J. Mark Meacham

Physical methods for intracellular delivery: practical aspects from laboratory use to industrial-scale processing.

Effective intracellular delivery is a significant impediment to research and therapeutic applications at all processing scales. Physical delivery methods have long demonstrated the ability to deliver cargo molecules directly to the cytoplasm or nucleus, and the mechanisms underlying the most common approaches (microinjection, electroporation, and sonoporation) have been extensively investigated. In this review, we discuss established approaches, as well as emerging techniques (magnetofection, optoinjection, and combined modalities). In addition to operating principles and implementation strategies, we address applicability and limitations of various in vitro, ex vivo, and in vivo platforms. Importantly, we perform critical assessments regarding (1) treatment efficacy with diverse cell types and delivered cargo molecules, (2) suitability to different processing scales (from single cell to large populations), (3) suitability for automation/integration with existing workflows, and (4) multiplexing potential and flexibility/adaptability to enable rapid changeover between treatments of varied cell types. Existing techniques typically fall short in one or more of these criteria; however, introduction of micro-/nanotechnology concepts, as well as synergistic coupling of complementary method(s), can improve performance and applicability of a particular approach, overcoming barriers to practical implementation. For this reason, we emphasize these strategies in examining recent advances in development of delivery systems.

Comparison of the internal energy deposition of Venturi-assisted electrospray ionization and a Venturi-assisted array of micromachined ultrasonic electrosprays (AMUSE).

The internal energy deposition of a Venturi-assisted array of micromachined ultrasonic electrosprays (AMUSE), with and without the application of a DC charging potential, is compared with equivalent experiments for Venturi-assisted electrospray ionization (ESI) using the “survival yield” method on a series of para-substituted benzylpyridinium salts. Under conditions previously shown to provide maximum ion yields for standard compounds, the observed mean internal energies were nearly identical (1.93–2.01 eV). Operation of AMUSE without nitrogen flow to sustain the air amplifier focusing effect generated energetically colder ions with mean internal energies that were up to 39% lower than those for ESI. A balance between improved ion transfer, adequate desolvation, and favorable ion energetics was achieved by selection of optimum operational ranges for the parameters that most strongly influence the ion population: the air amplifier gas flow rate and API capillary temperature. Examination of the energy landscapes obtained for combinations of these parameters showed that a low internal energy region (≤1.0 eV) was present at nitrogen flow rates between 2 and 4 L min−1 and capillary temperatures up to 250°C using ESI (9% of all parameter combinations tested). Using AMUSE, this region was present at nitrogen flow rates up to 2.5 L min−1 and all capillary temperatures (13% of combinations tested). The signal-to-noise (S/N) ratio of the intact p-methylbenzylpyridinium ion obtained from a 5 µM mixture of thermometer compounds using AMUSE at the extremes of the studied temperature range was at least fivefold higher than that of ESI, demonstrating the potential of AMUSE ionization as a soft method for the characterization of labile species by mass spectrometry.

Micromachined Ultrasonic Print-Head for Deposition of High-Viscosity Materials

The recent application of inkjet printing to fabrication of three-dimensional, multilayer and multimaterial parts has tested the limits of conventional printing-based additive manufacturing techniques. The novel method presented here, termed as additive manufacturing via microarray deposition (AMMD), expands the allowable range of physical properties of printed fluids to include important, high-viscosity production materials (e.g., polyurethane resins). AMMD relies on a piezoelectrically driven ultrasonic print-head that generates continuous streams of droplets from 45 μm orifices while operating in the 0.5-3.0 MHz frequency range. The device is composed of a bulk ceramic piezoelectric transducer for ultrasound generation, a reservoir for the material to be printed, and a silicon micromachined array of liquid horn structures, which make up the ejection nozzles. Unique to this new printing technique are the high frequency of operation, use of fluid cavity resonances to assist ejection, and acoustic wave focusing to generate the pressure gradient required to form and eject droplets. We present the initial characterization of a micromachined print-head for deposition of fluids that cannot be used with conventional printing-based rapid prototyping techniques. Glycerol-water mixtures with a range of properties (surface tensions of ∼58-73 mN/m and viscosities of 0.7-380 mN s/m 2 ) were used as representative printing fluids for most investigations. Sustained ejection was observed in all cases. In addition, successful ejection of a urethane-based photopolymer resin (surface tension of ∼25-30 mN/m and viscosity of 900-3000 mN s/m 2 ) was achieved in short duration bursts. Peaks in the ejection quality were found to correspond to predicted device resonances. Based on these results, we have demonstrated the printing of fluids that fall well outside of the accepted range for the previously introduced printing indicator. The micromachined ultrasonic print-head achieves sustained printing of fluids up to 380 mN s/m 2 , far above the typical printable range.

Enhanced intracellular delivery via coordinated acoustically driven shear mechanoporation and electrophoretic insertion

Delivery of large and structurally complex target molecules into cells is vital to the emerging areas of cellular modification and molecular therapy. Inadequacy of prevailing in vivo (viral) and in vitro (liposomal) gene transfer methods for delivery of proteins and a growing diversity of synthetic nanomaterials has encouraged development of alternative physical approaches. Efficacy of injury/diffusion-based delivery via shear mechanoporation is largely insensitive to cell type and target molecule; however, enhanced flexibility is typically accompanied by reduced gene transfer effectiveness. We detail a method to improve transfection efficiency through coordinated mechanical disruption of the cell membrane and electrophoretic insertion of DNA to the cell interior. An array of micromachined nozzles focuses ultrasonic pressure waves, creating a high-shear environment that promotes transient pore formation in membranes of transmitted cells. Acoustic Shear Poration (ASP) allows passive cytoplasmic delivery of small to large nongene macromolecules into established and primary cells at greater than 75% efficiency. Addition of an electrophoretic action enables active transport of target DNA molecules to substantially augment transfection efficiency of passive mechanoporation/diffusive delivery without affecting viability. This two-stage poration/insertion method preserves the compelling flexibility of shear-based delivery, yet substantially enhances capabilities for active transport and transfection of plasmid DNA.

Reduced Order Modeling and Experimental Investigation of Acoustic Particle Manipulation in Complex 3D Geometries

A reduced order computational model and imaging experiments are presented as a combined method to investigate migration and trapping of microscale particles within an ultrasonic droplet generator. Use of two-dimensional (2D) cross-sectional representations of the three-dimensional (3D) device enables observation of acoustic focusing phenomena that are otherwise visually inaccessible. Our approach establishes relationships between system operating parameters and particle retention due to acoustic radiation forces that arise during atomization of heterogeneous particle suspensions. The droplet generator consists of a piezoelectric transducer for ultrasonic actuation, a resonant fluid-filled chamber, and an array of microscopic pyramidal nozzles. 2D visualization chips were produced through anodic bonding of glass to microfluidic reservoirs deep reactive ion etched in silicon. Open nozzle orifices of the 3D microarray were sealed in its 2D representation to facilitate filling and testing. Finite element analysis was used to model the harmonic response of the 2D assembly from 500 kHz to 2 MHz. The average nozzle tip pressure amplitude across the 2D array was then used to identify operating frequencies that correspond to optimal droplet ejection from the 3D device (ejection modes). The pressure field at these resonant frequencies predicts the equilibrium distribution of polymeric beads suspended in the reservoirs of the 2D chips. To qualitatively assess the accuracy of the model results, visualization experiments were performed at the first three ejection modes of the system (fn1 ≈ 620–680 kHz, fn2 ≈ 1.14 MHz, and fn3 ≈ 1.63 MHz) using 10 μm polystyrene beads. The model demonstrates a remarkable ability to capture the overall shape, as well as specific details of the terminal particle distributions, defined as the state with no further movement toward a pressure node or antinode. Finally, time course trials of acoustic focusing of heterogeneous particle suspensions were used to observe the influence of particle volume on the magnitude of the acoustic radiation force. A mixture of 5 μm and 20 μm diameter polystyrene beads was subjected to a standing acoustic field in the 2D chips. Particle position was recorded at 5 ms intervals until an equilibrium distribution was achieved. As expected, the larger beads focused much more rapidly than smaller beads, acquiring their final positions in seconds (versus 10s of seconds for the 5 μm particles). The method and results reported here serve as building blocks toward translation of an existing ultrasonic droplet generator into a high-throughput particle separation and isolation platform.Copyright

Fuel Atomization From a Micromachined Ultrasonic Droplet Generator: Visualization, Scaling, and Modeling

Existing battery technologies have become a major obstacle to advances in the performance of portable energy-intensive devices primarily due to a limited lifetime between charge cycles.1,2 Fuel-cell-based energy sources are a viable alternative due to the high energy density of liquid fuels and the potential for high efficiency power generation. The focus of recent work has been the development of two types of fuel cells for portable applications, hydrogen-based fuel cells with external fuel reformation, i.e., conversion to hydrogen, and direct-methanol fuel cells that oxidize methanol directly at the cell anode.1,3 Regardless of whether internal or external fuel reformation is used, power-efficient atomization of liquid fuels ranging from methanol to higher hydrocarbons and diesel to kerosene and logistic fuels, e.g., JP-8, is an essential processing step for conversion of a fuel from liquid to gas phase. We present the experimental characterization and theoretical modeling of the fluid mechanics underlying the operation of a micromachined ultrasonic atomizer. This droplet generator utilizes fluid cavity resonances in the 0.5 to 3 MHz range along with acoustic wave focusing for low power atomization of liquids for fuel processing. The device comprises a fuel reservoir located between a bulk ceramic piezoelectric transducer for ultrasound generation and a silicon micromachined array of liquid horn structures as the ejection nozzles. The array size can be scaled to meet flow rate requirements for any application because a single piezoelectric actuator drives ejection from multiple nozzles. The atomizer is particularly well-matched to fuel processing applications because it is capable of highly controlled atomization of a variety of liquid fuels at low flow rates. This low-flow-rate requirement intrinsic to small-scale, portable power applications is especially challenging since one cannot rely on the conventional jet-instability-based atomization approach. Further, the planar configuration of the nozzle array is suited to integration with the planar design of fuel cells. Experimentally-validated finite element analysis (FEA) simulations of the acoustic response of the device are used to estimate the fraction of the electrical input power to the piezoelectric transducer that is imparted to the ejected fluid. Results of this efficiency analysis indicate that it is not optimal to design the ejector such that a cavity resonance (corresponding to acoustic wave focusing at the tips of the pyramidally-shaped nozzles and thus fluid ejection) coincides with the longitudinal resonance of the piezoelectric transducer. It also appears that the efficiency of the device increases with decreasing frequency. Atomization of methanol and kerosene from 5 to 25 μm diameter orifices is demonstrated at multiple frequencies between 0.5 and 3 MHz. In addition, high-resolution visualization of the ejection process is performed to investigate whether or not the proposed atomizer is capable of operating in either the discrete-droplet or continuous-jetting mode (see Figure 1). The results of the visualization experiments provide a basic understanding of the physics governing the ejection process and allow for the establishment of simple scaling laws that prescribe the mode of ejection; however, it is likely that the phenomena that dictate the mode of ejection (i.e., discretedroplet vs. continuous-jet) do not occur within the field of view of the camera. Further, the most important features that determine the initial interface evolution occur within the nozzle orifice itself. A detailed computational fluid dynamics (CFD) analysis of the interface evolution during droplet/jet ejection yields additional insight into the physics of the ejection process and provides further validation of the scaling laws. Figure 2 provides examples of simulation of both discrete-droplet and continuous-jet mode ejection.Copyright

Micromachined Ultrasonic ElectroSpray Source Array for High Throughput Mass Spectrometry

According to the recent Laboratory News’ Proteomics Special article Mass Spectroscopy (MS) has become the technology of choice to meet today’s unprecedented demand for accurate bioanalytical measurements, including protein identification. Although MS can be used to analyze any biological sample, it must be first converted to gas-phase ions before it can be introduced into a mass spectrometer for analysis. It is transfer of a very small liquid sample (proteins are very expensive and often very difficult to produce in sizable quantities) into a gas-phase ions that is currently considered to be a bottleneck to high throughput proteomics. Electrospray ionization (ESI) is a technique developed in early 1990th to generate a spray gas-phase ions by applying high voltage (from several hundreds volts and up to a few thousands kilovolts relative to the ground electrode of the MS interface) to a small capillary through which the liquid solution is pumped. The high electric field ionizes the fluid forming the converging Taylor cone of the exiting jet which eventually breaks into many small droplets when the repulsive Coulombic forces overcome the surface tension. Because of the focusing effect associated with the spraying the electrically charged fluid, the size of the electrospray cone and thus of the formed droplets is in a few tens of nanometers range although the inner diameter of the capillary is in the micrometer range.Copyright