IE20050335A1 - Piezoelectric configurations for real time ultrasound dose measurement in vitro and in vivo - Google Patents

Abstract

The invention relates to a system/configuration/kit that may be employed to measure, in real time, the dose of ultrasound delivered to tissue culture samples in vitro and/or to a site on the body in vivo. Furthermore the device/configuration/kit may be employed to measure the dose of ultrasound delivered in real time directly to samples/tissues, delivered through a liquid to the samples/tissues, delivered through air to the samples/tissues and/or delivered through an aerosol to the samples/tissues.

Description

Background Although ultrasound is exploited by some animals (bats and crickets) in nature, it was only in the early part of the 20th century that man began to exploit it in earnest. With the advent of piezoelectric materials and vacuum-tube electronics it became possible to generate ultrasound in media such as water, oil and other liquids in the megahertz frequency range at acoustical intensities sufficient for the detection of submarine objects. In addition to its exploitation in detection, it was soon discovered that ultrasound could also be bring about changes in physical and chemical characteristics of materials through which it passes and it was these early observations that have shaped our current view of US and its potential.

Over the past 70 years major advances in the field of ultrasound for applications in medicine have concentrated on its development as an imaging tool for diagnostics and its development as a tool for use in physical therapy. More recently however, medical applications of ultrasound have expanded to include its exploitation in other areas of and these include its application in: 1. Tumour ablation: This application involves the use of high intensity focussed ultrasound (HIFU) and involves the application of higher ultrasound intensities (KW range) for very short periods of time. The ultrasound beam if focussed to a point at the target and excessive heating brings about an ablative effect at the focal point. This treatment modality has been exploited in the treatment of liver tumours and promising results have been reported recently [Li etal., 2004; World J. Gastroenterol. 10, 2201-2204; WueZa/., 2004; Ann. Surg. Oncol. 11, 1061-1069]. 2. Sonoporation and gene therapy: This application involves the use of ultrasound at relatively low intensities to induce transient poration of biological membranes and it has been suggested and indeed demonstrated that this phenomenon can be exploited to facilitate the entry of exogenous materials into cells. In a variation of this technology it has also been demonstrated that sonoporation or ultrasound-induced transient poration of biological membranes may be exploited in facilitating transport of genetic information into cells and this has led to the development of a relatively novel form of gene transfection. It has been suggested that this may be exploited in vivo as a non-viral gene transfer modality for use in a variety of gene therapies thereby circumventing many of the problems associated with IEU 00 3 05 virus-based gene therapies [Tachibana K, 2004; Hum Cell 17, 7-15; Miller et al., 2002, Somat Cell Mol Genet. 27, 115-134]. 3. Sonophoresis/phonophoresis: This is a technology that employed relatively low frequency ultrasound (circa 20KHz) to facilitate or enhance transport of drugs or active agents across skin. Although the exact mechanism is not known, it has been found that transport of test substances such as fluroescein across the skin may be enhanced by between 2 and 9 fold [Vranic E, 2004, Bosn J Basic Med Sci. 4,2532; Cancel et a/., 2004, JPharm Pharmacol. 56,1109-1118]. 4. Sonodynamic therapy: This exploits the ultrasound dependent enhancement of certain cytotoxic activities of compounds known as sonosensitisers and forms the basis of a novel ultrasound-mediated treatment modality for conditions such as cancer. Although the mechanism of action is unclear it does appear that ultrasound induced production of free radicals does play a role in achieving a cytotoxic effect [Rosenthal et al., 2004. Ultrason. Sonochem. 11, 349-363].

Ultrasound is a mechanical wave with periodic vibrations of particles in a continuous, elastic medium at frequencies that are equal to or greater than 20kHz. In liquids the speed of ultrasound is about 1000-1600 ms'1 and as such its wavelength ranges from the micrometers to the centimetres. Therefore, the acoustic field cannot couple directly with the energy levels of molecules (λ = nanometers) including biological milieu at the molecular level. This has led to the perception that ultrasound is a safe form of energy that has the ability to penetrate deep into the body.

Hence its attractiveness as a tool in both diagnostic imaging and the therapeutic areas described above.

In terms of the use of ultrasound in physical therapy, in 1930 Harvey [Harvey EN, 1930, Biol. Bull. 59, 306-325] reported that ultrasound could bring about physiological change in animals by raising tissue temperatures. Subsequently, Freundlich et al. suggested that this phenomenon might be exploited in therapy and demonstrated that heat could be generated preferentially in bones and joints [Freundlich et al., 1932; Einge biologische wirkungen von ultraschallwellen. Klin Worhschr; 11,1512-1513]. In the first application of ultrasound physical therapy, in 1938, a patient with sciatica was treated. The frequency chosen was 800kHz and 2 IE °50 335 the intensity ranged from 4-5Wcm . In those experiments the intensity was adjusted w to be ‘only a little less than a value that was found to cause unacceptable pain. This was evidently the first use of the method, sometimes called the “ouch!” technique, which is still used, to some extent, in setting the acoustic level’ [Nyborg, WL, 2001, Ultrasound in Med. Biol. 27, 301-333]. This rather antiquated observation particularly in terms of pain induction, suggests that, although ultrasound may radiate a tissue or sample at wavelengths that are incompatible with direct interaction at a molecular level and may, on that basis, perceived to be relatively harmless, tissues or biological samples are indeed susceptible to change/damage during irradiation with ultrasound.

Research over the past decade has suggested that many of the adverse effects resulting from ultrasound over-dose arise from thermal mechanisms. Basically, as ultrasound propagates through mammalian tissues, it becomes attenuated and this attenuation is caused by absorption of the beam energy as it passes through the relevant tissues and by scattering of the energy out of the beam by inhomogeneities.

In terms of the former, the absorption of the ultrasound beam energy by tissues results in temperature increases suggesting that the beam energy is converted into heat. The conversion of the acoustical energy into heat may in one case provide a therapeutic effect such as that exploited in physical therapy or alternatively give rise to severe tissues damage such as that exploited in HIFU treatment [Nyborg, 2001, Ultrasound in Med. Biol. 27, 301-322].

Deleterious effects of ultrasound overdose may also be mediated by acoustic cavitation phenomena in biological samples. Acoustic cavitation in the term applied to the study of the interaction of ultrasound with gas or vapour filled bodies. These interactions may vary from gentle “breathing2 oscillations to violent implosions and it is the latter that brings about significant damage in biological samples. It has been suggested that such violent implosions mediate transient poration in biological membranes and this is exploited in sonoporation-based gene transfer In any case a means of measuring the ultrasound dose is essential treatment [Nyborg, 2001, Ultrasound in Med. Biol. 27, 301-322], ΙΕ ο 5 Ο 3 35 Although many of the effects of ultrasound on biological materials can often be attributed to either temperature increases or to cavitation events, some effects have been reported that cannot be attributed to either of those two mechanisms. In some cases the effects have been explained in terms of radiation pressure, force and torque and acoustic streaming. Many of these phenomena have been studied for over a century with much of the product of that study being theoretical and derived from experimentation with non-biological systems. One striking example of the influence of such phenomena was demonstrated in 1971 where an experimental system involving the use of a chick embryo coupled to a microscope visualisation system demonstrated that blood cells could be aggregated into banding patterns in that system and indeed cessation of flow (i.e. blood flow stasis) was demonstrated when the ultrasound was employed above a certain threshold [Dyson et al., 1971, Nature, 232, 572-573].

With the ever-increasing range of ultrasound applications and the potential for damage resulting from over-dose exposures during ultrasound application either in vivo or in vitro there is an urgent requirement for a convenient, inexpensive means of measuring the dose delivered and relating that to potential damaging endpoints (overheating during in vivo treatment [e.g. physical therapy] and cell destruction during in vitro treatments [e.g. sonoporation-mediated gene transfer]). To date only sophisticated methods have been available in order to measure the output of ultrasound probes and this is rarely accomplished during the actual exposure event.

One objective of the present invention is to provide a convenient and inexpensive means of measuring the dose of ultrasound delivered to a site either in vitro or in vivo in real time during the ultrasound exposure event.

According to the present invention, a flexible piezoelectric PVDF-based element coupled with an analogue to digital converter (ADC)which is, in turn connected to a PC, is provided that has the ability to measure the intensity and frequency of ultrasound emitted from an ultrasound probe during real time emission into a target. Coupling of the ultrasound-emitting surface of the probe to the target may be facilitated by ultrasound gel. ^°50 3 35 In a further embodiment coupling of the ultrasound-emitting surface of the probe may be facilitated using a solid carrageenan based gel slap into which the piezoelectric element has been cast.

According to the present invention, the piezoelectric element may be in direct contact with the target and the ultrasound-emitting device may transmit the ultrasound through air and/or an aerosol interface. Alternatively and/or occasionally the piezoelectric element may be in direct contact with the emitting surface of the ultrasound head and the ultrasound may transmit through air and/or an aerosol interface. In a further embodiment two piezoelectric elements may be employed where one is in direct contact with the emitting surface of the ultrasound head, the other may be in contact with the target and the intervening space may comprise air and/or an aerosol. This could be employed for comparative purposes and could also be employed to facilitate feedback control on the ultrasound signal generator.

In a further embodiment the piezoelectric measuring device may be calibrated by relating peak voltages to power output using a watt meter and employed to provide automatic feedback control to prevent overdose particularly where automatic ultrasound scanning devices may be employed.

IE 050 3 35 Examples The invention will be described with reference to the following examples.

Fig. I- Measurement of minimum (-) () and maximum (+) (A) amplitude (peak voltage) during ultrasound treatment of human forearm (brachioradialis region) at increasing ultrasound intensity settings.

Fig.2B. Integrity of ultrasound pulses delivered from a 1MHz ultrasound head with settings of 1,4 and 7 Wcm'2 at a duty cycle of 25% (100 Hz).

Fig.2. Measurement of minimum (-) () and maximum (+) (A) amplitude (peak voltage) following treatment ultrasound treatment to the outside of a tissue culture well and measurement of minimum (-) (·) and maximum (+) (□) amplitude (peak voltage) of the ultrasound that penetrated through the plastic of the tissue culture well into the liquid (water) contained within that well.

Example 1 Measuring ultrasound treatment dose in vitro in real time during treatment As mentioned above, one of the major in vitro applications of ultrasound is in the area of gene transfer or transfection. In all cases, cells to be treated are grown in tissue culture vessels and subsequently treated in those vessels in situ. The types of vessels normally employed to treat large numbers of samples with various ultrasound parameters include tissue culture well plates and the wells of these come in varying sizes ranging from 96-well plates to the more convenient 6-well plates. The latter are usually more convenient because they have larger well and facilitate ultrasound exposure either through the plastic well bottom or directly into the medium contained in the well. It has been suggested that because the volumes contained in wells of even the larger plates are so small ( circa 5 ml) that distortions and reverberations may occur either from passage of the ultrasound through the plastic bottom of the well or from the air-liquid surface of the very small volume employed in the plates. Such distortions and reverberation events will inevitably lead to variations in treatment and irreproducibi lity of results and the latter can be a noted problem in the field of gene transfer in vitro. To date, no convenient, inexpensive method has been available to ,Ε 0 5 Ο 3 35 measure either the dose of ultrasound emitted from the probe surface when it is in direct contact with the bottom of a tissue culture well or indeed to measure the dose and nature of the ultrasound passing through the plastic bottom of the well into the tissue culture medium (i.e., the nature and dose of the ultrasound to which the cells are actually exposed. In an effort to measure both ultrasound probe output when in contact with the plastic bottom of a tissue culture well and to determine the nature and relative dose of ultrasound actually treating the target cells during a sonoporative gene transfer experiment, sensor configurations exploiting PVDF piezoelectric outputs have been exploited. Initially, the ultrasound emitted from the surface of the ultrasound probe directly in contact with the plastic bottom of a tissue culture well and striking the bottom of that well was measured by placing PVDF film (0.64 x 1.63 x 28 micron thick piezo element with silver ink electrodes and 12 of 26 AWG wire, e.g. product DT1-028K/L, product name DT1-028K/L MSI Sensors, USA) between an ultrasound-emitting head (2cm2) and the plastic bottom of a tissue culture well containing 5 ml of distilled water. Contact between the PVDF film, the emitting surface of the probe and the plastic bottom of the well was facilitated using ultrasound gel. The PVDF film was connected to an analogue to digital converter system (ADC 200 Picoscope system , Pico Technologies, UK) and this was connected to the parallel port of a PC with the relevant software to drive the system and facilitate data capture. The average positive and negative peaks from the relevant emissions were monitored using the oscilloscope function of the Picoscope system at various ultrasound intensity settings on the ultrasound generator. Ultrasound was generated using a CRM ultrasound device, custom built by Rich Mar Corp,, USA and a 1MHz ultrasound head was employed. The ultrasound emission was set on a 25% duty cycle (100 Hz) and emissions at a variety of intensity settings were viewed using the configuration.

In addition to measuring the ultrasound that was emitted from the surface of the probe and striking the outside of the plastic well, it was also possible, using this system, to conveniently measure the ultrasound that was emitted into the liquid contained in the well, i.e. the ultrasound that would actually be treating cells during a sonoporative gene transfection experiment. In order to facilitate this, the ultrasound probe was placed in direct contact with the plastic well of the tissue culture plate and contact was mediated using ultrasound gel. The PVDF film was placed directly into ,E°S0 3 35 the liquid contained in the tissue culture well and positive and negative peaks were recorded at various ultrasound intensity settings.

Results 1 When measurement of the positive and negative peaks of ultrasound striking the outside of the plastic tissue culture well was carried out at different intensities the data shown in Fig. 1 was obtained. The data demonstrate as the intensity increased from 0 to 2.5 Wcm'2, the amplitude peaks (+) increased steeply and at intensities ranging from 2.5 to 7.5 cm'2, the amplitude peaks (+) increased less steeply. The data show a clear and expected relationship between the intensity of ultrasound striking the bottom surface of the tissue culture well and the amplitude peaks of the ultrasound measured by the simple PVDF device employed. This suggests that measured output from the PDVF sensor may be employed to determine the intensity of the ultrasound striking the surface of the tissue culture well and further suggests that, if required, this electronic output could be employed to provide a controlled feedback function for the ultrasound generator. In addition to providing the above data, Fig. 1A (first 3 panels) demonstrates that the nature of the ultrasound emissions could also be measured. This suggests that in treatment scenarios where distortions would be expected, the device could be employed to monitor these phenomena.

When the device was used to measure the ultrasound emitted directly into the liquid in the well, i.e., the ultrasound transmitted through the well bottom and that which would strike any sample to be treated during a sonoporative transfection experiment, the data obtained in Fig. 1 was obtained. In this case, at intensities ranging from 0-2.5Wcm'2 , the increase in the amplitude peaks (+) exhibited a linear, but somewhat erratic increase and this continues in a linear fashion up to 6 Wcm' .

Using the output from the monitoring system, it was found that the energy entering the well was much lower than that going in as shown in Fig. 1. Indeed the data suggest that at 2 Wcm'2 only approximately 10% ofthe ultrasound energy appears to have entered the tissue culture well and this demonstrates one of the major advantages associated with this novel, convenient and inexpensive monitoring system for tissue culture studies and this could be measured in real time during an actual treatment. It also provides an ideal means of relating in vitro studies to in vivo studies where such J 35 relationships were difficult to establish in the past. In terms of the nature of the ultrasound delivered into the well contents, the recorded amplitude peaks (+} suggested that the ultrasound was behaving in a rather erratic manner. Using the convenient monitoring configuration described here, it was possible to visualise the ultrasound emissions and in doing so, the data shown in Fig.l A (last three panels) were derived. Whilst the data demonstrate that ultrasound intensity could be related to the amplitude peaks (+), confirming the data in Fig.l, the three panels also demonstrate the erratic behaviour of the ultrasound, particularly at 4 and 7 Wcm', where clear distortions of the ultrasound pulses were visible. This further demonstrates the utility range of the novel, convenient and inexpensive monitoring system described here and suggests wide application in scenarios where ultrasound dose monitoring and its integrity may be required to facilitate extrapolation to in vivo studies and to enhance the reproducibility of in vitro treatments. The monitoring system could also be employed in order to provide electronic feedback control on ultrasound-generating system currently employed with tissue culture systems.

Example 2 Measuring ultrasound treatment dose in vivo in real time during treatment.

In order to determine whether or not the dose of ultrasound delivered during physical therapy could be monitored in real time and whether or not the nature of that ultrasound could be examined during treatment, it was decided to perform a treatment on the human forearm (muscle: brachioradialis region) at various intensities.

Ultrasound gel was placed on the site to be treated and this mediated contact between the PVDF film and the skin. Gel, when placed on top of the film, mediated contact between the film sensor and the emitting surface of the ultrasound head. The PVDF film was connected to an analogue to digital converter system (ADC 200 Picoscope system , Pico Technologies, UK) and this was connected to the parallel port of a PC with the relevant software to drive the system and facilitate data capture. The average positive and negative peaks from the relevant emissions were monitored using the oscilloscope function of the Picoscope system at various ultrasound intensity settings on the ultrasound generator. Ultrasound emissions were set at the intensities indicated in Fig.2 at a frequency of 1 MHz and at a duty cycle of 25% (100 Hz). Ultrasound was delivered to a single site without movement of the ultrasound ΙΕλ50 3 35 head, although the monitoring system is compatible with treatments involving movement of the ultrasound head.

Results 2 The target site on the forearm was treated at various ultrasound intensities and the data obtained are shown in Fig.2. Again, the data demonstrate that a clear relationship exists between ultrasound intensity and amplitude peak (+') and this relationship may be exploited to facilitate measurement of the dose received at the target site. In addition, the nature of the ultrasound delivered to the skin could also be monitored and the panels shown in Fig.2A are demonstrative of this and no aberrations are apparent in the scans shown. In overall terms the data demonstrate that ultrasound delivered to a site in vivo may be monitored in real time and this offers the advantages of safer, more reliable and more reproducible physical therapy. In addition the ability to monitor the accumulated ultrasound dose delivered to tissues in vivo in real time could also be exploited in providing electronic safety feedback control features to physical therapy and other ultrasound treatment devices (e.g. ultrasound devices for use in cancer therapies such as CEFUS therapy).

In overall terms the above-described examples provide evidence for the fact that the invention provides for PVDF-based sensor configurations suitable for use with either treatment of cells or tissues in either in vitro and/or in vivo scenarios. The invention may comprise any physical configuration that is compatible with presenting a PVDF membrane to an ultrasonic field for the purposes of measuring delivery of ultrasound to and/or through cells or tissues.

This invention is not limited to the embodiments described above which may clearly be modified and/or varied without departing from the scope of the invention.

Claims (23)

Claims

1. A system comprising a PVDF film-based sensor (metallized piezoelectric Film) connected to a PC-controlled analogue digital converter (ADC) that may be employed to monitor ultrasound treatment of a target.

2. A system comprising a PVDF film-based sensor (metallized piezoelectric film) connected to a PC-controlled analogue digital converter (ADC) that may be employed to monitor ultrasound treatment of a target in real time.

3. A system as described in claims 1 and 2 in which the average minimum and maximum amplitude (voltage) peaks may be determined and recorded during treatment of a target.

4. A system as described in claims 1 and 2 in which the integrity of ultrasound pulses may be monitored and recorded during real time treatment of a target site (s).

5. A system as described in claims 1 to 4 in which the ultrasound target may be a cell and/or tissue culture system in suspension.

6. A system as described in claims 1 to 4 where the ultrasound target may be a tissue in vivo.

7. A system as described in claims I to 4 where the ultrasound target may be an artificial organ system in vitro.

8. A system as described in claims 1 to 4 in which the ultrasound target may be an artificial organ system in vivo.

9. A system as described in claims 1 to 4 where the ultrasound target may be a living and/or non-living ultrasound responsive element embedded in tissues.

10. A system as described in claims 1 to 4 where contact between the ultrasound head and the target is interfaced with ultrasound gel and the PVDF sensor.

11. A system as described in claims 1 to 4 where contact between the ultrasound emitting surface and the target/sensor combination is interfaced with air.

12. A system as described in claims 1-4 in which contact between the ultrasound emitting-surface and the target/sensor combination is interfaced with an aerosol.

13. A system as described in claims 1-4 in which contact between the ultrasound emitting-surface and the target/sensor combination is interfaced with a liquid. IE 050 3 35

14. A system as described in claims 11 to 13 in which a PVDF sensor is fitted to the emitting surface of the ultrasound head to facilitate simultaneous monitoring of head emissions and target exposure.

15. A system as described in claims 11 to 14 in which the PVDF sensor comprises a single sheet of film with continuous metal layers.

16. A system as described in claims 11 to 14 in which the PVDF sensor comprises a single sheet of film with interconnected strips of metal layers.

17. A system as described in claims 15 and 16 in which the PVDF sensor comprises multiple sheets spread in a planar manner over the emission and target surfaces.

18. A kit to facilitate real-time monitoring of ultrasound exposures in vivo and in vitro which comprises: i. the PVDF sensor housed in a holding frame of various shapes and sizes, ii. an ADC converter iii. the relevant driving software iv. the relevant written instruction

19. A system as described in claims 1-4 in which the measured peak voltages may be employed to facilitate electronic feedback inhibition of an ultrasoundgenerating device for the purposes of preventing overdose.

20. A system as described in claim 19 in which the measured peak voltages may be employed to facilitate PC-controlled calculation of total dose received.

21. A system as described in claims 19 and 20 in which the ultrasound peak voltages (amplitude), the wave form and/or the pulse frequency may be displayed for the purposes of determining the integrity of both ultrasound head emission and/or ultrasound reception at the target site.

22. A system as described in claim 18 in which the PVDF sensor may be housed in a needle ‘window’.

23. A system as described in claim 22 in which the needle window may be employed to measure reception of ultrasound at deep sites in the body.