Advances in pulmonary drug delivery via high frequency acoustic nebulization
2017-02-28T03:05:40Z (GMT) by
There has been significant interest in the potential of pulmonary-delivered genetic vaccination in treating pulmonary diseases to mitigate vaccine safety issues and to obviate the requirement for needles and skilled, expensive personnel to handle them. Plasmid DNA(pDNA) offers a rapid production route to vaccines without significant side effects nor an extensive cold chain, which is especially important in being prepared for a pandemic caused by a highly infectious agent such as influenza. However, delivering therapeutics such as pDNA to the lung is challenging. Conventional methods including jet and ultrasonic nebulizers for the pulmonary DNA delivery of gene therapeutics are currently ineffective, as they largely fail to maintain the viability of large biomolecules such as pDNA due to the large shear stresses induced during the nebulization process. This thesis proposes a novel platform for the production of monodispersed aerosol-laden pDNA within a defined size range (0.5-5 μm) suitable for efficient pulmonary delivery to the lower respiratory airways for optimal dose efficacy, based on SAW (Surface Acoustic Wave) nebulization. SAWs are 10 nm order amplitude sound waves that originate as a result of the application of an alternating voltage onto an interdigital transducer patterned on a piezoelectric substrate. The megahertz (>10 MHz) order SAW vibration frequencies facilitate fluid and particle manipulation at a much finer scale, allowing extremely efficient transfer of acoustic radiation from the substrate into a drop comprising the drug solution. The acoustic energy is concentrated within a thin region in the drop adjacent to the substrate, which causes the drop interface to rapidly destabilize and breakup to form micron-dimension aerosol droplets containing the therapeutic molecule. The shear gradient generated within such a short period is not sufficient to degrade biomolecules such as pDNA, since the oscillation period of the SAW vibration at these frequencies is far shorter than the typical macromolecular relaxation time-scale in liquids. Extensive experimental studies were carried out to investigate the effect of SAW waves on pDNA. First, during in vitro studies, a solution containing a pDNA vector encoding a potential malaria vaccine candidate, merozoite surface protein 4/5, was nebulized using both 20 and 30 MHz SAW devices and the condensed mist was collected carefully. High levels of gene expression was observed in Western blots from in vitro experiments conducted using immortalized African green monkey kidney cells that were transfected with the post-nebulized pDNA. Next, in vivo studies were carried out using a pDNA encoding a yellow fluorescent protein (YFP) which was collected following 30 MHz SAW nebulization. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to a male Swiss mouse via intratracheal instillation. Subsequently, in vivo immunization trials were carried out using pDNA vector encoding an influenza A virus surface antigen, human hemagglutinin ((H1N1) strain) that was nebulized using a 30 MHz SAW device. Powerful pharmacodynamic responses were detected following the pDNA vaccination in sera of female Sprague-Dawley rats (n=8 per group) delivered via intratracheal instillation and female Merino-cross ewe lambs (n=4) delivered via nebulized mist inhalation. Moreover, these immunization trials demonstrated antibody responses with high functional activity as shown by the successful inhibition of viral agglutination of chicken red blood cells. These observations validate the use of SAW nebulization as a viable delivery platform for aerosol gene therapy. To enable miniaturization, the hand-held nebulizer system required the optimization of the usage of available power systems in the simplest manner. Amplitude modulation (AM) was investigated as a simple yet effective means for optimizing the power requirement for the SAW nebulizer. The effect of AM on shear-sensitive biomolecules was shown to be minimal. By employing AM less than 10 kHz, more efficient atomization was achieved and energy savings of around 40% were obtained. In addition, AM had little effect on the mean aerosol diameter, which is particularly important when therapies are targeted to the deep lung regions. Thus, AM holds great promise for use in SAW nebulizers for non-invasive inhalation therapy. SAW technology offers an in-home and clinical nebulizer which can be used to administer biologically-based medications to the lungs, with a broad ability to control droplet size through formulation to target specific regions of the lung most affected by disease. This research clearly demonstrates the potential of SAW technology as a needle-free, portable pulmonary delivery platform for gene therapy and DNA vaccination.