System for inducing sonoporation of a drug into cancer cells and method thereof

Abstract

System for inducing sonoporation of a drug into cancer cells in a tumor and method thereof, the system comprising a generator configured to provide electrical energy at an ultrasound frequency; an ultrasound probe electrically connected to the generator and configured to convert the electrical energy into low intensity pulsed ultrasonic waves defined by operation parameters, said operation parameters comprising the frequency, the duty cycle, the operation time of the ultrasonic waves; an input device enabling an operator to enter configuration data comprising: type of tumor, type of drug, localization of secondary tumor, anthropometric measurements and grade of tumor, and a processor configured to determine the values of the operation parameters on the basis of the entered configuration data and control the generator and the ultrasound probe to operate according to said determined values, wherein the value of the frequency is determined on the basis of the type of tumor, the localization of the tumor, the grade of tumor and the anthropometric measurements, the value of the duty cycle is determined on the basis of the drug, the type of tumor and the grade of the tumor, and the value of said operation time being determined on the basis of at least the type of tumor and the type of drug.

Claims

1. A system for inducing sonoporation of a drug into cancer cells in a tumor, comprising: a generator configured to provide electrical energy at an ultrasound frequency; at least one ultrasound probe electrically connected to the generator and configured to convert the electrical energy into low intensity non-focalized pulsed ultrasonic waves defined by operation parameters, said operation parameters comprising the frequency and the duty cycle of the ultrasonic waves; characterized in that the system further comprises: an input device enabling an operator to enter configuration data comprising: type of tumor and type of drug; and a processor configured to: determine the values of said operation parameters on the basis of the entered configuration data, wherein the value of the frequency is determined on the basis of at least the type of tumor, and the value of the duty cycle is determined on the basis of at least the type of drug and the type of tumor; and control the generator and the ultrasound probe to operate according to said determined values.

2. The system according to claim 1, wherein the operation parameters comprise the amplitude of the ultrasonic waves and the system is configured to automatically tune the frequency and the amplitude of the ultrasound probe to compensate the attenuation of the ultrasound waves caused by any medium interposed between the probe and the cancer cells.

3. The system according to claim 2 wherein the system is configured to automatically tune the frequency and the amplitude synchronously with the duty cycle.

4. The system according to claim 3 wherein the probe comprises a plurality of ultrasound transducers, a first subset of said transducers being configured as ultrasound emitters for converting the electrical energy into the ultrasonic waves for inducing sonoporation, and a second subset of said transducers being configured as ultrasound receivers for converting ultrasound waves reflected the any medium interposed into an electric signal readable by the processor, the processor being programmed to: calculate the differences of frequency and amplitude between the ultrasound waves emitted by the first subset of transducers and the ultrasound waves received by the second subset of transducers; and adjust the values of the amplitude and the frequency of the ultrasound emitted, in such a way to compensate said differences.

5. The system according to any claim 1 wherein the probe comprises one ultrasound transducer, configured as an ultrasound emitter for converting the electrical energy into the ultrasonic waves for inducing sonoporation and as an ultrasound receiver for converting ultrasound waves reflected the any medium interposed into an electric signal readable by the processor, the processor being programmed to: calculate the differences of frequency and amplitude between the ultrasound waves emitted and the ultrasound waves received by said transducer; and adjust the values of the amplitude and the frequency of the ultrasound emitted, in such a way to compensate said differences.

6. The system according to claim 1 comprising a computer readable memory storing a list of values of frequency and a list of values of duty cycle and wherein the processor is configured to: assign a value of frequency to a type of tumor; and assign a value of duty cycle to a couple of configuration data comprising: a type of tumor and a type of drug.

7. The system according to claim 1, wherein the type of the tumor is selected from the group consisting of human breast ductal carcinoma, estrogen independent human breast adenocarcinoma, human pancreatic adenocarcinoma, human melanoma; human lentiginous melanoma, human lentigo maligna melanoma; human superficial spreading melanoma; human acral lentiginous melanoma, human mucosal melanoma, human nodular melanoma, human polypoid melanoma, human small cell melanoma; human Spitzoid melanoma, human uveal melanoma and human desmoplastic melanoma, hepatocellular carcinoma.

8. The system according to claim 2, wherein the configuration data comprise anthropometric measurements of the patient to which the cancer cells belongs, said anthropometric measurements being selected from the group consisting of: abdominal circumference, body mass index, breast circumference, thorax circumference, and body fat percentage.

9. The system according to claim 8, wherein the value of the frequency is determined also on the basis of the anthropometric measurements.

10. The system according to claim 9, wherein the processor is configured to assign a value of frequency to a set of configuration data comprising the type of tumor and at least one anthropometric measurement selected from the group consisting of: abdominal circumference, body mass index, breast circumference, thorax circumference, and body fat percentage.

11. The system according to claim 1 wherein the configuration data comprise a grade of the tumor, said grade of the tumor being selected from the group consisting of: primary, or primitive, tumor, and secondary tumor, or metastasis.

12. The system according to claim 2 wherein the configuration data comprise a localization of the secondary tumor, if said grade of the tumor is secondary tumor, or metastasis.

13. The system according to claim 12, wherein the value of the duty cycle is determined also on the basis of the localization of the secondary tumor.

14. The system according to claim 12, wherein the processor is configured to assign a value of duty cycle to a set of configuration data comprising the type of tumor, the type of drug and the grade of the tumor.

15. The system according to claim 14, wherein the processor is configured to assign a value of duty cycle to a set of configuration data comprising the type of tumor, the type of drug and the localization of the secondary tumor, if the grade of tumor is secondary tumor, or metastasis.

16. The system according to claim 1 wherein the type of drug is selected from the group consisting of: paclitaxel, paclitaxel albumine, doxorubicin, liposomal doxorubicin, irinotecan, liposomal irinotecan and fluorouracil.

17. The system according to claim 1 wherein the operation parameters comprise an operation time of the ultrasonic waves, the value of said operation time being determined on the basis of at least the type of tumor and the type of drug.

18. The system according to claim 17, wherein the processor is configured to assign a value of operation time to a couple of configuration data comprising: a type of tumor and a type of drug.

19. The system according to claim 18 wherein, the operation time consists in: one temporal window in which ultrasounds are administered; or at least two temporal windows in which ultrasounds are administered, said two at least two temporal windows being interspaced by one temporal window in which no ultrasound are administered.

20. The system according to claim 1, wherein the determined value of the frequency is comprised between 0.6 MHz and 3 MHz and/or the determined value of the duty cycle is below 12%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The description refers to the accompanying drawings, in which:

(2) FIG. 1a shows the percentage of cell death obtained by in-vitro administering paclitaxel to human breast ductal carcinoma cells, said administration being performed simultaneously to the administration of ultrasound waves. The figure shows the results obtained with different total concentrations (50 μM, 10 μM, 0.5 μM);

(3) FIG. 1b shows the percentage of cell death obtained by in-vitro administering paclitaxel albumine to human breast ductal carcinoma cells, said administration being performed simultaneously to the administration of ultrasound waves. The figure shows the results obtained with different total concentrations (50 μM, 10 μM, 0.5 μM);

(4) FIG. 2a shows the percentage of cell death obtained by in-vitro administering doxorubicin to human breast ductal carcinoma cells, said administration being performed simultaneously to the administration of ultrasound waves. The figure shows the results obtained with different total concentrations (1000 μg/ml, 500 μg/ml, 100 μg/ml);

(5) FIG. 2b shows the percentage of cell death obtained by in-vitro administering liposomal doxorubicin to human breast ductal carcinoma cells, said administration being performed simultaneously to the administration of ultrasound waves. The figure shows the results obtained with different total concentrations (1000 μg/ml, 500 μg/ml, 100 μg/ml);

(6) FIG. 3a shows the percentage of cell death obtained by in-vitro administering paclitaxel to estrogen independent human breast adenocarcinoma cells, said administration being performed simultaneously to the administration of ultrasound waves. The figure shows the results obtained with different total concentrations (50 μM, 10 μM, 0.5 μM);

(7) FIG. 3b shows the percentage of cell death obtained by in-vitro administering paclitaxel albumine to estrogen independent human breast adenocarcinoma cells, said administration being performed simultaneously to the administration of ultrasound waves. The figure shows the results obtained with different total concentrations (50 μM, 10 μM, 0.5 μM);

(8) FIG. 4a shows the percentage of cell death obtained by in-vitro administering doxorubicin to estrogen independent human breast adenocarcinoma cells, said administration being performed simultaneously to the administration of ultrasound waves. The figure shows the results obtained with different total concentrations (1000 μg/ml, 500 μg/ml, 100 μg/ml);

(9) FIG. 4b shows the percentage of cell death obtained by in-vitro administering liposomal doxorubicin to estrogen independent human breast adenocarcinoma cells, said administration being performed simultaneously to the administration of ultrasound waves. The figure shows the results obtained with different total concentrations (1000 μg/ml, 500 μg/ml, 100 μg/ml);

(10) FIG. 5a shows the percentage of cell death obtained by in-vitro administering paclitaxel to estrogen independent human breast adenocarcinoma cells, said administration being performed simultaneously to the administration of ultrasound waves. The figure shows the results obtained with different total concentrations (50 μM, 10 μM, 0.5 μM);

(11) FIG. 5b shows the percentage of cell death obtained by in-vitro administering paclitaxel albumine to human pancreatic adenocarcinoma cells, said administration being performed simultaneously to the administration of ultrasound waves. The figure shows the results obtained with different total concentrations (50 μM, 10 μM, 0.5 μM);

(12) FIG. 6a shows the percentage of cell death obtained by in-vitro administering irinotecan to human pancreatic adenocarcinoma cells, said administration being performed simultaneously to the administration of ultrasound waves. The figure shows the results obtained with different total concentrations (50 μM, 10 μM, 0.05 μM);

(13) FIG. 6b shows the percentage of cell death obtained by in-vitro administering liposomal irinotecan together with fluorouracil to human pancreatic adenocarcinoma cells, said administration being performed simultaneously to the administration of ultrasound waves. The figure shows the results obtained with different total concentrations (50 μM, 10 M, 0.05 μM);

(14) FIG. 7 shows the results relative to an experiment of internalization of a fluoresceinamine (FA) in cancer cells. FIG. 7 shows in particular the uptake of FA into human pancreatic adenocarcinoma cells in five experimental conditions, said conditions being specifically designed in order to investigate the effect of the administration of ultrasounds;

(15) FIG. 8a shows an image obtained by Confocal Laser Scanning Microscopy (CLSM) of the intake of the FA into human pancreatic adenocarcinoma cells, said FA being administered alone, i.e. without administration of ultrasound and of any drug;

(16) FIG. 8b shows an image obtained by Confocal Laser Scanning Microscopy (CLSM) of the intake of the FA into human pancreatic adenocarcinoma cells, said FA being administered together with liposomal irinotecan without administration of ultrasounds;

(17) FIG. 8c shows an image obtained by Confocal Laser Scanning Microscopy (CLSM) of the intake of the FA into human pancreatic adenocarcinoma cells, said FA being administered together with liposomal irinotecan and simultaneously with the administration of pulsed ultrasounds with a duty cycle of 12%;

(18) FIG. 8d shows an image obtained by Confocal Laser Scanning Microscopy (CLSM) of the intake of the FA into human pancreatic adenocarcinoma cells, said FA being administered together with liposomal irinotecan after the administration of pulsed ultrasounds with a duty cycle of 1%; and

(19) FIG. 8e shows an image obtained by Confocal Laser Scanning Microscopy (CLSM) of the intake of the FA into human pancreatic adenocarcinoma cells, said FA being administered together with liposomal irinotecan and simultaneously with the administration of pulsed ultrasounds with a duty cycle of 1%;

(20) FIG. 9a shows the kinetics of cellular membrane pore's closure with Prohance (Gd.sup.3+);

(21) FIG. 9b shows the kinetics of cellular membrane pore's closure with Prohance and liposomes;

(22) FIG. 9c shows the kinetics of cellular membrane pore's closure with Tetramer; and

(23) FIG. 9d shows the kinetics of cellular membrane pore's closure with Tetramer and liposomes

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(24) A preferred embodiment of the system of the present invention comprises: a generator configured to provide electrical energy at an ultrasound frequency; an ultrasound probe electrically connected to the generator and configured to convert the electrical energy into low intensity non-focalized pulsed ultrasonic waves defined by operation parameters, said operation parameters comprising the frequency, the duty cycle and the operation time of the ultrasonic waves; an input device enabling an operator to enter configuration data comprising: type of tumor, type of drug, anthropometric measurements of the patient to which cancer cells belongs and grade of the tumor; and a processor configured to: determine the values of said operation parameters on the basis of the entered configuration data, wherein the value of the frequency is determined on the basis of the type of tumor and of the anthropometric measurements and the value of the duty cycle is determined on the basis of at least the type of drug and the type of tumor; and control the generator and the ultrasound probe to operate according to said determined values.

(25) More particularly, the system of the present invention comprises a computer readable memory storing a list of values of frequency and a list of values of duty cycle and the processor is thus configured to: assign a value of frequency to a type of tumor; and assign a value of duty cycle to the couple of configuration data comprising: a type of tumor and a type of drug.

(26) The power density of ultrasound waves generated by the preferred embodiment of the system is less than 3 W/cm.sup.2.

(27) The input device is preferably a touch screen integrated with the system. Other examples of input device are a laptop, a tablet and a smartphone.

(28) In the preferred embodiment of the present invention, the first configuration data that the operator is requested to enter, is the type of drug. The type of drug is selected from the group consisting of: paclitaxel, paclitaxel albumine, doxorubicin, liposomal doxorubicin, irinotecan, liposomal irinotecan and fluorouracil.

(29) After selecting from a menu list or entering by typing the type of drug, an operator can be requested to choose the type of tumor. The type of tumor is selected from the group consisting of human breast ductal carcinoma, estrogen independent human breast adenocarcinoma and human pancreatic adenocarcinoma. The grade of tumor has then to be entered by selecting the following two options: primary tumor (i.e. primitive tumor), and secondary tumor (i.e. metastasis). If the selected grade of tumor is: primary tumor, the duty cycle is determined on the basis of the type of drug and the type of tumor. secondary tumor, also the localization of the secondary tumor have to be entered by the operator. In this case the duty cycle is determined on the basis of the type of drug, the type of tumor and the localization of the secondary tumor.

(30) In this case, the processor is configured to assign a value of duty cycle to the set of configuration data comprising the type of tumor, the type of drug, the grade of the tumor and, eventually, the localization of the secondary tumor.

(31) For example, if the entered grade of tumor is primary, the entered type of tumor is human breast ductal carcinoma or estrogen independent human breast adenocarcinoma and the entered type of drug is paclitaxel or paclitaxel albumin or doxorubicin or liposomal doxorubicin, the assigned duty cycle is 9%.

(32) For example, if the entered grade of tumor is primary, the entered type of tumor is human pancreatic adenocarcinoma and the entered type of drug is paclitaxel or paclitaxel albumin or irinotecan or liposomal irinotecan or fluorouracil, the assigned duty cycle is 1%.

(33) After selecting the grade of tumor, anthropometric measurements have to be entered. The anthropometric measurement that have to be specified by the operator are different according to the type of tumor if the grade of tumor is a primary tumor and according to localization of the secondary tumor if the grade of tumor is secondary tumor. For example, anthropometric measurements can comprise abdominal circumference if the type of tumor is human pancreatic adenocarcinoma, and breast circumference if the type of tumor is human breast ductal carcinoma. The frequency is then determined on the basis of such anthropometric measurements.

(34) In this case, the processor is thus configured to assign a value of frequency to the set of configuration data comprising the type of tumor and at least one anthropometric measurement selected from the group consisting of: abdominal circumference, body mass index, breast circumference, thorax circumference, and body fat percentage.

(35) If the tumor extends to different depths, the system of the present invention can also comprise two probes operating at different frequencies corresponding to the different depths.

(36) After having completed the entering of the aforementioned configuration data, it is given to the operator also the possibility to choose another set of parameters relative to another tumor. For example, if the first set of configuration data is referred to a primary tumor, it is possible to set also configuration data relative to a methastasis of said primary tumor. The system of the present invention can, indeed, to provide two probes, one first probe for the primary tumor and a second probe for the metastasis. The ultrasounds emitted from the first probe will have a duty cycle dependent on the type of drug and on the type of the primary tumor, whereas the ultrasounds emitted from the second probe will have a duty cycle dependent on the type of drug, on the type of primary tumor, and of the localization of the second tumor.

(37) The operation time for each probe will be also determined on the basis of the configuration data entered. More particularly, according to the type of tumor and the type of drug, the operation time can comprise: one temporal window in which ultrasounds are administered; or two or more temporal windows in which ultrasounds are administered, said windows being interspaced by one temporal window in which no ultrasounds are administered. The operator is asked for accepting this minimum operation time or to enter another configuration data consisting in an integer number that multiplied by the minimum operation time, results in the operation time.

(38) In this case, n the processor is configured to assign a value of operation time to the couple of configuration data comprising: a type of tumor and a type of drug.

(39) Finally, in a preferred embodiment, of the system of the present invention, the probe can comprise a plurality of ultrasound transducers, a first subset of said transducers being configured as ultrasound emitters for converting the electrical energy into the ultrasonic waves for inducing sonoporation, and a second subset of said transducers being configured as ultrasound receivers for converting ultrasound waves reflected the any medium interposed into an electric signal readable by the processor, the processor being programmed to: calculate the differences of frequency and amplitude between the ultrasound waves emitted by the first subset of transducers and the ultrasound waves received by the second subset of transducers; and adjust the values of the amplitude and the frequency of the ultrasound emitted, in such a way to compensate said differences.

(40) The first and the second subset of transducers can be also overlap in an only one transducer. In this case, the probe comprises one ultrasound transducer, configured as an ultrasound emitter for converting the electrical energy into the ultrasonic waves for inducing sonoporation and as an ultrasound receiver for converting ultrasound waves reflected the any medium interposed into an electric signal readable by the processor, the processor being programmed to: calculate the differences of frequency and amplitude between the ultrasound waves emitted and the ultrasound waves received by said transducer; and adjust the values of the amplitude and the frequency of the ultrasound emitted, in such a way to compensate said differences.

EXAMPLES

(41) The system of the present invention was tested in vitro using two cellular lines of human breast cancer and one cellular line of human pancreatic adenocarcinoma.

(42) In particular the following cellular lines were employed: MCF-7: human breast ductal carcinoma; MDA-MB-231: estrogen independent human breast adenocarcinoma; MiaPaCa-2: human pancreatic adenocarcinoma.

(43) The following table summarized the tested combinations of cellular lines and type of drug:

(44) TABLE-US-00001 Cellular Lines Type of tumor Type of drug MCF -7 Human breast ductal carcinoma Paclitaxel MCF -7 Human breast ductal carcinoma Paclitaxel albumine MCF -7 Human breast ductal carcinoma Doxorubicin MCF -7 Human breast ductal carcinoma Liposomal Doxorubicin MDA-MB-231 Estrogen indipendent Human breast Paclitaxel adenocarcinoma MDA-MB-231 Estrogen indipendent Human breast Paclitaxel adenocarcinoma albumine MDA-MB-231 Estrogen indipendent Human breast Doxorubicin adenocarcinoma MDA-MB-231 Estrogen indipendent Human breast Liposomal adenocarcinoma Doxorubicin MiaPaCa-2 Human pancreatic adenocarcinoma Paclitaxel MiaPaCa-2 Human pancreatic adenocarcinoma Paclitaxel albumine MiaPaCa-2 Human pancreatic adenocarcinoma Irinotecan MiaPaCa-2 Human pancreatic adenocarcinoma Liposomal irinotecan + Fluorouracil

(45) For each combination the percentage of cell death was measured in three main experimental conditions: administration of the drug (hereinafter named “NO-US” condition); administration of the drug and simultaneous administration of non focalized pulsed low-intensity ultrasound with a frequency of 1 MHz and a period of 1 second, said drug being administered in a unique dose; administration of non focalized pulsed low-intensity ultrasound with a frequency of 1 MHz and a period of 1 second, followed by administration of the drug; and administration of the drug and simultaneous administration of non focalized pulsed low-intensity ultrasound with a frequency of 1 MHz and a period of 1 second, said drug being administered in two doses of equal concentration (hereinafter termed “US-DC” condition).

(46) All the experimental conditions, other than NO-US, were replicated for different values of duty cycle and each experimental condition were replicated for three different total concentrations of drugs. Several operation times were also tested for each cellular lines and for each type of drug. The better results were obtained in US-DC condition by a first administration of a first dose of drug and simultaneous administration of ultrasounds for 20 seconds followed by a second administration of a second dose of drug and simultaneous administration of ultrasound for other 20 seconds.

(47) The results are listed in tables below and showed in the FIGS. 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b.

(48) The tables in particular summarize the results obtained for the US condition and US-DC condition for the three different total concentration of drugs listed above. For the latter condition two values of duty cycle are chosen to be showed as better representative of the efficacy of the treatment. In particular, for the cellular lines MCF-7 and MDA-MB-231, the results relative to the following values of duty cycle are showed: duty cycle=12% (US-DC=12%) duty cycle=9% (US-DC=9%)

(49) For the cellular line MiaPaCa-2, the results relative to the following values of duty cycle are showed: duty cycle=12% (US-DC=12%) duty cycle=1% (US-DC=1%).

Example 1

(50) In the following table the percentage of cell death are reported for three different concentration of pacitaxel administered to cells of MCF-7 in three conditions: NO-US: a first administration of a first dose of paclitaxel without administration of ultrasounds (NO-US) followed by a second administration of a second dose of paclitaxel; US-DC=12%: first administration of a first dose of paclitaxel and simultaneous administration of pLINFUs (with duty cycle=12%) for 20 seconds followed by a second administration of a second dose of paclitaxel and simultaneous administration of pLINFUs for other 20 seconds; US-DC=9%: first administration of a first dose of paclitaxel and simultaneous administration of pLINFUs (with duty cycle=9%) for 20 seconds followed by a second administration of a second dose of paclitaxel and simultaneous administration of pLINFUs for other 20 seconds.

(51) TABLE-US-00002 Paclitaxel - MCF -7 Cell Increasing % Cell Increasing % Cell Increasing % Death % vs NO-US Death % vs NO-US Death % vs NO-US Total concentration Total concentration Total concentration 50 μM 10 μM 0.5 μM NO - US 7.2% —  4.1% — 1.0% — US - DC = 12% 17.8% +147.3% 13.2% +221.6% 3.4% +242.5% US - DC = 9% 32.7% +354.2% .sup. 24% +484.7% 9.1% +807.5%

(52) The results of the table above are graphically represented in FIG. 1a.

Example 2

(53) In the following table the percentage of cell death are reported for three different concentration of pacitaxel albumine administered to cells of MCF-7 in three conditions: NO-US: a first administration of a first dose of pacitaxel albumine without administration of ultrasounds (NO-US) followed by a second administration of a second dose of paclitaxel albumine; US-DC=12%: first administration of a first dose of paclitaxel albumine and simultaneous administration of pLINFUs (with duty cycle=12%) for 20 seconds followed by a second administration of a second dose of paclitaxel albumin and simultaneous administration of pLINFUs for other 20 seconds; US-DC=9%: first administration of a first dose of paclitaxel albumine and simultaneous administration of pLINFUs (with duty cycle=9%) for 20 seconds followed by a second administration of a second dose of paclitaxel albumin and simultaneous administration of pLINFUs for other 20 second

(54) TABLE-US-00003 Paclitaxel albumine - MCF -7 Cell Increasing % Cell Increasing % Cell Increasing % Death % vs NO-US Death % vs NO-US Death % vs NO-US Total concentration Total concentration Total concentration 50 μM 10 μM 0.5 μM NO - US 23.7% — 12.9% —  2.6% — US - DC = 12% 39.9% +68.3% 26.5% .sup. +106% 25.24% +491.3% US - DC = 9% 51.7% +118.1% 33.7% +161.8%   24% .sup. +830%

(55) The results of the table above are graphically represented in FIG. 1b.

Example 3

(56) In the following table the percentage of cell death are reported for three different concentration of doxorubicin administered to cells of MCF-7 in three conditions: NO-US: a first administration of a first dose of doxorubicin without administration of ultrasounds (NO-US) followed by a second administration of a second dose of doxorubicin; US-DC=12%: first administration of a first dose of doxorubicin and simultaneous administration of pLINFUs (with duty cycle=12%) for 20 seconds followed by a second administration of a second dose of doxorubicin and simultaneous administration of pLINFUs for other 20 seconds; US-DC=9%: first administration of a first dose of doxorubicin and simultaneous administration of pLINFUs (with duty cycle=9%) for 20 seconds followed by a second administration of a second dose of doxorubicin and simultaneous administration of pLINFUs for other 20 seconds.

(57) TABLE-US-00004 Doxorubicin - MCF -7 Cell Increasing % Cell Increasing % Cell Increasing % Death % vs NO-US Death % vs NO-US Death % vs NO-US Total concentration Total concentration Total concentration 1000 μg/ml 500 μg/ml 100 μg/ml NO - US 59% — 23.3% — 17.1% — US - DC = 12% 82% .sup. +39% 49.9% +114.2% 28.2% +64.9% US - DC = 9% 70.6%.sup.  +19.7% 47.2% +102.5% 29.8% +74.4%

(58) The results of the table above are graphically represented in FIG. 2a.

Example 4

(59) In the following table the percentage of cell death are reported for three different concentration of liposomal doxorubicin administered to cells of MCF-7 in three conditions: NO-US: a first administration of a first dose of liposomal doxorubicin without administration of ultrasounds (NO-US) followed by a second administration of a second dose of liposomal doxorubicin; US-DC=12%: first administration of a first dose of liposomal doxorubicin and simultaneous administration of pLINFUs (with duty cycle=12%) for 20 seconds followed by a second administration of a second dose of liposomal doxorubicin and simultaneous administration of pLINFUs for other 20 seconds; US-DC=9%: first administration of a first dose of liposomal doxorubicin and simultaneous administration of pLINFUs (with duty cycle=9%) for 20 seconds followed by a second administration of a second dose of liposomal doxorubicin and simultaneous administration of pLINFUs for other 20 seconds.

(60) TABLE-US-00005 Liposomal doxorubicin - MCF -7 Cell Increasing % Cell Increasing % Cell Increasing % Death % vs NO-US Death % vs NO-US Death % vs NO-US Total concentration Total concentration Total concentration 1000 μg/ml 500 μg/ml 100 μg/ml NO - US 75.4% — 35.5% — 16.2% — US - DC = 12% .sup. 82% +8.7% 49.8% +40.4% 28.2%   +74% US - DC = 9% 92.8%  +23% 47.2% +97.4% .sup. 37% +128.7%

(61) The results of the table above are graphically represented in FIG. 2b.

Example 5

(62) In the following table the percentage of cell death are reported for three different concentration of pacitaxel administered to cells of MDA-MB-231 in three conditions: NO-US: a first administration of a first dose of paclitaxel without administration of ultrasounds (NO-US) followed by a second administration of a second dose of paclitaxel; US-DC=12%: first administration of a first dose of paclitaxel and simultaneous administration of pLINFUs (with duty cycle=12%) for 20 seconds followed by a second administration of a second dose of pacitaxel and simultaneous administration of pLINFUs for other 20 seconds; US-DC=9%: first administration of a first dose of paclitaxel and simultaneous administration of pLINFUs (with duty cycle=9%) for 20 seconds followed by a second administration of a second dose of paclitaxel and simultaneous administration of pLINFUs for other 20 seconds.

(63) TABLE-US-00006 Paclitaxel - MDA-MB-231 Cell Increasing % Cell Increasing % Cell Increasing % Death % vs NO-US Death % vs NO-US Death % vs NO-US Total concentration Total concentration Total concentration 50 μM 10 μM 0.5 μM NO - US 7.9% — 6.4% — 6.4% — US - DC = 12% 12.3% +55.3% 7.4%   +15% 5.9% −7.8% US - DC = 9% 25.5% +221.9% 14.7% +128.4% 11.3% +76.6%

(64) The results of the table above are graphically represented in FIG. 3a.

Example 6

(65) In the following table the percentage of cell death are reported for three different concentration of paclitaxel albumine administered to cells of MDA-MB-231 in three conditions: NO-US: a first administration of a first dose of paclitaxel without administration of ultrasounds (NO-US) followed by a second administration of a second dose of paclitaxel albumine; US-DC=12%: first administration of a first dose of paclitaxel albumine and simultaneous administration of pLINFUs (with duty cycle=12%) for 20 seconds followed by a second administration of a second dose of paclitaxel albumin and simultaneous administration of pLINFUs for other 20 seconds; US-DC=9%: first administration of a first dose of pacitaxel albumine and simultaneous administration of pLINFUs (with duty cycle=9%) for 20 seconds followed by a second administration of a second dose of paclitaxel albumin and simultaneous administration of pLINFUs for other 20 seconds.

(66) TABLE-US-00007 Paclitaxel albumine - MDA-MB-231 Cell Increasing % Cell Increasing % Cell Increasing % Death % vs NO-US Death % vs NO-US Death % vs NO-US Total concentration Total concentration Total concentration 50 μM 10 μM 0.5 μM NO - US 17.8% — 9.4% —   5% — US - DC = 12% 21.6% +21.3% 11.3% +20.2%  7.8%  +56% US - DC = 9% 45.1% +153.4% 31.9% +239.4% 80.9% +282%

(67) The results of the table above are graphically represented in FIG. 3b.

Example 7

(68) In the following table the percentage of cell death are reported for three different concentration of doxorubicin administered to cells of MDA-MB-231 in three conditions: NO-US: a first administration of a first dose of paclitaxel without administration of ultrasounds (NO-US) followed by a second administration of a second dose of doxorubicin; US-DC=12%: first administration of a first dose of doxorubicin and simultaneous administration of pLINFUs (with duty cycle=12%) for 20 seconds followed by a second administration of a second dose of doxorubicin and simultaneous administration of pLINFUs for other 20 seconds; US-DC=9%: first administration of a first dose of doxorubicin and simultaneous administration of pLINFUs (with duty cycle=9%) for 20 seconds followed by a second administration of a second dose of doxorubicin and simultaneous administration of pLINFUs for other 20 seconds.

(69) TABLE-US-00008 Doxorubicin - MDA-MB-231 Cell Increasing % Cell Increasing % Cell Increasing % Death % vs NO-US Death % vs NO-US Death % vs NO-US Total concentration Total concentration Total concentration 1000 μg/ml 500 μg/ml 100 μg/ml NO - US 54.5% — 22.8% — 10.9% — US - DC = 12% 54.6% +0.3% .sup. 27% +18.4% .sup. 12% +11.8% US - DC = 9% 60.5% +11.1% 38.8% +70.5% 23.5%  +116%

(70) The results of the table above are graphically represented in FIG. 4a.

Example 8

(71) In the following table the percentage of cell death are reported for three different concentration of liposomal doxorubicin administered to cells of MDA-MB-231 in three conditions: NO-US: a first administration of a first dose of paclitaxel without administration of ultrasounds (NO-US) followed by a second administration of a second dose of liposomal doxorubicin; US-DC=12%: first administration of a first dose of liposomal doxorubicin and simultaneous administration of pLINFUs (with duty cycle=12%) for 20 seconds followed by a second administration of a second dose of liposomal doxorubicin and simultaneous administration of pLINFUs for other 20 seconds; US-DC=9%: first administration of a first dose of liposomal doxorubicin and simultaneous administration of pLINFUs (with duty cycle=9%) for 20 seconds followed by a second administration of a second dose of liposomal doxorubicin and simultaneous administration of pLINFUs for other 20 seconds.

(72) TABLE-US-00009 Liposomal doxorubicin - MDA-Mb-231 Cell Increasing % Cell Increasing % Cell Increasing % Death % vs NO-US Death % vs NO-US Death % vs NO-US Total concentration Total concentration Total concentration 1000 μg/ml 500 μg/ml 100 μg/ml NO - US 65.7% — .sup. 29% — 11.1% — US - DC = 12% 72.4% +10.2% 37.8% +30.4% 17.1% +53.9% US - DC = 9% 81.3% +23.7% 51.2% +76.6% 29.4% +164.6%

(73) The results of the table above are graphically represented in FIG. 4b.

Example 9

(74) In the following table the percentage of cell death are reported for three different concentration of paclitaxel administered to cells of MiaPaCa2 in three conditions: NO-US: a first administration of a first dose of paclitaxel without administration of ultrasounds (NO-US) followed by a second administration of a second dose of paclitaxel; US-DC=12%: first administration of a first dose of paclitaxel and simultaneous administration of pLINFUs (with duty cycle=12%) for 20 seconds followed by a second administration of a second dose of paclitaxel and simultaneous administration of pLINFUs for other 20 seconds; US-DC=1%: first administration of a first dose of pacitaxel and simultaneous administration of pLINFUs (with duty cycle=1%) for 20 seconds followed by a second administration of a second dose of paclitaxel and simultaneous administration of pLINFUs for other 20 seconds.

(75) TABLE-US-00010 Paclitaxel - MiaPaCa2 Cell Increasing % Cell Increasing % Cell Increasing % Death % vs NO-US Death % vs NO-US Death % vs NO-US Total concentration Total concentration Total concentration 50 μM 10 μM 0.5 μM NO - US 80.2% — 65.3% — 45.5% — US - DC = 12% 87.6% +9.2% 9.2% +12.7% 12.7% 17.1% US - DC = 1% 90.5% +12.8% 12.8% +22.7% 22.7% 40.1%

(76) The results of the table above are graphically represented in FIG. 5a.

Example 10

(77) In the following table the percentage of cell death are reported for three different concentration of pacitaxel albumine administered to cells of MiaPaCa2 in three conditions: NO-US: a first administration of a first dose of pacitaxel albumine without administration of ultrasounds (NO-US) followed by a second administration of a second dose of paclitaxel albumine; US-DC=12%: first administration of a first dose of paclitaxel albumine and simultaneous administration of pLINFUs (with duty cycle=12%) for 20 seconds followed by a second administration of a second dose of paclitaxel albumine and simultaneous administration of pLINFUs for other 20 seconds; US-DC=1%: first administration of a first dose of paclitaxel albumine and simultaneous administration of pLINFUs (with duty cycle=1%) for 20 seconds followed by a second administration of a second dose of paclitaxel albumine and simultaneous administration of pLINFUs for other 20 seconds.

(78) TABLE-US-00011 Paclitaxel albumine - MiaPaCa2 Cell Increasing % Cell Increasing % Cell Increasing % Death % vs NO-US Death % vs NO-US Death % vs NO-US Total concentration Total concentration Total concentration 50 μM 10 μM 0.5 μM NO - US 7.9% — 6.4% — 6.4% — US - DC = 12% 12.3% +55.3% 7.4%   +15% 5.9% −7.8% US - DC = 1% 25.5% +221.9% 14.7% +128.4% 11.3% +76.6%

(79) The results of the table above are graphically represented in FIG. 5b.

Example 11

(80) In the following table the percentage of cell death are reported for three different concentration of irinotecan administered to cells of MiaPaCa2 in three conditions: NO-US: a first administration of a first dose of irinotecan without administration of ultrasounds (NO-US) followed by a second administration of a second dose of irinotecan; US-DC=12%: first administration of a first dose of irinotecan and simultaneous administration of pLINFUs (with duty cycle=12%) for 20 seconds followed by a second administration of a second dose of irinotecan and simultaneous administration of pLINFUs for other 20 seconds; US-DC=1%: first administration of a first dose of irinotecan and simultaneous administration of pLINFUs (with duty cycle=1%) for 20 seconds followed by a second administration of a second dose of irinotecan and simultaneous administration of pLINFUs for other 20 seconds.

(81) TABLE-US-00012 Irinotecan - MiaPaCa2 Cell Increasing % Cell Increasing % Cell Increasing % Death % vs NO-US Death % vs NO-US Death % vs NO-US Total concentration Total concentration Total concentration 50 μM 1 μM 0.05 μM NO - US 56.7% — 21.1% — 11.5% — US - DC = 12% .sup. 66% +16.5% .sup. 30% +42.5% 18.5% +60.6% US - DC = 1% 74.3% +31.1% 34.3% +62.9% 25.4% +120.8%

(82) The results of the table above are graphically represented in FIG. 6a.

Example 12

(83) In the following table the percentage of cell death are reported for three different concentration of liposomal irinotecan and fluorouracil (FU) administered to cells of MiaPaCa2 in three conditions: NO-US: a first administration of a first dose of a solution containing liposomal irinotecan and fluorouracil, without administration of ultrasounds (NO-US) followed by a second administration of a second dose a solution containing liposomal irinotecan and fluorouracil; US-DC=12%: first administration of a first dose of a solution containing liposomal irinotecan and fluorouracil, and simultaneous administration of pLINFUs (with duty cycle=12%) for 20 seconds followed by a second administration of a second dose of a solution containing liposomal irinotecan and fluorouracil, and simultaneous administration of pLINFUs for other 20 seconds; US-DC=1%: first administration of a first dose of a solution containing liposomal irinotecan and fluorouracil, and simultaneous administration of pLINFUs (with duty cycle=1%) for 20 seconds followed by a second administration of a second dose of a solution containing liposomal irinotecan and fluorouracil, and simultaneous administration of pLINFUs for other 20 seconds.

(84) TABLE-US-00013 Liposomal irinotecan + Fluorouracil (FU) - MiaPaCa2 Cell Increasing % Cell Increasing % Cell Increasing % Death % vs NO-US Death % vs NO-US Death % vs NO-US Total concentration Total concentration Total concentration 50 μM + 5 FU 100 μM 1 μM + 5 FU 10 μM 0.05 μM + 5 FU 1 μM NO - US 85% — 67% — 25% — US - DC = 12% 89% +4.7% 80.5%.sup.  +20.1% 39%  +56% US - DC = 1% 98% +11.8% 88% +31.3% 53.5%.sup.  +114%

(85) The results of the table above are graphically represented in FIG. 6b.

Example 13

(86) This example is relative to an experiment of internalization of a fluoresceinamine (FA). FIG. 7 shows in particular the uptake of FA into human pancreatic adenocarcinoma cells in five experimental conditions, said conditions being specifically designed in order to compare the effect of the administration of ultrasounds. The tested experimental condition are: FA-NO-US-NO DRUG: administration of FA alone, without administration of any drug and without administration of ultrasounds; FA-LI-NO-US: administration of FA and liposomal irinotecan (LI), without administration of ultrasounds; US-DC=12%: administration of FA and liposomal irinotecan (LI), with the simultaneous administration of pLINFUs with a duty cycle of 12%; US-PRE-DC=1%: administration of pLINFUs with a duty cycle of 1% followed by subsequent administration of FA and liposomal irinotecan (LI); and US-DC=1%: administration of FA and liposomal irinotecan (LI), with the simultaneous administration of pLINFUs with a duty cycle of 1%.

(87) In FIG. 7, it is graphically represented the FA Internalization (%), i.e. the percentage of FA measured after the removal of the surfactant liquid from the cell culture wells, i.e. the percentage of FA inside the cell.

(88) The results of the experiment of this example were also analyzed by Confocal Laser Scanning Microscopy (CLSM) after 2 hours of incubation. FIGS. 8a, 8b, 8c, 8d, 8e show the intake of the FA into human pancreatic adenocarcinoma cells, in the five aforementioned experimental conditions. From FIG. 7, it can be deducted that the higher FA internalization % is obtained with the condition US-DC=1%.

(89) From these images it is possible to see how FA alone (represented in white) is not accumulated in the cells (represented in grey) (FIG. 8a). When FA is administered together with liposomal irinotecan (FIG. 8b) a greater fluorescence density is observed, but only at an extracellular perimembrane level. It can be explained with a greater chemical interaction between the polymeric chains of polyethylene glycol of the liposomal membrane and the FA. In the FIG. 8c and FIG. 8d (US-DC=12% and US-PRE-DC=1%, respectively), the presence of FA inside the cells became evident and the highest level of FA internalization is showed in FIG. 8e corresponding to the US-DC=1% condition. In the condition US-PRE-DC=1% a certain grade of FA internalization is achieved, probably, due to the fact that after administration of ultrasounds, the cells membranes need some tens of seconds to return to the permeability that they have before sonoporation.

Example 14

(90) This example is relative to the study of the opening and the closing of pores in breast cancer cells.

(91) For each point of the graph (FIG. 9a, 9b, 9c, 9d) a sample is sonoporated. All the samples contains the same amount of cells.

(92) On the X axis are reported the times between the end of the cell sonoporation and the addition of the substance whose internalization must be measured in the cells.

(93) The experiment of internalization is the following: Time 0 min: as soon as 1 minute of sonoporation has been completed, a fixed amount of Prohance (contrast medium for magnetic resonance) is added to the cell culture; after 1 minute the cells are isolated. Time 1 min: 1 minute of sonoporation, then after 1 minute a fixed amount of Prohance is added to the cell culture, after another minute the cells are isolated Time n min: 1 minute of sonoporation, then after n minutes a fixed amount of Prohance is added to the cell culture; after another minute the cells are isolated.

(94) These tests, in addition to those performed on other types of cells, have shown that the closing time of cell pores depends on: type of cells; type of drug molecule (Prohance and Tetramer simulated the presence of pharmacological molecules of different composition and size) presence or absence of vector agents (liposomes)