SYSTEM FOR INDUCING SONOPORATION OF A DRUG INTO CANCER CELLS AND METHOD THEREOF

Abstract

A method for controlling ultrasonic waves to induced sonoporation of a drug into cancer cells in a tumor. The method may include accessing configuration data that may include a type of the tumor and a type of drug, and determining values of operational parameters that may be based on the configuration data to define determined values. The operational parameters may include frequency value and duty cycle value. The method may also include determining frequency value which may be based on the type of tumor that may define a determined frequency value, and determining the duty cycle value which may be based on the type of drug and the type of tumor that may define a determined duty cycle value.

Claims

1. A method for controlling ultrasonic waves to induced sonoporation of a drug into cancer cells in a tumor, the method comprising: accessing configuration data that includes a type of the tumor and a type of drug; determining values of operational parameters based on the configuration data to define determined values, the operational parameters including frequency value and duty cycle value; determining frequency value based on the type of tumor to define a determined frequency value; and determining the duty cycle value based on the type of drug and the type of tumor to define a determined duty cycle value.

2. The method according to claim 1, further comprising automatically tuning the determined frequency value and an amplitude of the ultrasonic waves to compensate for attenuation of the ultrasonic waves caused by a medium interposed between at least one ultrasound probe used to emit the ultrasonic waves and the cancer cells.

3. The method according to claim 2, wherein the determined frequency value and the amplitude of the ultrasonic waves are automatically tuned synchronously with the duty cycle value.

4. The method according to claim 3, wherein the at least one ultrasound probe comprises a plurality of ultrasound transducers comprising a first subset of ultrasound transducers and a second subset of ultrasound transducers; wherein the first subset of ultrasound transducers are configured as ultrasound emitters for converting electrical energy into the ultrasonic waves for inducing sonoporation; and wherein the second subset of ultrasound transducers are configured as ultrasound receivers for converting ultrasonic waves reflected by the medium interposed into an electric signal readable by a processor; and wherein the method further comprises: calculating a difference of frequency and amplitude between the ultrasonic waves emitted by the first subset of transducers and the ultrasonic waves received by the second subset of transducers to define calculated differences; and adjusting the amplitude and the frequency of the ultrasonic waves to compensate for the calculated differences.

5. The method according to any claim 3, wherein the at least one ultrasound probe comprises an ultrasound transducer; wherein the ultrasound transducer is configured as an ultrasound emitter for converting electrical energy into the ultrasonic waves for inducing sonoporation; and wherein the ultrasound transducer is further configured as an ultrasound receiver to convert the ultrasonic waves reflected by the medium interposed into an electric signal readable by a processor; and wherein the method further comprises: calculating a difference of frequency and amplitude between the ultrasonic waves emitted and the ultrasonic waves received by the ultrasound transducer to define a calculated difference; and adjusting the amplitude and the frequency of the ultrasonic waves to compensate for the calculated difference.

6. The method according to claim 1, further comprising storing a list of values of frequency and a list of values of duty cycle on a computer readable memory; assigning a value of frequency to a type of tumor; defining a coupled configuration data that includes a type of tumor and a type of drug; and assigning a value of duty cycle to the coupled configuration data.

7. The method according to claim 1, wherein 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 method according to claim 2, wherein the configuration data includes anthropometric measurements being selected from a group consisting of abdominal circumference, body mass index, breast circumference, thorax circumference, and body fat percentage.

9. The method according to claim 8, wherein the frequency value of the operational parameter is determinable based on the anthropometric measurements.

10. The method according to claim 9, further comprising defining a set of coupled configuration data that includes a type of tumor and at least one anthropometric measurement; and assigning a value of the frequency to the set of coupled configuration data.

11. The method according to claim 1, wherein the configuration data includes a grade of the tumor; and wherein the grade of the tumor comprises at least one of primary tumor, primitive tumor, and secondary tumor, and metastasis.

12. The method according to claim 2, wherein the configuration data includes a localization of a secondary tumor.

13. The method according to claim 12, wherein the duty cycle value is determinable based on the localization of the secondary tumor.

14. The method according to claim 12, defining a set of coupled configuration data that includes the type of tumor, the type of drug, the grade of the tumor, and the localization of the secondary tumor; and assigning a value of the duty cycle to the set of coupled configuration data.

15. The method according to claim 1, wherein the type of drug comprises at least one of paclitaxel, paclitaxel albumine, doxorubicin, liposomal doxorubicin, irinotecan, liposomal irinotecan and fluorouracil.

16. The method according to claim 1, wherein the operational parameters comprise an operation time of the ultrasonic waves; wherein a value of the operation time is determinable based on at least one of the type of tumor and the type of drug; and wherein the method further comprises defining a coupled configuration data that includes the type of tumor and the type of drug; and assigning the value of the operation time to the coupled configuration data.

17. The method according to claim 16, wherein the operation time comprises at least one of: a single temporal window in which ultrasonic waves are administered; and at least two temporal windows in which during a first one of the at least two temporal windows, ultrasonic waves are administered, and during a second one of the at least two temporal windows, no ultrasonic waves are administered, so that ultrasonic waves are administered in alternating temporal windows and separated by a single temporal window where no ultrasonic waves are administered.

18. The method according to claim 1, wherein the determined frequency value is between 0.6 MHz and 3 MHz; wherein the determined duty cycle value is below 12%.

19. A computer program product comprising code to control ultrasonic waves to induced sonoporation of a drug into cancer cells in a tumor, the computer program product being operable to: access configuration data that includes a type of the tumor and a type of the drug; determine values of operational parameters based on the configuration data to define determined values, the operational parameters including frequency value and duty cycle value; determine frequency value based on the type of tumor to define a determined frequency value; and determine the duty cycle value based on the type of drug and the type of tumor to define a determined duty cycle value.

20. A computer readable medium to store a computer program code to control ultrasonic waves to induced sonoporation of a drug into cancer cells in a tumor, the computer program product being executable to: access configuration data that includes a type of the tumor and a type of the drug; determine values of operational parameters based on the configuration data to define determined values, the operational parameters including frequency value and duty cycle value; determine frequency value based on the type of tumor to define a determined frequency value; and determine the duty cycle value based on the type of drug and the type of tumor to define a determined duty cycle value.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Some embodiments of the present invention are illustrated as an example and are not limited by the figures of the accompanying drawings, in which like references may indicate similar elements.

[0043] 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 ?M, 10 ?M, 0.5 ?M);

[0044] 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 ?M, 10 ?M, 0.5 ?M);

[0045] 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 ?g/ml, 500 ?g/ml, 100 ?g/ml);

[0046] 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 ?g/ml, 500 ?g/ml, 100 ?g/ml);

[0047] 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 ?M, 10 ?M, 0.5 ?M);

[0048] 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 ?M, 10 ?M, 0.5 ?M);

[0049] 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 ?g/ml, 500 ?g/ml, 100 ?g/ml);

[0050] FIG. 4b shows the percentage of cell death obtained by in-vitro administering liposomal doxorubicin 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 ?g/ml, 500 ?g/ml, 100 ?g/ml);

[0051] 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 ?M, 10 ?M, 0.5 ?M);

[0052] 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 ?M, 10 ?M, 0.5 ?M);

[0053] 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 ?M, 10 ?M, 0.05 ?M);

[0054] 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 ?M, 10 ?M, 0.05 ?M);

[0055] 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;

[0056] 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;

[0057] 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;

[0058] 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%;

[0059] 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%;

[0060] 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%;

[0061] FIG. 9a shows the kinetics of cellular membrane pore's closure with Prohance (Gd3+);

[0062] FIG. 9b shows the kinetics of cellular membrane pore's closure with Prohance and liposomes;

[0063] FIG. 9c shows the kinetics of cellular membrane pore's closure with Tetramer;

[0064] FIG. 9d shows the kinetics of cellular membrane pore's closure with Tetramer and liposomes.

DETAILED DESCRIPTION OF THE INVENTION

[0065] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Those of ordinary skill in the art realize that the following descriptions of the embodiments of the present invention are illustrative and are not intended to be limiting in any way. Other embodiments of the present invention will readily suggest themselves to such skilled persons having the benefit of this disclosure. Like numbers refer to like elements throughout.

[0066] Although the following detailed description contains many specifics for the purposes of illustration, anyone of ordinary skill in the art will appreciate that many variations and alterations to the following details are within the scope of the invention. Accordingly, the following embodiments of the invention are set forth without any loss of generality to, and without imposing limitations upon, the claimed invention.

[0067] In this detailed description of the present invention, a person skilled in the art should note that directional terms, such as above, below, upper, lower, and other like terms are used for the convenience of the reader in reference to the drawings. Also, a person skilled in the art should notice this description may contain other terminology to convey position, orientation, and direction without departing from the principles of the present invention.

[0068] Furthermore, in this detailed description, a person skilled in the art should note that quantitative qualifying terms such as generally, substantially, mostly, and other terms are used, in general, to mean that the referred to object, characteristic, or quality constitutes a majority of the subject of the reference. The meaning of any of these terms is dependent upon the context within which it is used, and the meaning may be expressly modified.

[0069] A preferred embodiment of the system of the present invention comprises: [0070] a generator configured to provide electrical energy at an ultrasound frequency; [0071] an ultrasound probe electrically connected to the generator and configured to [0072] 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; [0073] 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 [0074] a processor configured to: [0075] 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 [0076] control the generator and the ultrasound probe to operate according to said determined values.

[0077] 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: [0078] 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.

[0079] The power density of ultrasound waves generated by the preferred embodiment of the system is less than 3 W/cm.sup.2. 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. 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.

[0080] 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: [0081] primary tumor, the duty cycle is determined on the basis of the type of drug and the type of tumor. [0082] 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.

[0083] 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. 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%.

[0084] 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%. 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.

[0085] 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. 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. 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.

[0086] 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. 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: [0087] one temporal window in which ultrasounds are administered; or [0088] 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.

[0089] In this case, 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. 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: [0090] 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 [0091] adjust the values of the amplitude and the frequency of the ultrasound emitted, in such a way to compensate said differences.

[0092] 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: [0093] calculate the differences of frequency and amplitude between the ultrasound waves emitted and the ultrasound waves received by said trasnducer; and [0094] adjust the values of the amplitude and the frequency of the ultrasound emitted, in such a way to compensate said differences.

EXAMPLES

[0095] 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. In particular the following cellular lines were employed: [0096] MCF-7: human breast ductal carcinoma; [0097] MDA-MB-231: estrogen independent human breast adenocarcinoma; [0098] MiaPaCa-2: human pancreatic adenocarcinoma.

[0099] The following table summarized the tested combinations of cellular lines and type of drug:

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

[0100] For each combination the percentage of cell death was measured in three main experimental conditions: [0101] administration of the drug (hereinafter named NO-US condition); [0102] 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; [0103] 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 [0104] 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).

[0105] 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 5 seconds.

[0106] The results are listed in tables below and showed in the FIGS. 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b. 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: [0107] duty cycle=12% (US-DC=12%) [0108] duty cycle=9% (US-DC=9%)

[0109] For the cellular line MiaPaCa-2, the results relative to the following values of duty cycle are showed: [0110] duty cycle=12% (US-DC=12%) [0111] duty cycle=1% (US-DC=1%).

Example 1

[0112] In the following table the percentage of cell death are reported for three different concentration of paclitaxel administered to cells of MCF-7 in three conditions: [0113] 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; [0114] 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; [0115] 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.

TABLE-US-00002 Paclitaxel - MCF -7 Cell Increasing % Cell Increasing % Cell Increasing % Death % vs NO-US Death % vs NO-aUS Death % vs NO-US Total concentration Total concentration Total concentration 50 ?M 10 ?M 0.5 ?M indicates data missing or illegible when filed

[0116] The results of the table above are graphically represented in FIG. 1a.

Example 2

[0117] In the following table the percentage of cell death are reported for three different concentration of paclitaxel albumine administered to cells of MCF-7 in three conditions: [0118] NO-US: a first administration of a first dose of paclitaxel albumine without administration of ultrasounds (NO-US) followed by a second administration of a second dose of paclitaxel albumine; [0119] 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; [0120] 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 seconds.

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 ?M 10 ?M 0.5 ?M 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%

[0121] The results of the table above are graphically represented in FIG. 1b.

Example 3

[0122] 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: [0123] 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; [0124] 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; [0125] 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.

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 ?g/ml 500 ?g/ml 100 ?g/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%

[0126] The results of the table above are graphically represented in FIG. 2a.

Example 4

[0127] 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: [0128] 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; [0129] 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

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 ?g/ml 500 ?g/ml 100 ?g/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%
administration of pLINFUs for other 20 seconds; [0130] 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.

[0131] The results of the table above are graphically represented in FIG. 2b.

Example 5

[0132] In the following table the percentage of cell death are reported for three different concentration of paclitaxel administered to cells of MDA-MB-231 in three conditions: [0133] 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; [0134] 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; [0135] 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.

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 ?M 10 ?M 0.5 ?M 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%

[0136] The results of the table above are graphically represented in FIG. 3a.

Example 6

[0137] 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: [0138] 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; [0139] 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; [0140] 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 seconds.

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 ?M 10 ?M 0.5 ?M 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%

[0141] The results of the table above are graphically represented in FIG. 3b.

Example 7

[0142] 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: [0143] 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; [0144] 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; [0145] 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.

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 ?g/ml 500 ?g/ml 100 ?g/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%

[0146] The results of the table above are graphically represented in FIG. 4a.

Example 8

[0147] 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: [0148] 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; [0149] 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; [0150] 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.

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 ?g/ml 500 ?g/ml 100 ?g/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%

[0151] The results of the table above are graphically represented in FIG. 4b.

Example 9

[0152] 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: [0153] 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; [0154] 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; [0155] US-DC=1%: first administration of a first dose of paclitaxel 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.

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 ?M 10 ?M 0.5 ?M 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%

[0156] The results of the table above are graphically representewd in FIG. 5a.

Example 10

[0157] In the following table the percentage of cell death are reported for three different concentration of paclitaxel albumine administered to cells of MiaPaCa2 in three conditions: [0158] NO-US: a first administration of a first dose of paclitaxel albumine without administration of ultrasounds (NO-US) followed by a second administration of a second dose of paclitaxel albumine; [0159] 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; [0160] 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.

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 ?M 10 ?M 0.5 ?M 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%

[0161] The results of the table above are graphically represented in FIG. 5b.

Example 11

[0162] 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: [0163] 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; [0164] 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; [0165] 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.

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 ?M 1 ?M 0.05 ?M 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%

[0166] The results of the table above are graphically represented in FIG. 6a.

Example 12

[0167] 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: [0168] 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; [0169] 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; [0170] 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.

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 ?M + 5 FU 100 ?M 1 ?M + 5 FU 10 ?M 0.05 ?M + 5 FU 1 ?M 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%

[0171] The results of the table above are graphically represented in FIG. 6b.

Example 13

[0172] 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: [0173] FA-NO-US-NO DRUG: administration of FA alone, without administration of any drug and without administration of ultrasounds; [0174] FA-LI-NO-US: administration of FA and liposomal irinotecan (LI), without administration of ultrasounds; [0175] US-DC=12%: administration of FA and liposomal irinotecan (LI), with the simultaneous administration of pLINFUs with a duty cycle of 12%; [0176] US-PRE-DC=1%: administration of pLINFUs with a duty cycle of 1% followed by subsequent administration of FA and liposomal irinotecan (LI); and [0177] US-DC=1%: administration of FA and liposomal irinotecan (LI), with the simultaneous administration of pLINFUs with a duty cycle of 1%.

[0178] 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. 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%. 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).

[0179] 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

[0180] This example is relative to the study of the opening and the closing of pores in breast cancer cells. For each point of the graph (FIG. 9a, 9b, 9c, 9d) a sample is sonoporated. All the samples contains the same amount of cells. 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. The experiment of internalization is the following: [0181] 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. [0182] 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 [0183] 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.

[0184] These tests, in addition to those performed on other types of cells, have shown that the closing time of cell pores depends on: [0185] type of cells; [0186] type of drug molecule (Prohance and Tetramer simulated the presence of pharmacological molecules of different composition and size) [0187] presence or absence of vector agents (liposomes)

[0188] Some of the illustrative aspects of the present invention may be advantageous in solving the problems herein described and other problems not discussed which are discoverable by a skilled artisan.

[0189] While the above description contains much specificity, these should not be construed as limitations on the scope of any embodiment, but as exemplifications of the presented embodiments thereof. Many other ramifications and variations are possible within the teachings of the various embodiments. While the invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best or only mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Also, in the drawings and the description, there have been disclosed exemplary embodiments of the invention and, although specific terms may have been employed, they are unless otherwise stated used in a generic and descriptive sense only and not for purposes of limitation, the scope of the invention therefore not being so limited. Moreover, the use of the terms first, second, etc. do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another. Furthermore, the use of the terms a, an, etc. do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item.

[0190] Thus the scope of the invention should be determined by the appended claims and their legal equivalents, and not by the examples given.

[0191] The claims in the instant application are different than those of the parent application or other related applications. Applicant therefore rescinds any disclaimer of claim scope made in the parent application or any predecessor application in relation to the instant application. Any such previous disclaimer and the cited references that it was made to avoid, may need to be revisited. Further, any disclaimer made in the instant application should not be read into or against the parent application.