Mechanism of Cellular Uptake to Optimized AuNP Beacon for Tracing mRNA Changes in Living Cells

Molecular beacon is a promising tool for mRNA detection in living cells. But the low detecting efficiency and narrow application range limited its development. In this study, we synthesized a novel gold nanoparticle (AuNP) beacon by optimizing the sequence amount and modified polyethylene glycol (PEG) and cell‐penetrating peptide (CPP) on the gold core. Then, the mechanism of beacon cell uptake was investigated. Lastly, we used the AuNP beacon to study the Akt‐mTOR‐HIF‐1 signaling pathway and the function and mechanism of miR‐7 in breast tumor cells. The results showed that the optimization obviously amplified the fluorescence signal of the AuNP beacon. The mechanism study described the process of AuNP beacon cellular uptake and confirmed amplifying the amount of beacon cellular uptake could obviously enhance the fluorescence signal. Compared to results, the accuracy of the gold nanoparticle beacon is similar to the results of real‐time‐Q‐PCR (RT‐PCR) and western blotting but that the operation is much simpler. Furthermore, in this study, we found that our Akt gold nanoparticle beacon had a similar function to that of the Akt small interfering RNA (siRNA). In summary, the gold nanoparticle beacon may be a promising method for the study of signaling pathways.     

Direct Synthesis of Gold Nanoparticle‐Over‐Nanosheet for Sensitive SERS Detection

A simple ethanol sol‐based method for the synthesis of gold nanosheets (AuNSs) and gold nanoparticle‐over‐nanosheet (AuNP/NS) is developed. Gold nanoparticles (AuNPs) with average sizes of ≈8 nm are grown in situ on the surface of the AuNS, which forms a NP/NS structure that obtains strong, significantly improved, surface‐enhanced Raman spectroscopy activity with the magnitude ≈2 and ≈6 orders higher than the simplex AuNP and AuNS, respectively. This performance is mainly attributed to uniform AuNPs that are closely packed over AuNS and coupled with NP–NS and NP–NP interactions. The NP–NS–GP (the gap between NP–NS) is narrower than NP–NP–GP in which much stronger and steadier plasmon resonance is obtained that can significantly enhance the Raman signal. The results show that single‐crystalline AuNS is an ideal substrate, which can be further coated with other metallic NPs to form a new flexible, high‐activity and AuNS‐based nanocomposite for a wide variety of applications.     

Design of Gold Nanoparticles in Dendritic Cell‐Based Vaccines

The design of effective cancer vaccines must be able to activate dendritic cells (DCs) of the innate immune system in order to induce immunity to pathogens and cancer. DCs patrol the body and once they encounter antigens, they orchestrate a complex mechanism of events and signals that can alert the adaptive immune system to action. However, DC‐based vaccines remain a challenge in part because the source and quality of antigens, the DC targeting molecule, type of adjuvant, and delivery vehicle must be optimized to induce a robust immune response. Gold nanoparticles (AuNPs) have now entered clinical trials as carriers due to their ease of functionalization with antigens, adjuvants, and targeting molecules. This progress report discusses how AuNPs can influence DC activation and maturation, as well as their potential impact on T helper (Th) differentiation. Ultimately, successful AuNP‐based DC vaccines are able to induce phagocytosis, activation/maturation, migration, T cell costimulation, and cytokine secretion, which is named AuNP‐induced DC tuning (AuNP‐DC tuning). Although at its infancy, understanding the processes of AuNP‐DC tuning will give a better understanding of how best to engineer AuNPs and will redefine the next generation of DC‐based vaccines.     

E224G Regulation of the PIP_2-Induced Gating Kinetics of Kir2.1 Channels

As a member of the inwardly rectifying K~+ channel(Kir) family, Kir2.1 allows K~+ to influx the cell more easily than to efflux, a biophysical phenomenon named inward rectification. The function of Kir2.1 is to set the resting membrane potential and modulate membrane excitability. It has been reported that residue E224 plays a key role in regulating inward rectification. The mutant Kir2.1(E224 G) displays weaker inward rectification than the WT channel. Gating of Kir2.1 depends on the membrane lipid, PIP2, such that the channel gates are closed in the absence of PIP_2. Here we perform electrophysiological and computational approaches, and demonstrate that E224 also plays an important role in the PIP2-dependent activation of Kir2.1 in addition to its influence on inward rectification. The E224 G mutant takes 4.5 times longer to be activated by PIP_2. To probe the mechanism by which E224 G slows the channel opening kinetics, we perform targeted molecular dynamics simulations and find that the mutant weakens the interactions between CD-loop and C-linker(H221-R189) and the adjacent G-loops(R312-E303) which are thought to stabilize the open state of the channel in our previous work. These data provide new insights into the regulation of Kir2.1 channel activity and suggest that a common mechanism may be involved in the distinct biophysical processes, such as inward rectification and PIP2-induced gating.     

The Effect of ζ‐Potential and Hydrodynamic Size on Nanoparticle Interactions in Hydrogels

The ζ‐potential and hydrodynamic size (d_h) of nanoparticles (NPs) are systematically controlled by capping gold NPs (AuNPs) with polymers having different charges and treating them in NaCl solutions of diverse concentrations. Interactions between AuNPs in hydrogel are caused by chemical reactions induced by 1,4‐dithiothreitol. The effect of ζ‐potential is clear, as negatively charged AuNPs can be aggregated in neutral agarose gel, but the amount of aggregation is significantly affected by the magnitude of the negative surface charge on the AuNPs. However, all positively charged AuNPs show negligible aggregation in agarose gel with slightly negative polarity. The effect of d_h on AuNP aggregation is different from that of ζ‐potential. Although AuNPs with small d_h generally show more aggregation than those with large d_h, the amount of AuNP capping layer is critical. Thus, the amount of polymer present on NP surface needs to be considered to investigate the effect of d_h on AuNP aggregation. Through extended Derjaguin, Landau, Verwey, Overbeek (XDLVO) theory, it is shown that the charges of the AuNPs and the hydrogel, as well as the d_h of the NPs, are related to electrostatic repulsion and steric hindrance, which affect AuNP aggregation in hydrogel.     

The Competitive Dynamic Binding of Some Blood Proteins Adsorbed on Gold Nanoparticles

Nanoparticle (NP) surfaces are modified immediately by the adsorption of proteins when injected into human blood, leading to the formation of a protein corona. The protein‐coated NPs may be recognized by living cells. Furthermore, the adsorption of serum proteins is a continuous competitive dynamic process that is the key to exploring the bioapplication and biosafety of NPs. In this study, the competitive dynamic adsorption of some serum proteins on gold nanoparticles (AuNPs) is investigated by fluorescence emission, dynamic light scattering, and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Serum proteins with different AuNPs binding affinities are used to address the competitive dynamic process of protein‐AuNP interactions in vitro. The results show that more abundant serum proteins, such as human serum albumin, adsorb on AuNPs first, and then the higher binding affinity and lower concentration serum proteins, such as fibrinogen (FIB), replace the abundant and lower binding affinity serum proteins. However, the lower binding affinity serum proteins, such as hemoglobin, do not replace the higher binding affinity proteins from the protein‐AuNP conjugates. During the dynamic exchange process, the larger the binding affinities difference between two proteins, the faster the exchange rate. This dynamic exchange process usually takes longer in inner protein‐AuNP conjugates (hard corona) than the external surface of protein‐AuNP conjugates (soft corona).     

Four channels mediate the mechanical aspects of touch

Although previous physiological and anatomical experiments have identified four afferent fiber types (PC, RA, SA II, and SA I) in glabrous (nonhairy) skin of the human somatosensory periphery, only three have been shown to mediate tactile (mechanoreceptive) sensation. Psychophysical evidence that four channels (P, NP I, NP II, and NP III) do, indeed, participate in the perceptual process is presented. In a series of experiments involving selective masking of the various channels, modification of the skin-surface temperature, and testing cutaneous sensitivity down to very low-vibratory frequencies, the fourth psychophysical channel (NP III) is defined. Based on these experiments and previous work from our laboratory, it is concluded that the four channels work in conjunction at threshold to create an operating range for the perception of vibration that extends from at least 0.4 to greater than 500 Hz. Each of the four channels appears to mediate specific portions of the overall threshold-frequency characteristic. Selection of appropriate neural-response criteria from previously published physiological data and correlation of their derived frequency characteristics with the four psychophysical channels indicates that each channel has its own physiological substrate: P channel and PC fibers, NP I channel and RA fibers, NP II channel and SA II fibers, and NP III channel and SA I fibers. These channels partially overlap in their absolute sensitivities, making it likely that suprathreshold stimuli may activate two or more of the channels at the same time. Thus the perceptual qualities of touch may be determined by the combined inputs from four channels.     

Raman spectroscopy of combinatory anticancer drug release from gold nanoparticles inside a single leukemia cell

Combinatory anticancer drug release from gold nanoparticles (AuNPs) in K562 human myeloid leukemia cells was performed using Raman spectroscopy. We fabricated the anticancer drug of imatinib as a BCR‐ABL tyrosine kinase inhibitor on AuNP surfaces along with a transferrin (Tf)‐targeting moiety to treat the leukemia cells. DNA topoisomerase I inhibitor topotecan was also assembled to monitor its fluorescence onto AuNPs. The linker group of 4‐carboxylic benzoic acid was used to conjugate to targeting the Tf protein. Our Raman data indicated that the drug molecules were not detached in the cell culture media but released after treatment with glutathione (2 mM). Intracellular distribution and release of the anticancer drug–AuNP conjugates in K562 cells were examined by both fluorescence microscopy and dark‐field microscopy with surface‐enhanced Raman scattering. Copyright © 2013 John Wiley & Sons, Ltd.     

Structure and Stability of PEG‐ and Mixed PEG‐Layer‐Coated Nanoparticles at High Particle Concentrations Studied In Situ by Small‐Angle X‐Ray Scattering

Ligand‐layer structure and stability of gold nanoparticles (AuNP) coated with α‐methoxypoly(ethylene glycol)‐ω‐(11‐mercaptoundecanoate) (PEGMUA) layers and mixed layers of PEGMUA and 11‐mercaptoundecanoic acid (MUA) at high AuNP concentrations are studied in situ by small‐angle X‐ray scattering (SAXS). The thickness of the ligand layer is modified by the molecular weight of the PEG‐ligands (2 and 5 kDa), and the PEG‐grafting density is decreased by coadsorption of MUA. The response of the conjugates to a pressure of up to 4 kbar is probed. The results indicate strongly hydrated PEG layers at high grafting densities. The stability of the mixed ligand‐layer conjugates is lower. This is most probably due to enhanced interparticle PEG–PEG interactions at lower grafting densities. The presented study demonstrates that a detailed structural characterization of polymer ligand layers in situ and in response to external stimuli is possible with SAXS.     

Peptide‐Targeted Gold Nanoparticles for Photodynamic Therapy of Brain Cancer

Targeted drug delivery using epidermal growth factor peptide‐targeted gold nanoparticles (EGF_pep‐Au NPs) is investigated as a novel approach for delivery of photodynamic therapy (PDT) agents, specifically Pc 4, to cancer. In vitro studies of PDT show that EGF_pep‐Au NP‐Pc 4 is twofold better at killing tumor cells than free Pc 4 after increasing localization in early endosomes. In vivo studies show that targeting with EGF_pep‐Au NP‐Pc 4 improves accumulation of fluorescence of Pc 4 in subcutaneous tumors by greater than threefold compared with untargeted Au NPs. Targeted drug delivery and treatment success can be imaged via the intrinsic fluorescence of the PDT drug Pc 4. Using Pc 4 fluorescence, it is demonstrated in vivo that EGF_pep‐Au NP‐Pc 4 impacts biodistribution of the NPs by decreasing the initial uptake by the reticuloendothelial system (RES) and by increasing the amount of Au NPs circulating in the blood 4 h after IV injection. Interestingly, in vivo PDT with EGF_pep‐Au NP‐Pc 4 results in interrupted tumor growth when compared with EGF_pep‐Au NP control mice when selectively activated with light. These data demonstrate that EGF_pep‐Au NP‐Pc 4 utilizes cancer‐specific biomarkers to improve drug delivery and therapeutic efficacy over untargeted drug delivery.     

Interfacial ketene via the photo‐Wolff rearrangement for the modification of monolayer protected gold nanoparticles

An interfacial diazoketone‐modified gold nanoparticle (AuNP) was prepared and characterized by ¹H NMR and IR spectroscopy, TGA and TEM. Irradiation of the diazoketone leads to loss of nitrogen and the formation an interfacial ketene–AuNP via the photo‐Wolff rearrangement, evidenced by the loss of the characteristic C = N = N signal at 2068 cm^?1 in the IR spectrum and the growth of a new signal at 2100 cm^?1 indicative of the ketene. This ketene is relatively stable in the absence of added nucleophiles, but reacts quickly with oxygen nucleophiles illustrating the potential use of this ketene–AuNP as a template for a wide range of surface modifications. Copyright © 2013 John Wiley & Sons, Ltd.     

Fabrication of periodical Ag–Au compound nanostructure films with controllable Ag nanoparticle aggregate patterns: a study on surface‐enhanced Raman scattering

The Ag–Au compound nanostructure films with controllable patterns of Ag nanoparticle (NP) aggregates were fabricated. A strategy of two‐step synthesis was employed toward the target products. Firstly, the precursor Au NP (17 nm) films were synthesized as templates. Secondly, the Ag NPs (45 nm) were deposited on the precursor films. Three types of Ag NP aggregates were obtained including discrete Ag NPs (discrete type), necklace‐like Ag NP aggregates (necklace type), and huddle‐like Ag NP aggregates (huddle type). The surface‐enhanced Raman scattering (SERS) property was studied on these nanostructures by using the probing molecule of rhodamine 6G under the excitation laser of 514.5 nm. Interestingly, the different types of samples showed different enhancement abilities. A statistical method was employed to assess the enhancement. The relative enhancement factor for each Ag NP was estimated quantitatively under the ratio of 1 : 25 : 18 for the discrete‐type, necklace‐type, and huddle‐type samples at the given concentration of 10⁻⁸ mol/l. This research shows that the enhancement ability of each Ag NP is dependent on the aggregate morphology. Moreover, the different enhancement abilities displayed different limit detection concentrations up to 10⁻⁸, 10⁻¹¹, and 10⁻⁹ mol/l, separately. The understanding of the relationship between the defined nanostructures and the SERS enhancement is very meaningful for the design of new SERS substrates with better performance. Copyright © 2015 John Wiley & Sons, Ltd.     

Binding of ciguatera toxins to the voltage‐gated Kv1.5 potassium channel in the open state. Docking of gambierol and molecular dynamics simulations of a homology model

Ciguatera poisoning is a toxinological syndrome from ingestion of seafood contaminated by dinoflagellate toxins which has serious social and economic consequences from the Indo‐Pacific to the Caribbean. These polyannealed ethereal‐ring toxins, which comprise ciguatoxins, maitotoxin, and gambierol, are known to affect ion channels. Reported here are the first indications at molecular level as to the mode of interaction of these toxins with ion channels. The study concerns gambierol, an eight‐ring ladder polyether which is known to affect TRPV1‐type of thermal and pain sensation channels, as well as to inhibit voltage‐gated currents in K⁺ channels of mouse taste cells. Automated docking of gambierol on a homology model of the voltage‐gated Kv1.5 potassium ion channel in implicit solvent is followed by molecular dynamics (MD) simulation of the complex in a POPC membrane solvated with water. It is found that gambierol binds to the internal helices of the channel, unequally to the different subunits of the tetramer. Such unequal binding is a novel observation that should stimulate and aid developing a much demanded medical treatment of ciguatera poisoning. Copyright © 2008 John Wiley & Sons, Ltd.     

Unravelling Malaria Antigen Binding to Antibody‐Gold Nanoparticle Conjugates

Conjugates formed by antibody adsorption to gold nanoparticles (AuNP) have found extensive utilization in immunoassays due to the high surface area and interesting optical and electronic properties of the nanomaterials. Nevertheless, the mechanism of formation of antibody‐AuNP conjugates and their antigen binding characteristics have not been sufficiently explored in terms of specificity and consequent clinical applicability. Dynamic light scattering and related techniques have been successfully employed to detect antigen binding to antibody‐AuNP complexes. Here, a range of different techniques from the bionanotechnology realm have been applied to obtain a detailed picture of a competitive immunoassay for malaria antigen detection, based on fluorescence‐quenching by AuNPs. Both agarose gel electrophoresis and differential centrifugal sedimentation (DCS) analyses provide binding constants in the same order of magnitude, for antibody binding to AuNP and for antigen binding to antibody‐AuNP conjugates. Both techniques are also able to reveal inhibition of antigen binding in the presence of a major blood plasma protein, transferrin (via competitive binding). DCS is further used to show inhibition of the binding of the antigen in the presence of human plasma, a realistic testing condition, of high relevance to the implementation of immunoassays at the clinical level.     

Q‐switched and modelocked all‐fiber lasers based on advanced acousto‐optic devices

The interest in all‐fiber lasers is stimulated by the inherent advantages they have over bulk lasers in aspects such as heat dissipation and robustness. The performance of Q‐switched and modelocked fiber lasers can benefit enormously from the development of all‐fiber configurations. A fiber laser with strictly all‐fiber components can fulfil the requirements of mechanical stability, low maintenance, enhanced power efficiency, simplified assembly process, and low cost. In this framework, recent developments infiber acousto‐optic devices are reviewed that have demonstrated new possibilities for actively Q‐switched distributed feedback fiber lasers, modelocking lasers and doubly active Q‐switched modelocked lasers. The aim is to demonstrate the great potential of infiber devices for the active control of different types of fiber lasers.     

In Situ Self-Assembly of Quantum Dots at the Plasma Membrane Mediates Energy Transfer-Based Activation of Channelrhodopsin

The use of nanoparticle (NP) bioconjugates to control the activity of membrane ion channels has recently emerged as a new paradigm for the activation of electrically excitable cells. An NP-based strategy is reported for the specific activation of channelrhodopsin C1V1 (ChR-C1V1) expressed in the plasma membrane of HEK 293T/17 cells. Hydrophilic CdSe/ZnS core–shell semiconductor quantum dots (QDs) are self-assembled to the exofacial face of recombinantly expressed ChR-C1V1 by metal affinity-driven interaction of the QD ZnS shell with an N-terminal hexahistidine tag displayed on ChR-C1V1. This configuration enables the Förster resonance energy transfer (FRET)-based excitation and activation of the 11-cis-retinal moiety of ChR-C1V1 using the QD as a light harvesting transducer/energy donor. It is shown that the specific laser-induced opening of the ChR-C1V1 channel wherein the photoexcited QD (405 nm excitation, 530 nm emission) iteratively activates ChR-C1V1 channels as confirmed using the voltage-sensitive dye (VSD) bis-(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC ₂ (3)). In the absence of the QD transducer, excitation of ChR-C1V1-expressing cells at 405 nm results in no activation of ChR-C1V1. The results demonstrate the ability to controllably interface QDs with living cells for the activation of ChR membrane proteins and detail a new NP-bioconjugate hybrid system for the specific activation of ion channels.     

Nanoscale C‐Rich SixC1−x Bus/Ring Waveguide Based Cross‐Wavelength Data Converter

The carbon‐rich silicon carbide (C‐rich Si_xC_1?x) micro‐ring channel waveguide with asymmetric core aspect is demonstrated for all‐optical cross‐wavelength pulsed return‐to‐zero on‐off keying (PRZ‐OOK) data conversion. Enhanced nonlinear optical Kerr switching enables 12‐Gbit per second data processing with optimized modulation depth. The inverse tapered waveguide at end‐face further enlarges the edge‐coupling efficiency, and the asymmetric channel waveguide distinguishes the polarization modes. To prevent data shape distortion, the bus/ring gap spacing is adjusted to control the quality factor (Q‐factor) of the micro‐ring. Designing the waveguide cross section at 500 × 350 nm² provides the C‐rich Si_xC_1?x channel waveguide to induce strong transverse electric mode (TE‐mode) confinement with a large Kerr nonlinearity of 2.44 × 10^?12 cm² W^?1. Owing to the trade‐off between the Q‐factor and the on/off extinction ratio, the optimized bus/ring gap spacing of 1400 nm is selected to provide a coupling ratio at 5–6% for compromising the modulation depth and the switching throughput. Such a C‐rich Si_xC_1?x micro‐ring with asymmetric channel waveguide greatly enhances the cross‐wavelength data conversion efficiency to favor its on‐chip all‐optical data processing applications for future optoelectronic interconnect circuits.     

Near‐field optical enhancement by lead‐sulfide quantum dots and metallic nanoparticles for SERS

There is a growing interest in using quantum dots (QDs) and metallic nanoparticles (NPs), both for luminescence enhancement and surface‐enhanced Raman scattering (SERS). Here, we study the electromagnetic‐field enhancement that can be generated by lead‐sulfide (PbS) QDs using three‐dimensional finite‐element simulations. We investigate the field enhancement associated with combinations of PbS QDs with metallic NPs and substrates. The results show that high field enhancement can be achieved by combining PbS QDs with metallic NPs of larger sizes. The ideal size for Ag NPs is 25 nm, providing a SERS enhancement factor of ~5*10⁸ for light polarization parallel to the NP dimer axis and a gap of 0.6 nm. For Au NPs, the bigger the size, the higher is the field for the studied diameters, up to 50 nm. The near‐field values for PbS QDs above metallic substrates were found to be lower compared to the case of PbS QD‐metal NP dimers. This study provides the understanding for the design and application of QDs for the enhancement of near‐field phenomena. Copyright © 2013 John Wiley & Sons, Ltd.     

Prominin‐1‐Specific Binding Peptide‐Modified Apoferritin Nanoparticle Carrying Irinotecan as a Novel Radiosensitizer for Colorectal Cancer Stem‐Like Cells

Resistance of cancer stem cells to radiotherapy remains a major obstacle to successful cancer management. Prominin‐1 (PROM1) is a cancer stem cell marker. Nanoparticle (NP) chemotherapeutics preferentially accumulate in tumors and are able to target cancer and cancer stem‐like cells through cancer cell‐specific ligands, making them uniquely suited as radiosensitizers for chemoradiation therapy. Using a biocompatible apoferritin NP, a PROM1‐targeted NP carrying irinotecan (PROM1‐NP) is engineered. The synergistic effect of the NP and irradiation is evaluated in PROM1‐overexpressing HCT‐116 colorectal cancer cell lines in vitro and in vivo. PROM1‐NP has a size of 17.2 ± 0.2 nm and surface charge of ?13.5 ± 0.2 mV. It demonstrates higher intracellular uptake than nontargeted NP or irinotecan alone. Treatment with PROM1‐NPs decreases HCT‐116 cell proliferation in a dose‐ and time‐dependent manner. In vitro radiosensitization reveals that PROM1‐NP is significantly more effective as a radiosensitizer than nontargeted NP or irinotecan. HCT‐116 tumor xenograft growth is markedly slower following treatment with PROM1‐NP plus irradiation, suggesting that PROM1‐NP is more effective as a radiosensitizer than irinotecan and nontargeted NP in vivo. This study provides the first preclinical evidence of the effectiveness of PROM1‐targeted NP formulation of irinotecan as a radiosensitizer.     

Field-effect transistor with recombinant potassium channels: fast and slow response by electrical and chemical interactions

Electrical interfacing of semiconductor devices with ion channels is the basis for a development of neuroelectronic systems and of cell-based biospecific electronic sensors. To elucidate the mechanism of cell–chip coupling, we studied the voltage-gated potassium channel Kv1.3 in HEK 293 cells on field-effect transistors in silicon with a metal-free gate of silicon dioxide. Upon intracellular depolarization there is a positive change of the effective extracellular voltage on the transistor with an amplitude that correlates with the gating of Kv1.3 channels, but with a dynamics that is far slower than channel gating. After repolarization there is a fast negative change of the transistor signal followed by a slow relaxation dynamics without any membrane current. To rationalize the involved transistor response, we propose a concept that combines the electrodiffusion of ions in the cell–chip junction with selective ion binding in the electrical double layer of silicon dioxide. The model implies (i) an electrical charging and discharging of the cell–chip capacitance within a microsecond, (ii) a changing K⁺ concentration in the cell–chip junction within a millisecond and (iii) a changing adsorption of K⁺ and Na⁺ ions within tens of milliseconds. The total transistor signal is a superposition of the changed electrical potential in the extracellular space between cell and chip and of the changed surface potential at the chip surface. PACS 73.40.Mr; 82.45.Vp; 85.30.Tv; 87.16.Uv; 87.19.Nn