Patent Grant
11998765
Not Applicable.
The invention generally relates to cancer imaging methods and cancer treatment methods using thermotherapy and drug delivery. More particularly, the invention relates to cancer imaging methods and cancer treatment methods using nanoparticles and photoacoustics, ultrasound, laser, and/or an alternating magnetic field for thermotherapy and drug delivery.
A real-time and non-invasive thermometry technique is essential in thermal therapies to monitor and control the treatment. The current clinical standard for temperature measurement in the body is performed by either temperature sensing probes or magnetic resonance imaging (MRI). However, temperature sensing probes are invasive and provide a temperature measurement only at a single point and MRI systems are expensive and require MRI-compatible heating modalities. Ultrasound (US) is an attractive thermometry modality due to its relatively high sensitivity to change in temperature and fast data acquisition and processing capabilities. In addition, US thermometry is inexpensive, portable and non-ionizing. For US thermometry, a temperature sensitive acoustic parameter is required to track the changes in that parameter during the treatment. Among existing US thermometry methods, the backscattered RF echo-shift is currently the most established one and its feasibility has been demonstrated in a small animal model in vivo. However, the echo-shift method is highly sensitive to tissue motion (mainly respiratory and cardiac motion). In addition, this method suffers from erroneous temperature estimates in the distal area to the heated region due to the distortion of the US beam as they traverse the region undergoing temperature change.
The echo-shift technique estimates the temperature change in a uniform medium as:
where c0 is the SOS at the initial temperature, α=(∂d(T)/∂T)/d(T) is the linear coefficient of thermal expansion of the medium, λ=(∂c(T)/∂T)/c(T) is the thermal coefficient of the SOS, and δt(z) is the cumulative time-shift at depth z. The first term in Eq. (1),
is a material-dependent parameter denoted by k, and the second term,
(δt(z)), is the axial gradient of the cumulative shifts in the RF echo signal.
Therefore, what is needed is an US thermometry method that is less susceptible to tissue motion. In addition, an US thermometry method is needed that is more sensitive to temperature compared to the echo-shift method.
Moreover, chemotherapy is a primary curative modality for many types of cancers. It involves administering pharmaceutical compounds that exert cytotoxic effects and disrupt the rapid overgrowth of malignant cells. However, the side effects of the chemotherapeutic agents caused by poor drug distribution and lack of confinement to the target region remain a problem. Nanocarriers, such as liposomes, are used to overcome the issues with conventional chemotherapeutic agents. Liposomes are spherical bilayer vesicles that are composed of phospholipid membranes and an aqueous core and are used as carriers for molecules. Liposomes are the most studied nanoparticle-based drug delivery systems for cancer treatment. Liposomes are generally biocompatible, and liposome-based drug delivery has several advantages compared to the administration of the free drug. Advantages include: (a) an increase in drug accumulation in the tumour tissue due to the enhanced permeability and retention (EPR) effect and (b) confining the cytotoxic molecules within a non-toxic carrier. This approach can minimize normal tissue exposure to the cytotoxic agents by selective focused ultrasound exposure. However, passive drug accumulation from the EPR effect can take days to achieve maximum effect, and the encapsulation of the drug can also limit the timely release and uptake of the drugs at the tumour site. Liposome systems that respond to external stimuli such as temperature, pH, electromagnetic fields, ultrasound, and light have been studied to overcome the limitations of timely release. Among the different stimuli, ultrasound poses an advantage as it can propagate into deep tissues as long as there is an acoustic path for the ultrasound to travel through.
The mechanism of drug release with ultrasound can broadly be divided into thermal and mechanical processes (or a combination of both). The thermal release requires an ultrasound-induced temperature increase at the target region from the absorption of acoustic energy. The thermal release using ultrasound generally requires moderate intensities of several W/cm2, high duty cycles up to 100%, moderate pressures of 100s of kPa to MPa range, and long treatment times, of the order of several seconds to 30 minutes. High-intensity focused ultrasound (HIFU) has a focal intensity of a few kW/cm2, and the temperature rise is 60-100° C. HIFU therapy is used to damage diseased tissue through ablation or cavitation. Low-intensity focused ultrasound has a focal intensity of a few tens of W/cm2 and results in the temperature rise of 41-45° C.
Therefore, what is needed is a method for releasing a drug from nanocarriers using both thermal and mechanical ultrasound processes. In addition, a method is needed for measuring the release of the drug from the nanocarriers using the thermal and mechanical ultrasound processes.
Furthermore, thermal therapy is currently used either as a stand-alone cancer treatment or in combination with other treatment modalities, such as radiation therapy, chemotherapy, and heat-activated drug release using thermosensitive liposomes. Despite the successful clinical application of some of these modalities, a major limitation remains the lack of simple real-time non-invasive thermal imaging for adaptive localization and control of the temperature.
The monitoring and control of temperature rise in a malignant lesion are imperative for assuring that the prescribed temperature is delivered to the tumour for a desired time while protecting the vital tissue and organs surrounding it. The current standard for temperature measurement and control during thermal therapy is either point temperature sensors or magnetic resonance thermal imaging (MRTI). The former is invasive and only provides spatially sparse thermometry while the latter is both expensive and requires MRI compatible equipment. Hence, there is a need for inexpensive and non-invasive temperature monitoring. Some of the emerging non-invasive thermometry methods that meet these needs are ultrasound (US) and photoacoustic (PA) thermometry.
Photoacoustic (PA) imaging is an emerging hybrid modality that has the contrast of optical imaging and the high resolution of ultrasound imaging. Nanosecond pulses of light illuminate the area of interest resulting in small rapid temperature increases due to light absorption, causing a subsequent thermoelastic expansion in the medium that produces acoustic waves that are measured at the surface of the imaged tissue using a US transducer. PA imaging, therefore, produces an image of optical absorbers distribution, making it complementary to US imaging, which produces an image of acoustic scatterers. The ability of photoacoustic imaging to provide structural and functional imaging has led to attempts to use it in diverse application areas, including temperature monitoring in tissues. For hyperthermia where heating does not induce significant changes in the tissue's optical properties, the heating-induced change of the PA emission depends mainly on the change in the Grüneisen parameter. Photoacoustic thermometry has resulted in encouraging outcomes using tissue phantoms, and recently, in some in-vivo studies. Nevertheless, improvements are still needed, especially in producing sufficient quality temperature images for reliable real-time non-invasive monitoring and control of temperature.
Accordingly, the present invention is directed to cancer imaging methods and cancer treatment methods using thermotherapy and drug delivery that substantially obviate one or more problems resulting from the limitations and deficiencies of the related art.
In accordance with one or more embodiments of the present invention, there is provided a non-invasive thermometry method for use in cancer treatment and/or imaging. The method comprises the steps of: (i) heating tissue using a thermal energy source at a site of a tumor so as to damage one or more tumor cell membranes and release antigenic material in vivo that activates and stimulates an immunogenic response of the patient at the site of the tumor; (ii) imaging the heated tissue at the site of the tumor using an imaging ultrasound transducer so as to acquire radio-frequency data; and (iii) determining, by using an ultrasound scanner, a temperature of the heated tissue at the site of the tumor from the radio-frequency data acquired by the imaging ultrasound transducer, the temperature being determined from the radio-frequency data based upon a change in backscattered energy of acoustic harmonics.
In a further embodiment of the present invention, the thermal energy source for heating the tissue is selected from the group consisting of ultrasound, laser, an alternating magnetic field, microwave radiation, and radiofrequency (RF) energy.
In yet a further embodiment, the thermal energy source for heating the tissue is a therapeutic ultrasound transducer operating in a low intensity focused ultrasound (LIFU) mode.
In still a further embodiment, the therapeutic ultrasound transducer has a central frequency of approximately 1 megaHertz; and the step of heating the tissue further comprises heating the tissue to a temperature in a range between about 37° C. and about 47° C. using an acoustic power of approximately 4.5 watts, a frequency of approximately 1 megaHertz, and a 50% duty cycle.
In yet a further embodiment, the imaging ultrasound transducer is a linear array-type transducer with a central frequency of approximately 4.2 megaHertz and a sampling rate of 31.25 megaHertz.
In still a further embodiment, the step of determining the temperature of the heated tissue further comprises determining, by using the ultrasound scanner, two-dimensional temperature maps of the heated tissue from the radio-frequency data.
In yet a further embodiment, the step of determining the temperature of the heated tissue further comprises determining, by using the ultrasound scanner, the temperature from the radio-frequency data based upon a change in backscattered energy of the fundamental acoustic harmonic (BEf0) and second acoustic harmonic (BEh2).
In accordance with one or more other embodiments of the present invention, there is provided a cancer treatment method using ultrasound energy for targeted drug delivery. The method comprises the steps of: (i) administering a plurality of nanocarriers to a patient in need thereof so as to target a tumor in the patient, the administered nanocarriers containing an anti-tumor medication; and (ii) exciting the nanocarriers in a first non-thermal ultrasound mode and/or a second thermal ultrasound mode using an ultrasound source that generates a non-thermal ultrasonic wave and/or a thermal ultrasonic wave so as to release the anti-tumor medication from one or more of the plurality of nanocarriers at a site of the tumor.
In a further embodiment of the present invention, the plurality of nanocarriers are selected from the group consisting of antibody-conjugated nanoparticles, aptamer-conjugated nanoparticles, liposomes, and micelles.
In yet a further embodiment, at least some of the plurality of nanocarriers are conjugated with cell penetrating peptides (CPPs).
In still a further embodiment, the ultrasound source is a therapeutic ultrasound transducer with a frequency of approximately 1 megaHertz.
In yet a further embodiment, the step of exciting the nanocarriers further comprises exciting the nanocarriers using the therapeutic ultrasound transducer in the first non-thermal ultrasound mode, the therapeutic ultrasound transducer emitting pulsed high intensity focused ultrasound (pHIFU) for mechanical excitation of the plurality of nanocarriers in the first non-thermal ultrasound mode so as to release the anti-tumor medication from the nanocarriers by rupturing membranes of the nanocarriers and/or peeling a polymer coating off the nanocarriers.
In still a further embodiment, the step of exciting the nanocarriers with the pulsed high intensity focused ultrasound (pHIFU) in the first non-thermal ultrasound mode further comprises generating approximately 25 millisecond pulses at a pulse repetition frequency of approximately 1 Hertz with a focal peak pressure of approximately 3.3 megapascals for a total exposure time duration of approximately 30 seconds.
In yet a further embodiment, in the first non-thermal ultrasound mode where the therapeutic ultrasound transducer is emitting the pulsed high intensity focused ultrasound (pHIFU), the therapeutic ultrasound transducer has an acoustic power of approximately 332 watts and an input voltage of approximately 500 mVpp.
In still a further embodiment, the step of exciting the nanocarriers further comprises exciting the nanocarriers using the therapeutic ultrasound transducer in the second thermal ultrasound mode, the therapeutic ultrasound transducer emitting low intensity focused ultrasound (LIFU) for thermal heating of the plurality of nanocarriers in the second thermal ultrasound mode so as to release the anti-tumor medication from the nanocarriers by thermally rupturing the nanocarriers and/or melting a polymer coating of the nanocarriers.
In yet a further embodiment, the step of exciting the nanocarriers with the low intensity focused ultrasound (LIFU) in the second thermal ultrasound mode further comprises generating a thermal ultrasonic wave with a frequency of approximately 1 megaHertz at a focal peak pressure of approximately 0.35 megapascals and a 50% duty cycle for a total exposure time duration of approximately 5 minutes.
In still a further embodiment, in the second thermal ultrasound mode where the therapeutic ultrasound transducer is emitting the low intensity focused ultrasound (LIFU), the therapeutic ultrasound transducer has an acoustic power of approximately 2.1 watts and an input voltage of approximately 160 mVpp.
In yet a further embodiment, in the second thermal ultrasound mode where the therapeutic ultrasound transducer is emitting the low intensity focused ultrasound (LIFU), the therapeutic ultrasound transducer thermally heats the plurality of nanocarriers to a temperature in a range between about 37° C. and about 47° C. under feedback control of the therapeutic ultrasound transducer by a feedback controller.
In still a further embodiment, the anti-tumor medication comprises doxorubicin.
In accordance with yet one or more other embodiments of the present invention, there is provided a cancer treatment method using nanoparticle-mediated thermal therapy using photoacoustic imaging. The method comprising the steps of: (i) administering a plurality of nanoparticles to tissue at a site of tumor in a patient; (ii) heating the tissue and at least some of the plurality of nanoparticles at the site of the tumor using a thermal energy source so as to generate photoacoustic signals, damage one or more tumor cell membranes, and release antigenic material in vivo that activates and stimulates an immunogenic response of the patient at the site of the tumor; (iii) performing photoacoustic imaging with a photoacoustic imaging unit so as to acquire the photoacoustic signals; and (iv) determining, by using the photoacoustic imaging unit, a temperature of the heated tissue at the site of the tumor from the photoacoustic signals.
In a further embodiment of the present invention, the cancer treatment method further comprises a proportional-integral-derivative (PID) controller operatively coupled to the thermal energy source and the photoacoustic imaging unit; and the step of heating the tissue and the at least some of the plurality of nanoparticles at the site of the tumor further comprises controlling the thermal energy source using the proportional-integral-derivative (PID) controller based on the temperature determined by the photoacoustic imaging unit in order to heat the tissue and the at least some of the plurality of nanoparticles to a prescribed temperature so as to provide real-time control of nanoparticle-mediated thermal therapy.
In yet a further embodiment, the thermal energy source for heating the tissue is selected from the group consisting of laser, ultrasound, an alternating magnetic field, microwave radiation, and radiofrequency (RF) energy.
In still a further embodiment, the photoacoustic imaging unit comprises an ultrasound transducer and a nanosecond excitation laser.
In yet a further embodiment, the ultrasound transducer of the photoacoustic imaging unit operates a frequency of approximately 21 megaHertz and the nanosecond excitation laser operates in a wavelength range of about 680 nanometers to about 930 nanometers.
In still a further embodiment, a first subset of the plurality of nanoparticles administered to the tissue at the site of the tumor in the patient comprises gold nanoparticles; and the step of heating the tissue and the at least some of the plurality of nanoparticles further comprises heating at least some of the gold nanoparticles at the site of the tumor, the heating of the gold nanoparticles resulting in an increased temperature rise at the site of the tumor that is five to seven times greater than a temperature rise achieved without the administration of gold nanoparticles.
In yet a further embodiment, a second subset of the plurality of nanoparticles administered to the tissue at the site of the tumor in the patient comprises liposomes containing an anti-tumor medication.
In still a further embodiment, the anti-tumor medication comprises doxorubicin.
It is to be understood that the foregoing general description and the following detailed description of the present invention are merely exemplary and explanatory in nature. As such, the foregoing general description and the following detailed description of the invention should not be construed to limit the scope of the appended claims in any sense.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The invention will now be described, by way of example, with reference to the accompanying drawings, in which:
In one embodiment, an ultrasound thermometry method based on change in backscattered energy (CBE) of the acoustic harmonics is used to estimate the temperature in ex vivo tissues. Also, the feasibility of using the CBE method in detecting localized heated region generated low intensity focused ultrasound and controlling the temperature in the heated region using a temperature control system are demonstrated.
Before the experiment, freshly excised ex vivo pork muscle tissue obtained from the local butcher shop was immersed in 0.9% degassed saline solution at 5° C. for 24 hours. The tissue sample was placed in a tissue holder and the dimensions of the tissue sample were 5×8×3 cm3 (axial×lateral×elevational). The tissue holder was placed in a water tank housing a single-element focused therapeutic transducer (Imasonic SAS, Voray sur l'Ognon, France) that operates in a LIFU (low intensity focused ultrasound) regime. A schematic diagram of the setup is shown in
The fundamental and second harmonic signals of the data were filtered from the acquired RF data by a bandpass filter. The backscattered energy of the fundamental (BEf
where tn is the time at which the n-th frame of data was acquired and the bar represents the average value. The cumulative CBE maps of the fundamental and second harmonic were then computed as:
The fundamental and second harmonics CBE maps were also combined to obtain a compounded CBE map with two different frequencies (i.e., 4.2 MHz and 8.4 MHz) as follows:
The CBEh2 and CBEcomb were filtered by 5×5 median and mean filters to reduce the noise in the CBE maps. Tissue motion is the main source of noise in the CBE maps. In order to reduce tissue motion between two consecutive frames, a block matching method was used. Typically, block matching methods pick a block of data from a frame and find the location where the selected block matches best with a block of data in a reference frame. In this work, a 2D normalized cross-correlation technique was used as a block matching algorithm to estimate the axial and lateral displacements between two consecutive frames using the RF data. Each frame was aligned to the previous frame based on the estimated axial and lateral displacements.
Temperature calibration was performed to determine the linkage between change in harmonic signal and change in temperature. In separate trials a calibrated needle type-K thermocouple was placed at the focus of the LIFU transducer and temperature was recorded as the tissue was being exposed to LIFU with the same parameters as the described above. This was repeated five times for different tissue samples. The linkage between percentage change in CBE and temperature was obtained from the 2D maps of CBE. A circular region with 1 mm diameter (which was determined from the simulated intensity field as shown in
The feasibility of using the CBE methods for controlling the temperature was evaluated. A temperature control algorithm was designed to control the output of the LIFU transducer based on the estimated temperature from a calibrated needle thermocouple. The software controlled the therapeutic transducer by changing the duty cycle and applied voltage. The block of the temperature control methodology is shown in
The temperature maps of the proposed second harmonic CBE (CBEh2) and combined CBE (CBEcomb) are shown in
In order to compare with our CBEh2 and CBEcomb methods, temperature maps calculated from the echo-shift technique are shown in
The plot of the temperature measurement from the thermocouple inserted in the focal point is shown in
The average percentage change in CBEh2 as a function of the average temperature reading from the thermocouple is shown in
For the temperature control system, the CBEcomb method was used since it was compounded of 2 different frequencies leading to noise reduction. The temperature maps of CBEcomb is shown in
In one embodiment, the CBEh2 and CBEcomb methods are used to produce 2D temperature maps. The change in temperature at the focal region of the therapeutic transducer is observed in the 2D temperature maps of the CBEh2 and CBEcomb methods. The increase and decrease in the size of the localized heated region was observed in the 2D temperature maps as the tissue was being heated and cooled. In the experiment, the temperature maps using the echo-shift technique appeared noisier compared to the CBEh2 and CBEcomb methods since the echo-shift technique is more sensitive to tissue motion.
In one embodiment, the feasibility of using the CBEcomb method in a closed-loop feedback system was also demonstrated, where the estimated temperature from the CBEcomb method correlated with the thermocouple measurement.
In one embodiment, an application of the CBEcomb method is the controlled release of drugs from thermosensitive liposomes.
In one embodiment, CBEcomb method is used with LIFU in a closed-loop feedback system to achieve a temperature of 39-43 degrees C. under software control and maintain the temperature for a desired time so as to be used to control the release of drugs from thermosensitive liposomes or antibody-coated liposomes to deliver antiproliferative agents to the cancer, such as brain tumors, skin cancer, eye cancer, mucosa cancers, genitourinary cancer, prostate cancer, lung cancer, breast cancer, mouth cancer, throat cancer, intestinal tract cancer, kidney cancer, liver cancer, urinary tract cancer, spinal cord cancer, bone cancer, spleen cancer, ovarian cancer, etc.
In one embodiment, using LIFU and controlled temperature, it is desirable to release the medication locally at a desired site and monitor and control the temperature in a range that is safe and does not induce any risk of thermal damage to tissue.
In one embodiment of thermotherapy in the treatment of a neoplasm, the rise of the temperature to about 43° C. for a period of time (e.g., 1-4 minutes) makes the tumour cells prone to damage by other adjuvant cancer treatments, such as radiation therapy, chemotherapy, gene therapy, and simultaneous immune therapy due to the fact that tumor cells, in general, have higher cell metabolism and are less efficient to cool themselves down compared to normal cells. The damage due to the controlled thermal application at about 39-43 C damages the tumor, thereby enhancing apoptosis of tumor cells and releasing tumor antigens that encourages a cellular immune response to the tumor antigens that attacks the damaged tumor cells more readily and does not give the tumor cells a chance to mount a chemical response to disguise the tumor.
In one embodiment of thermotherapy in the treatment of a neoplasm, the rise of the temperature to about 39-43° C. for a period of time causes a slight tissue expansion due to the leaky vessels during the thermotherapy leading to the collapse of the capillaries when the thermal energy is maintained beyond a few minutes that, in turn, collapses the capillaries and strangulates further the tumor cells, thereby preventing their cooling by the circulation and reduces their oxygenation and nutrition.
In one embodiment of thermotherapy in the treatment of a neoplasm, the rise of the temperature to about 39-43° C. for a period of time causes the peripheral capillaries of the tumor to become leakier and permits the medication to diffuse easier inside the tumor cells which are being damaged more precisely by the combination of thermotherapy and drug delivery using antibody-coated liposomes or nanoparticles to release the conjugated medication with the nanoparticles.
In one embodiment, the thermotherapy is combined with simultaneous imaging of the lesion before or after the treatment to observe the progression of the disease or disappearance of the tumor.
As with other non-invasive ultrasound thermometry, the method described herein requires calibration for each tissue type and transducer parameters (e.g., imaging frequency and tissue depth). The calibration becomes more challenging for heterogeneous tissues since variation between subjects is generally larger.
In one embodiment, the challenges of ultrasonic imaging may be addressed by repeating the calibration for a larger number of subjects to reduce the inter-subject variability and obtain reliable calibration information.
In one embodiment, the implementation of ultrasonic controlled thermotherapy benefits from 2D temperature maps with a high enough frame rate (at least tens of frames per minute) to control hyperthermia therapy. In order to produce a 2D temperature, the acquired backscattered data is beamformed, motion compensated and bandpass filtered (to obtain 2nd harmonic signals), then backscattered energy and change in backscattered energy is determined via the software. In some embodiments, parallel computing and/or a graphic processing unit (GPU) is used for processing the data.
In one embodiment, the method described herein with the temperature control system is used to control hyperthermia therapies.
In one embodiment of controlled thermotherapy, one can use other heating modalities, such as laser and alternative magnetic field combined with photoacoustic imaging and thermometry.
In one embodiment, for example, in veterinary applications, one can use high-frequency ultrasound scanner, such as Vevo LAZR (FUJIFILM Visualsonics, Inc., Toronto, ON, Canada) and drug-loaded thermosensitive liposomes that are activated with LIFU, laser or alternative magnetic field while the temperature is imaged and controlled by the proposed method.
In another embodiment, as described in the example provided hereinafter, an ultrasound thermometry method based on the change in backscattered energy (CBE) in a high-frequency ultrasound scanner is used to produce localized 2D temperature maps induced by an interstitial laser heating source and to control the tissue temperature non-invasively and in real-time using a closed-loop controller.
Before the experiment, freshly excised ex vivo pork muscle tissue obtained from the local butcher shop was immersed in 0.9% degassed saline solution at 5° C. for 24 h. The tissue sample was placed in a tissue holder and the dimensions of the tissue sample were 3×4×1.5 cm3 (axial×lateral×elevational). The tissue holder was placed in a temperature-controlled water bath (Haake DC10, Thermo Electron Corp., Newington, NH) to maintain the baseline temperature at 37° C. A schematic diagram of the setup is shown in
The imaging plane of the transducer was perpendicular to the fibers, and it was about 1 mm away from the temperature fiber. RF echo data was exported to the PC workstation for real-time processing with a transfer rate of approximately one frame per 6 seconds. In order to reduce the tissue motion between consecutive frames, a motion compensation algorithm based on 2D normalized cross-correlation was used to compensate in the axial and lateral directions. The backscattered energy (BE) of the received RF data was calculated by taking the envelope of the signal using Hilbert transfer and squaring the enveloped signals. The incremental change in backscattered energy CBEincr of each pixel (x,y) was estimated as:
where tn is the time at which the n-th frame of data was acquired and the BEavg is the average of BE data. Then the cumulative CBE maps were computed as:
The CBE maps were filtered by 15×15 pixel2 median and mean filters to reduce the noise in the CBE maps. Then CBE maps were converted to temperature maps by using calibration data. The calibration data were obtained in separate experiments, where ex vivo tissue samples were gradually heated monotonically from 37° C. to 46° C. using the same experimental setup. The correlation between the CBE map and fiber-optic temperature measurement from the average of five different trials was estimated to be:
Temperature(x,y)=0.268×CBE(x,y)+36.45 (7)
The temperature control software was a custom controller with three discrete power levels. It was designed to rapidly increase the temperature from 37° C. (baseline temperature) to 43° C. (target temperature) and to maintain the target temperature for 6 minutes. This temperature profile is able to be used in targeted drug delivery applications with thermosensitive liposomes.
The temperature map overlaid on the B-mode image at various times is shown in
Some of the artifacts that appear in the temperature could be due to tissue motion. Even though the ex vivo experiment was well-controlled and tissue motion was kept minimal, tissue motion could not be completely eliminated. The main source of motion in the experiment was the circulating water in the water bath that was used to maintain the background temperature at 37° C. A 2D motion compensation algorithm was applied to the RF data to correct the translational motion in the axial and lateral directions. The average motions between each frame (measured by the motion compensation algorithm) in the axial and lateral directions were about 0.1 mm and 0.2 mm, respectively. Translational motion in the elevational direction and other more sophisticated motions were not compensated. Thus, tissue motion is one of the main issues that remain to be addressed.
The average temperature in the heated region measured from the temperature maps as a function of time is shown in
The results of this example show that the ultrasound thermometry based on CBE method generated by a high-frequency ultrasound scanner is able to be used to produce 2D temperature maps of a localized heating region in the hyperthermia temperature range (˜43° C.). The estimated temperature varied by an average of ±0.8° C. compared to a calibrated fiber-optic temperature measurement. The controlled experiment showed that the target temperature could be maintained in a localized region of interest in ex vivo tissue for as long as it is required.
In one embodiment, a robust real-time non-invasive ultrasound thermometry method is used to enhance the effectiveness and safety of the thermal therapy either as a direct treatment modality or as an adjuvant to other therapeutic modalities, such as chemotherapy and radiation therapy. Moreover, in one or more embodiments, it can be used in targeted drug delivery systems such as heat-activated drug release using thermosensitive liposomes.
In one embodiment, the system described in the above example is used to control a heated tissue temperature in real-time during thermal therapy.
In one embodiment, a non-invasive ultrasound thermometry method based on the CBE technique is used for real-time monitoring and control of hyperthermia treatments, using interstitial laser heating source with acceptable accuracy.
The mechanisms of drug release from thermosensitive liposomes depend on the thermal and mechanical effects of ultrasound. As described hereinafter, an in vitro setup with custom 3D-printed sample holders embedded in tissue was used to evaluate the drug release from thermosensitive liposomes upon ultrasound exposure with exposure parameters that would induce either thermal and/or mechanical effects. In the thermal heating exposure experiments, the output of the 1 MHz transducer was controlled with a feedback controller based on temperature control. The temperature was measured using a calibrated needle thermocouple to maintain a 43° C. temperature for 5 minutes, which resulted in a fractional release of 0.96±0.27. Water-bath thermal heating at 43° C. temperature for 5 minutes was also performed for comparison, resulting in a similar fractional release (0.88±0.26). For the mechanical non-thermal ultrasound exposure parameters, a 1 MHz transducer was used to generate 25 ms pulses at a pulse repetition frequency of 1 Hz and with a focal peak pressure of 3.3 MPa for 30 seconds. The exposure to mechanical parameters also resulted in significant fractional release of 0.76±0.37. The exemplary experiment described hereinafter demonstrates how 3D-printed sample holders can be used to measure ultrasound-mediated drug release, and how both thermal and non-thermal methods could be used to achieve the release.
Prior to conducting the experiment, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Brij® S2, HEPES were purchased from Sigma-Aldrich (St Louis, MO, USA). Isopropanol was obtained by Caledon Laboratories Ltd (Halton Hills, ON, Canada). Doxorubicin hydrochloride was purchased from LC Laboratories (Woburn, MA, USA) and was dissolved in DMSO at 100 mg/ml stock solution concentrations, and the aliquots were kept at −20° C. The aliquots were then de-frosted and were diluted in Milli-Q water at 1 mg/ml working solutions that were kept at 4° C., which was further diluted at appropriate dilutions prior to use. RPMI1640, Phosphate Buffered Saline (PBS), Fetal bovine serum (FBS), Penicillin-streptomycin, Trypsin/EDTA 0.25% were obtained by Wisent Inc. (St-Bruno, QC, Canada).
Preparation of the thermosensitive liposome formulation with encapsulated doxorubicin was modified with liposomal lipid, DPPC, and a non-ionic surfactant consisting of PEGylated acyl chains, Brij® S2, at a composition of 96:4 mol %. The liposomes were formed using the thin film hydration method. Briefly, 100 mg of lipids were dissolved in 10 mL of isopropanol. The solution was aliquoted to 1 mL batches in glass vials, and the solution was dried by a stream of nitrogen gas, followed by overnight desiccation under vacuum. The lipid film was hydrated with doxorubicin at a 0.05 drug:lipid ratio at a temperature above the main phase transition temperature of 41.3° C. for complete lipid swelling and hydration. The lipid solution was extruded 9 times through a polycarbonate disk, and two stacked 0.1 μm filter supports at a temperature above the main phase transition temperature. Non-encapsulated doxorubicin was removed using dialysis against the HEPES loading buffer. The particle size and concentration were determined by resonant mass measurement using Archimedes (Malvern Panalytical Ltd., Malvern, UK). Doxorubicin fluorescence assay was used for the quantification of loaded doxorubicin. Briefly, Triton X-100 was added to the liposomal doxorubicin to produce a 1% v/v final concentration of the detergent. Then the fluorescence (Ex 485 nm/Em 590 nm) was measured with a multimode plate reader (Molecular Devices, San Jose, USA) and compared to a doxorubicin standard curve. The loaded liposomes were either used immediately or stored at room temperature and then used within 48 h.
Doxorubicin release was determined using a fluorescence de-quenching assay. The fractional release of doxorubicin was calculated as:
where R is the fractional release, Ix is the fluorescence of the treated sample, I0 is the fluorescence of a non-treated sample, and I100 is the fluorescence of the sample treated with 1% Triton X-100 (vol/vol). Triton X-100 is a detergent that lyses the liposomes and therefore corresponds to the completely released drug fluorescence reference. The fluorescence (Ex 485 nm/Em 590 nm) was measured with a multimode plate reader (Molecular Devices, San Jose, USA).
Freshly excised ex vivo porcine muscle tissue was obtained from the local butcher shop and was immersed in a 0.9% de-gassed saline solution at 4° C. for 24 hours. Then, the tissue was pre-heated to 37° C. by immersing the tissue-containing beaker in a water bath. The heated tissue was placed in a 3D printed tissue holder with the dimensions of 3×8×5 cm3 (axial×lateral×elevational) which is designed to be suspended on the upper axial edges of the water tank. The surface parallel to the transducer was previously enclosed with a transparent cellophane film with a thickness of approximately 40 μm, which reduces the acoustic impedance compared to using relatively thicker polystyrene tubes. The doxorubicin-loaded liposomes were placed in a 3D printed sample holder, with a loading dimension of 0.5×1×1 cm3 (axial×lateral×elevational), where the surface that will be placed parallel to the transducer was previously enclosed with transparent cellophane film. On the lateral side of the sample holder, a port was designed for sample loading/unloading, and miniature screws were used enclose the port. The sample containing holder was then inserted in between the ex vivo porcine muscle tissue containing holder. A diagram of the sample and tissue holders are shown in
The samples were exposed to focused ultrasound in two different exposure regimes: (a) low intensity focused ultrasound (LIFU) for thermal, and (b) pulsed high intensity focused ultrasound (pHIFU) for mechanical exposures. A 1 MHz single-element therapeutic transducer was used (IMASONIC SAS, Voray sur l'Ognon, France) with a focal length and diameter of 10 cm and 1.25 cm, respectively. The temperature of the water in the tank was controlled and maintained at 37° C. by a circulating water bath (Haake DC10, Thermo Electron Corp., Newington, NH). Calibrated needle type-K thermocouples and a digital thermometer (Omegaette HH360, Omega Eng. Inc., Stamford, CT) were also placed in the sample holder and in the ex vivo tissue sample close to the LIFU focal zone for temperature measuring, monitoring and control purposes. The setup is shown in
Compared to continuous HIFU, pulsed HIFU (pHIFU) with low duty cycles (<10%) can significantly reduce the temperature rise and may be used to promote the mechanical effects such as acoustic cavitation. To limit unspecific heat damage to normal tissues, low-intensity focused ultrasound (LIFU) can be used in combination with drug carriers (such as thermosensitive liposomes). In this approach, the payloads are delivered at a few degrees above physiological temperatures. Thermosensitive liposomes are drug delivery systems that release the encapsulated drug when heated to hyperthermia temperatures at 41-43° C. At these temperatures, it has also been shown that nanoparticle extravasation increases, likely due to increased pore sizes between endothelial cells and increased intravascular blood flow. The mechanism for mechanical release in the absence of a temperature increase is not well known. Mechanical release can be caused from high mechanical stresses on the drug carrier shell using short ultrasound pulses causing membrane rupture and generation of membrane pores. In this experiment, doxorubicin was released in response to LIFU (thermal) and pHIFU (mechanical) exposure in a custom 3D-printed sample holder that is embedded in tissue.
The final size of the liposomes was 212±19 nm with a polydispersity index (PDI) of 0.25±0.1 (n=8 independently synthesized batches) measured with Archimedes (Malvern Panalytical, Malvern, UK) a particle metrology system using a resonant mass measurement method. The drug to lipid ratio after removing the non-encapsulated doxorubicin was 0.01, achieving approximately 20% of doxorubicin passive loading. This loading is within the expected range of passive encapsulation efficiency of water-soluble drugs in liposomes. The liposomes were freshly prepared for each of the experiments.
The liposome sample was inserted in the tissue that was pre-heated to 37° C. The output of the LIFU transducer was controlled based on the temperature measured from a calibrated needle thermocouple. The software controlled the transducer by changing the duty cycle and applied voltage. Once the liposome sample also reached 37° C., the designed temperature control software was used to maintain the temperature of the liposome samples at a pre-determined temperature (43° C.) and time period (5 min). The temperature was measured for another 5 minutes after the LIFU was turned off to record the cool-down trend. A representative temperature profile measured with the calibrated thermocouple placed inside the liposome sample holder is shown in
The release of doxorubicin from the liposomes upon exposure to the water bath, LIFU, and pHIFU is shown in
Significant drug release of 0.76±0.37 (n=3) was also observed from specific non-thermal ultrasound exposures where the bulk temperature increase was less than 0.5° C. These results indicate that the combined drug-loaded liposome therapy with ultrasound exposures can lead to enhanced efficacy even without bulk temperature increase. The mechanism for mechanical release is not well understood. It has been hypothesized that the mechanical release of drugs can be caused by high mechanical stresses on the drug carriers using short ultrasound pulses. The short, intense pulses could cause membrane rupture and generation of membrane pores induced by acoustic cavitation, the formation of gas or vapour filled cavities in a medium exposed to high oscillating pressure. Although bubbles were not initially injected, gas bodies dispersed in the sample may serve as cavitation nuclei and form natural microbubbles under the high-intensity ultrasound waves. The formed bubbles can collapse within the focal point of the ultrasound and disrupt the lipid bilayer and produce temporary pores in the liposome membrane. The transient permeabilization of the lipid bilayer is referred to as sonoporation, and from these temporary pores, the contents of the liposomes can be released.
The unique setup allowed the quantification of drug release in freshly excised ex vivo porcine muscle tissue that was exposed to ultrasound. The ultrasound exposures created bulk tissue heating or induced non-thermal drug release due to the mechanical perturbation of the liposomal formulation. Several aspects of the 3D-printed holder have been modified since the initial design and form the basis for future work. For example, initially the holder was rectangular with a loading window but it evolved to become the current T-shaped with a narrowing bottom edge to place the sample holder more accurately within the focal region. Holders with different dimensions and/or designs may be required to use with different transducers. The use of a 3D printer allows the customization of the holder to the target dimensions.
A unique setup with 3D printed sample holders enclosed with cellophane film was designed to measure the release of doxorubicin from thermosensitive liposomes in response to both thermal and mechanical (non-thermal) parameters of ultrasound with a 1 MHz transducer. A complete release of the drug was observed when the bulk temperature was increased to 43° C. by both water bath heating and LIFU exposure, indicating that at 43° C., the lipid bilayer exists in the liquid phase and the membrane is permeable throughout. Significant drug release was also induced by using exposure parameters that led to non-thermal drug release. These results indicate that the mechanical stresses from the ultrasound pulses may have ruptured the liposomal membrane causing the release of the drug content.
In one embodiment, significant drug release was induced by exposing the drug-loaded thermosensitive liposomes with both thermal and non-thermal mechanical parameters of ultrasound with a 1 MHz transducer.
In one embodiment, one can implement the non-invasive temperature measurements and control for the hyperthermal treatments in which the methodologies are currently in development for drug release for any medication used in any part of the body, for drug delivery in any drug, such as bacterial, viral, fungal, or parasitic infection, or for delivery or antibodies, or medications at a precise location of the internal of external part of the body.
In one embodiment, the nanoparticles can be of any size 1 nm to micron size, ball, cylindrical rod shape hollow, of filled metallic, non-metallic, organic, synthetic, magnetic, paramagnetic, coated with antibodies for specific cell or tumor, etc. or micelles, liposomes, etc. filled up with other nanoparticles or medications or genes, hormones, poison, venom, immune stimulators or cell pathway inhibitors, anti-inflammatory or vaccines needed to be delivered at a specific body part to stimulate humoral or cellular immune response locally or systemically.
In one embodiment other heating modalities, including laser and alternating magnetic field (AMF), microwave, radiofrequency, or any electromagnetic radiation can be used as sources of external stimuli.
In one embodiment, the biological effect of the combined drug-filled thermosensitive liposome and external stimuli will be observed through imaging, sensor or electrophysiology or liquid biopsy, chemical or non-invasive optical or non-optical imaging, etc. means.
In one embodiment, low-intensity focused ultrasound has a focal intensity of a few tens of W/cm2 and results in the temperature rise of 41-45° C. To limit unspecific heating damage, LIFU can be used in combination with drug carriers that deliver the payloads at a few degrees above physiological temperatures, such as thermosensitive liposomes. Thermosensitive liposomes are drug delivery systems that release the encapsulated drug when heated to hyperthermal temperatures at 41-43° C.
In one embodiment, at low hyperthermal temperatures, during the controlled thermotherapy, the nanoparticle extravasation increases due to increased pore sizes between endothelial cells and increased intravascular blood flow.
In one embodiment, the focused ultrasound is used as a short pulse of milliseconds with high power to cause mechanical damage to the wall of the liposomes producing breakdown/pores in the liposome's wall or the nanoparticle polymeric coating, carrying the medication, thereby releasing the medication, or liposome membrane rupture with generation of membrane pores being induced by acoustic cavitation, and the formation of gas or vapor-filled cavities in a medium exposed to oscillating pressure.
A proportional-integral-derivative (PID) controller (
The Vevo LAZR system was used with a 21 MHz ultrasound transducer and a nanosecond excitation laser that could operate in the wavelength range of 680 nm-930 nm. The system produces high-resolution PA and ultrasound images and is widely used in small animal experiments. The data is usually saved on the system during imaging and later exported for post-processing and analysis using the VevoLAB software. Co-registered PA and ultrasound frames were exported in real-time to the computer running the control system. The transfer rate was approximately one frame per three seconds. MATLAB code was written to be run on the controlling computer in order to read the PA images in real-time for the feedback controller. The PA (and ultrasound) images are used by an operator to identify the region-of-interest for which the temperature needs to be monitored prior to the treatment.
A high-power continuous-wave (808 nm, up to 15 W) diode laser system (BWT Beijing Ltd, Beijing, China) was used as the heating laser. This laser system could be accessed either through its front control panel or through a serial port connected to a computer. MATLAB code was created that activated and turned on and off the laser system, and controlled the driving current or the output power of the laser in real-time through its serial port interface.
Point thermometry was used to verify and calibrate the PA-based thermometry and to conduct primary PID controller testing, validation, and tuning. A fluoroptic thermometry system (3100 Series Fluoroptic Thermometer, Luxtron Corporation, Santa Clara, CA) was used for this purpose in order to avoid artifacts caused by the interaction of the laser with traditional metal thermocouples. The fluoroptic thermometry system was also interfaced to the controlling computer through a serial port connection. MATLAB code was used to activate the system, acquire temperature measurements in real-time, and deactivate the system.
The ability of photoacoustic imaging to provide both structural and functional imaging at the same time has led to investigations of its use in diverse application areas, such as temperature monitoring of tissue. Understanding how this is done requires a mathematical model of photoacoustic imaging.
The initial pressure generated at location x in tissue due to the photoacoustic effect is given by:
p0(x)=Γ(x)μa(x)φ(x), (9)
where μa(x) is the absorption coefficient, φ(x) is the light fluence (note that μaφ(x) is the absorbed optical power density), and F(x) is the Grüneisen parameter. The Grüneisen parameter relates the initial pressure increase to the absorbed power density and is given by:
where β(x) is the thermal coefficient of expansion, c(x) is the velocity of sound, and Cp(x) is the specific heat capacity at constant pressure. Since the Grüneisen parameter has been found to be temperature dependent the initial PA signal is also temperature dependent. The Grüneisen parameter's dependence on temperature can be described by an affine function:
Γ(x,T)=a0(x)+a1(x)T(x). (11)
We assume that the PA image intensity at location x is proportional to the initial pressure at that location: I(x) p0(x). We also assume in the hyperthermic temperature range only the Grüneisen parameter varies with temperature while the optical absorption coefficient does not. The ratio of the PA image intensity at some temperature, I(x, T), to the image intensity at the baseline temperature, Ib(x, Tb) is given by:
Hence, the temperature value based on PA thermometry is given by:
T(x)=[a(x)+Tb(x)]R(x,T)−a(x), (13)
where a(x)=a0(x)/a1(x) is the only parameter that needs to be determined when calibrating the PA thermometry system for a certain medium or tissue type.
In the experiment, gold nanorods (AuNR) are prepared by a modified version of the seeded growth method.
Seed preparation: The synthesis is performed in a water bath at 30° C. After adding 50 μL of 1% HAuCl4 solution to 4.7 mL of 0.1 M of CTAB, the mixture is slowly stirred for 5 min until no signs of turbidity. Then 300 L of freshly prepared 10 mM of NaBH4 under rapid stirring. The solution is then stirred mildly before use.
Growth: A solution of 200 μL, 1% HAuCl4 is added to 10 mL of 0.1 M stirred gently for 10 min to ensure complexation between the gold salts and CTAB. Then, 75 μL of 0.1 M ascorbic acid is added to the mixture so that the solution turns colourless. A solution of 80 μL of 5 mM AgNO3 is added to the growth solution. Finally, 120 μL of seeds solution is added to the mixture and the solution is stirred vigorously and then left undisturbed in the water bath.
The CTAB stabilized AuNRs filtered twice and were purified by centrifugation (10,000×g for 50 min) and re-dispersed in ultrapure water (MilliQ), followed by second centrifugation at 7,000×g for 30 min. The concentration of the GNR is estimated to be 0.286 mg/ml measured with the ICP-AES.
Two types of experiments were performed. One involved heating the surface of tissue using an end-cut fibre radiating onto the tissue surface and the other involved heating deep in tissue using an interstitial heating fibre. These two types of experiments were conducted to study the performance of the control system until varying conditions.
(1) Surface heating: The surface laser heating experimental setup is shown in
(2) Deep-tissue heating: The experimental setup used for PA thermometry calibration and thermal therapy control is shown in
Results for the effect of GNRs on laser heating is presented first followed by control of laser heating with GNRs using the Luxtron point-thermometry system. This is followed by results from the PA thermometry that was developed and finally by results of the control of GNR based laser thermal therapy using PA thermometry.
The enhancement in tissue laser heating by GNRs at various concentrations was investigated. Using the surface laser heating setup (
The control of heating at the surface and below the surface of pork loin tissue was investigated.
(1) Surface laser-heating control: The results were obtained using the setup shown in
(2) Deep-tissue laser-heating control: The ability to control laser heating deep in tissue using interstitial fibre light delivery and changing the desired temperature, Tr(t), in various stages is demonstrated in
PA imaging and PA thermometry of deep heating was tested using the experimental setup shown in
The same setup (
The above-described experiments investigated real-time control of GNR mediated laser thermal therapy using PA imaging. Specifically, ex-vivo experiments were performed to establish the feasibility of using the VisualSonics Vevo LAZR system for the monitoring and control of temperature rise in hyperthermia treatments. While the experiments focused on GNR mediated laser thermal therapy, they introduced a simple, practical framework to build a non-invasive controller for a wide range of thermal therapy modalities using the Vevo LAZR system. This system is widely used for small animal pre-clinical research. The system produces high-resolution photoacoustic and ultrasound images which, when combined, produce a variety of anatomical, functional and molecular information. These capabilities, along with other features like compensation for respiratory motion, make the Vevo LAZR system a versatile tool for the monitoring and control of many types of treatments that are based on heating.
The use of GNRs both enhances the laser heating process and improves the SNR of the PA signal. Both of these enhancements are only possible if the photoacoustic pulsed laser and the heating continuous-wave (CW) laser operating near the absorption peak of the GNRs (808 nm in this work). This two-fold benefit motivates the inclusion of GNRs in targeted drug delivery using thermosensitive liposomes. The embedding GNRs into the liposomes will facilitate both imaging of the liposome accumulation using PA imaging and the release of the drug, using a CW laser directed at the target region.
The above-described experiments demonstrate the use of PA thermometry, obtained from a commercially available imaging system, in controlling thermal therapy. The results obtained also confirm that the magnitude of the PA signal increases when the temperature rises and that a single calibration parameter could be used to obtain an absolute temperature from the relative changed in the PA intensity.
As described above, a PID controller for laser thermal therapy using based on invasive point temperature measurement was developed and tested, and it was demonstrated that this approach could achieve the prescribed temperature profile for both surface heating and deep-tissue heating. Subsequently, these results were used to perform non-invasive temperature control experiments, where the heating power was controlled by a PID controller using PA thermometry. The performance of the PA-based PID controller (in
In one embodiment, PA thermometry obtained from a photoacoustic imaging system was used for feedback in PID control of GNR mediated laser thermal therapy. The first completely non-invasive real-time controller for hyperthermia based on PA thermometry was utilized. This was done using the VisualSonics Vevo LAZR system that produces high-resolution PA and ultrasound images. The system is easy to use and comes with a wide range of modalities and real-time features. Introducing thermal therapy monitoring and control based on such a system can lead to the widespread use of the non-invasive PA thermometry in effectively controlling thermal therapies. The monitoring and control methods can also be used in other thermal therapy related treatments and applications.
In one embodiment, an automated control system for hyperthermia therapy that can accurately achieve a prescribed temperature for both surface heating and deep-tissue heating was developed and tested. A non-invasive PA thermometry using a high-frequency PA imaging platform (e.g., the Vevo LAZR system from FUJIFILM Visualsonics, Inc., Toronto, ON, Canada) was implemented. The Vevo LAZR system is useful for oxymetry, nanoparticle-mediated imaging and therapies, vascular imaging, and nanoparticle drug delivery systems. Advantageously, the ability to perform PA thermometry in real-time using the Vevo LAZR system in order to control heating (using any modality) enables a broad range of new possibilities in the pre-clinical setting, such as targeted thermal therapy, drug delivery using thermosensitive liposomes, thermal radiosensitization and thermal enhancement of chemotherapy.
In one embodiment, the PA imaging system was interfaced to obtain PA images while scanning ex-vivo tissue. These images were then used to obtain temperature maps in real-time during heating. Validation and calibration of the PA thermometry were done using a flouroptic thermometer. This thermometer was also used to develop and tune a software-based proportional integral derivative (PID) controller. Finally, a PA-based PID closed-loop controller was used to control gold nanorod (GNR) mediated laser thermotherapy for the desired temperature and time period.
In one embodiment, the use of GNRs substantially enhanced laser heating; the temperature rise increased to 7-fold by injecting a GNR solution with a concentration of 0.0286 mg/mL. The control experiments demonstrated that the desired temperature could be achieved and maintained at a targeted location in the ex-vivo tissue. The steady-state mean absolute deviations (MAD) from the targeted temperature during control were between 0.16° C. and 0.5° C., depending on the experiment.
In one embodiment, hyperthermia treatments were controlled using a software-based PID controller and a PA imaging system. The monitoring and control of the temperature in thermal-based therapies are important for assuring a prescribed temperature to the target tissue while minimizing the temperature of the surrounding healthy tissue. Advantageously, the easily implemented non-invasive control system described above facilitates the realization of a broad range of hyperthermia treatments.
Any of the features, attributes, or steps of the above described embodiments and variations can be used in combination with any of the other features, attributes, and steps of the above described embodiments and variations as desired.
Although the invention has been shown and described with respect to a certain embodiment or embodiments, it is apparent that this invention can be embodied in many different forms and that many other modifications and variations are possible without departing from the spirit and scope of this invention.
Moreover, while exemplary embodiments have been described herein, one of ordinary skill in the art will readily appreciate that the exemplary embodiments set forth above are merely illustrative in nature and should not be construed as to limit the claims in any manner. Rather, the scope of the invention is defined only by the appended claims and their equivalents, and not, by the preceding description.
This patent application claims priority to, and incorporates by reference in its entirety, U.S. Provisional Patent Application No. 63/085,007, entitled “Cancer Imaging Methods And Cancer Treatment Methods Using Thermotherapy And Drug Delivery”, filed on Sep. 29, 2020.
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| Number | Date | Country | |
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| 20220096873 A1 | Mar 2022 | US |
| Number | Date | Country | |
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| 63085007 | Sep 2020 | US |
