Electroactive nanoinjection platform for intracellular delivery and gene silencing.

BACKGROUND: Nanoinjection-the process of intracellular delivery using vertically configured nanostructures-is a physical route that efficiently negotiates the plasma membrane, with minimal perturbation and toxicity to the cells. Nanoinjection, as a physical membrane-disruption-mediated approach, overcomes challenges associated with conventional carrier-mediated approaches such as safety issues (with viral carriers), genotoxicity, limited packaging capacity, low levels of endosomal escape, and poor versatility for cell and cargo types. Yet, despite the implementation of nanoinjection tools and their assisted analogues in diverse cellular manipulations, there are still substantial challenges in harnessing these platforms to gain access into cell interiors with much greater precision without damaging the cell's intricate structure. Here, we propose a non-viral, low-voltage, and reusable electroactive nanoinjection (ENI) platform based on vertically configured conductive nanotubes (NTs) that allows for rapid influx of targeted biomolecular cargos into the intracellular environment, and for successful gene silencing. The localization of electric fields at the tight interface between conductive NTs and the cell membrane drastically lowers the voltage required for cargo delivery into the cells, from kilovolts (for bulk electroporation) to only ≤ 10 V; this enhances the fine control over membrane disruption and mitigates the problem of high cell mortality experienced by conventional electroporation. RESULTS: Through both theoretical simulations and experiments, we demonstrate the capability of the ENI platform to locally perforate GPE-86 mouse fibroblast cells and efficiently inject a diverse range of membrane-impermeable biomolecules with efficacy of 62.5% (antibody), 55.5% (mRNA), and 51.8% (plasmid DNA), with minimal impact on cells' viability post nanoscale-EP (> 90%). We also show gene silencing through the delivery of siRNA that targets TRIOBP, yielding gene knockdown efficiency of 41.3%. CONCLUSIONS: We anticipate that our non-viral and low-voltage ENI platform is set to offer a new safe path to intracellular delivery with broader selection of cargo and cell types, and will open opportunities for advanced ex vivo cell engineering and gene silencing.

Shear Assisted Electrochemical Exfoliation of Graphite to Graphene.

The exfoliation characteristics of graphite as a function of applied anodic potential (1-10 V) in combination with shear field (400-74 400 s(-1)) have been studied in a custom-designed microfluidic reactor. Systematic investigation by atomic force microscopy (AFM) indicates that at higher potentials thicker and more fragmented graphene sheets are obtained, while at potentials as low as 1 V, pronounced exfoliation is triggered by the influence of shear. The shear-assisted electrochemical exfoliation process yields large (∼10 µm) graphene flakes with a high proportion of single, bilayer, and trilayer graphene and small ID/IG ratio (0.21-0.32) with only a small contribution from carbon-oxygen species as demonstrated by X-ray photoelectron spectroscopy measurements. This method comprises intercalation of sulfate ions followed by exfoliation using shear induced by a flowing electrolyte. Our findings on the crucial role of hydrodynamics in accentuating the exfoliation efficiency suggest a safer, greener, and more automated method for production of high quality graphene from graphite.

Oscillating high aspect ratio micro-channels can effectively atomize liquids into uniform aerosol droplets and dial their size on-demand.

Ultrasonic atomization of liquids into micrometer-diameter droplets is crucial across multiple fields, ranging from drug delivery, to spectrometry and printing. Controlling the size and uniformity of the generated droplets on-demand is crucial in all these applications. However, existing systems lack the required precision to tune the droplet properties, and the underlying droplet formation mechanism under high-frequency ultrasonic actuation remains poorly understood due to experimental constraints. Here, we present an atomization platform, which overcomes these current limitations. Our device utilizes oscillating high aspect ratio micro-channels to extract liquids from various inlets (ranging from sessile droplets to large beakers), bound them in a precisely defined narrow region, and, controllably atomize them on-demand. The droplet size can be precisely dialled from 3.6 µm to 6.8 µm by simply tuning the actuation parameters. Since the approach does not need nozzles, meshes or impacting jets, stresses exerted on the liquid samples are reduced, hence it is gentler on delicate samples. The precision offered by the technique allows us for the first time to experimentally visualise the oscillating fluid interface at the onset of atomization at MHz frequencies, and demonstrate its applications for targeted respiratory drug delivery.

Engineering Efficient CAR-T Cells via Electroactive Nanoinjection.

Chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising cell-based immunotherapy approach for treating blood disorders and cancers, but genetically engineering CAR-T cells is challenging due to primary T cells' sensitivity to conventional gene delivery approaches. The current viral-based method can typically involve significant operating costs and biosafety hurdles, while bulk electroporation (BEP) can lead to poor cell viability and functionality. Here, a non-viral electroactive nanoinjection (ENI) platform is developed to efficiently negotiate the plasma membrane of primary human T cells via vertically configured electroactive nanotubes, enabling efficient delivery (68.7%) and expression (43.3%) of CAR genes in the T cells, with minimal cellular perturbation (>90% cell viability). Compared to conventional BEP, the ENI platform achieves an almost threefold higher CAR transfection efficiency, indicated by the significantly higher reporter GFP expression (43.3% compared to 16.3%). By co-culturing with target lymphoma Raji cells, the ENI-transfected CAR-T cells' ability to effectively suppress lymphoma cell growth (86.9% cytotoxicity) is proved. Taken together, the results demonstrate the platform's remarkable capacity to generate functional and effective anti-lymphoma CAR-T cells. Given the growing potential of cell-based immunotherapies, such a platform holds great promise for ex vivo cell engineering, especially in CAR-T cell therapy.

The effect of clinical face shields on aerosolized particle exposure.

Background: Face shields protect healthcare workers (HCWs) from fluid and large droplet contamination. Their effect on smaller aerosolized particles is unknown. Materials & methods: An ultrasonic atomizer was used to simulate particle sizes equivalent to human breathing and forceful cough. Particles were measured at positions correlating to anesthetic personnel in relation to a patient inside an operating theatre environment. The effect of the application of face shields on HCW exposure was measured. Results & Conclusion: Significant reductions in particle concentrations were measured after the application of vented and enclosed face shields. Face shields appear to reduce the concentration of aerosolized particles that HCWs are exposed to, thereby potentially conferring further protection against exposure to aerosolized particles in an operating theatre environment.

Face shields protect health workers from splash contamination. We do not know if they protect against smaller invisible aerosol drops that can carry diseases like coronavirus 2019/COVID-19. The authors tested whether face shields can stop floating droplets using different types of face shields. This included one that was designed and made by a 3D printer, and traditional face shields. The shields were tested in a hospital operating room. A machine was designed that made invisible saltwater droplets. A monitor was used to measure the droplets present at a doctor's or nurse's mouth and then if this changed when a face shield was used. The face shield might be helpful in stopping health workers from catching diseases by stopping the flow of aerosol drops.

A Lotus shaped acoustofluidic mixer: High throughput homogenisation of liquids in 2 ms using hydrodynamically coupled resonators.

This paper presents an acoustically actuated microfluidic mixer that uses an array of hydrodynamically coupled resonators to rapidly homogenise liquid solutions and synthesise nanoparticles. The system relies on 8 identical oscillating cantilevers that are equally spaced on the perimeter of a circular well, through which the liquid solutions are introduced. When an oscillatory electrical signal is applied to a piezoelectric transducer attached to the device, the cantilevers start resonating. Due to the close proximity between the cantilevers, their circular arrangement and the liquid medium in which they are immersed, the vibration of each cantilever affects the response of its neighbours. The streaming fields and shearing rates resulting from the oscillating structures were characterised. It was shown that the system can be used to effectively mix fluids at flow rates up to 1400 µl.min-1 in time scales as low as 2 ms. The rapid mixing time is especially advantageous for nanoparticle synthesis, which is demonstrated by synthesising Poly lactide-co-glycolic acid (PLGA) nanoparticles with 52.2 nm size and PDI of 0.44.

X-ray compatible microfluidics for in situ studies of chemical state, transport and reaction of light elements in an aqueous environment using synchrotron radiation.

This paper presents an X-ray compatible microfluidic platform for in situ characterization of chemical reactions at synchrotron light sources. We demonstrate easy to implement techniques to probe reacting solutions as they first come into contact, and study the very first milliseconds of their reaction in real-time through X-ray absorption spectroscopy (XAS). The devices use polydimethylsiloxane (PDMS) microfluidic channels sandwiched between ultrathin, X-ray transparent silicon nitride observation windows and rigid substrates. The new approach has three key advantages: i) owing to the assembly techniques employed, the devices are suitable for both high energy and tender (1-5 keV) X-rays; ii) they can operate in a vacuum environment (a must for low energy X-rays) and iii) they are robust enough to survive a full 8 hour shift of continuous scanning with a micro-focused beam, providing higher spatial and thus greater time resolution than previous studies. The combination of these opens new opportunities for in situ studies. This has so far not been possible with Kapton or glass-based flow cells due to increased attenuation of the low energy beam passing through these materials. The devices provide a well-defined mixing region to collect spatial maps of spatially stable concentration profiles, and XAS point spectra to elucidate the chemical structure and characterize the chemical reactions. The versatility of the approach is demonstrated through in situ XAS measurements on the mixing of two reactants in a microfluidic laminar flow device, as well as a segmented droplet based system for time resolved analysis.

On-demand sample injection: combining acoustic actuation with a tear-drop shaped nozzle to generate droplets with precise spatial and temporal control.

An on-demand droplet injection method for controlled delivery of nanolitre-volume liquid samples to scientific instruments for subsequent analysis is presented. We employ pulsed focussed surface acoustic waves (SAW) to eject droplets from an enclosed microfluidic channel into an open environment. The 3D position of individual droplets and their time of arrival can be precisely controlled to within 61 µs in a 500 µm square target region 40 µm wide. The continuous ejection rate of 16 000 droplets per second can be tuned to produce pulsed trains of droplets from 0 up to 357 Hz. The main benefit of this technique is its ease of integration with complex microfluidic processing steps, such as droplet merging, sorting, and encapsulation, prior to sample delivery. With its ability to precisely deliver a small quantity of fluid to a pre-defined location this technology is applicable in X-ray based molecular studies, including the rapidly expanding field of X-ray free electron lasers. Fabrication procedures for this device, the underlying forcing mechanism, the role of nozzle design, and demonstration of the performance in both continuous and on-demand modes are reported.

The importance of travelling wave components in standing surface acoustic wave (SSAW) systems.

The use of ultrasonic fields to manipulate particles, cells and droplets has become widespread in lab on a chip (LOC) systems. There are two dominant actuation methods, the use of bulk acoustic waves (BAW) or surface acoustic waves (SAW). The development of BAW actuated systems have been underpinned by a robust understanding of the link between the ultrasonic field and forces which can be generated. In this work, we examine this link for standing surface acoustic waves (SSAW) comparing the relative strengths of streaming induced drag and acoustic radiation forces on suspended particles. To achieve this we have employed boundary conditions which accurately capture the travelling wave components of the pseudo-standing wave field, describe the key features of the acoustic radiation force fields and the acoustic streaming fields which can be generated, and finally we show that the relative importance of these two mechanisms is spatially dependant within a fluid chamber. The boundary condition used models the SSAW as two counter-propagating travelling waves, rather than assuming a standing wave directly. This allows the accurate inclusion of energy decay as the SAW couples into the fluid chamber and the resulting travelling wave component. This study shows that this previously neglected complexity of the SAW field is a critical factor in the nature of the resultant streaming field, as it gives rise to strong streaming rolls at the channel walls, which we validate experimentally. These rolls result in spatial variations of the dominant forces which in turn varies particle migration patterns spatially across the fluid domain.

On-chip droplet production regimes using surface acoustic waves.

Aqueous droplets suspended in an immiscible carrier fluid are a key tool in microfluidic chemical analysis platforms. The approaches for producing droplets in microfluidic devices can be divided into three general categories: batch emulsification, continuous production and tailored on-demand production. The major distinctions between each category are the rate of production and the degree of control over the droplet formation process in terms of the size and quantity. On-demand methods are highly desirable when, for example, small numbers or even single droplets of one sample type are required at a time. Here, we present a method for the on-demand production of femtolitre droplets, utilising a pressure source generated by high frequency surface acoustic waves (SAW). An increase in the continuous phase flow rate is enabled by a quasi-3D feature at the droplet production nozzle. A wide range of accessible flow rates permits the identification of different physical regimes in which droplets of different dimensions are produced. In the system investigated droplets measuring as little as 200 fl have been produced, ∼1/60th of the minimum volume previously reported. The experimental findings are supported by a numerical model which demonstrates the link between the number of droplets formed and the pulse length used.