Enhancing the production of adeno-associated virus (AAV)2 and AAV9 with high full capsid ratio in HEK293 cells through design-of-experiment optimization of triple plasmid ratio.

The recombinant adeno-associated virus (rAAV) vectors used in gene therapy are usually produced by transfecting three different plasmids (Adenoviral helper plasmid (pHelper), AAV rep/cap plasmids (pRepCap), and Transgene plasmid (pAAV-GOI)) into human embryonic kidney 293 (HEK293) cells. However, the high proportion of unwanted empty capsids generated during rAAV production is problematic. To simultaneously enhance the genome titer and full capsid ratio, the ratio of the three plasmids transfected into HEK293 cells was optimized using design-of-experiment (DoE). AAV2 and AAV9, which have different production kinetics, were selected as cell-associated and secreted model AAVs, respectively. In 125 mL Erlenmeyer flasks, the genome titers of rAAV2 and rAAV9 at DoE-optimized plasmid weight ratios (pHelper:pRep2Cap2:pAAV-GOI = 1:3.52:0.50 for rAAV2 and pHelper:pRep2Cap9:pAAV-GOI = 1:1.44:0.27 for rAAV9) were 2.23-fold and 2.26-fold higher than those in the widely used plasmid weight ratio (1:1:1), respectively. In addition, compared with the plasmid ratio of 1:1:1, the relative VP3 band intensities of rAAV2 and rAAV9, which represent the relative empty capsid ratios, were reduced by 26% and 25%, respectively, at the DoE-optimized plasmid ratio. Reduced empty capsid ratios in the DoE-optimized plasmid ratios were also confirmed using transmission electron microscopy (TEM). Taken together, regardless of the AAV serotype, DoE-aided optimization of the triple plasmid ratio was found to be an efficient means of improving the production of rAAV with a high full capsid ratio.

Distinguishing between DNA-Loaded Full and Empty Capsids of Adeno-Associated Virus with Atomic Force Microscopy Imaging.

Recently, miraculous therapy approaches involving adeno-associated virus (AAV) for incurable diseases such as spinal muscular atrophy and inherited retinal dysfunction have been introduced. Nonreplicative, nonpathogenic, low rates of chromosome insertional properties and the existence of neutralizing antibodies are main safety reasons why the FDA approved its use in gene delivery. To date, AAV production always results in a mixture of nontherapeutic (empty) and therapeutic (DNA-loaded) full capsids (10-98%). Such existence of empty viral particles inevitably increases viral doses to human. Thus, the rapid monitoring of empty capsids and reducing the empty-to-full ratio are critical in AAV science. However, transmission electron microscopy (TEM) is the primary tool for distinguishing between empty and full capsids, which creates a research bottleneck because of instrument accessibility and technical difficulty. Herein, we demonstrate that atomic force microscopy (AFM) can be an alternative tool to TEM. The simple, noncontact-mode imaging of AAV particles allows the distinct height difference between full capsids (∼22 nm) and empty capsids (∼16 nm). The sphere-to-ellipsoidal morphological distortion observed for empty AAV particles clearly distinguishes them from full AAV particles. Our study indicates that AFM imaging can be an extremely useful, quality-control tool in AAV particle monitoring, which is beneficial for the future development of AAV-based gene therapy.

Effects of Top and Bottom Electrodes Materials and Operating Ambiance on the Characteristics of MgFx Based Bipolar RRAMs.

The effects of electrode materials (top and bottom) and the operating ambiances (open-air and vacuum) on the MgFx-based resistive random-access memory (RRAM) devices are studied. Experiment results show that the device's performance and stability depend on the difference between the top and bottom electrodes' work functions. Devices are robust in both environments if the work function difference between the bottom and top electrodes is greater than or equal to 0.70 eV. The operating environment-independent device performance depends on the surface roughness of the bottom electrode materials. Reducing the bottom electrodes' surface roughness will reduce moisture absorption, minimizing the impact of the operating environment. Ti/MgFx/p+-Si memory devices with the minimum surface roughness of the p+-Si bottom electrode show operating environment-independent electroforming-free stable resistive switching properties. The stable memory devices show promising data retentions of >104 s in both environments with DC endurance properties of more than 100 cycles.

Tracking Efficiency Improvement According to Incident Beam Size in QPD-Based PAT System for Common Path-Based Full-Duplex FSO Terminals.

Free space optical (FSO) communication can support various unmanned aerial vehicles' (UAVs) applications that require large capacity data transmission. In order to perform FSO communication between two terminals, it is essential to employ a pointing, acquisition, and tracking (PAT) system with an efficient and optimal performance. We report on the development of a common optical-path-based FSO communication system, tailored for applications in UAVs. The proposed system is equipped with a quadrant photodiode (QPD)-based PAT system without an additional beacon beam subsystem. The presented approach reduced the structural complexity and improved the tracking efficiency for the same size, weight, and power (SWaP). To achieve a robust FSO link in a dynamic UAV environment, the observability and controllability were obtained based on the linearized control according to the incident beam size on the QPD, which was verified by optical simulation and experiments. As a result, the QPD-based PAT system for implementing FSO links demonstrated an up to 4.25 times faster tracking performance. Moreover, the FSO link experimentally confirmed the 1.25 Gbps full-duplex error-free communication at a 50 m distance.

Vacuum and Low-Temperature Characteristics of Silicon Oxynitride-Based Bipolar RRAM.

This study investigates the switching characteristics of the silicon oxynitride (SiOxNy)-based bipolar resistive random-access memory (RRAM) devices at different operating ambiances at temperatures ranging from 300 K to 77 K. The operating ambiances (open air or vacuum) and temperature affect the device's performance. The electroforming-free multilevel bipolar Au/Ni/SiOxNy/p+-Si RRAM device (in open-air) becomes bilevel in a vacuum with an on/off ratio >104 and promising data retention properties. The device becomes more resistive with cryogenic temperatures. The experimental results indicate that the presence and absence of moisture (hydrogen and hydroxyl groups) in open air and vacuum, respectively, alter the elemental composition of the amorphous SiOxNy active layer and Ni/SiOxNy interface region. Consequently, this affects the overall device performance. Filament-type resistive switching and trap-controlled space charge limited conduction (SCLC) mechanisms in the bulk SiOxNy layer are confirmed.

Heterogeneous and Monolithic 3D Integration of III-V-Based Radio Frequency Devices on Si CMOS Circuits.

Next-generation wireless communication such as sixth-generation (6G) and beyond is expected to require high-frequency, multifunctionality, and power-efficiency systems. A III-V compound semiconductor is a promising technology for high-frequency applications, and a Si complementary metal-oxide-semiconductor (CMOS) is the never-beaten technology for highly integrated digital circuits. To harness the advantages of these two technologies, monolithic integration of III-V and Si electronics is beneficial, so that there have been everlasting efforts to accomplish the monolithic integration. Considering that the on horizon 6G wireless communication requires faster and more energy-efficient system-on-chip technologies, it is imperative to realize a radio frequency (RF) system in which III-V technology and Si CMOS technology are integrated at a device level. Here we report heterogeneous and monolithic three-dimensional (3D) analog/RF-digital mixed-signal integrated circuits that contain two types of InGaAs high-electron-mobility transistors (HEMTs) designed for high fT and fMAX in the top and Si CMOS mixed-signal circuits consisting of an analog-to-digital converter and digital-to-analog converter in the bottom. A high unity current gain cutoff frequency of 448 GHz and unity power gain cutoff frequency of 742 GHz have been achieved by the fT oriented and fMAX oriented InGaAs HEMTs, respectively, without being affected by mixed-signal interference. At the same time, the bottom Si CMOS circuits provide valid signals without any performance degradation by the integration process.

Effects of the Operating Ambiance and Active Layer Treatments on the Performance of Magnesium Fluoride Based Bipolar RRAM.

This study investigates switching characteristics of the magnesium fluoride (MgFx)-based bipolar resistive random-access memory (RRAM) devices at different operating ambiances (open-air and vacuum). Operating ambiances alter the elemental composition of the amorphous MgFx active layer and Ti/MgFx interface region, which affects the overall device performance. The experimental results indicate that filament type resistive switching takes place at the interface of Ti/MgFx and trap-controlled space charge limited conduction (SCLC) mechanisms is dominant in both the low and high resistance states in the bulk MgFx layer. RRAM device performances at different operating ambiances are also altered by MgFx active layer treatments (air exposure and annealing). Devices show the better uniformity, stability, and a higher on/off current ratio in vacuum compared to an open-air environment. The Ti/MgFx/Pt memory devices have great potential for future vacuum electronic applications.

Low-temperature characteristics of magnesium fluoride based bipolar RRAM devices.

This study investigates the temperature-independent switching characteristics of magnesium fluoride (MgFx) based bipolar resistive memory devices at temperatures ranging from 300 K down to 77 K. Filament type resistive switching at the interface of Ti/MgFx and the trap-controlled space charge limited conduction (SCLC) mechanism in the bulk MgFx layer are confirmed. The experimental results indicate that the operating environment and temperature critically control the resistive switching performance by varying the non-stoichiometry of the amorphous MgFx active layer and Ti/MgFx interface region. The gaseous atmosphere (open air or vacuum) affects device performances such as the electroforming process, on-state current, off-state current, on/off ratio, SET/RESET voltage and endurance of resistive-switching memory devices. After electroforming, the device performance is independent of temperature variation. The Ti/MgFx/Pt memory devices show promising data retention for >104 s in a vacuum at room temperature and 77 K with the DC endurance property for more than 150 cycles at 77 K. The devices have great potential for future temperature-independent electronic applications.

Fabrication and Characterization of In0.53Ga0.47As/InAs/In0.53Ga0.47As Composite Channel Metamorphic HEMTs (mHEMTs) on a GaAs Substrate.

In this work, we successfully demonstrated In0.53Ga0.47As/InAs/In0.53Ga0.47As composite channel metamorphic high electron mobility transistors (mHEMTs) on a GaAs substrate. The fabricated mHEMTs with a 100 nm gate length exhibited excellent DC and logic characteristics such as VT = -0.13 V, gm,max = 949 mS/mm, subthreshold swing (SS) = 84 mV/dec, drain-induced barrier lowering (DIBL) = 89 mV/V, and Ion/Ioff ratio = 9.8 × 103 at a drain-source voltage (VDS) = 0.5 V. In addition, the device exhibited excellent high-frequency characteristics, such as fT/fmax = 261/304 GHz for the measured result and well-matched modeled fT/fmax = 258/309 GHz at VDS = 0.5 V, which is less power consumption compared to other material systems. These high-frequency characteristics are a well-balanced demonstration of fT and fmax in the mHEMT structure on a GaAs substrate.

Electroforming-Free Bipolar Resistive Switching Memory Based on Magnesium Fluoride.

Electroforming-free resistive switching random access memory (RRAM) devices employing magnesium fluoride (MgFx) as the resistive switching layer are reported. The electroforming-free MgFx based RRAM devices exhibit bipolar SET/RESET operational characteristics with an on/off ratio higher than 102 and good data retention of >104 s. The resistive switching mechanism in the Ti/MgFx/Pt devices combines two processes as well as trap-controlled space charge limited conduction (SCLC), which is governed by pre-existing defects of fluoride vacancies in the bulk MgFx layer. In addition, filamentary switching mode at the interface between the MgFx and Ti layers is assisted by O-H group-related defects on the surface of the active layer.

Pastable, Adhesive, Injectable, Nanofibrous, and Tunable (PAINT) Biphasic Hybrid Matrices as Versatile Therapeutic Carriers.

A critical challenge in many pharmaceutical fields is developing versatile adjuvant devices that can reduce the off-target delivery of therapeutic materials to target lesions. Herein, a biphasic hybrid fibrous system that can manipulate the spatial and temporal delivery of various therapeutic agents to target lesions by integrating multiple distinct systems and technologies such as fluffy coaxial electrospun polycaprolactone (PCL)/polystyrene (PS) fibers, cyclohexane-mediated leaching to remove PS layers selectively, amine display on PCL fibers, conjugation of naturally occurring adhesive gallol molecules onto hyaluronic acid (HA-g), and electrostatically complexing the aminated PCL fibers with the gallol-conjugated HA. In the context of "paintable" systems on target lesions, the resulting system is called a PAINT matrix (abbreviated according to the initial letter of its features: pastable, adhesive, injectable, nanofibrous, and tunable). Its viscoelastic property, which was attributed by coalescing aminated PCL fibers with viscous HA-g, enabled it to be noninvasively injected and fit into any cavity in the body with various morphologies, manually pasted on tissue surfaces, and adhered onto moisture-rich surfaces to ensure the secure delivery of therapeutics toward the target lesions. The PAINT matrix efficiently supplied immunomodulatory human neural stem cells (hNSCs) at rat hemisectioned spinal cord injury (SCI) sites and promoted both locomotive and sensory recovery in SCI models, presumably by protecting hNSCs against host immunosurveillance. The PAINT matrix will be broadly utilized for efficiently delivering therapeutics to difficult-to-reach target lesions by direct infusion or conventional biomaterial-mediated approaches due to their locations, wet surfaces, or complicated ambient environments.

Chitosan-Alginate-Pectin-coated Suspended-Liquid-Encapsulating (CAPSuLE) marbles for therapeutic agent storage and delivery.

Developing a cutting-edge system capable of ensuring long-lasting functionality of therapeutic agents and implementing diverse delivery modes is challenging. A quasi-spherical triple-layered capsule containing suspended liquid droplets and allowing multi-modal delivery of therapeutic agents in the aqueous phase was developed, primarily by adopting the core principles for creating liquid marbles. A naturally derived wettable polysaccharide-pectin-was utilized as a liquid-air interfacial barrier to keep the liquid droplets in the core zone. To tailor the pectin-coated droplet as a therapeutic agent carrier, anionic alginate and cationic chitosan layers were sequentially formed via additional interactions: physically stacking substances with structural chirality (pectin-alginate) and inducing electrostatic association to create the reversible complex coacervates (alginate-chitosan). The resulting system, which is called a Chitosan-Alginate-Pectin-coated Suspended-Liquid-Encapsulating (CAPSuLE) marble, had sufficient mechanical strength to resist external harsh environments and exhibited unique features: ecofriendly sustainability, responsiveness to external stimuli, coacervate-driven coalescence for linking adjacent marbles, and a self-repairing ability. The proposed CAPSuLE system can facilitate the adoption of the liquid-marble concept to biomedical fields, extending its applicability in the fields of biology and applied engineering.

Soft subdermal implant capable of wireless battery charging and programmable controls for applications in optogenetics.

Optogenetics is a powerful technique that allows target-specific spatiotemporal manipulation of neuronal activity for dissection of neural circuits and therapeutic interventions. Recent advances in wireless optogenetics technologies have enabled investigation of brain circuits in more natural conditions by releasing animals from tethered optical fibers. However, current wireless implants, which are largely based on battery-powered or battery-free designs, still limit the full potential of in vivo optogenetics in freely moving animals by requiring intermittent battery replacement or a special, bulky wireless power transfer system for continuous device operation, respectively. To address these limitations, here we present a wirelessly rechargeable, fully implantable, soft optoelectronic system that can be remotely and selectively controlled using a smartphone. Combining advantageous features of both battery-powered and battery-free designs, this device system enables seamless full implantation into animals, reliable ubiquitous operation, and intervention-free wireless charging, all of which are desired for chronic in vivo optogenetics. Successful demonstration of the unique capabilities of this device in freely behaving rats forecasts its broad and practical utilities in various neuroscience research and clinical applications.

Impact of Na Doping on the Carrier Transport Path in Polycrystalline Flexible Cu2ZnSn(S,Se)4 Solar Cells.

It is well-known that the alkali doping of polycrystalline Cu2ZnSn(S,Se)4 (CZTSSe) and Cu(In,Ga)(Se,S)2 has a beneficial influence on the device performance and there are various hypotheses about the principles of performance improvement. This work clearly explains the effect of Na doping on the fill factor (FF) rather than on all of the solar cell parameters (open-circuit voltage, FF, and sometimes short circuit current) for overall performance improvement. When doping is optimized, the fabricated device shows sufficient built-in potential and selects a better carrier transport path by the high potential difference between the intragrains and the grain boundaries. On the other hand, when doping is excessive, the device shows low contact potential difference and FF and selects a worse carrier transport path even though the built-in potential becomes stronger. The fabricated CZTSSe solar cell on a flexible metal foil optimized with a 25 nm thick NaF doping layer achieves an FF of 62.63%, thereby clearly showing the enhancing effect of Na doping.

Ultra-High Energy Density Hybrid Supercapacitors Using MnO2/Reduced Graphene Oxide Hybrid Nanoscrolls.

Manganese oxide (MnO2) is a promising material for supercapacitor applications, with a theoretical ultra-high energy density of 308 Wh/kg. However, such ultra-high energy density has not been achieved experimentally in MnO2-based supercapacitors because of several practical issues, such as low electrical conductivity of MnO2, incomplete utilization of MnO2, and dissolution of MnO2. The present study investigates the potential of MnO2/reduced graphene oxide (rGO) hybrid nanoscroll (GMS) structures as electrode material for overcoming the difficulties and for developing ultra-high-energy storage systems. A hybrid supercapacitor, comprising MnO2/rGO nanoscrolls as anode material and activated carbon (AC) as a cathode, is fabricated. The GMS/AC hybrid supercapacitor exhibited enhanced energy density, superior rate performance, and promising Li storage capability that bridged the energy-density gap between conventional Li-ion batteries (LIBs) and supercapacitors. The fabricated GMS/AC hybrid supercapacitor demonstrates an ultra-high lithium discharge capacity of 2040 mAh/g. The GMS/AC cell delivered a maximum energy density of 105.3 Wh/kg and a corresponding power density of 308.1 W/kg. It also delivered an energy density of 42.77 Wh/kg at a power density as high as 30,800 W/kg. Our GMS/AC cell's energy density values are very high compared with those of other reported values of graphene-based hybrid structures. The GMS structures offer significant potential as an electrode material for energy-storage systems and can also enhance the performance of the other electrode materials for LIBs and hybrid supercapacitors.

Cu(In,Ga)Se2 Solar Cells Integrated with Subwavelength Structured Cover Glass Fabricated by One-Step Self-Masked Etching.

We report an anti-reflective cover glass for Cu(In,Ga)Se2 (CIGS) thin film solar cells. Subwavelength structures (SWSs) were fabricated on top of a cover glass using one-step self-masked etching. The etching method resulted in dense whiskers with high aspect ratio. The produced structure exhibited excellent anti-reflective properties over a broad wavelength range, from the ultraviolet to the near infrared. Compared to a flat-surface glass, the average transmittance of the glass integrated with the SWSs improved from 92.4% to 95.2%. When the cover glass integrated with the SWSs was mounted onto the top of a CIGS device, the short-circuit current and the efficiency of the solar cell were enhanced by 4.38 and 6%, respectively, compared with a CIGS solar cell without cover glass.

TNF-α Pretreatment Improves the Survival and Function of Transplanted Human Neural Progenitor Cells Following Hypoxic-Ischemic Brain Injury.

Neural progenitor cells (NPCs) therapy offers great promise in hypoxic-ischemic (HI) brain injury. However, the poor survival of implanted NPCs in the HI host environment limits their therapeutic effects. Tumor necrosis factor-alpha (TNF-α) is a pleiotropic cytokine that is induced in response to a variety of pathological processes including inflammation and immunity. On the other hand, TNF-α has protective effects on cell apoptosis and death and affects the differentiation, proliferation, and survival of neural stem/progenitor cells in the brain. The present study investigated whether TNF-α pretreatment on human NPCs (hNPCs) enhances the effectiveness of cell transplantation therapy under ischemic brain. Fetal brain tissue-derived hNPCs were pretreated with TNF-α before being used in vitro experiments or transplantation. TNF-α significantly increased expression of cIAP2, and the use of short hairpin RNA-mediated knockdown of cIAP2 demonstrated that cIAP2 protected hNPCs against HI-induced cytotoxicity. In addition, pretreatment of hNPCs with TNF-α mediated neuroprotection by altering microglia polarization via increased expression of CX3CL1 and by enhancing expression of neurotrophic factors. Furthermore, transplantation of TNF-α-treated hNPCs reduced infarct volume and improved neurological functions in comparison with non-pretreated hNPCs or vehicle. These findings show that TNF-α pretreatment, which protects hNPCs from HI-injured brain-induced apoptosis and increases neuroprotection, is a simple and safe approach to improve the survival of transplanted hNPCs and the therapeutic efficacy of hNPCs in HI brain injury.

A Versatile Adeno-Associated Viral Vector Cross-Linking Platform Capable of Tuning Cellular Tropisms and Simultaneously Inducing Solid-Phase Gene Delivery.

Developing gene carriers with improved affinities for target cells and the simultaneous diversification of their delivery modes will be pivotal for upgrading gene therapy technologies. In this study, a simple and versatile adeno-associated virus (AAV) conjugation platform using the cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) is proposed. Depending on the quantity of the DTSSP molecules, the AAV-DTSSP complexes could either be linked with the relevant biomolecules for altering cellular tropisms or further form a self-assembled AAV-DTSSP pellet capable of mimicking a polymeric gene delivery system. At lower quantities of DTSSP, the AAV-DTSSP complexes were conjugated with aminated l-fucose molecules, whose levels are typically upregulated in pancreatic cancer cells, resulting in enhanced gene delivery efficiencies in pancreatic cancer cells. At higher concentrations of DTSSP, visible solid forms of the AAV-DTSSP pellets were formed, and the AAV pellets demonstrated the capability to induce a localized, sustained gene expression pattern comparable to that of conventional biomaterial-based approaches. Thus, a multipurpose AAV cross-linking platform, which can enable AAV vector systems that are capable of altering cellular tropisms and simultaneously inducing solid-phase delivery, will provide crucial insights into vector design for further upgrading of gene delivery technologies.

Luminescent down-shifting CsPbBr3 perovskite nanocrystals for flexible Cu(In,Ga)Se2 solar cells.

To overcome the parasitic absorption of ultraviolet (UV) light in the transparent conductive oxide (TCO) layer of flexible Cu(In,Ga)Se2 (CIGS) thin film solar cells, a CsPbBr3 perovskite nanocrystal based luminescent down-shifting (LDS) layer was integrated on CIGS solar cells fabricated on a stainless steel foil. The CsPbBr3 perovskite nanocrystal absorbs solar irradiation at wavelengths shorter than 520 nm and emits photons at a wavelength of 532 nm. These down-shifted photons pass the TCO layer without parasitic absorption and are absorbed in the CIGS absorber layer where they generate photocurrent. By minimizing the parasitic absorption in the TCO layer, the external quantum efficiency (EQE) of the CIGS solar cell with the CsPbBr3 perovskite nanocrystal layer is highly improved in the UV wavelength range between 300 and 390 nm. Additionally, in the wavelength range between 500 and 1100 nm, the EQE is improved since the surface reflectance of the CIGS device with the CsPbBr3 perovskite LDS layer was reduced. This is because the CsPbBr3 perovskite nanocrystal layer, which has an effective refractive index of 1.82 at a wavelength of 800 nm, reduces the large refractive index mismatch between air (nair = 1.00) and the TCO layer (nZnO = 1.96 at a wavelength of 800 nm). Both the short circuit current density and power conversion efficiency of the flexible CIGS solar cell integrated with the CsPbBr3 perovskite are improved by 4.5% compared with the conventional CIGS solar cell without the CsPbBr3 perovskite LDS layer.

AAVR-Displaying Interfaces: Serotype-Independent Adeno-Associated Virus Capture and Local Delivery Systems.

Interfacing gene delivery vehicles with biomaterials has the potential to play a key role in diversifying gene transfer capabilities, including localized, patterned, and controlled delivery. However, strategies for modifying biomaterials to interact with delivery vectors must be redesigned whenever new delivery vehicles and applications are explored. We have developed a vector-independent biomaterial platform capable of interacting with various adeno-associated viral (AAV) serotypes. A water-soluble, cysteine-tagged, recombinant protein version of the recently discovered multi-AAV serotype receptor (AAVR), referred to as cys-AAVR, was conjugated to maleimide-displaying polycaprolactone (PCL) materials using click chemistry. The resulting cys-AAVR-PCL system bound to a broad range of therapeutically relevant AAV serotypes, thereby providing a platform capable of modulating the delivery of all AAV serotypes. Intramuscular injection of cys-AAVR-PCL microspheres with bound AAV vectors resulted in localized and sustained gene delivery as well as reduced spread to off-target organs compared to a vector solution. This cys-AAVR-PCL system is thus an effective approach for biomaterial-based AAV gene delivery for a broad range of therapeutic applications.