CN100340302C - Apparatus and method for treating liquid media with ultrasound to prevent hyperproliferative or infected cell growth - Google Patents

Abstract

The invention discloses a device and a method for treating, preventing the growth of and suppressing over-proliferation, undifferentiation or virus-infected cells in a liquid medium by using high-frequency, low-energy ultrasound.

Description

Apparatus and method for treating liquid media with ultrasound to prevent hyperproliferative or infected cell growth

Technical field

The present invention relates to the use of high frequency, low energy ultrasound to treat liquid media. In specific embodiments, the devices and methods of the present invention are capable of significantly inducing apoptosis in cells suspended in physiological fluids.

Background technique

Cells can be damaged by exposure to ultrasound. For example, ultrasound can cause irreversible cell damage and induce destructive changes in cell membranes. Some reports have suggested that cavitation caused by the collapse of bubbles generated from acoustic pressure fields may be responsible for the cell damage induced by ultrasonic radiation. It has also been suggested that single-strand DNA breaks can be induced by cavitation through the action of residual hydrogen peroxide.

The application of ultrasound in cancer therapy has become an important aspect. Ultrasound is often used in combination with heat, as well as light, radiation, and chemotherapy. Malignant cells are known to be more sensitive to these combination treatments than their normal counterparts. Direct radiative action (e.g., ultrasound, laser, light) on certain molecules (e.g., classical photosensitizers and sonosensitizers) produces highly reactive species such as singlet oxygen, superoxide radicals, hydrogen peroxide, and fatty acid radicals Oxygen species that selectively act on malignant cells play an important role in cancer therapy.

Depending on the source of the radiation, the above treatment is called PDT (photoenergy therapy) or SDT (sound energy therapy) if by ultrasound or sonoluminescence. Both treatments require prior addition of a photosensitizer. Although the overall effects produced by SDT and PDT differ in terms of cell viability, both SDT (especially involving ultrasonic cavitation activity) and PDT produce reactive oxidative species and lead to a reduction in intracellular thiol levels. During PDT with ultraviolet-A (UVA), singlet oxygen generation can induce apoptosis of T helper cells, but this effect must depend on the initial concentration of photosensitizer (PS) and the local oxygen content. For SDT, cell lysis is the dominant phenomenon due to the high energy involved, which may mask other effects on viable cells.

US Patent No. 4,971,991 to Umemura et al. discloses the use of ultrasound to treat tumor cells, but relies on high ultrasound power levels and does not describe the use of microbubbles. Other patents, such as U.S. Patent No. 5,215,680 to D'Arrigo, describe ultrasound and microbubbles that rely on the cavitation and thermal effects of ultrasound to treat tumors, as opposed to treating cancer cells alone, by treatment duration and number to determine the scope of its treatment. This type of treatment uses high energy and prolonged radiation, mainly producing lysis and necrosis of cells. See Kondo, Cancer Letters 178(1), 63-70, (2002).

A brief description of the drawings

Fig. 1 shows an embodiment of the ultrasonic treatment device according to the present invention.

Figure 2 shows three views of the device for treating hyperproliferative cells in suspension using ultrasound and microbubbles. The leftmost figure is a top view of the device, the middle figure is a main view, and the rightmost figure is a side view of the device.

Figure 3 is a bar graph showing the effect of sonication on cellular glutathione levels. Data are expressed as percentage of cells exhibiting glutathione levels relative to untreated cells. Values are mean ± standard error of 3 independent experiments.

Figure 4 is a bar graph showing the effect of high-frequency ultrasound on cellular caspase-3 activity.

Figure 5 is a histogram showing the effect of radiation on the cloning efficiency of K562 cells. The results are expressed as the mean ± standard error of 3 independent experiments.

Fig. 6 is a histogram showing the percentage of K562 apoptotic cells 5 hours after 1 or 3 times of ultrasonic treatment. After labeled with Annexin-V, the apoptosis ratio was measured by flow cytometry, and the results were expressed as the mean ± standard error of 7 independent experiments.

Figure 7 shows a dot plot of phosphatidylserine distribution as a function of time and successive sonication treatments. Results from one representative experiment are expressed as percentage of cells labeled with Annexin-V-FITC.

Figure 8 is a histogram showing the apoptotic effects of ultrasonic treatment on normal monocytes (MNC) and leukemia cells (Nalm-6, KGla, HL-60 cells and primary leukemia cells from 5 patients). Results are mean ± standard error of 5 independent experiments.

Detailed description of the invention

Apoptosis, or programmed cell death, is a normal part of the development and health of multicellular organs. Apoptosis ensures tissue homeostasis during development, host defense, aging and in response to a variety of signals including gamma radiation and UV exposure. The death of cells in response to various stimuli, especially in the process of apoptosis, which is carried out in a controlled manner. This makes apoptosis different from another form of cell death called necrosis, in which uncontrolled cell death leads to the lysis of cells, an inflammatory response and potentially various health problems. In contrast, apoptosis is a process by which a cell can play an active role in its own death, which is why apoptosis is often referred to as cell suicide.

When specific signals are received indicating apoptosis, a series of specific biochemical and morphological changes usually occur in the cell. For example, a family of proteins called caspases are often activated early in apoptosis. These proteins break down or cleave key cellular substrates necessary for normal cellular function, including structural proteins in the cytoskeleton and nuclear proteins such as DNA repair enzymes. Caspases also activate other degrading enzymes such as DNase, which cleaves DNA in the nucleus. Typically, the death of apoptotic cells is characterized by early changes in the nuclear envelope, chromatin condensation, and DNA fragmentation. These biochemical changes result in morphological changes in the cells.

The teachings of the present invention relate to devices and methods for suppressing, preventing the growth of, or eliminating hyperproliferative cells (eg, tumor cells) present in a liquid medium. In more specific embodiments, the present invention provides methods and devices that induce apoptosis of hyperproliferative cells in suspension, such as physiological fluids. Physiological fluids that may be processed include blood, plasma, serum, and cerebrospinal fluid, which can be extracted from and/or administered to animals including mammals, humans, and the like.

Low-energy, high-frequency ultrasonic treatment according to the teachings of the present invention can induce apoptosis in hyperproliferative cells. These effects include, for example, effects on mitochondrial membranes (decrease in mitochondrial potential), loss of phosphatidylserine asymmetry, stimulated oxidation of membrane lipids (decrease in cellular GSH levels), morphological changes, DNA fragmentation, plasma membrane loss etc. In addition, low-energy ultrasound-induced apoptosis can be involved in the activation of caspase-3 in cells, the protein degradation of the caspase substrate PARP, and the regulation of the bcl-2/bax ratio.

Specific experiments (see Examples) have demonstrated rapid induction of apoptosis with a limited amount of necrosis.

Equipment and methods

Embodiments of the apparatus that can be used to carry out the methods of the present invention can be found in U.S. Provisional Application No. 60/423,368, U.S. Application No. 10/358,445, and U.S. Patent No. 6,540,922 to Cordemans et al., each of which is All are incorporated herein by reference. Methods of treating hyperproliferative cells can be performed using the devices disclosed in the present invention. A particular embodiment of an apparatus that can be used to treat liquid media such as aqueous media (eg, physiological fluids) is shown in FIG. 1 . In certain embodiments, the treated fluid contains hyperproliferative cells. In other embodiments, the fluid being treated can be, for example, a physiological fluid suspected of containing hyperproliferative cells after diagnosis. Incompletely differentiated cells such as stem cells and solutions containing viruses and/or virus-infected cells can also be treated. Examples of viruses that can be treated include eg HIV, HCV, HBV, herpes virus, hantavirus, influenza virus and Ebola virus.

Referring to Figure 1, the device according to the invention comprises a compartment 2, preferably of cylindrical or rectangular cross-section. In a particular embodiment, said compartment 2 is connected to a reservoir (not shown) filled with a liquid medium to be treated. In other embodiments (eg, when human or animal physiological fluids are handled), the devices provided herein do not contain a reservoir directly attached to the human or animal body. These embodiments include those in which the physiological fluid is extracted and/or administered (eg, reinjected) during ex vivo treatment of the human and other animal body. Accordingly, animals such as humans can be replaced by any "reservoir" referred to in the present invention.

In other embodiments, the apparatus shown in Figure 2 is capable of processing hyperproliferative cell suspensions. In this embodiment, the air inlet tube 3 is used as a microbubble emitter 3 to emit microbubbles 5 into said hyperproliferative cell suspension 22 contained in a compartment (or poach) 20 . The compartment (or poach) 20 containing the cell suspension 22 may be immersed in a water bath 24 such as an incubator.

In a further embodiment, the compartment 2 contains (e.g., along its walls or near its bottom) one or more high-frequency ultrasonic transmitters 1 that transmit ultrasonic waves 4 into the compartment 2 (favorably The ground should be towards the center of this compartment 2). In other embodiments, the container also has one or more microbubble emitters 3 to emit gas microbubbles 5, which are designed to emit said gas microbubbles 5 into the ultrasonic waves 4 emitted into the compartment 2 area.

The term "microbubble" used in the present invention specifically refers to bubbles with an average diameter of less than 1 mm. In some embodiments, the diameter is less than or equal to 50 μm. In yet other embodiments, the microbubbles are less than 30 μm in diameter. In certain embodiments, the microbubbles are selected from air, oxygen and ozone or mixed microbubbles thereof. To reduce operating costs, it would be advantageous to be able to use non-ozone microbubbles such as air microbubbles. Advantageous embodiments of the invention do not rely on generating a thermal effect to treat cells. Although in certain embodiments the use of stabilized microvesicles can effectively treat cells, in preferred embodiments the use of stabilized microvesicles is not required. An example of a stable microbubble is a lipid boundary microbubble.

The term "hyperproliferative cell" means a cell that differentiates, regenerates, or proliferates at a relatively high level, and may include cancer cells (eg, leukemia cells), precancerous cells, tumor cells, myeloid cells, and totipotent cells.

In certain embodiments, the term "fluid medium" relates to a physiological fluid that can be administered to and/or extracted from a human or animal. In specific embodiments, physiological fluids are processed prior to reinjection (eg, extracorporeal processing followed by reinfusion). In certain embodiments, the term "physiological fluid" includes, but is not limited to, blood, serum, cerebrospinal fluid, cerebrospinal fluid, plasma, and the like. US Patent No. 5,401,237 to Tachibana et al., which is hereby incorporated by reference in its entirety, describes the extraction and readministration of physiological fluids.

In specific embodiments, the methods and devices described herein comprise treating hyperproliferative cells with low energy high frequency ultrasound. The term "high frequency" means frequencies above 100 kHz and up to several MHz. In certain embodiments, the high frequency used is between 200 kHz and 20 MHz. In various embodiments, the ultrasonic frequency is selected between 200 kHz and 10 MHz. In a preferred embodiment, the frequency used is between 200 kHz and 1.8 MHz.

In various embodiments of the apparatus according to the invention, the microbubble emitter 3 for emitting gas microbubbles 5 is placed at the bottom 11 of the compartment 2 (i.e. at the base of the compartment 2) such that the microbubbles Bubbles move by natural rise or by entrainment of gas in the liquid flow.

In further embodiments, the devices and methods described herein induce apoptosis in hyperproliferative cells. Healthy cells have been found to be less sensitive to high-frequency ultrasound than leukemia cells. This behavioral difference between healthy and leukemic cells is not related to differences in the distribution of endogenous photosensitizers, but may be caused by changes in underlying cellular mechanisms such as p53 status, signaling pathways, and tolerance to oxidative stress. In particular, apoptosis of cancer cells (eg, leukemia cells), precancerous cells, tumor cells, myeloid cells, totipotent cells, and the like can be induced.

While the teachings of the present invention are not to be limited in any way by their precise mechanism of action, in more specific embodiments, the devices and methods described herein are capable of producing such substances as ROS (reactive oxygen species), H ^{, .} Free radicals of OH and HOO ^· which can also form H ₂ O ₂ , this molecule and/or these free radicals are toxic to hyperproliferative cells and thus inactivate and/or destroy them. Lipid peroxidation products resulting from oxidative stress in the ultrasound regime may also be potential participants in this biological mechanism.

Although the evidence does not support the formation of singlet oxygen during acoustic energy therapy, these data are only consistent with prolonged and "high energy" ultrasound exposure, which can be directly due to pyrolysis or due to the formation of H ^· or ^· OH radical reaction leads to accumulation of sensitizer derived free radicals.

The substances produced using the disclosed method and apparatus are understood to be derived from reactions induced by the action of high frequency ultrasound on water molecules, most likely the following reactions (especially in the presence of oxygen):

H ₂ O→H ^· +OH ^·

H ⁺ O ₂ → ^HOO

HOO ^{+ HOO} → H ₂ O ₂ +O ₂ ,

^· OH+ ^· OH→H ₂ O ₂

Advantageously, the energy required to produce these toxic substances is reduced if microbubbles are present during the process, as described in the present invention. In certain embodiments, the generator is designed to be able to provide an energy of less than 1 W/cm ² to said ultrasound transmitter. In preferred embodiments, the energy provided is about 0.5 W/ ^cm2 or less, or in many more advantageous embodiments, the energy is about 0.25 W/ ^cm2 or less. In an advantageous embodiment, this power level applied to the transmitter dissipates less than 30 mW/cm ³ of energy in said volume of physiological fluid. In some embodiments, the energy consumption is about 7 mW/cm ³ .

While in some embodiments the ultrasound can be administered continuously, in other embodiments the ultrasound is administered intermittently in an on/off cycle. Based on cell volume, cell type, and other relevant variables, one skilled in the art can determine effective on/off cycle times.

Further embodiments of the teachings of the present invention relate to the treatment of hyperproliferative cells in suspension, rather than tumors or tumor bodies. In these embodiments, the teachings of the present invention do not rely on microvesicles to concentrate or pool at specific tumor sites. This enables the treatment of unwanted hyperproliferative cells that do not clump together.

Another advantage of the method and device provided by the present invention is that the hyperproliferative cells can be effectively treated in a relatively short period of time. In a specific embodiment, hyperproliferative cells can be treated within 1 minute. In a more specific embodiment, said cells are treated within 30 seconds, eg including between 5-20 seconds.

As is known in the art, the biophysical modes of ultrasound action are classified as either thermal, cavitational, or athermal and non-cavitational. It should be noted that using the above energy ranges and shortening the treatment time avoids significant heating of the fluid and/or cells, which allows thermally induced cell death not to occur. By way of example, athermal treatments include treatments performed at less than 40°C, 35°C, and 30°C. The power level is likewise a level at which no appreciable degree of cavitation occurs, thereby substantially avoiding loss of cell membranes caused by ultrasound.

Recently, it was found that injection of microbubbles into the ultrasonic region at higher ultrasonic energies increases the sonoluminescence phenomenon through the superposition of microbubbles on ultrasonically induced cavitation bubbles, which can increase the number of excited and toxic substances. This phenomenon can be observed at the macroscopic level when sonication is synergistically combined with the presence of appropriately sized microbubbles.

In a further embodiment, since it was observed that the treatment system functions by the diffusion of in situ formed products (such as formed free radicals _{and H2O2} ) to the reservoir 6 of the aqueous medium to be treated, the present The device and method provided by the invention have the advantage of not needing to inject ultrasonic waves into specific areas.

In a further embodiment, said one or more ultrasound 4 transmitters 1 in said device of the invention are oriented so as not to generate any standing wave phenomena. For example, in certain embodiments, one or more ultrasound emitters are oriented obliquely (acute angles not perpendicular to this axis 9) with respect to the axis 9 of the compartment 2 and with respect to the flow of liquid and microbubbles 5 (See Figure 1). This feature allows all microvesicles 5 in compartment 2 to be treated in a statistically consistent manner without creating a zone of quiescence in compartment 2 .

The apparatus and method according to the present invention may include transmitting gas microbubbles having an average diameter of less than 1mm into the high frequency ultrasonic region of the liquid medium being treated. In some embodiments, the diameter of the microbubbles is less than or equal to 50 μm. In other embodiments, the microvesicles are less than 30 μm in diameter. In certain embodiments, the microbubbles are selected from air, oxygen and ozone microbubbles. In other embodiments, the microbubbles are non-ozone microbubbles.

According to other embodiments, the devices and methods of the present invention comprise a light emitter 12 (ie an electromagnetic radiation emitter) that emits radiation, mainly of frequencies in the visible range, into the region of the ultrasound waves 4 in the compartment 2 . However, for certain applications, in order to remove certain hyperproliferative cells, it is advantageous to emit electromagnetic radiation of predominantly non-visible frequencies, such as ultraviolet (e.g., UVA, UVB or UVC types), infrared, laser, microwave, etc. .

It has recently been unexpectedly discovered that radiation comprising microbubbles emitted into the region, combined with ultrasound and optionally light radiation, is particularly effective in inactivating and removing hyperproliferative cells in liquid media such as physiological fluids. The luminescence phenomenon can promote the production of hyperactive oxidative species such as peroxide radicals or singlet oxygen, which can lead to a series of biochemical reactions that are hypertoxic to certain hyperproliferative cells. In an advantageous embodiment, said radiation is emitted intermittently in an on/off cycle. In a more specific embodiment, the on/off cycle period is about 5.5 ms/3 ms.

It is known that luminescence can be produced in the presence of so-called sensitizing molecules (eg, photosensitizers and sonosensitizers), leading to an antitumor effect in certain cancer cells. Such molecules include: porphyrins, chlorine, tetracyclines, methylene blue, fluorescein, acridine, rhodamine, and the like. These active agents can be injected into the organism or administered orally and subsequently activated by sonoluminescence. Upon activation, these agents generate singlet oxygen, which in turn plays a fundamental role, especially in biochemical processes arising from oxidative stress. In particular, singlet oxygen is capable of oxidizing various cellular components such as proteins, lipids, amino acids and nucleic acids.

In other embodiments, solid particles and solid surfaces can be used to synergistically enhance luminescence and/or emission of radiation. These solids include, for example, _TiO2 , clays and ceramics.

Many embodiments relate to devices and methods that do not require additional chemical products such as photosensitizers and/or sonosensitizers to suppress, prevent growth of and/or remove hyperproliferative cells in physiological media. Due to the unexpected discovery that certain hyperproliferative cells (eg leukemia cells) in physiological fluids (eg blood) that already contain these photosensitizing molecules are capable of producing luminescence in situ, adding photosensitizers or sonosensitizers to said fluids Medium is not always necessary.

Although the device and method described in the present invention can be used in combination with other drugs such as photosensitizers, sonosensitizers, chemotherapeutic agents, antibiotics, and antiviral drugs, it should be pointed out that the method for treating hyperproliferative cells provided by the present invention And the effectiveness of the device is not dependent on the use of other chemicals, reagents or drugs. Thus, the methods and devices of the present invention can be used in the absence of other substances, including chemicals, reagents, hormones, peptides, proteins, nucleic acids, hydrocarbons, DNA vaccines, angiogenesis stimulators or drugs. In more specific embodiments, the teachings of the present invention do not rely on cellular uptake of these substances.

In particular, the effects obtained by the teachings of the present invention can be achieved without the need for typical photosensitizers and sonosensitizers. The physiological effects obtained with techniques such as PDT depend simultaneously on the dose of radiation used, the nature of the photosensitizers used, their content and their distribution. While sensitizers are required to perform conventional treatments, the teachings provided herein do not require sensitizers, thus relatively simplifying the methods and apparatus described.

In certain embodiments, the net effect of sonication is associated with locally higher levels of endogenous photosensitizer in the structure. For example, endogenous photosensitizers are mainly distributed in membrane structures of microsomes such as lysosomes, mitochondria, nuclear membranes, Golgi apparatus, and endoplasmic reticulum, whose opposite surfaces account for nearly 50% of the cell membrane surface.

In some embodiments, the apparatus and methods of the present invention include a pump for circulating the liquid medium, and one or more means for restoring the liquid medium present in the liquid medium, preferably by filtration, centrifugation or sedimentation (eg, spinning, etc.). device of hyperproliferative cells. In certain embodiments, said pump for recovery and/or recovery device is placed between said reservoir (or animal) containing said liquid medium to be treated and said compartment 2 .

In certain embodiments, the devices and methods described herein can be used to extract physiological fluids (eg, blood) from individuals suspected of having (eg, diagnosed with) cancer (eg, leukemia). After extraction, the physiological fluid can be treated with high-frequency low-energy ultrasound and gas microbubbles with a diameter of less than 1 mm. In certain embodiments, the methods induce apoptosis of the cancerous cells (eg, leukemia). After processing the physiological fluid such that the hyperproliferative cells are sufficiently suppressed, prevented from growing, or removed, the fluid can then be administered to the individual. These methods are performed in a similar manner to other methods of extracorporeal treatment and reinfusion, such as hemodialysis.

An individual undergoing blood treatment may be connected to one of the devices according to the invention. According to certain embodiments, the individual's blood flow may be connected to the ultrasound device of the present invention through a fistula inside the individual's arm. This involves the surgical connection between arteries and veins. When they are connected, the stronger blood flow from the arteries causes the veins to thicken. A needle may be inserted into the enlarged vein to connect the individual to the ultrasound device.

Another way to provide access to blood flow is to insert an internal graft. In this procedure, the artery is surgically connected to the vein with a short special tube placed under the skin through which a needle can be inserted.

In other embodiments, certain intravenous catheters are used when rapid access to the blood stream is necessary, or when, for example, the veins in the arm are too small to provide sufficient blood for sonication . During this procedure, a flexible tube is surgically inserted into a large vein in the neck or near the collarbone. In some embodiments, the method is temporary until a permanent entry site is in place.

Individuals that can be treated according to the methods of the present invention include any animal, such as mammals including humans, mice, monkeys, dogs, pigs, and the like.

In a further embodiment, the devices and methods of the present invention utilize low-energy, high-frequency ultrasound to prevent, treat or suppress hyperproliferative cells by inducing apoptosis of cells (eg, leukemia cells). Induction of apoptosis in hyperproliferative cells can lead to a series of characteristic consequences, including reduction of mitochondrial potential, loss of phosphatidylserine asymmetry, morphological changes, DNA fragmentation, loss of plasma membrane, etc. In addition, apoptosis induced by low-energy ultrasound can include activation of caspase-3 in cells, protein degradation of caspase substrate PARP, and adjustment of bcl-2/bax ratio.

Additional approaches include the use of ultrasound-induced sonochemiluminescence to initiate apoptosis by excitation of photosensitized singlet oxygen production by direct light radiation. Under typical ultrasonic irradiation conditions, the direct destructive cavitation plays a dominant role in the sonoluminescence, which is rather weak in the absence of intervening air/liquid interfaces in the medium. Accordingly, to enhance luminescence in cavitation, it is advantageous to use microbubbles in combination with ultrasonic waves.

The following examples describe the use of high frequency low energy sonication and various assays showing the presence of apoptosis.

Example 1

      Cell preparation and high-frequency ultrasonic treatment

Human leukemia cell lines (K562, Nalm-6, KGla, and HL-60) obtained from the American Type Culture Collection (ATCC, Rockville, MD, U.S.) were cultivated in a medium containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% L-glutamine (Gibco) in RPMI-1640 (Bio Whittaker, Walkersville, MD, USA). Leukemic cells were collected, resuspended in phosphate buffered saline (PBS, pH=7.2, Gibco), and used immediately for experiments. Heparinized venous blood was obtained from healthy volunteers and leukemia patients. Monocytes were isolated by Ficoll-Hypaque density gradient centrifugation (International Medical Products, Brussels, Belgium).

Sonication of the cells is described below. Human leukemia cell lines (K562, HL-60, KGla and Nalm-6 were treated at different exposure times by ultrasonic waves with a frequency of 1.8MHz, acoustic energy of 7mW/mL, and a radiation cycle (on/off) of 5.5ms/3ms. ), primary leukemia cells and normal monocytes.

After 18 hours of incubation in an incubator (37°C and 5% CO2 ₎ , the viability of the cells can be successfully detected by trypan blue exclusion assay. Additional experiments performed on the treated cells are described in detail in the Examples below. Apoptosis was assessed by cell morphology, phosphatidylserine exposure, and DNA fragmentation. Mitochondrial potential, glutamine content, caspase-3 activation, PARP degradation and bcl-2/bax ratio were detected by flow cytometry. Cloning efficiency was assessed in methylcellulose.

Example 2

The effect of high-frequency ultrasound on DNA fragmentation

DNA fragmentation is associated with apoptosis. Cells containing degraded DNA were quantified according to the method described by Nicoletti, I. et al. J Immunol Methods 139(2):271 (1991) and Apotarget Quick DNA Ladder Detection Kit (Biosource). Cell pellets ( ¹⁰⁶ cells) were resuspended in 20 L of lysis buffer and DNA was extracted according to the manufacturer's instructions. DNA was analyzed after separation by gel electrophoresis (1% agarose). As a positive control, cells were irradiated with UV light by placing the plate directly under a UV illuminator for 10 minutes (intensity 5 mW/cm ² ). Subsequently, cells were incubated at 37°C for 5 and 18 hours before assessing apoptosis.

After permeabilization, cells were incubated in a solution containing PI and RNAse (Coulter DNA-prep reagent). The vials were left overnight at 4°C in the dark prior to flow cytometry to identify the sub-GO peak corresponding to apoptosis.

A typical nucleosomal DNA ladder pattern was observed in DNA samples from cells treated with UV light (positive control) and sonication. There was little visible internal nucleosomal DNA degradation after 5 hours of sonication but became clearly visible after 18 hours (results not shown). Furthermore, an increase in the number of nuclei with fragmented DNA was observed by staining with PI after 5 hours of treatment. Specifically, 15% of the treated cells had fragmented DNA compared to 2% of untreated cells (data not shown).

Example 3

Effect of high frequency ultrasound on mitochondrial transmembrane potential

In some types of apoptosis, an early disruption of the mitochondrial transmembrane potential (ΔYm) prior to the development of DNA fragmentation is observed. The effect of high-frequency ultrasonic treatment on ΔYm was determined by the following analysis.

Immediately after cell treatment, the mitochondrial potential was estimated by binding to the cationic fluorescent dye _DiOC6 according to the procedure published by A. Macho, et al., Blood. 86(7):2481 (1995).

Briefly, K562 cells (10 ⁶ /mL) were incubated with 2.5 nmol/L 3,3′-dihexyloxycarbocyanine (DiOC6; Molecular Probes, Eugene, OR) at 37°C for 15 min, followed by Analyzed by flow cytometry.

Sonication was accompanied by an increase in cell populations showing lower ΔYm (results not shown). A population of cells showing reduced binding to _DiOC6 was found 30 min after treatment, and the reduction in mitochondrial potential was very clear 5 h after sonication, with more than 50% of the cells having a lower ΔYm. These results provide evidence of apoptosis.

Example 4

The effect of high-frequency ultrasound on the level of glutathione in cells

It has been shown that there is a loss of glutathione during apoptosis. Analysis for the determination of cellular glutathione content was performed after sonication. Intracellular glutathione levels were determined using the cell tracer green CMFDA (5-chloromethylfluorescein diacetate; Molecular Probes) as described by D.W. Hedley et al. Cytometry 15:349 (1994).

As shown in Figure 3, a subpopulation showing lower GSH levels (>50% of cells showing low levels of GSH) appeared immediately after sonication compared to untreated cells. The results of continuous treatment showed that more GSH was lost after 5 hours. These results, expressed as the percentage of cells showing GSH levels compared to untreated cells, clearly show that high frequency sonication is associated with GSH loss.

Example 5

The effect of high-frequency ultrasound on the activity of caspase-3 in cells

Caspase-3 has been shown to play an important role in chemotherapy-induced apoptosis. Specifically, activation of caspases can lead to cell death through the degradation of cellular substrates such as actin, gelsolin or PARP. To directly confirm the involvement of caspase-3 in ultrasound-induced apoptosis, the caspase activity was measured by flow cytometry and colorimetric analysis.

Specifically, a polyclonal rabbit antibody (BD-Pharmingen, San Diego, CA, U.S.) that has anti-caspase-3 monoclonal antibody activity and phycoerythrin (PE) was used to measure caspase-3 by flow cytometry. 3. Cells were fixed and permeabilized using a fixation and permeabilization kit (Caltag, Burlingame, CA) for 15 minutes at room temperature. Cells were subsequently labeled with anti-caspase-3 antibody and incubated for 15 minutes. Cells were washed and analyzed by flow cytometry. Caspase-3 enzyme activity was measured using Apotarget's caspase-3/cpp32/colorimetric protease assay kit (Biosource) according to the manufacturer's recommendations. Caspase-3 activity can also be evaluated indirectly through PARP degradation, using rabbit anti-PARP degradation site AB combined with FITC (Biosource).

As shown in Figure 4, high-frequency sonication resulted in the activation of caspase-3. Furthermore, the activity of this protease was highest 1 hour after treatment. Furthermore, degradation of PARP was evident 2 hours after treatment, with 40% of cells labeled with rabbit anti-PARP FITC compared to only 5% of untreated cells (results not shown).

Example 6

     The effect of high-frequency ultrasound on the ratio of BCL-2/BAX in cells

Various proteins of the bcl-2 family have been shown to play a role in initiating and preventing apoptosis. The following analysis was performed to determine whether the two main members of the bcl-2 family, bcl-2 and bax, were involved in the induction of apoptosis by ultrasound. After permeabilization, cells were incubated with an isotype-matched negative control, FITC-labeled mouse anti-human bcl-2 (Dako, Glostrup, Denmark), and a polyclonal rabbit antibody against bax. FITC-labeled secondary antibody (Dako) was subsequently added to bax. To determine the expression levels of bcl-2 and bax, the cytometer was standardized with known amounts of fluorochrome-labeled particle mixtures (Dako). The mean fluorescence intensity (MFI) was then converted to moles of equivalent soluble fluorescent dye (MESF) using a standard curve.

The results (data not provided) showed that untreated cells expressed a high level of anti-apoptotic protein bcl-2 protein ((47±4)×10 ³ MESF), and this expression appeared in the form of a fluorescent single peak. After 1 hour of sonication, the expression of bcl-2 protein has been downregulated ((40±0.9)×10 ³ MESF and (32±0.9)×10 ³ MESF in K562 cells treated 1 or 3 times with sonication, respectively) . Two hours after treatment, bcl-2 expression showed a clear double peak, and the cells showed either a high bcl-2 (compared to untreated cells) phenotype or a low bcl-2 ((11±2)×10 ³ MESF) Phenotype.

In contrast to bcl-2, the level of the pro-apoptotic protein bax was significantly increased in sonicated cells compared with untreated cells ((85±0.5)×10 ³ MESF and (48±5 ×10 ³ MESF). Therefore, the bcl-2/bax ratio decreased significantly during high-frequency sonication, providing evidence of apoptosis (0.98 for control cells and 0.38 for sonicated cells).

Example 7

The effect of high-frequency ultrasound on the level of phosphatidylserine in cells

In apoptosis, phosphatidylserine residues are flipped from the inside to the outside of the plasma membrane, and this change can be measured using Annexin-FITC that binds PS residues. The Annexin V binding assay performed is described below. Flow-through of Annexin-V-fluorescein isothiocyanate (FITC)- and iodopyridine (PI)-labeled cells was performed using kits purchased from Biosource International (Camarillo, CA, USA) according to the manufacturer's recommendations. Cytometer analysis. Data are presented as dot plots showing the change in mean fluorescence intensity of Annexin-V-FITC/iodopyridine (not shown).

The results showed that, depending on time, the phosphatidylserine distribution varied over time. In particular, the results showed a low percentage of plasma membrane damage induced by sonication in the cells, indicating that the necrotic effect of the sonication tested was very weak. Interestingly, after two hours of treatment, an increase in apoptotic cells was observed. After five hours of treatment, 35% of the cells were Annexin-V-positive, indicating the effect of ultrasound on the induction of apoptosis in K562 cells.

Example 8

Effect of high frequency ultrasound on colony formation

Important evidence for a contribution to cell viability is the inability of cells to proliferate and form colonies. A clonogenic assay is described below, which was performed on the K562 cell line to determine the effect of high-frequency ultrasound radiation on clonogenic efficiency. Briefly, media consisted of IMDM containing 20% FCS and methylcellulose at a final concentration of 4%. Cultures were performed at 37°C in an atmosphere of 5% _CO2 and colonies (>20 cells) were counted after 5 days. The colony formation efficiency of K562 cell line was 16%. As shown in Figure 5, after 1 and 3 treatments, a significant decrease in the clonogenic efficiency of K562 cells was observed (25% and 42% inhibition, respectively), demonstrating the sensitivity of leukemia cells to high-frequency ultrasound.

Example 9

The effect of scavenger on apoptosis

The following procedure was performed to determine the induction of apoptosis by reactive oxygen scavengers generated by high-frequency ultrasound. K562 cells were incubated with L-histidine (10 mM) and/or mannitol (100 mM). Some cells were treated with high frequency sonication while others were not. Apoptosis was determined by Annexin-V/PI assay. The results are shown in Table 1, which indicated that the apoptosis induced by ultrasound was significantly reduced in the presence of histidine and mannitol (apoptosis induced by three consecutive treatments was inhibited by 43% and 47%, respectively).

Table 1 Effect of active scavenger on cell apoptosis induced by ultrasound No US treatment 1 US treatment 3 US treatments Reagent-free Histidine Mannitol Histidine + Mannitol 18±615±511±411±2 42±824±821±516±3 63±536±1130±325±5

Results are expressed as the percentage of cells showing phosphatidylserine superficialization after 5 hours of treatment (mean ± standard error from 4 independent experiments).

The combination of mannitol and histidine resulted in over 60% inhibition of apoptosis. The effectiveness of these agents in reducing apoptosis induced by sonication provides evidence that sonication-induced singlet oxygen and hydroxyl radicals are important mediators of apoptosis induction.

Example 10

The effect of continuous high-frequency ultrasonic treatment on cells

Flow cytometry analysis was repeated for K562 cells cultured for 0.5, 2 and 5 hours after sonication. After one treatment, three times the level of apoptotic cells was observed compared to the control (26% and 8% of treated and untreated cells, respectively, after 5 hours of incubation). A 5-10% necrosis was also observed, which was significantly lower than that observed with drug treatment and photoenergy treatment (PDT). By continuous irradiation, the apoptosis of K562 cells increased to 37±3% (p<0.02 ) and 49±5% (p<0.02) ( FIG. 6 ). Morphological changes (eg, cell shrinkage, membrane blebbing, chromatin condensation) were also observed after serial treatments (results not shown). Figure 7 shows the increase in cellular phosphatidylserine content detected by Annexin-V assay after continuous high-frequency ultrasonic treatment.

Example 11

The effect of high-frequency ultrasound on different cell lines

In addition to K562, the effect of sonication on other normal and malignant cell lines was examined, including KGla (minimally differentiated naive acute myeloid leukemia blasts), HL-60 (promyeloid leukemia cells) and Nalm-6 (ALL cell line ). The results, shown in Figure 8, indicate that sensitivity to ultrasound is cell type dependent, but that continuous treatment resulted in a significant increase in the number of apoptotic cells in all cell lines evaluated.

Mononuclear cells from 5 patients (1 refractory anemia with excess blast cells [RAEB], 1 secondary acute myeloid leukemia [AML], and 3 patients classified as M3, M4, and M4Eo AML French-American-British [FAB]), and with reference to the previous description of F. Lacombe, F. et al. Leukemia 11: 1878 (1997), blasts can be distinguished from contaminating normal cells based on their CD45 expression . Exposed phosphatidylserine in these cells can also be labeled by its recognition by FITC-Annexin. This method enables the comparison of the respective apoptotic behaviors of sonicated leukemic blasts and normal cells. The results shown in Figure 8 indicated that primary leukemia cells were sensitive to ultrasonic treatment, and the number of apoptotic cells observed 5 hours after 3 treatments exceeded 37±18%.

The foregoing description details certain embodiments of the teachings of the present invention. It should be understood, however, that no matter how detailed the foregoing description may appear, the apparatus and methods described herein can be implemented in a variety of ways. Also as mentioned above, it should be pointed out that the specific terms used in describing certain features or aspects of the teachings of the present invention should not be regarded as redefinitions in the present invention, and are used to limit the terms related to the terms that include the teachings of the present invention. any particular characteristics of the features and aspects of the Accordingly, the scope of the present teachings should be construed in accordance with the appended claims and any equivalents thereof.

Claims (26)

1. handle the equipment of cell suspending liquid, comprising:

The compartment that contains liquid, described liquid contain hyper-proliferative, the not differentiation or the cell of viral infection;

Be designed to be lower than 30mW/cm ³Power level, and be enough to cause the programmed death of cell in the described cell and do not cause significant cavitation or the persistent period of significantly heating described liquid that emission has the hyperacoustic emitter that is higher than 100kHz; And

The emitting microbubbles device of average diameter less than the gas microbubbles of 1mm launched in the described ultrasonic zone that is designed in described compartment.

2. equipment as claimed in claim 1, wherein said gas microbubbles is selected from air micro-bubbles and bubbles of oxygen.

3. equipment as claimed in claim 1, wherein said compartment contains the physiological fluid that extracts from mammal.

4. equipment as claimed in claim 3, wherein said physiological fluid is selected from one or more blood, blood plasma, serum or cerebrospinal fluid.

5. equipment as claimed in claim 1, the average diameter of wherein said gas microbubbles is less than 50 μ m.

6. equipment as claimed in claim 5, the average diameter of wherein said gas microbubbles is less than 30 μ m.

7. equipment as claimed in claim 1, wherein said emitting microbubbles device is positioned at the bottom of described compartment.

8. equipment as claimed in claim 1, wherein said compartment comprise and are designed to continuously or are interrupted the hyperacoustic a plurality of ultrasonic transmitters of emission.

9. equipment as claimed in claim 1 further comprises emitter of electromagnetic radiation.

10. equipment as claimed in claim 9, wherein said emitter of electromagnetic radiation is selected from the radiation of the frequency transmission of interrupted of ultraviolet, infrared ray and microwave with one or more.

11. equipment as claimed in claim 10, wherein said ultraviolet are UVA, UVB or UVC.

12. equipment as claimed in claim 1 further comprises hyper-proliferative in the cell suspending liquid that recovers to be present in processing, the device of the cell of differentiation or viral infection not.

13. equipment as claimed in claim 12 wherein saidly is used for collecting hyper-proliferative, the device of the cell of differentiation or viral infection is not selected from filter and spin separator.

14. further comprising being designed to provide to described ultrasonic transmitter, equipment as claimed in claim 1 is lower than 1W/cm ²The generator of energy.

15. compacting, remove and/or prevent to be suspended in hyper-proliferative in the external physiological fluid, the method for the growth of differentiation or virus infected cell not, comprising:

To be lower than 30mw/cm ³Power level in the compartment that contains described pending external physiological fluid, launch and have the ultrasound wave that is higher than the 100kHz frequency; And

Described ultrasonic zone emission in the described compartment that contains described external physiological fluid has the gas microbubbles of average diameter less than 1mm, make the emission of described ultrasound wave and gas microbubbles induce described hyper-proliferative, not differentiation or virus infected cell remarkable programmed cell death and do not cause the significant cavitation in described zone and the remarkable described liquid of heating.

16. method as claimed in claim 15, wherein said gas microbubbles are not ozone microbubbles.

17. method as claimed in claim 15, wherein said gas microbubbles is selected from air micro-bubbles and bubbles of oxygen.

18. method as claimed in claim 15, wherein said external physiological fluid are given mammal and/or extract from mammal.

19. method as claimed in claim 15, wherein said external physiological fluid is selected from blood, blood plasma, serum and cerebrospinal fluid.

20. method as claimed in claim 15, the average diameter of wherein said gas microbubbles is less than 50 μ m.

21. method as claimed in claim 15, the average diameter of wherein said gas microbubbles is less than 30 μ m.

22. method as claimed in claim 15, wherein the described ultrasound wave of launching in described compartment does not produce the stationary field phenomenon.

23. method as claimed in claim 15 comprises that further the light that emission has mainly in the electromagnetic radiation of visible range enters described supersonic zone.

24. method as claimed in claim 15, wherein said excessive proliferated cell are selected from tumor cell, medullary cell, tumor stem cell and preceding cancerous cell.

25. method as claimed in claim 15, wherein said excessive proliferated cell are the leukaemia.

26. as method as described in the claim 15 further comprise to as described in ultrasonic transmitter provide and be lower than 1W/cm ²Energy.

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