chemistry, topography and substrate compliance will provide versatile “test‐beds” for fundamental studies in cell biology as well as allow the discovery of rational design principles for the development of biomaterials and tissue‐engineering scaffolds.     

Layer‐by‐Layer Fabrication and Characterization of Gold‐Nanoparticle/Myoglobin Nanocomposite Films

Multilayer thin films of ～ 7 nm diameter gold nanoparticles (GNPs) linked with horse heart myoglobin (Mb) are fabricated, for the first time, by layer‐by‐layer (LbL) assembly on glass slides, and silicon and plastic substrates. The GNP/Mb nanocomposite films show sharp surface plasmon resonance (SPR) absorption bands that are used to follow the LbL growth of the film and to determine the kinetics of GNP adsorption on the Mb‐modified surface. The GNP/Mb nanocomposite films are characterized using atomic force microscopy, transmission electron microscopy, polarized UV‐vis spectroscopy, and spectroscopic ellipsometry. The GNPs in the multilayer films are spatially separated from one another, and interparticle interactions remain in the film, making it optically anisotropic. The GNP/Mb nanocomposite films are stable in air at temperatures up to 100 °C, and can withstand successive immersions in strongly acidic and basic solutions. The SPR absorption band of the GNP/Mb nanocomposite film in air exhibits a red‐shift in the wavelength maximum and an increase in the maximum absorbance relative to that in water. This result, which is in contrast to that observed with a GNP monolayer on an aminosilane‐functionalized substrate, suggests the shrinkage in air and swelling in water of Mb molecules embedded in the nanocomposite film.     

Porous Silicon Nanodiscs for Targeted Drug Delivery

There is a strong demand for techniques that allow the fabrication of biocompatible porous nanoparticles for drug delivery applications. In this work, a new method to fabricate size‐ and shape‐controlled porous silicon (pSi) nanodiscs is described. The process relies on a combination of colloidal lithography and metal‐assisted chemical etching. Height and diameter of the pSi nanodiscs can be easily adjusted. The nanodiscs are degradable in physiological milieu and are nontoxic to mammalian cells. In order to highlight the potential of the pSi nanodiscs in drug delivery, an in vitro investigation that involved loading of nanodiscs with the anticancer agent camptothecin and functionalization of the nanodisc periphery with an antibody that targets receptors on the surface of neuroblastoma cells is carried out. The thus‐prepared nanocarriers are found to selectively attach to and kill cancer cells.     

Multiarray Nanopattern Electronic Nose (E‐Nose) by High‐Resolution Top‐Down Nanolithography

An electronic nose (E‐nose) is an artificial sensing device that mimics the human olfactory system using a multiarray sensor system. However, since the design and fabrication of multiarray sensing channels are significantly limited because of the requirement of time‐consuming and nonuniversal processes, the development of commercializable and high‐throughput fabrication approaches are critically required. Herein, high‐resolution top‐down lithography is developed for E‐nose fabrication for the first time. Five different metal oxide semiconductor (MOS) nanopattern channels (NiO, CuO, Cr₂O₃, SnO₂, and WO₃) are fabricated into multiarray sensors with high‐throughput using a unique lithographic approach that utilizes the sputtering of grains of the metals via low‐energy ion plasma bombardment. The nanopattern channels show i) high‐resolutions (15 nm scale), ii) high‐aspect‐ratios (11; 14 nm width and 150 nm height), and iii) ultrasmall grains (5.1 nm) with uniformity on a cm² scale, resulting in high sensitivity toward the target analytes. The E‐nose system, which is composed of five MOS nanopattern channels, can successfully distinguish seven different hazardous analytes, including volatile organic compounds and nitrogen‐containing compounds. It is expected that this unique lithography approach can provide a simple and reliable method for commercializable channel fabrication, and the E‐noses can have further applications in real‐life situations.     

3D‐Printed Soft Magnetoelectric Microswimmers for Delivery and Differentiation of Neuron‐Like Cells

Neurodegenerative diseases generally result in irreversible neuronal damage and neuronal death. Cell therapy shows promise as a potential treatment for these diseases. However, the therapeutic targeted delivery of these cells and the in situ provision of a suitable microenvironment for their differentiation into functional neuronal networks remain challenging. A highly integrated multifunctional soft helical microswimmer featuring targeted neuronal cell delivery, on‐demand localized wireless neuronal electrostimulation, and post‐delivery enzymatic degradation is introduced. The helical soft body of the microswimmer is fabricated by two‐photon lithography of the photocurable gelatin–methacryloyl (GelMA)‐based hydrogel. The helical body is then impregnated with composite multiferroic nanoparticles displaying magnetoelectric features (MENPs). While the soft GelMA hydrogel chassis supports the cell growth, and is degraded by enzymes secreted by cells, the MENPs allow for the magnetic transportation of the bioactive chassis, and act as magnetically mediated electrostimulators of neuron‐like cells. The unique combination of the materials makes these microswimmers highly integrated devices that fulfill several requirements for their future translation to clinical applications, such as cargo delivery, cell stimulation, and biodegradability. The authors envision that these devices will inspire new avenues for targeted cell therapies for traumatic injuries and diseases in the central nervous system.     

Photoresponsive Soft‐Robotic Platform: Biomimetic Fabrication and Remote Actuation

Biomimetic microsystems, which can be driven by various stimuli, are an emerging field in micro/nano‐technology and nano‐medicine. In this study, a soft and fast‐response robotic platform, constituted by PDMS/graphene‐nanoplatelets composited layer (PDMS/GNPs) and pristine PDMS layer, is presented. Due to the differences in coefficient of thermal expansion and Young's modulus of the two layers, the bilayer platform can be driven to bend to the PDMS/GNPs side by light irradiation. The robotic platform (1 mm in width and 7 mm in length) can be deflected about 1500 μm by near infrared irradiation (nIR)(808 nm in wavelength) within 3.4 s, and excellent reversibility and repeatability in actuation are also revealed by sweeping and multicycle light irradiation. The experiments also show that, the presented bilayer platform in various shapes, that is, fish‐like shapes, can float and swim to perspective location in fluid (i.e., water), whose moving directions and velocities can be remotely adjusted by light, indicating an excellent light‐actuation ability and well controllability. The results may be not only hopeful in developing light‐driven drug‐delivery platform, but also the bio‐robotic microgrippers applying in vivo and in vitro.     

Nanoparticle Growth via Concentration Gradients Generated by Enzyme Nanopatterns

Biomineralizing organisms can grow nanomaterials with unexpected morphologies in an organic matrix where temporal and vectorial gradients of crystal growth precursors are established. Here, concentration gradients for the crystallization of gold nanoparticles are generated and applied on silicon substrates. Gradients of crystal growth precursors are generated by enzymes patterned as lines that are separated by distances ranging from the micro‐ to the nanoscale. The concentration of crystallization precursors around the lines separated by nanometric distances is not only determined by mass transport and enzyme activity but also by the nanoscale organization of biocatalysts. This nanoscale organization favors non‐classical crystal growth conditions that lead to the formation of nanoparticle clusters containing nanocrystals that are highly crystallographically aligned. The combination of bottom‐up crystal growth with top‐down electron beam lithography enables the fabrication of micrometric patterns containing gold nanoparticles of different size, shape, and surface density. These are all critical parameters that determine the physical properties of these nanomaterials.