Apparatus and Method for Sonoporation

The present invention relates to an apparatus and method for sonoporation and in particular to an apparatus and method that allows a surface to be etched, punctured or marked by the application of ultrasound to particles. The surface to be etched, punctured or marked may be a blank or patterned surface used in printing, pattern forming or lithography, a cell membrane or other suitable surface.

The cell membrane represents the outer extremity of all eukaryotic cells. In mammals, it is essentially constituted by a thin (5nm) bi-layer film of lipids which enclose the cell, defining its boundary and maintaining the essential physical and chemical differences between the internal cytoplasm and the extra-cellular environment. Under normal circumstances, the lipid nature of the cell membrane acts as an impermeable barrier to

the passage of most water soluble molecules. Thus, the selective introduction of therapeutic agents to the inside of dysfunctional or diseased cells is challenging.

In addressing this challenge, biophysical approaches offer an attractive route for generic drug delivery in that they typically offer wider applicability when compared with their viral or biochemical counterparts, which tend to be cell/tissue selective and often have serious side-effects .

It is known that the level of contrast in an ultrasound image can be increased in the presence of microbubbles . Microbubbles are typically hollow capsules that can be filled with a suitable gas and which have a diameter of the order of a few microns. The injection of microbubbles into a patient around the area where an ultrasound image is to be viewed is known to improve the contrast between different features contained in the image .

It is also known that ultrasound exposure (insonation) in the presence of contrast agent microbubbles will enhance membrane permeability and lead to molecular uptake from the locale. Under higher ultrasound pressures (>0.2MPa), this process (sonoporation) can elicit a number of clinically relevant biological effects. Two specific therapeutic applications of sonoporation have been identified. Firstly, sonoporation has been used to kill cells by either direct physical lysis (lethal sonoporation) or by initiation of programmed cell death and secondly to deliver therapeutic agents and plasmid DNA whilst retaining cell viability. In addition, promising observations of tumour regression have been

demonstrated in murine studies . Sonoporation thus offers significant therapeutic potential across the spectrum of disorders.

Attempts to produce controlled sonoporation via Bjerknes forces have geometrical constraints. In addition, laser nucleated free bubbles have a limited lifespan and therefore place a temporal constraint on their use.

Lithography is the art of making patterns in or on surfaces and is widely used in for example, microelectronics, microfluidics, and in the creation of high density data storage media.

In general, printed circuits are manufactured in a well known manner using a photolithographic process. In this process, copper-backed fibreglass resin board is coated with a patterned photoresist, washed, etched with an acid and the excess copper removed with acid. Ion etching can also be used in conjunction with a patterned photoresist. Laser ablation of conductive inks is also used for circuit formation.

Alternatives to known circuit printing techniques must be able to etch, mark or puncture a surface in accordance with a predetermined pattern.

The use of sonoporation has been limited by the inability to understand and therefore control the mechanism by which sonoporation occurs .

It is an object of the present invention to provide improvements in and relating to sonoporation.

In accordance with a first aspect of the invention there is provided an apparatus for sonoporation, the apparatus comprising: an ultrasound source; and positioning means for positioning one or more microbubbles at a predetermined distance (Z axis) from a surface wherein the ultrasound source is adapted to produce an ultrasound signal capable of rupturing the one or more microbubbles when the one or more microbubbles is held at said predetermined position.

Preferably, the positioning means • controls the position of the one or more microbubbles in the x and y axes.

Preferably, the positioning means is an optical trap.

Preferably, the optical trap comprises a well or annulus in which the optical intensity is at a minimum in the centre of the well or annulus and at a maximum at positions surrounding the centre.

Preferably, the optical trap is a Laguerre-Gaussian optical trap.

Preferably, the positioning means provides an array of positions at which the microbubbles are held to allow a plurality of sonoporation events to occur substantially simultaneously.

Optionally, dynamic holographic methods may be used to porate several tens of cells.

Optionally, the dynamic holographic methods employ spatial light modulators (SLM) .

The user may select the cells at will on a computer monitor and the predetermined hologram will then steer the laser to each cell site causing sonoporation to occur with no moving parts .

Preferably, one or more rαicrobubbles are trapped at or near the position of the minimum optical intensity.

Optionally, the positioning means is an electric field.

Optionally, the positioning means is a magnetic field.

Preferably, the microbubbles are f illed with a gas .

Preferably, the microbubbles are ultrasound contrast agents .

Preferably, the microbubbles have a diameter of between 1 and 10 μ m .

Preferably, the microbubbles have an albumin shell .

Optionally, the microbubbles are electrically chargeable .

Optionally, the microbubbles are magnetized .

Preferably, the microbubbles emit a microjet of gas which etches, punctures, or marks the surface.

Preferably, the microbubbles undergo quasi-spherical expansion to etch, puncture or mark the surface.

Preferably, the ruptured shells of the microbubbles etch, puncture, or mark the surface.

Preferably, the ultrasound source is capable of emitting ultrasound at a frequency of 0.9 to 1.1 MHz.

Preferably, the ultrasound source is capable of emitting ultrasound at a peak negative pressure of 0.1 to 10 MPa.

More preferably, the ultrasound source is capable of emitting ultrasound at a peak negative pressure of 0.5 to 5MPa.

Most preferably, the ultrasound source is capable of emitting ultrasound at a peak negative pressure of 1.2 to 1.6MPa .

Preferably, the depth of etch, mark, or perforation is determined by the magnitude of the peak negative pressure.

Preferably, the ultrasound source is capable of emitting ultrasound in pulses.

Preferably, the ultrasound pulses have a period of less than 250ms.

Preferably, the ultrasound source is coupled to the microbubbles by means of a liquid.

Preferably, the liquid is water.

Preferably, the microbubbles are present in the liquid at a concentration of between 10 ⁴ particles/ml and 10 ⁶ particles/ml.

The concentration of the microbubbles is set to minimise the extent of hydrodynamic coupling of the microbubbles.

Preferably, the apparatus is further provided with sensing means to measure the distance between the surface and the microbubble which has been positioned by the positioning means.

In accordance with a second aspect of the invention there is provided a method for etching, puncturing or marking a surface, the method comprising the steps of: positioning one or more microbubbles at a distance (Z axis) from a surface; and coupling an ultrasound source to said one or more microbubbles; rupturing the one or more microbubbles using the ultrasound signal with the one or more microbubbles positioned a predetermined distance from the surface, the rupturing of the one or more microbubbles causing the surface to be etched, punctured or marked.

Preferably, the position of the one or more microbubbles may be controlled in the x and y axes.

Preferably, an optical trap is used to position said one or more microbubbles .

Preferably, the optical trap comprises a well or annulus in which the optical intensity is at a minimum in the

centre of the well or annulus and at a maximum at positions surrounding the centre.

Preferably, the optical trap is a Laguerre-Gaussian optical trap.

An array of optical traps can be used to allow a plurality of sonoporation events to occur simultaneously in a predetermined pattern.

Preferably, the one or more microbubbles are trapped at or near the position of the minimum optical -intensity.

Optionally, the one or more microbubbles are trapped using an electric field.

Optionally, the one or more microbubbles are trapped using a magnetic field.

Optionally, the microbubbles are electrically charged.

Optionally, the microbubbles are magnetized .

Preferably, the microbubbles emit a microjet of gas which etch, puncture or mark the surface.

Preferably, the microbubbles undergo quasi-spherical expansion to etch, puncture or mark the surface.

Preferably, the ruptured shells of the microbubbles etch, puncture or mark the surface.

Preferably, the ultrasound signal is emitted at a frequency of 0.9 to 1.1 MHz .

Preferably, the ultrasound signal is emitted at a peak negative pressure of 0.1 to 10 MPa.

More preferably, the ultrasound signal is emitted at peak negative pressure of 0.5 to 5MPa

Most preferably, the ultrasound signal is emitted at peak negative pressure of 0.5 to 1.6MPa.

Preferably, the depth of etch, mark, or perforation is determined by the magnitude of the peak negative pressure .

Preferably, the ultrasound signal is emitted in pulses.

Preferably, the ultrasound pulses have a period of less than 100 // s .

Preferably, the ultrasound source is coupled to the microbubbles by means of a liquid.

Preferably, the liquid is water.

Preferably, the method further comprises measuring the distance between the surface and the microbubble to determine that at least one microbubble is at the predetermined distance from the surface and switching on the ultrasound signal to rupture the microbubble.

In accordance with a third aspect of the invention there is provided an apparatus for forming patterns on a substrate, the apparatus comprising sonoporation means as

defined with respect to the first aspect of the invention.

Preferably, the substrate is a semiconductor.

Preferably, the substrate is a plastic adapted to function as a micro fluidic or nano fluidic device.

The present invention will now be described by way of example only with reference to the accompanying drawings in which:

Fig. 1 is a schematic drawing of an optical trap positioning means in accordance with the present invention;

Fig.2 is perspective schematic drawing of the sonoporation apparatus of an embodiment of the present invention;

Fig.3 is a frame showing an optical trap positioning means used in the embodiment of Fig.l;

Fig.4 is a series of frames showing a trapped microbubble;

Fig.5 is a series of frames showing microbubble cavitation;

Fig.6 is a frame showing microbubble cavitation;

Fig.7 is a frame of a pit formed in a surface caused by a microjet at high pressure;

Fig.8 is a frame of a pit formed in a surface caused by a microjet at low pressure;

Fig.9 is a series of frames showing a pit formed in a surface caused by a microjet at low pressure;

Fig.10 is a series of frames showing microbubble cavitation in proximity to a cell monolayer;

Figs.11a to Hf show the correlation of specific cavitation events with membrane damage;

Fig.12a shows a 4 x 4 array of hologram generated Laguerre-Gaussian optical traps and Figs.12b to 12d are a series of photomicrographs demonstrating optical manipulation of 8 ultrasound contrast agent micro-bubbles simultaneously in solution; and

Fig.13a and Fig.13b show a side view and a plan view of a substrate and sonoporation microbubbles .

Fig.l shows an apparatus 1 that provides an optical trap for use in accordance with the present invention. The apparatus 1 comprises a laser 3 situated at one end of an optical path 2. The optical path is defined by a series of lenses 5, 9, 11, 13 and 15 and a series of mirrors 7. In addition a first hologram 19 is located in the optical path which alters the intensity distribution of the laser light to form an optical trap. In this example a holographic Laguerre-Gaussian optical trap is used. A second hologram 20 modifies the output of the first hologram 19 to produce an array of Laguerre-Gaussian optical traps. As will be described below, the optical traps provide positioning means that allow microbubbles

to be held at a predetermined distance from a surface. A high speed camera 25 is positioned in the optical path 2 remote from an infra red filter 28. A cell 21 is positioned in the optical path and is adapted to accommodate a sample surface and a sample of microbubbles which is typically suspended in a liquid such as water.

Fig.2 and Fig.3 describe sonoporation apparatus in accordance with the present invention in more detail and shows the configuration of the optically trapped itiicrobubble 39 in relation to coverslip/monolayer system 40.

For orientational purposes we designate the coverslip surface to occupy the xy plane with the trapped microbubble 39 displaced in the orthogonal z direction. A x60 objective lens 31 (NA=O.9) acts as both the focussing lens for the laser trapping beam, and also constitutes the principal magnification element. Illumination 27 is provided by two Xe-W flashlamps coupled via 3mm optical fibre bundles and condenser lenses. Observations were undertaken within a precalibrated (via standard traceable needle hydrophone) sonoporation chamber 35. The transducer 37, which is submerged in degassed water at room temperature and pressure, generates a focussed region of ultrasound within the sonoporation chamber 35, into which the optically trapped microbubble is translocated after selection from a resident population.

Fig.3 is the view obtained from the objective lens 31 showing a trapped microbubble 39 held at controlled displacement from the coverslip 40. The optical trap is maintained during insonation and does not interfere with the cavitation dynamics of the system.

Advantageously, the system described in relation to Fig. 2 facilitates observation without geometric constraint or interference to the natural hydrodynamic environment in the vicinity of the cavitating ultrasound contrast agent. This is achieved by the use of optical trapping to spatially control individual microbubbles relative to a substrate, together with ultra-high speed imaging to monitor the cavitation dynamics.

The ultrasound contrast agent microbubbles employed (Optison®) consist of denatured albumin shells surrounding gas [octafluoropropane] cores, with diameters ranging from 2-7μm or 2-5μm range microbubbles with lipid shells encapsulating sulphur hexafluoride gas cores (Sonovue)

In the above example of the present invention, a Laguerre-Gaussian profile is provided via a phase modulating static hologram 19 (Fig. 1) for a diftractive optic element. This maximises intensity (and thus trapping force) and redefines the intrinsic TEM _0O (1=0) mode emanating from a Nd:YVO ₄ solid state laser (at 1064 nm) to a TEMoi (1=3) LG mode with intensity distribution orthogonal to the laser axis, I(r) , thus:

(1)

Here, α> ₀ corresponds to the precursor Gaussian beam width, and ω _v is the characteristic core size selected to accommodate ultrasound contrast agents (microbubbles) of diameter *» 5μm ¹⁴ . Once trapped, the microbubbles are guided to a controllable z-displacement (Fig. 2) from the coverslip plane, and an insonation protocol applied as a

20μs burst of IMHz ultrasound at peak negative pressure 1.28MPa (±14%), with synchronous triggering of the UHS data acquisition.

In Figs. 4 to 8, representative ultra high speed sequences acquired at a framing rate of 50OkHz showing microbubble cavitation in proximity to naked coverslips (frames 43, 45, 47, 49, 51, 53, 55 and 57). Timings (in microseconds) relative to the instant of cavitation inception are indicated at each frame. Images were spatially calibrated by observing βμm calibration beads (12 such beads have been superimposed on the scene to provide scale in frame 47) . Each frame measures 163 μm x 110 μm. Initially (t<0) a 4.5μm diameter microbubble 44 is trapped and manipulated to a displacement (D) 40 26.5μm from the coverslip 42 (darker region to the right) . The cavitating bubble 44 expands and microjet formation initiates with subsequent collapse (46,48,50) .

In the sequence of Fig. 5, by controlled displacement of a similarly sized microbubble 54 to a location some 19μm farther from the substrate than shown in Fig.4, then insonating, only the microjet is seen to touch-down at the surface 52 (at 6μs) .

A 10 x lOμm image of a sono-lithography pit 59 formed by microjet touchdown at high pressure is shown in Fig. 6. At lower pressures, much smaller features can be controllably written to the substrate. Fig.7 shows a lOOnm width pit 61 formed with a depth of just 25nm. Fig.8 shows this feature 63 in cross-section 65.

As is apparent in Figs. 4 to 8, upon cavitation inception, the microbubble 44,54 expands rapidly to give

the expanded microbubble 46,56 with spontaneous formation of a thin (micrometer width) linear involuted microjet traversing its breadth and directed orthogonally towards the plane of the coverslip. The maximum distance in the z-axis direction at which the microjet will impinge upon the surface is defined by R _MAX . Accordingly, control of the z-axis direction displacement of the microbubble is used to refine the microbubble' s interaction with the substrate, so that microjet touchdown without contact of the expanded UCA shell can occur (Fig. 5) . This fine z- control could be used over a range of substrate moduli including cells, lithographic plates or the like. It is the control of the proximity of the microbubble with respect to the surface that allows the microjets to be preferentially directed towards the surface.

In the examples shown in Figs. 4 and 5, microjetting was relatively common on naked coverslips, occurring in 39% of this data. In the remaining cases the microbubbles engaged in quasi-spherical expansion and coverslip contact, without the formation of a clearly visible microjet.

Figs. 9 and 10 show ultra high speed sequences (frames 71, 73, 75, 79 and 81 (Fig.9) and Fig.10 91, 93, 95, and 97) acquired at a framing rate of 50OkHz showing microbubble cavitation in proximity to cell monolayers.

In Figs. 9 and 10 the coverslips supported cultured monolayers of adherent live MCF7 (human breast cancer) cells at 70-80% confluence, in the sonoporation chamber containing Dulbeccos modified Eagle's medium (DMEM). The dominant finding here was quasi-spherical expansion with contact, and compression of underlying cells 48%.

In Fig.10, core ejection of gas was also observed during cavitation near cells. In this case, a 6μm diameter microbubble 99 was initially trapped at a z-axis displacement of 19.5μm from a live cell and undergoes core ejection with resultant compression (frame 73) of the proximal cell as the shell remnant 107 recoils into it.

At later times the cell appears to partially recover its original morphology whereas the shell remnant 107 rebounds away from the cell and the ejected gas eventually reforms into a free bubble 105.

Core-ejection was observed in 35% of data acquired on monolayers. Here, the microbubble shell 99 appeared to rupture during the initial phase of cavitation, rapidly (within 2μs) giving rise to an emergent jet directed away from the coverslip plane. To conserve momentum, the shell remnant 107 is ballistically propelled towards the monolayer, compressing any intervening cells.

Assuming the entire membrane is stretched during this deformation process, and estimating the membrane areal increase δA, and its initial area A ₀ , the resultant areal strain can be calculated as (δA/Ao) . Approximating Ao « 2πR ² (l-cos θ) , where R is the radius of curvature of the cell (53μm in Fig. 2d) ) , and θ is the angle the cell membrane makes with the coverslip (θ «32 ^a from Fig. 10, initial frame) , then δA introduced by the impacting microbubble shell is given by πR _s ² , where R _s =10μm (estimated from indentation depth in Fig. 10 at 4μs) is the radius of the hemispherical compression zone, recognised as the topological deformation of a circular

patch of membrane also of radius R ₃ . δA/A ₀ is thus calculated at 11.7%, exceeding the critical areal strain of between 2-5% previously reported to cause membrane rupture. A cell may thus be rendered temporarily permeable, by this process.

Active microjetting into monolayers was observed in 17% of such sequences. Here, microbubbles are seen to experience inertial cavitation and collapse, with the outermost hemisphere undergoing involution to form a central reentrant jet directed at the cell (Fig. lib) .

With inter-frame times on the order of 0.5-1. Oμs, a lower-bound estimate for the jet velocities, V _j , can be made, equating to circa 5.5ms ^"1 (distance traversed (22μm) over traversal time (4μs) in Figs. 3a-b) . Membrane breaching may occur if any induced tension exceeds the critical rupture stress, τ _cr i _t , related to the local elastic modulus, E, thus:

τ _crit = E . ε _r (1) .

Here ε _r is the relative deformation (critical areal strain) needed for rupture to occur, a value commonly accepted ²³ as 3%, hence ε _r =0.03. Measurements of E on live cells are consistently below lOOkPa leading to an approximate upper limit of τ _cr it ~ 3kPa. Consideration of two distinct candidate mechanisms: development of a water hammer pressure, P _m ; and momentum exchange due to fluid flow, P _f , allows estimates of their respective induced pressures to be ascertained.

As P _m * 0.5 p c Vj , where p is the fluid density (998 kg irf ³ ) and- c is the speed of sound in the medium (1480ms ^"1 )

we thus estimate Pm ~ 4MPa. We estimated the flow induced pressure at the stagnation point as 0.5 p Vj ² , and therefore P _f * 15kPa, so that both pressure estimates exceed τ _cr i _t/ confirming that either (or both) are viable breaching mechanisms in this high MI regime.

In Fig.11, correlation of specific cavitation events with membrane damage was observed using ultra high speed imaging and atomic force microscopy (AFM) . Fig.11a is a 163μm x llOμm frame showing a quiescent 4μm diameter microbubble 114 trapped 17μm from a cell membrane 116. The shadow to the lower right of a (labelled 'N') is a notch 115 on the edge of the coverslip that facilitates rapid registration of the region of interest for post- insonation microscopy. Fig. lib shows that at t=8μs after cavitation inception, the microbubble has developed an involution to form a central jet which contacts the membrane 116 over a region some 15μm wide. By subsequent extraction of the insonated coverslip (within circa 20 seconds) , and chemically fixing in 4% paraformaldehyde (on ice) , the instantaneous membrane topography was preserved.

Fig. lie shows an atomic force micrograph frame of fixed cells. In this case, the local topography near to the notched coverslip edge 115 (labelled 'N') aids identification of the cell that had been contaced by the microjet. The resultant sonopore 123 measures 16μm, as indicated on the respective cross-sections of Figs. Hd and lie. A perspective view of another sonopore, the cross-section through which 125 is shown in Fig. Hf .

Pits (sonopores) were consistently observed on all cells previously located directly below the hypocentre of

trapped microbubble that underwent microjetting into the respective cell. Fig. lie shows that sonopore formed due to the jetting event depicted in Figs, lla-b. The cross- section through this entity is similar to that observed during UHS imaging of the contact phase of the microjet (Fig. lib) .

Moreover, the jet's penetration power was such that sonopore depth extends to the underlying coverslip. Figure Hd shows a perspective AFM image of a representative sonopore 125 formed on a different cell monolayer and exhibiting a distinct peripheral lip of raised material .

The spatial extent of these microjet induced sonopores suggests that such events probably lead to cell lysis. However, if damage to the nucleus, and vital organelles is avoided, then even large membrane disruptions, up to lOOOμm ² , can reseal, and a cell might remain viable after microjet penetration.

In this latter circumstance, it is pertinent to consider the level of molecular uptake possible during microjet injection. The volume of ambient fluid within a jet, Vj can be approximated as Vj * 0.1R _C ? , where R _c is the radius of a microbubble that is on the verge of collapsing. In our observations (e.g. Fig. 10), R _c « 27μm, leading to a microinjection volume of 2.0 picolitres .

In the case of the present invention micro-injection into the cells occurs and the membrane breach caused by the micro-injection can remain open for several seconds, allowing cytosol borne material to leak out of the cell .

The use of optical trapping for initial positioning of the cells in a holographically generated array together with their microbubble counterparts which can be held in a separate LG trap array is further described below. The Gaussian traps are filled with cells within an optically gated reservoir (adjoining a main fluidics channel) , then the LG traps are filled with microbubbles from a separate optically gated reservoir.

Figure 12a shows a 4 x 4 array of hologram generated Laguerre-Gaussian optical traps and figures 12b to 12d are a series of photomicrographs demonstrating optical manipulation of 8 UCA micro-bubbles simultaneously in solution. The trapped ultrasound contrast agent can be redistributed at will.

A hologram generated trap array (Figure 12a) is swept through a population of particles (in this case microbubbles) and a percentage of the traps are filled. The optical gates will then be switched off to allow the guide arrays to manipulate the trapped cells and microbubbles out from the reservoirs and into a main flow channel. The two arrays will then be spatially arranged to within a predetermined displacement and then stimulated sonoporation will be initiated by a short (microsecond range) burst of ultrasound.

Dynamic holographic methods may be used to porate several tens of cells in unison using a spatial light modulator (SLM) to allow an in situ method for user interfacing for cell poration. The user may select the cells at will on a computer monitor and the predetermined hologram will then steer the laser to each cell site causing sonoporation to occur with no moving parts.

The present invention provides an apparatus and method by which the energetic micrometer scale interactions between individual cells and cavitating microbubbles can be controlled by controlling the z-axis displacement of the microbubbles from the surface. The term microbubble is used herein to describe any suitable sonoporation agent which ruptures upon the application of ultrasound in a manner suitable for use in the present invention.

Of the handful of feasible biophysical approaches devised thus far, the use of ultrasound to mediate molecular delivery (i.e. sonoporation) has particular promise for clinical implementation as it offers the following possibilities:

(i) the treatment may not require open surgical access to the target tissues and thus patients would not be exposed to the trauma associated with incision nor the enhanced probability for subsequent infection; (ii) therapy may be implemented using standard hospital equipment with minor modifications; (iii) exposure is localised so that a target region only is affected.

In addition, the present invention provides an apparatus and method that allows a surface to be perforated, marked, or scored in a highly controllable manner through the control of the distance of the microbubbles with respect to the surface. In addition, the pressure applied to the microbubbles using ultrasound controls the energy with which an emergent microjet will contact the surface and therefore the depth of the perforation, etch, or mark made in the surface .

It is also possible to arrange the microbubbles in predetermined geometrical patterns by means of an optical trap, or otherwise using magnetic or electric fields in order to provide a pattern of markings on a surface .

In another example of the present invention, a lithography system is provided which allows a surface to be etched in order to produce a circuit pattern. Accordingly, the holes can be used to provide functional gateways in micro/nano fluidic or micro/nano electronic devices. Advantageously, the present invention can achieve this without the need of a photomask.

The use of the present invention for marking or etching a surface is shown in Fig.13a and Fig.13b. Fig.13a is a side view of a surface 90 where an arrangement of microbubbles 92 is held in a predetermined pattern at a predetermined distance from the surface 90. Fig.13b shows the two-dimensional pattern. The application of ultrasound causes the microbubbles to burst which etches, or marks the surface . The microbubbles may be held in position using an optical trap arrangement, a magnetic field or an electric field. Advantageously, as the pattern of microbubbles can be prescribed and the microbubbles positioned simultaneously, the pattern can be made with a single application of the ultrasound signal to cause sonoporation of all of the microbubbles.

Improvements and modifications may be incorporated herein without deviating from the scope of the invention.