Assessment of the potential of a high frequency acoustomicrofluidic nebulisation platform for inhaled stem cell therapy.

Despite the promise of stem cell therapy for lung therapeutics and repair, there are few viable means for directly delivering stem cells to locally target the respiratory airways via inhalation. This is not surprising given the significant challenges in aerosolising stem cells, particularly given their susceptibility to damage under the large stresses involved in the nebulisation process. Here, we present promising results using a microfluidic acoustic nebulisation platform that is not only low cost and portable, but also its high MHz order frequencies are effective for preserving the structural and functional integrity of mesenchymal stem cells (MSCs) during the nebulisation process. This is verified through an assessment of the viability, structure, metabolic activity, proliferation ability and genetic makeup of the nebulised MSCs using a variety of assays, including cell viability staining, flow cytometry, reverse transcription and quantitative polymerase chain reaction, and immunophenotyping, thus demonstrating the platform as a promising method for efficient pulmonary stem cell delivery.

Rapid Enhancement of Cellular Spheroid Assembly by Acoustically Driven Microcentrifugation.

Intense acoustically driven microcentrifugation flows are employed to enhance the assembly of cellular spheroids in the microwell of a tissue culture well plate. This ability to interface microfluidics with commonly used tissue culture plasticware is a significant advantage as it can potentially be parallelized for high throughput operation and allows existing analytical equipment designed to fit current laboratory formats to be retained. The microcentrifugation flow, induced in the microwell coated with a low adhesive hydrogel, is shown to rapidly enhance the concentration of cells into tight aggregates within a minute-considerably faster than the conventional hanging drop and liquid overlay methods, which typically require days-while maintaining their viability. The proposed method also affords better control of the compaction force and hence the spheroid dimension simply by tuning the input power, which is a significant improvement over other microfluidic methods that require the fabrication of different geometries and microstructures to generate spheroids of different sizes. The spheroids produced are observed to exhibit the concentric heterogeneous cell populations and tight cell-cell interfaces typical of in vivo tumors, and are potentially useful in a broad spectrum of cancer biology and drug screening studies.

Pulmonary monoclonal antibody delivery via a portable microfluidic nebulization platform.

Nebulizers have considerable advantages over conventional inhalers for pulmonary drug administration, particularly because they do not require coordinated breath actuation to generate and deliver the aerosols. Nevertheless, besides being less amenable to miniaturization and hence portability, some nebulizers are prone to denature macromolecular drugs due to the large forces generated during aerosolization. Here, we demonstrate a novel portable acoustomicrofluidic device capable of nebulizing epidermal growth factor receptor (EGFR) monoclonal antibodies into a fine aerosol mist with a mass median aerodynamic diameter of approximately 1.1 µm, optimal for deep lung deposition via inhalation. The nebulized monoclonal antibodies were tested for their stability, immunoactivity, and pharmacological properties, which confirmed that nebulization did not cause significant degradation of the antibody. In particular, flow cytometry demonstrated that the antigen binding capability of the antibody is retained and able to reduce phosphorylation in cells overexpressing the EGFR, indicating that the aerosols generated by the device were loaded with stable and active monoclonal antibodies. The delivery of antibodies via inhalation, particularly for the treatment of lung cancer, is thus expected to enhance the efficacy of this protein therapeutic by increasing the local concentration where they are needed.

Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization.

BACKGROUND: Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization. METHODS: In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep. RESULTS: The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation. CONCLUSION: Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA with a size range suitable for delivery to the lower respiratory airways.

Enabling practical surface acoustic wave nebulizer drug delivery via amplitude modulation.

A practical, commercially viable microfluidic device relies upon the miniaturization and integration of all its components--including pumps, circuitry, and power supply--onto a chip-based platform. Surface acoustic waves (SAW) have become popular in microfluidic manipulation, in solving the problems of microfluidic manipulation, but practical applications employing SAW still require more power than available via a battery. Introducing amplitude modulation at 0.5-40 kHz in SAW nebulization, which requires the highest energy input levels of all known SAW microfluidic processes, halves the power required to 1.5 W even while including the power in the sidebands, suitable for small lithium ion batteries, and maintains the nebulization rate, size, and size distributions vital to drug inhalation therapeutics. This simple yet effective means to enable an integrated SAW microfluidics device for nebulization exploits the relatively slow hydrodynamics and is furthermore shown to deliver shear-sensitive biomolecules--plasmid DNA and antibodies as exemplars of future pulmonary gene and vaccination therapies--undamaged in the nebulized mist. Altogether, the approach demonstrates a means to offer truly micro-scale microfluidics devices in a handheld, battery powered SAW nebulization device.

Hydroxypropyl cellulose methacrylate as a photo-patternable and biodegradable hybrid paper substrate for cell culture and other bioapplications.

In addition to the choice of appropriate material properties of the tissue construct to be used, such as its biocompatibility, biodegradability, cytocompatibility, and mechanical rigidity, the ability to incorporate microarchitectural patterns in the construct to mimic that found in the cellular microenvironment is an important consideration in tissue engineering and regenerative medicine. Both these issues are addressed by demonstrating a method for preparing biodegradable and photo-patternable constructs, where modified cellulose is cross-linked to form an insoluble structure in an aqueous environment. Specifically, hydroxypropyl cellulose (HPC) is rendered photocrosslinkable by grafting with methylacrylic anhydride, whose linkages also render the cross-linked construct hydrolytically degradable. The HPC is then cross-linked via a photolithography-based fabrication process. The feasibility of functionalizing these HPC structures with biochemical cues is verified post-fabrication, and shown to facilitate the adhesion of mesenchymal progenitor cells. The HPC constructs are shown to be biocompatible and hydrolytically degradable, thus enabling cell proliferation and cell migration, and therefore constituting an ideal candidate for long-term cell culture and implantable tissue scaffold applications. In addition, the potential of the HPC structure is demonstrated as an alternative substrate to paper microfluidic diagnostic devices for protein and cell assays.

Uniform mixing in paper-based microfluidic systems using surface acoustic waves.

Paper-based microfluidics has recently received considerable interest due to their ease and low cost, making them extremely attractive as point-of-care diagnostic devices. The incorporation of basic fluid actuation and manipulation schemes on paper substrates, however, afford the possibility to extend the functionality of this simple technology to a much wider range of typical lab-on-a-chip operations, given its considerable advantages in terms of cost, size and integrability over conventional microfluidic substrates. We present a convective actuation mechanism in a simple paper-based microfluidic device using surface acoustic waves to drive mixing. Employing a Y-channel structure patterned onto paper, the mixing induced by the 30 MHz acoustic waves is shown to be consistent and rapid, overcoming several limitations associated with its capillary-driven passive mixing counterpart wherein irreproducibilities and nonuniformities are often encountered in the mixing along the channel--capillary-driven passive mixing offers only poor control, is strongly dependent on the paper's texture and fibre alignment, and permits backflow, all due to the scale of the fibres being significant in comparison to the length scales of the features in a microfluidic system. Using a novel hue-based colourimetric technique, the mixing speed and efficiency is compared between the two methods, and used to assess the effects of changing the input power, channel tortuousity and fibre/flow alignment for the acoustically-driven mixing. The hue-based technique offers several advantages over grayscale pixel intensity analysis techniques in facilitating quantification without limitations on the colour contrast of the samples, and can be used, for example, for quantification in on-chip immunochromatographic assays.

Template-free synthesis and encapsulation technique for layer-by-layer polymer nanocarrier fabrication.

The encapsulation of therapeutic molecules within multiple layers of biocompatible and biodegradable polymeric excipients allows exquisite design of their release profile, to the extent the drug can be selectively delivered to a specific target location in vivo. Here, we develop a novel technique for the assembly of multilayer polyelectrolyte nanocarriers based on surface acoustic wave atomization as a rapid and efficient alternative to conventional layer-by-layer assembly, which requires the use of a sacrificial colloidal template over which consecutive polyelectrolyte layers are deposited. Polymer nanocarriers are synthesized by atomizing a polymer solution and suspending them within a complementary polymer solution of opposite charge subsequent to their solidification in-flight as the solvent evaporates; reatomizing this suspension produces nanocarriers with a layer of the second polymer deposited over the initial polymer core. Successive atomization-suspension layering steps can then be repeated to produce as many additional layers as desired. Specifically, we synthesize nanocarriers comprising two and three, and up to eight, alternating layers of chitosan (or polyethyleneimine) and carboxymethyl cellulose within which plasmid DNA is encapsulated and show in vitro DNA release profiles over several days. Evidence that the plasmid's viability is preserved and hence the potential of the technique for gene delivery is illustrated through efficient in vitro transfection of the encapsulated plasmid in human mesenchymal progenitor and COS-7 cells.

The extraction of liquid, protein molecules and yeast cells from paper through surface acoustic wave atomization.

Paper has been proposed as an inexpensive and versatile carrier for microfluidics devices with abilities well beyond simple capillary action for pregnancy tests and the like. Unlike standard microfluidics devices, extracting a fluid from the paper is a challenge and a drawback to its broader use. Here, we extract fluid from narrow paper strips using surface acoustic wave (SAW) irradiation that subsequently atomizes the extracted fluid into a monodisperse aerosol for use in mass spectroscopy, medical diagnostics, and drug delivery applications. Two protein molecules, ovalbumin and bovine serum albumin (BSA), have been preserved in paper and then extracted using atomized mist through SAW excitation; protein electrophoresis shows there is less than 1% degradation of either protein molecule in this process. Finally, a solution of live yeast cells was infused into paper, which was subsequently dried for preservation then remoistened to extract the cells via SAW atomization, yielding live cells at the completion of the process. The successful preservation and extraction of fluids, proteins and yeast cells significantly expands the usefulness of paper in microfluidics.

Miniature inhalation therapy platform using surface acoustic wave microfluidic atomization.

Pulmonary drug administration requires direct delivery of drug formulations into the lower pulmonary tract and alveoli of the lung in the form of inhaled particles or droplets, providing a distinct advantage over other methods for the treatment of respiratory diseases: the drug can be delivered directly to the site of inflammation, thus reducing the need for systemic exposure and the possibility of adverse effects. However, it is difficult to produce droplets of a drug solution within a narrow monodisperse size range (1-10 microm) needed for deposition in the lower pulmonary tract and alveoli. Here, we demonstrate the use of surface acoustic wave microfluidic atomization as an efficient means to generate appropriate aerosols containing a model drug, the short-acting beta2 agonist salbutamol, for the treatment of asthma. The mean aerosol diameter produced, 2.84+/-0.14 microm, lies well within the optimum size range, confirmed by a twin-stage impinger lung model, demonstrating that approximately 70 to 80% of the drug supplied to the atomizer is deposited within the lung. Our preliminary study explores how to control the aerosol diameter and lung delivery efficiency through the surface tension, viscosity, and input power, and also indicates which factors are irrelevant-like the fluid density. Even over a modest power range of 1-1.5 W, SAW atomization provides a viable and efficient generic nebulization platform for the delivery of drugs via the pulmonary route for the treatment of various diseases. The control offered over the aerosol size, low power requirements, high delivery efficiency, and the miniaturization of the system together suggest the proposed platform represents an attractive alternative to current nebulizers compatible with microfluidic technologies.