WO2012028898A1

WO2012028898A1 - Device for delivering a substance inside a medium, and method for delivering the substance

Info

Publication number: WO2012028898A1
Application number: PCT/IB2010/002451
Authority: WO
Inventors: Jean-Yves Chapelon; Cyril Lafon; Jean-Louis Mestas; Alexei Moussatov
Original assignee: Inserm (Institut National De La Sante Et De La Recherche Medicale)
Priority date: 2010-08-31
Filing date: 2010-08-31
Publication date: 2012-03-08

Abstract

A device (1) for delivering a substance inside a medium, comprising first ultrasound means (11) adapted to generate a first beam of a first ultrasound wave inside the medium towards a first direction (D1), the first beam being directed to a target point (P) inside the medium, and second ultrasound means (12) adapted to generate a second beam of a second ultrasound wave inside the medium towards a second direction (D2), the second beam being directed to the target point (P). The second direction is inclined relative to the first direction.

Description

Device for delivering a substance inside a medium, and method for delivering the substance

FIELD OF THE INVENTION

The present invention concerns a device and a method for delivering a substance inside a medium.

BACKGROUND OF THE INVENTION

The present invention concerns more precisely a device for delivering a substance inside a medium.

It is known to generate and to focus a single ultrasound beam to a point inside a medium, so that to produce cavitation inside a region around said point and to make a delivery compound to switch from a holding state in which the substance is held by the delivery compound, to a releasing state in which the substance is released by the delivery compound.

OBJECTS AND SUMMARY OF THE INVENTION

One object of the present invention is to provide a more efficient device for delivering a substance inside a medium.

To this effect, the substance is held by a delivery compound in a holding state, and the device comprises:

- first ultrasound means adapted to generate a first beam of a first ultrasound wave inside said medium towards a first direction, said first beam being directed to a target point inside said medium,

- second ultrasound means adapted to generate a second beam of a second ultrasound wave inside said medium towards a second direction, said second beam being substantially directed to said target point, and

wherein the second direction is inclined relative to the first direction for inducing the cavitation inside a region around said target point and for switching the delivery compound from the holding state to a releasing state inside said region.

Thanks to these features, the region wherein the substance is released and delivered has a reduced volume and size. Moreover, the cavitation inside said region is increased compared to the cavitation outside said region. Moreover, the inventors have observed that the cavitation inside the region is more stable.

In various embodiments of the device, one and/or other of the following features may optionally be incorporated:

- the region has a size in all directions lower than 5 mm, and preferably lower than 2 mm;

- the device further comprises third ultrasound means adapted to produce acoustic images of the medium;

- the third ultrasound means produce an image of the medium to define a region of interest inside said medium, and the device comprises control means to move sequentially the target point to a plurality of determined points inside said region of interest for controlling the cavitation inside said determined region of interest;

- the first beam is focused to the target point, the second beam is substantially focused to the target point, and the target point is a focal point;

- the second direction is inclined relative to said first direction of an angle, said angle being comprised between 30° and 150°, and preferably between 60° and 120°;

- the first and second ultrasound waves have different frequencies;

- the first and second ultrasound waves have frequencies comprised between 100 kHz and 10 MHz, and preferably between 0.5 MHz and 1 MHz;

- the delivery compound is chosen in the list of a nanoparticle and a microparticule;

- the delivery compound is chosen in the list of a liposome and a micelle. Another object of the invention is to provide a method for delivering a substance inside a medium, wherein the substance is held by a delivery compound in a holding state, and the method comprises the steps of:

- generating by first ultrasound means, a first beam of a first ultrasound wave inside said medium towards a first direction, said first beam being directed to a target point inside said medium,

- generating by second ultrasound means, a second beam of a second ultrasound wave inside said medium towards a second direction, said second beam being substantially directed to said target point, and

wherein the second direction is inclined relative to the first direction for inducing the cavitation inside a region of the medium around said target point and for switching the delivery compound from the holding state to a releasing state inside said region.

In preferred embodiments of the method proposed by the invention, one and/or the other of the following features may optionally be incorporated:

- the method further comprises the steps of:

- imaging the medium to determine a region of interest inside the medium,

- moving sequentially the target point to a plurality of determined points inside the region of interest ;

- each determined point inside the region of interest is controlled with a predetermined elapsed time or predetermined intensity of said first and second beams;

- the medium comprises cells, the substance comprises at least a plasmid, the target point is positioned near at least one cell, so that the cavitation releases the substance and open the one cell to transfer the plasmid inside said cell;

- the substance is a drug intended to be delivered to a tumor inside said medium, and the target point is positioned inside said tumor;

- the medium comprises fat cells, said fat cells being substantially destroyed by positioning the target point on these fat cells;

- the first beam is focused to the target point, the second beam is substantially focused to the target point, and the target point is a focal point.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the invention will be apparent from the following detailed description of embodiments given by way of non-limiting examples, with reference to the accompanying drawings. In the drawings:

- Figure 1 and 3 are a schematic views of first and second embodiments of a device for delivering a substance according to the invention,

- Figure 2 is a perspective view of the first embodiment of figure 1.

MORE DETAILLED DESCRIPTION

Figure 1 represents a first embodiment of a device 1 for delivering a substance inside a medium.

The medium may be any medium that can propagate ultrasound waves and have cavitation phenomenon. For example, the medium may be an aqueous medium. The medium may be a human or animal body.

The medium is susceptible to the cavitation phenomenon. The cavitation is the creation or formation of vapour bubbles in a medium in a region where the pressure falls bellow a pressure threshold, said threshold corresponding to the called cavitation level. Additionally, said pressure threshold may be the vapour pressure of said medium. During cavitation the cavitation bubbles may dynamically oscillates. Then, the cavitation bubbles may rapidly collapse, producing a localised shock wave in the medium, an increase of local temperature, some mechanical stresses and/or chemical effects. The cavitation may be produced when the medium comprises some impurities. The cavitation may be also produced by adding microbubbles or bubbles inside the medium, or by adding ultrasound contrast agents.

The substance may be a chemical substance, a radioactive substance, a colour substance, a gene, a plasmide or a drug.

The substance is held by a delivery compound in a holding state when there is no cavitation inside the medium in near proximity of the delivery compound. In that state, the substance is not free to move into the medium independently of the delivery compound, and is not able to be active and to react with another substance inside the medium.

The substance is released by the delivery compound in a releasing state when there is cavitation inside the medium in near proximity of the delivery compound. In that state, the substance is free to move into the medium independently of the delivery compound, and is able to be active and to combine with other substance.

The delivery compound is therefore sensible to the cavitation, and releases the substance if it is in near proximity of cavitation bubbles. Near proximity means a distance between the delivery compound and a cavitation bubble smaller than 1 mm.

If cavitation bubbles are localized inside a region V of the medium, i.e. if the cavitation occurs inside said region V, the delivery compound releases the substance substantially inside said region V. The substance is transported through the medium to the localized region V inside said medium and released inside said localized and reduced region V.

The delivery compound may be considered as a means of transportation and delivering for the substance inside the medium from any origin to the localized region V. Many delivery compounds are well-known. It may be a microparticules or nanoparticle, and for example a liposome, a micelle, or the like.

As used herein, the term "particles" refers to an aggregated physical unit of solid material. The particles according to the invention may be micro- or nanoparticles .

Microparticles are understood as particles having a median diameter d₅₀ ranging from 500 ym to 1 ym and more preferably from 100 ym to 1 ym, and most preferably from 10 ym to 1 ym.

Nanoparticles are understood as particles having a median diameter d₅₀ inferior to 1 ym and notably ranging from 0.1 ym and 0.01 ym.

As used herein, the term "median diameter d₅₀" refers to the particle diameter so that 50 % of the volume or of the number of the particles population have a smaller diameter .

More specifically, the microparticles or nanoparticles may be microspheres or microcapsules, nanospheres or nanocapsules respectively, containing an active substance.

The device 1 comprises:

- first ultrasound means 11 adapted to generate a first beam Bi of a first ultrasound wave inside the medium towards a first direction Di, said first beam Bi being focused to a focal point P inside said medium,

- second ultrasound means 12 adapted to generate a second beam B₂ of a second ultrasound wave inside the medium towards a second direction D₂, said second beam B₂ being substantially focused to said same focal point P, that is to say focused to an other focal point P' (not shown) at a distance from said focal point P lower than 5 mm, and preferably lower than 1 mm. For simplicity, the focal points P and P' are supposed superposed in the following description, and the focal point P is only referred . The focal point P (or P' ) of focused beams is the effective focal point, that is to say the point into the medium where a pressure inside the medium has a minimum value, i.e. where the acoustical power or intensity inside the medium has a maximum value.

The first ultrasound means 11 comprises at least a first transducer receiving a first signal Sj from a first signal generator 21 and generating the first beam Bi .

The second ultrasound means 12 comprises at least a second transducer receiving a second signal s₂ from a second signal generator 22 and generating the second beam B₂.

The device 1 also comprises control means 30 adapted to control, synchronise and tune the characteristics of the first and second signal generators

21, 22. The control means 30 may be a computer with a keyboard, and a display for interfacing with a user.

The first signal Si and the second signal s₂ may be time continuous signals, or burst signals or a combination of continuous and burst sine signals. These signals comprise for example a sine wave or a plurality of sine waves or any other wave.

In case of first and second burst signals, the control means 30 synchronise these first and second signals so that the first and second waves arrive near the focal point P at the same time. The waves add their amplitudes.

The local pressure amplitude (acoustic power or intensity) may be increased.

The first and second transducers also have a geometric focal point, respectively marked with the references Pi and P₂ on figure 1. These geometric focal points are localized at a predetermined distance from a surface of each transducer, said surface producing the ultrasound wave into the medium.

As shown on figure 1, the geometric focal points

PI, P2 are usually situated on the respective direction, first or second direction Dl, D2, and they are situated at a distance from the transducer surface preferably higher than the focal point P, P' of the ultrasound beams Bl, B2.

The first beam Bi produces a first zone Zi of low pressure inside the medium, i.e. a first zone of maximum acoustic power or intensity. The first zone Zi is typically centred on the focal point P and has an elongated shape along the first direction Di . The first zone Zi is for example a region of the medium having a length of 13 mm in the first direction Di and a width of 2 mm in orthogonal directions perpendicular to the first direction Di .

The second beam B₂ produces a second zone Z₂ of low pressure inside the medium, i.e. a second zone of maximum acoustic power or intensity. The second zone Z₂ is substantially centred on the focal point P and has an elongated shape along the second direction D₂. The second zone Z₂ is similar to the first zone Zi, and it has for example the same size as the first zone Zi, but it is elongated in the second direction D₂.

In case of a single beam focused to the focal point

P, the first zone Zi corresponds to a region of the medium wherein the cavitation occurs if the first signal Sj has an amplitude greater than a predetermined amplitude. This first zone is quite large and elongated. Moreover, the cavitation and cavitation thresholds inside this region are not stable: Cavitation bubbles appear and collapse at various positions inside the volume. These positions of the cavitation bubbles seem to move inside the region, and not to be equally spatially spread inside said region.

In case of non coaxial and confocal dual beams, corresponding to the present invention, the first and second zones Zi, Z₂ intersect in a region V around the focal point P, said region V having a reduced size compared to the size of said first zone Zi or said second zone Z₂.

Thanks to these features, the region V has a reduced size and the cavitation inside this region V is increased compared to the cavitation outside said region.

For example, the region V may have a size in all directions lower than 5 mm, and preferably lower than 2 mm.

The second direction D₂ is inclined relative to the first direction Di of an angle a. The second direction D2 is therefore non coaxial to the first direction Dl . The angle can be for example of 90°. The region V has therefore the smallest size.

The angle a may be comprised between 30° and 150°, and preferably between 60° and 120°. A reduced angle a may be useful to have more than two ultrasound means in the device, to define a more precise shape of the region V, to reduce the size of the region V and to better stabilize the bubbles inside said region V.

If the first signal Sj has an amplitude greater than a first predetermined amplitude and if the second signal s₂ has an amplitude greater than a second predetermined amplitude, the cavitation occurs inside the region V that is substantially the intersection of the first and second zones Zi, Z₂, and there are no cavitation outside of said region V.

The first signal Sj has an amplitude tuned so that the first beam does not induce the cavitation near the focal point P in the absence of the second beam. Reciprocally, the second signal s₂ has an amplitude tuned so that the second beam does not induce the cavitation near the focal point P in the absence of the first beam. But, the amplitude of the first and second signals are tuned so that to induce the cavitation inside the region V around the focal point P, when both beams are present or at least when both of the first and second waves arrive inside said region V of the medium.

The first and second signals may comprise same or different frequencies, to respectively produce first and second ultrasound waves.

Usually, the signals and waves have main frequencies comprised between 100 kHz and 10 MHz. Preferably, these frequencies are comprised between 0.5 MHz and 1 MHz.

Inside the region V, the delivery compound switches from the holding state to the releasing state. The substance is released and delivered inside said region V.

Figure 2 represents a more detailed view of the first embodiment of the device 1 comprising a water tank 3 filled with a medium 4 of degassed water.

In this device 1, the first ultrasound means comprises a spherical transducer 11 having a emitting surface 11a for emitting the first ultrasound wave in the first direction Di and for focussing the first ultrasound wave at a first distance from the emitting surface 11a.

The first transducer 11 is a piezoelectric ceramic transducer having a nominal frequency of 1 MHz, a first distance of 50 mm and an emitting surface 11a of 50 mm diameter .

The second ultrasound means comprises:

- a second transducer 12 having an emitting surface

12a for emitting the second ultrasound wave in the second direction D2 and for focussing the second ultrasound wave at a second distance from the emitting surface 12a, and

- an imaging transducer 12b in order to produce images of the medium 4.

The images produced with the imaging transducer 12b are used to visualize the medium 4 and to localize the cavitation bubbles inside the medium 4. These images may be used to determine the first and second signal amplitudes to control the cavitation level, in such a way as a closed controlled loop.

The second transducer 12 is for example a transducer, having a nominal frequency of 1 MHz, a second distance of 45 mm and an emitting surface area of 56.9x34.6 mm². The imaging transducer 12b is for example a transducer, having a nominal frequency of 5 MHz and a comprising multi-element array of piezoelectric elements.

The first signal Sj is a pulsed burst sine wave of 1 MHz, with repetition frequency of 200 Hz, and a duty cycle of 5 %. The first predetermined amplitude corresponds to a power of generated first ultrasound wave of 5 Watts.

In this embodiment, designed for testing, the angle a between the first direction Dl and the second direction D2 is substantially of 90°.

The tests performed with this device 1 confirmed that the region V where the cavitation is produced is a small region having a size approximately of 2 mm x 2 mm x 2 mm in three orthogonal directions (X, Y, Z) .

Such region V having cavitation bubbles is observed with the imaging transducer 12b at a position relative to the device 1 that is stable, not moving in time during.

Inside said region V, the cavitation bubbles have in time during a constant density and are uniformly spread inside the region V.

Outside said region V, no cavitation is observed. An embodiment of the device 1 may comprise control means to sequentially move the focal point P to a plurality of predetermined points inside a region of interest inside the medium. The region of interest may be determined by an image produced by the imaging transducer 12b.

It is well understood that the focal point P may be moved by moving the first and second ultrasound means relative to the medium or by moving the medium relative to the ultrasound means.

The control means comprises additional mechanical devices to move the medium or a portion of the medium relative to the ultrasound means. For example, the mechanical devices comprises displacement drives according to one, two or a plurality of linear directions or one, two or another plurality of rotational directions, or any combination of linear or rotational directions. For example, the mechanical devices comprise three displacement drives according to three orthogonal directions X, Y, Z.

Alternatively the ultrasound means are able to produce a moveable beam of ultrasound wave, having a beam focused to a focal point having a controlled position inside the medium, without moving any mechanical parts. Such technique, is well known and use beam forming technology with an ultrasound mean having a plurality of transducers, each of them precisely controlled according a beam forming algorithm.

Figure 3 represents a second embodiment of a device 1 for delivering a substance inside a medium. In this second embodiment, the first and second beams are not focused to the focal point P, but only directed to a similar point P now named, a target point P.

Consequently, for this second embodiment, the first beam is directed to a target point P inside the medium, the second beam is substantially directed to said target point P, and the second direction is inclined relative to the first direction.

The first and second ultrasound waves are not converging waves, but substantially plane waves that propagate along the first and second directions respectively .

For the other features, the second embodiment of this invention is similar to the first embodiment.

The first and second beams intersect inside a region V of the medium around the target point P. These features are inducing the cavitation inside said region and the switching of the delivery compound from the holding state to the releasing state inside said region.

Thanks to these features, the region V has a reduced size and the cavitation inside this region V is increased and more stable compared to the cavitation outside said region.

In other embodiments, the device 1 may comprise more than two ultrasound means to generate a plurality of beams intersecting to a target point P. The region V has therefore a more reduced size and the cavitation inside this region V is increased and more stable. In relation to Figure 1, the implementation of the above described device 1 in a method for delivering a substance inside a medium is now disclosed.

The method for delivering a substance inside a medium, wherein the substance is held by a delivery compound in a holding state, comprises the steps of:

- generating by first ultrasound means, a first beam of a first ultrasound wave inside said medium towards a first direction, said first beam being focused to a focal point inside said medium,

- generating by second ultrasound means, a second beam of a second ultrasound wave inside said medium towards a second direction, said second beam being substantially focused to said focal point, and

wherein the second direction is inclined relative to the first direction for producing the cavitation inside a region of the medium around said focal point and for switching the delivery compound from the holding state to a releasing state inside said region.

Moreover, the method may implement the scanning of a region of interest inside the medium, by the added following steps:

- imaging the medium to determine a region of interest inside the medium,

- moving sequentially the focal point to a plurality of determined points inside the region of interest . Firstly, the device and method may be used for drug delivery inside a body comprised inside the medium. The body is for example an animal or a human body. Thanks to the above-specified method the drug may be delivered inside the body to a predetermined region inside the body, and not somewhere else.

Moreover, the needed quantity of drug for the treatment of the predetermined region is greatly reduced compared to known methods without such delivery.

Such application has a great interest for the treatment of cancer tumor. The drug is released by the delivery compound only inside the tumor. The cancer tumor may be treated without releasing the drug everywhere inside the body. The drug is often harmful and toxic for organs inside the body. Many bad effects of a global treatment of the body may be therefore avoided.

Additionally, the quantity of drug injected inside the body is much smaller than the quantity for known methods.

Secondly, the device and method may be used for sonoporation or transfection applications, wherein plasmids are transferred inside a cell.

In the method, the substance comprises at least a plasmid. The substance may be a plasmid, a gene, or a plasmid graft on a liposome. The focal point is positioned near at least one cell. The cavitation phenomenon releases the substance and simultaneously opens the cell to transfer the plasmid inside said cell.

Tests were conducted to verify the efficiency of the combination of the transfection method and the present method for delivering a substance. Surprisingly, the cavitation generated for delivering the substance is also able to open the cells to transfer plasmids into the cells and without destroying said cells. The test was done on mice. Two injections were done on these mice with RL cells so that 24 days after injection they have two tumors, each having a volume of at least 1000 m³.

The first tumor is insonified with dual confocal ultrasound beams as described above.

A substance siRNA Bcl2Ll comprising genes coupled to an Alexa Fluor is injected into the second tumor. The Alexa Fluor is an example of marker (fluorescence marker) adapted to detect the presence of siRNA added genes. The injected volume is 30 μΐ, and the substance concentration is of 7.5 yg/ml .

Then, the second tumor is also insonified with the same dual confocal ultrasound beams.

Both tumor are analysed to detect intracellular fluorescence. The second tumor comprises 16 % of fluorescent cells, that is to say 16 % of transfected cells. Higher percentages may be obtained. Thirdly, the device and method may be used for destroying fat cells or adipocytes, inside a body. In that case, the substance may by a catalyst substance so that the adipocytes destruction is improved by the combination of the cavitation and the catalyst effects.

Claims

1. A device for delivering a substance inside a medium, wherein the substance is held by a delivery compound in a holding state, and the device comprises:

2. A device according to claim 1, wherein said region has a size in all directions lower than 5 mm, and preferably lower than 2 mm.

3. A device according to any one of the preceding claims, further comprising third ultrasound means adapted to produce acoustic images of the medium.

4. A device according to claim 3, wherein the third ultrasound means produce an image of the medium to define a region of interest inside said medium, and said device comprises control means to move sequentially the target point to a plurality of determined points inside said region of interest for controlling the cavitation inside said determined region of interest.

5. A device according to any one of the preceding claims, wherein the first beam is focused to the target point, the second beam is substantially focused to the target point, and the target point is a focal point.

6. A device according to any one of the preceding claims, wherein the second direction is inclined relative to said first direction of an angle, said angle being comprised between 30° and 150°, and preferably between 60° and 120°.

7. A device according to any one of the preceding claims, wherein the first and second ultrasound waves have different frequencies.

8. A device according to any one of the preceding claims, wherein the first and second ultrasound waves have frequencies comprised between 100 kHz and 10 MHz, and preferably between 0.5 MHz and 1 MHz.

9. A device according to any one of the preceding claims, wherein the delivery compound is a compound chosen in the list of a nanoparticle and a microparticle.

10. A device according to any one of the preceding claims, wherein the delivery compound is chosen in the list of a liposome and a micelle.

11. A method for delivering a substance inside a medium, wherein the substance is held by a delivery compound in a holding state, and the method comprises the steps of:

12. A method according to claim 11, further comprising the steps of:

- imaging the medium to determine a region of interest inside the medium,

- moving sequentially the target point to a plurality of determined points inside the region of interest .

13. A method according to claim 12, wherein each determined point inside the region of interest is controlled with a predetermined elapsed time or predetermined intensity of said first and second beams.

14. A method according to claim 11, wherein the medium comprises cells, the substance comprises at least a plasmid, the target point is positioned near at least one cell, so that the cavitation releases the substance and opens the one cell to transfer the plasmid inside said cell .

15. A method according to claim 11, wherein the substance is a drug intended to be delivered to a tumor inside said medium, and the target point is positioned inside said tumor.

16. A method according to claim 11, wherein the medium comprises fat cells, said fat cells being substantially destroyed by positioning the target point on these fat cells .

17. A method according to any one of the claims 11 to 16, wherein the first beam is focused to the target point, the second beam is substantially focused to the target point, and the target point is a focal point.