WO2013166542A1

WO2013166542A1 - Microfluidic apparatus for the atomisation of a liquid using surface acoustic waves

Info

Publication number: WO2013166542A1
Application number: PCT/AU2013/000316
Authority: WO
Inventors: James Friend; Leslie Yu-Ming Yeo; Aisha Qi; Anushi Erandica RAJAPAKSA; Ming Tan
Original assignee: Monash University
Priority date: 2012-05-11
Filing date: 2013-03-28
Publication date: 2013-11-14

Abstract

An apparatus for the atomisation of a liquid including: a piezoelectric substrate having at least one working surface, at least one electrode supported on the piezoelectric substrate; a signal generating means for applying an ultrasonic signal to said electrode for generating a surface acoustic wave (SAW) in the working surface of the piezoelectric substrate, and a liquid delivery arrangement for delivering liquid to the working surface, wherein liquid delivered to the working surface is atomised by SAW irradiation, and wherein the ultrasonic signal is amplitude modulated.

Description

MICROFLUIDIC APPARATUS FOR THE ATOMISATION OF A LIQUID USING SURFACE ACOUSTIC WAVES

TECHNICAL FIELD

The present invention is directed to a microfluidic apparatus. More particularly, the present invention is directed to a micofluidic apparatus for the atomisation of a liquid. While the invention is described with respect to its use as a pulmonary delivery apparatus, it is to be appreciated that the invention is not restricted to this application, and that other applications are also envisaged.

BACKGROUND TO THE INVENTION

Pulmonary drug delivery has for several decades constituted a highly effective and widely administered form of therapy, particularly for the treatment of respiratory diseases such as asthma and chronic obstructive pulmonary disease. The pressurized metered dose inhalers, dry powder inhalers and nebulisers, that are widely available and commonly used to date, have evolved little from the original push-and-breathe concept introduced in the 1950s. This is in no part due to a dearth of advances in inhaler and nebulizer technology— on the contrary, there have been a number of significant advances such as the development of adaptive delivery technology which synchronizes delivery of the aerosol to the patient's breathing pattern in order to optimize delivery and reduce the amount of drug wasted during exhalation. Such state-of-the-art technologies, nevertheless, often involve a combination of in-built sensors, electronic control schemes and active aerosol generation mechanisms. There are however technological barriers to practical and widespread implementation of recent advanced technology. In relation to active aerosol generation mechanisms, the power supply and ancillary equipment required to drive such devices is large and complex, thus rendering them cumbersome and hence impractical and unattractive for personal daily use, and restricting their use, for example, in hospitals. These considerations also have a wider consequence, acting as an impediment to broader implementation of inhalation therapy for vaccination, gene therapy and the treatment of other diseases such as diabetes, cystic fibrosis and lung cancer, all of which represent potential new therapeutic applications given the suitability of pulmonary drug administration for efficient, reproducible, non-invasive, safe and low-cost systemic delivery of peptides, proteins and pDNA (Plasmid DNA). A microfluidic aerosol delivery platform based on surface acoustic wave (SAW) atomization technology such as disclosed in International Patent Publication No. WO 2010/129994, the content of which is herein incorporated by reference in its entirety, offers many advantages over conventional nebulization devices. Cost, size, low dose efficiency (typically between 10-30%), possible drug degradation and the necessity for large and cumbersome power supplies or compressed air sources are amongst the many constraints that limit the use of conventional nebulization devices. In contrast, a typical SAW platform offers the attractive flexibility of tuning the administered dose according to the patient's physiological profile (size, gender, age) and disease severity together with rapid, reproducible and controllable aerosol generation, high dose efficiency (around 80%) and the absence of nozzles and orifices that have a tendency to clog and hence require regular cleaning or replacement, all in the form of a portable and reusable microfluidic device with the benefit of exploiting the economies of scale associated with advanced top-down nanofabrication technology for low-cost device mass manufacture. In addition, high frequency (10-100 MHz) SAW operation minimizes shear and cavitational molecular lysis— a critical consideration to ensure the structural integrity of the drug molecules are preserved during atomization.

Whilst the original concept of a SAW microfluidic aerosol delivery platform was intended to be driven by a palm-sized battery-powered circuit (Figure la), the limited power output available from the circuit imposed a constraint on the aerosol production rate. Increasing the power available to the device requires the use of larger driver circuits (Figure lb) that prohibit a complete miniaturized package for portable use.

It would be desirable to provide an apparatus for SAW atomization of a liquid which has reduced power requirements without impacting on the aerosol delivery rate.

Any discussion of documents, devices, acts or knowledge in this specification is included to explain the context of the invention. It should not be taken as an admission that any of the material formed part of the prior art base or the common general knowledge in the relevant art in Australia on or before the priority date of the claims herein.

Comprises/comprising and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. SUMMARY OF THE INVENTION

In accordance with the present invention in its broadest form, there is provided a microfluidic device including:

a piezoelectric substrate having at least one working surface;

at least one electrode supported on the piezoelectric substrate, and

a signal generating means for applying an ultrasonic signal to said electrode for generating a surface acoustic wave (SAW) in the working surface of the piezoelectric substrate,

wherein the ultrasonic signal is amplitude modulated.

In a more specific form, there is provided in a first aspect of the invention an apparatus for the atomisation of a liquid including:

a piezoelectric substrate having at least one working surface, at least one electrode supported on the piezoelectric substrate;

a signal generating means for applying an ultrasonic signal to said electrode for generating a surface acoustic wave (SAW) in the working surface of the piezoelectric substrate, and

a liquid delivery arrangement for delivering liquid to the working surface, wherein liquid delivered to the working surface is atomised by SAW irradiation, and wherein the ultrasonic signal is amplitude modulated.

The ultrasonic signal may be amplitude modulated at a frequency below approximately 100 kHz.

Preferably, the ultrasonic signal is amplitude modulated at a frequency below approximately 40 kHz.

In particular, the ultrasonic signal may be amplitude modulated at a frequency between approximately 500 Hz and 40 kHz. More preferably, the ultrasonic signal is amplitude modulated at a frequency between approximately 1kHz and 10 kHz.

The electrode may be an elliptical, electrode width controlled single phase unidirectional transducer (EWC-SPUDT).

The liquid delivery arrangement preferably includes a wick in contact with the working surface for delivery of the liquid. Further, the wick of the liquid delivery arrangement is preferably provided by at least one paper strip or string.

The liquid delivery arrangement may further include a liquid reservoir for containing the liquid to be delivered to the apparatus. The apparatus may further include a capillary tube extending from the liquid reservoir, the wick receiving the liquid via the capillary tube.

In accordance with a second aspect of the present invention there is provided a method for the pulmonary delivery of a drug composition using the apparatus of the first aspect of the invention.

By introducing amplitude modulation to the ultrasonic signal, the present invention is advantageously able to reduce the power requirements for SAW atomization. For example, with amplitude modulation at approximately 1 kHz, it is possible to maximize the atomization production rate with 40% less power compared with using no amplitude modulation. Amplitude modulation is accordingly an effective means for reducing the power requirement in surface acoustic wave (SAW) microfluidic platforms. This is particularly advantageous as practical implementation and commercial realization of microfluidic devices hinges on the miniaturization and integration of all components onto a chip-based platform. This includes ancillary equipment such as pumps as well as power supplies and electronic circuitry.

Due to amplitude modulation, the SAW microfluidic aerosol delivery platform of the present invention is advantageously able to be driven by a palm-sized, battery- powered circuit that may be integrated with the chip-scale SAW atomization device to constitute a low cost portable handheld nebulization platform for pulmonary drug administration with many advantages over conventional inhalation and nebulization systems.

BRIEF DESCRIPTION OF THE DRAWINGS

It will be convenient to further describe the invention with reference to the accompanying drawings. In the drawings:

FIG. 1 (a) is an initial prototype of the battery-powered circuit to be used to drive the SAW microfluidic aerosol delivery platform (scale bar = 1 cm).

FIG. 1(b) is a typical signal generator and amplifer used in laboratory settings to provide sufficient power for the SAW atomization (scale bar = 3 cm).

FIG. 2(a) is an image of a working 30 MHz SPUDT SAW device. The fluid is delivered to the SAW substrate through a paper strip embedded in a capillary tube which is connected to a reservoir. In order to record the aerosol production rate, the capillary tube is marked along its length at 0.5 cm intervals. FIG. 2(b) is an enlarged view of the SPUDT. The width and gap of the interleaving finger electrodes determine the SAW wavelength which is equal to 132 μηι for a 30 MHz device.

FIG. 2(c) is a side view illustration of the SAW atomization mechanism. The SAW (not shown to scale) propagates along the substrate and leaks energy into the liquid film to drive the destabilization of its free surface.

FIG. 2(d) schematically illustrates the setup used to condense and collect aerosolized DNA or antibodies within a 50 ml conical Falcon tube for further in vitro characterization of their post-atomization viability.

FIG. 3 is a representation of pVR1020 with vector encoding the haemagglutinin

(HA) gene sequence. Gene encoding HA is inserted in plasmid VR1020 that contains secretion signal of tissue plasminogen activator (TPA), human cytomegalovirus (CMV) early promoter, CMV intron A, bovine growth hormone (BGH) terminator and kanamycin resistance gene.

FIG. 4(a) is a gel electrophoresis of post-atomized plasmid DNA showing the effect of the amplitude modulation at various frequencies on the structural integrity of the pDNA.

FIG. 4(b) is a graph showing the percentage retention of post-atomized supercoiled (shaded) and open circular (unshaded) pDNA compared to that in the unatomized sample. The results are the mean of triplicate atomization runs where the error bars represent standard deviation.

FIG. 5 is an image of post-atomized YFP antibody samples spotted onto a representative dot blot showing the preservation of the bioactivity of protein molecules in samples with and without amplitude modulation.

DETAILED DESCRIPTION OF THE INVENTION

In the application of amplitude modulation to SAW atomization, it is important to appreciate that the aerosols are formed due to destabilization and break-up of the parent drop's free surface that is governed by a capillary- viscous mechanism. A dominant force balance between the capillary and viscous stresses then suggests that the capillary-viscous resonant frequency scales as which implies that a parent drop with a characteristic dimension R ~ 10^~3 m and comprised of typical fluids with surface tension γ ~ 10^~2 kg/s² and viscosity μ ~ 10^~2 -10^"3 kg/ms, typically vibrates under resonance at 1-10 kHz order frequencies. Such intuitive considerations of the underlying fundamental physical mechanism governing the atomization process is useful as it suggests that driving a carrier signal at these resonant frequencies supplies acoustic energy directly to efficiently drive drop vibration at resonance. Consequently, one is able to exploit the combined advantages of both high (MHz order) frequency and low (kHz order) frequency operation simultaneously to drive more efficient atomization.

One of the common concerns, however, of low frequency excitation is the potential molecular damage that this causes due to large shear stresses exerted on the molecule either directly by the hydrodynamics or indirectly through cavitational processes. For this reason, a large consideration in our study reported below is devoted to the investigation of the viability of biomolecules, in particular, DNA and antibodies, given the context of pulmonary gene and vaccine delivery, during the atomization process when amplitude modulation at kHz order frequencies are employed. In addition, it is also necessary to verify that the aerosol drop size distribution still lies within the optimum respirable size range (1-10 μιη) necessary for oropharyngeal and deep lung delivery.

Whilst we have chosen to demonstrate the use of amplitude modulation for reducing the power requirements in SAW nebulization devices, given that atomization requires by far the highest amount of power in SAW microfluidic devices, the same scheme can be extended to drive the entire range of SAW microfluidics at reduced power, therefore enabling the power supply to these platforms and hence the devices themselves to be miniaturized and integrated for true miniaturized on-chip functionality. A myriad of SAW devices have been proposed for applications ranging from fluid manipulation, e.g., droplet transport, microchannel pumping, mixing and jetting, particle sorting and micro centrifugation to chip-scale chemical and biochemical synthesis, biosensing, and microfluidic chip interfacing with mass spectrometry; in addition, it is also possible to exploit the SAW to drive these microfluidic manipulations on disposable superstates. (I) EXPERIMENTS AND MATERIALS

Here, we specifically demonstrate the use of the amplitude modulation scheme for reducing the power requirements for SAW atomization, by far requiring the highest energy input levels of all SAW microfluidic processes. We show that amplitude modulation of 10-100 MHz order SAW at 1-10 kHz order frequencies can approximately halve the power required while maintaining monodisperse aerosol droplet sizes in the 1- 10 micron range and aerosol production rates commensurate with the prerequisites of practical nebulization devices for inhalation therapy. In addition, we also verify that the superposition of the kHz order carrier signal does not cause appreciable damage to shear- sensitive biomolecules in contrast to sonoporation and cell lysis processes which are carried out at similar frequencies. In particular, we show that with amplitude modulation at approximately 1 kHz, it is possible to maximize the aerosol production rate with 40% less power while minimizing denaturing of plasmid DNA or antibodies, both of which represent exciting future therapeutic targets for pulmonary gene and vaccine delivery. FIG. 2(a) is an image of a working 30 MHz SPUDT SAW device. Fluid is delivered to a SAW substrate through a paper strip embedded in a capillary tube which is connected to a reservoir. In order to record the aerosol production rate, the capillary tube is marked along its length at 0.5 cm intervals. FIG. 2(b) is an enlarged view of the SPUDT. The width and gap of the interleaving finger electrodes determine the SAW wavelength )SAW, which is equal to 132 μιη for a 30 MHz device. FIG. 2(c) is a side view illustration of the SAW atomization mechanism. The SAW (not shown to scale) propagates along the substrate and leaks energy into the liquid film to drive the destabilization of its free surface. The 30 MHz SAW device employed in the experiment consisted of a low-loss piezo-electric substrate material, 127.86° F-X-rotated single-crystal lithium niobate (LiNbC ), patterned with a pair of chromium-aluminum single-phase unidirectional transducers (SPUDT) via standard UV photolithography processes. The width and gap of the interlaced finger patterns of the SPUDT determines the SAW wavelength λsAw; in this study, SAW = 132 μηι corresponding to a SAW frequency of 30 MHz (carrier frequency). The curved SPUDTs focus the acoustic energy to which fluid is delivered from a reservoir via a pre-wetted paper wick embedded at the tip of a capillary tube which allows continuous flow of fluid without the need of a syringe pump. Images of the devices are shown in Figs. 2(a) and 2(b). As a sinusoidal electrical input at the SAW frequency (together with the corresponding amplitude modulation) is applied to the SPUDT, SAWs are generated that propagate along the LiNbC^ substrate across to the leading edge of the paper wick, where they continuously draw liquid out from the paper onto the substrate to form a thin film. Since acoustic energy also leaks into the liquid film, the free surface of the film destabilizes and beyond a critical input power, breaks up, to form aerosol droplets (Fig. 2(c)). There is no observable flow onto the device in the absence of the SAW (for example, that might which be present due to evaporation), and hence the flow rate through the capillary tube, measured using the graduation scale marked on the tube, is a good estimate of the aerosol production rate given that the film dimensions can be assumed to be fairly constant at steady-state.

To study the effect of amplitude modulation on SAW atomization, the following sets of experiments were performed:

Effect of amplitude modulation on the aerosol size

The aerosol size distributions of the deionised water droplets generated via SAW nebulization were measured using laser diffraction (Spraytec, Malvern Instruments, Malvern, UK) for measurement. Here, we measure a single aerosol size parameter D_V5₀, representing the mean diameter across the 50^th percentile within the volume size distribution. Effect of amplitude modulation on the atomization rate

The paper wick and capillary tube (without the reservoir) was filled with deionized water as the model fluid. As the water was atomized, the time was recorded as the meniscus retracted past consecutive graduation marks on the capillary tube shown in Fig. 2(a). Experiments were repeated three times at each sinusoidal amplitude modulation frequency: 500 Hz, 1 kHz, 5 kHz, 10 kHz, 20 kHz and 40 kHz. Since the amplitude modulation gave rise to more efficient atomization through lower energy consumption, the power input was kept lower at 1.5 W and 2 W, compared to our control experiments without amplitude modulation, where power levels of 1.5 W, 2 W, 3 W and 4 W were used.

Effect of amplitude modulation on naked plasmid DNA delivery

Plasmid DNA (pDNA) was prepared from an influenza A virus surface antigen, human haemagglutinin (A/Solomon Islands/3/2006 (egg passage) (H1N1) strain), once cloned into the mammalian expression vector VR1020 (Vical Inc., San Diego, CA) (Fig. 3). The entire coding sequence of HA was amplifed by PCR using primers forward and reverse that incorporated a BamHI site at the 5° end and a EcoRI site at the 3° end, forward: 5°-CGCGGATCCATGAAAGTAAAACTACTGGTCCTGTTATG-3°; reverse: 5^°-CCGGAATTCTTGTTTGTAATCCCATTAATGGCATTTTGT-3°. The PCR product was digested with BamHI / EcoRI and ligated into the 3°C plasmid, VR1020, resulting in plasmid VR1020-HA. A colony of E. coli DH5 transformed with the plasmid VR1020- HA (6 kbp) was picked from a streaked selective plate and inoculated in 10 ml of LB medium containing 100.0 g/ml of kanamycin. The starter culture was incubated at 37°C and agitated at 200 rpm for 8 h before being transferred to five separated 200 ml LB media, and further cultured for 12 h. The cell cultures were stored at -70°C for subsequent use. The plasmids were purified from cells using an endotoxin-free plasmid purification kit (Plasmid Mega Kit, QIAGEN Pty. Ltd., Doncaster, VIC, Australia) according to the manufacturer's instructions. Atomization was confined in a 50 ml conical Falcon tube (BD Bioscience, Franklin Lakes, NJ), as depicted in Fig. 2(d). Plasmid DNA aerosols were collected after condensing on the tube wall. Both control and atomized pDNA samples were analysed for potential alterations in the plasmid structure with 1 % agarose gel electrophoresis that used GelRed™ staining, and a 1 kbp DNA ladder was employed as a size marker. The gel was made up of 5 g agarose at lx dilution of TAE buffer (242.0 g Tris base, 57.1 ml CH₃COOH, 9.3 g of EDTA). The electrophoresis was carried out under 120 V for 60 min. The resulting gel was analysed and imaged in an automated gel imaging and documentation system (Molecular Imager® Gel Doc XR, Bio- Rad Laboratories Inc., Hercules, CA). The intensity of the bands for each structure corresponds to the number of DNA molecules. The percentage of supercoiled (sc) and open circular (oc) to fragmented DNA was quantified via densitometry software (Quantity One®, Bio-Rad Laboratories Inc., Hercules, CA ) by comparing pre- and post-atomized samples.

Effect of amplitude modulation on protein delivery

Atomisation of rabbit anti-YFP (Yellow Florescent Protein) antiserum solution (diluted at 1 :20) obtained from collaborators at the Coppel Lab (Department of Microbiology, Monash University) was carried out in the same manner as that for the pDNA. The antibody was then detected using dot blot analysis when 5μ1 of YFP protein solution was dotted onto a transfer membrane (PolyScreen® PVDF, PerkinElmer Inc., Waltham, MA) for immunoblotting. The membranes were incubated in TBS-T buffer (0.05 M Tris-HCl pH 7.4, 0.15 M NaCl, 0.05% Tween 20) containing 5% non-fat milk powder overnight at 4°C. Subsequently, the membranes were probed with the atomized anti-YFP antiserum solutions for 1 h at room temperature, and washed three times in TBS-T for 10 min each time. Primary antibody reactivity to immunoblotted proteins was detected with anti-rabbit immunoglobulin conjugated to horseradish peroxidase (HRP; Silenus Laboratories Pty. Ltd., Hawthorn, VIC, Australia), visualized by Renaissance® Western Blot Chemiluminescence Reagent (NEN Life Science, PerkinElmer Inc., Waltham, MA).

Statistical Analysis

Statistical analysis was performed by a one-way ANOVA with a Tukey's post hoc test, using the SPSS (statistics 19) software program, whenever the data survives Levene's Test of Equality of Error Variances. In the instance when the Levene's Test of Equality of Error Variances was significant, the non-parametric test Kruskal-Wallis Test was used to test the significance. All data are expressed the mean ± SD. The results were considered significant if P was less than 0.05.

(II) RESULTS AND DISCUSSION

A. Aerosol Size and Production Rate

TABLE I: Effect of amplitude modulation at various frequencies on the aerosol volume mean diameter D_V5₀. For each experiment, the fluid was atomized for 20 seconds with 50 data points sampled every second. This was repeated four times for every parameter set, i.e., 4000 data points were collected for each parameter set.

Table I is a compilation of measured aerosol sizes showing the effect of amplitude modulation and the effect of the amplitude modulation frequency. Other than an increase in the aerosol size with the application of amplitude modulation at 1kHz AM (p=0.03) at 2W compared to that when no modulation was applied at the same power level,there was no significant change in the atomization due to amplitude modulation at both 2W and 1.5 W(Tukey's post hoc test and Kruskal-Wallis Test respectively). An increase in aerosol size is also observed at the higher 3 W (p=0.016)and 4 W (p=0.028) power levels compared to the application of a low 1.5W (Tukey's post hoc test). These observations are not entirely surprising when interpreted in light of the existing theory, which suggests that the aerosol diameter D can be estimated from the wavelength λ associated with the interfacial instability, which, from a balance between the dominant axial capillary stress and the viscous stress, reads

where H and L are characteristic height and length scales of the liquid drop or film, suggesting that the geometry of the parent liquid plays a role in the aerosol size. This was reported in our previous work where we observed higher input powers to deform the liquid into a conical drop with a H/L ratio close to 1 , therefore leading to the production of larger aerosol diameters upon atomization, consistent with that seen in the data of Table I for the higher power levels. This is possibly true, although to a much weaker extent, with the slight increase in the aerosol diameter upon application of the amplitude modulation, since this serves to magnify the capillary resonance by concentrating the acoustic energy at the carrier frequency, which is sufficiently close to the capillary resonant frequency. Nevertheless, since the capillary wave resonant frequency in itself is independent of either the excitation frequency or the carrier frequency, we therefore do not expect the carrier frequency at which the amplitude modulation signal is driven to give rise to appreciable differences in the aerosol size, as is observed here. In any case, the absence of any significant change in the aerosol size with amplitude modulation of the SAW signal is encouraging from a practical perspective given the necessity for the aerosol diameters to lie in the 1-10 μηι range for optimal deposition in the oropharyngeal and lower respiratory tract region.

Table II shows the atomization rate at each power level, with and without application of the amplitude modulation. The rate at which aerosols are produced at 1.5 W and 2 W is roughly quadrupled and trebled, respectively, when amplitude modulation is employed. Alternatively, we observe the power required to achieve satisfactory aerosol delivery for a portable handheld nebulization device, typically above 100 μΖ/min, to be halved with the use of the amplitude modulation scheme. From Table II, we note that the atomization rate is reduced slightly as the frequency at which the amplitude modulation is driven is increased, particularly beyond 10 kHz, possibly since the capillary viscous resonant frequency which magnifies the capillary resonance estimated from Eq. (1) is around 1-10 kHz order.

When comparing the optimal aerosol production rate to the results obtained with similar SAW devices, driven at 9.8 MHz, employing pulse width modulation (1 kHz) of the input signal, voltage of around 120-140 V_p._p were required to achieve production rates of 200 μΐ/min. For the SAW devices in the current study, voltages of around 50 V_p._p are required to achieve similar aerosol production rates. Furthermore, employing amplitude modulation reduces the voltage to as little as 35 V_p._p or 45V_P._P, which is considerable reduction in driving voltage. TABLE II; Sifeci of am litu e modulation at various ire¾ue£5ck<» on the Atamfearton rale

B. Post-Atomization Biomolecular Integrity and Viability

The integrity of cells and proteins under 10-100 MHz order SAW irradiation, both at intensity levels below and at which atomization occurs, has been previously investigated. In these studies, little damage due to shear lysis of these biomolecules have been found. This was primarily attributed to the short time scales associated with the periodic reversal linked to the electro acoustic field oscillating in the fluid at 10-100 MHz order oscillating signal, which are at least several orders of magnitude smaller than the characteristic hydrodynamic time scale. Consequently, there is typically insufficient time to cause hydrodynamic shear unfolding of biomolecular structures. Clearly, these investigations did not account for lower kHz order frequency effects associated with the amplitude modulation, which naturally would be a concern since cell and biomolecular disruption is commonly carried out using ultrasonication at frequencies typically between 20 and 50 kHz, both through hydrodynamic shear as well as cavitation damage. Here, we carry out a brief study employing two model molecules to span the spectrum of potential applications for the SAW inhalation therapy platform across gene and vaccine delivery, namely plasmid DNA (pVR1020-HA) and an antibody (rabbit anti-YFP antisera). Figure 4(a) is a gel electrophoresis of post-atomized plasmid DNA showing the effect of the amplitude modulation at various frequencies on the structural integrity of the pDNA. Lane M: 1 kbp DNA ladder; Lane 1 : Atomization at 1.5 W without amplitude modulation; Lanes 2-9: Atomisation at 1.5 W with amplitude modulation at 500 Hz, 1 kHz, 5 kHz, 10 kHz, 20 kHz and 40kHz, respectively. Each lane was loaded with 250 ng pDNA and a representative gel from three independent experiments are shown. Arrows indicate the position of the open circular (OC) and supercoiled (SC) forms of the pDNA.

We note that pDNA fragmentation largely occurs under typical strain rates of 10^"5— 10^"6 s^"1, which correspond to oscillations in the 100 kHz to 1 MHz range. Although this appears fortunately to be in between the 10-100 MHz order SAW carrier and the 1- 10 kHz order modulation frequencies, the lower and upper bound of the quoted strain rates that lead to large pDNA structural damage is unclear. In fact, we observe the integrity of supercoiled pDNA retained is almost 80% in the absence of amplitude modulation (Figure 4b). This is seen to gradually decrease to around 50% at around 10 kHz, sharply dropping to 30% as the carrier frequency is increased to 40 kHz, closer to our estimate of the 100 kHz order frequencies corresponding to the shear rates at which pDNA uncoiling occurs and consistent with the typical 20-50 kHz applied frequencies at which ultrasonication is carried out to effect cell and molecular poration. This therefore provides a rationale for driving the amplitude modulation at the lower 1 kHz spectrum to minimize pDNA denaturation. It is also interesting to note that while there is less effect of the amplitude modulation frequency on the open circular conformation of the pDNA, the increase (>100%) in the number of such structures in the absence of amplitude modulation is beyond that in the feedstock prior to atomization, suggesting that some of the supercoiled pDNA relaxed into an open circular form rather than being fragmented, which is encouraging given that biological function is usually retained even with open circular structures.

Similarly, antibodies constitute both sensitive and fragile proteins and hence their post-atomized bioactivity is required to be verified in order to demonstrate the SAW microfluidic aerosol delivery device as a viable platform for pulmonary administration of protein and peptide based vaccines.

Figure 5 is an image of post-atomized YFP antibody samples spotted onto a representative dot blot showing the preservation of the bioactivity of protein molecules in samples with and without amplitude modulation. Lane 1 : No atomization; Lane 2: Atomization at 1.5W in the absence of amplitude modulation; Lanes 3-8: Atomization at 1.5 W with amplitude modulation at frequencies of 500Hz, 1 kHz, 5 kHz, 10 kHz, 20 kHz and 40 kHz, respectively.

Due to the small quantities of antibody present in the post-atomized samples, it was not possible to obtain quantitative measurements of the post-atomized antibody bioactivity; nevertheless, the qualitative results from the dot blot test in which YFP was spotted using post-atomized rabbit anti-YFP antisera (Figure 5) confirms that there is no observed degradation of the active sites of the protein molecule during amplitude modulation at all carrier frequencies investigated. These results therefore confirm that the application of amplitude modulation at kHz order frequencies to the SAW causes no significant damage to active sites on protein molecules which therefore retain their bioactivity.

(III) CONCLUSIONS

We have demonstrated the feasibility of applying amplitude modulation for approximately doubling the power while maintaining the aerosol production rate in SAW atomization— an important consideration that addresses current issues hindering the miniaturization and integration of the power supply into a practical and commercially realizable portable handheld nebulizer platform for the pulmonary administration of a wide variety of therapeutic targets. We have verified that the introduction of amplitude modulation in the system does not significantly alter the aerosol droplet diameter from the respirable size range for optimal dose administration to the oropharyngeal and lower respiratory tract. Judiciously limiting the amplitude modulation carrier frequency to 1 kHz simultaneously optimizes the atomization rate while limiting loss of bioactivity through pDNA fragmentation as well as protein denaturation, both of which represent important therapeutic targets for gene and vaccine delivery. These results therefore lend confidence to the attractiveness and feasibility of the SAW atomization platform as a true miniaturized and integrated handheld platform for portable inhalation therapy from a practical and commercial standpoint for applications as ubiquitous as asthma and chronic obstructive pulmonary diseases to exciting future possibilities in non-invasive gene and vaccine administration to treat a variety of diseases. In addition, the results of the study can also be extrapolated to reduce the power requirements and hence afford the miniaturization and integration of the power supply with the existing chip-based SAW microfluidic platform to drive a whole range of microscale and nanoscale fluid actuation and bioparticle manipulation processes on a truly integrated chip-scale device.

Modifications and variations as would be deemed obvious to the person skilled in the art are included within the ambit of the present invention as claimed in the appended claims.

Claims

CLAIMS:

1. An apparatus for the atomisation of a liquid including:

2. An apparatus according to claim 1, wherein the ultrasonic signal is amplitude modulated at a frequency below approximately 100 kHz.

3. An apparatus according to claim 1 or 2 wherein the ultrasonic signal is amplitude modulated at a frequency below approximately 40 kHz.

4. An apparatus according to any one of claims 1 to 3 wherein the ultrasonic signal is amplitude modulated at a frequency between approximately 500 Hz and 40 kHz.

5. An apparatus according to claim 4 wherein the ultrasonic signal is amplitude modulated at a frequency between approximately lkHz and 10 kHz.

6. An apparatus according to any one of claim 1 to 5 wherein the electrode is an elliptical, electrode width controlled single phase unidirectional transducer (EWC-

SPUDT).

7. An apparatus according to any one of claim 1 to 6, wherein the liquid delivery arrangement includes a wick in contact with the working surface for delivery of the liquid.

8. An apparatus according to claim 7 wherein the wick of the liquid delivery arrangement is provided by at least one paper strip or string.

9. An apparatus according to any one of claims 1 to 8, wherein the liquid delivery arrangement further includes a liquid reservoir for containing the liquid to be delivered to the apparatus.

10. An apparatus according to claim 9, further including a capillary tube extending from the liquid reservoir, the wick receiving the liquid via the capillary tube.

11. A method including the pulmonary delivery of a drug composition using the apparatus as claimed in any one of the preceding claims.