Nanobubble targeted therapy

Abstract

To provide a method for inducing cell death in a subject, and system for treating cancer in a subject.SOLUTION: A method for inducing cell death in a subject includes: administering a plurality of cell-targeting nanobubbles internalized by a target cell to a subject; and resonating nanobubbles internalized into the target cell by using inertia cavitation of internalized nanobubbles and ultrasonic energy effective for promoting apoptosis and / or necrosis of the target cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 988,832, filed March 12, 2020, the subject matter of which is incorporated herein by reference in its entirety.

[0002] government funds This invention was made with government support under Grant Nos. 5R01EB025741-02 and 1-R01-EB028144-01A1 awarded by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0003] Technical Field This application relates to diagnostic and therapeutic compositions, and more particularly to nanobubbles for diagnostic, therapeutic, and theranostic applications. [Background technology]

[0004] background Ultrasound contrast agents (UCAs) are small, gas-filled bubbles with a stabilizing shell made from a variety of materials, including polymers, proteins, or lipids. Beyond the traditional use of these agents in diagnostic ultrasound imaging, UCAs have found relevance in therapeutic applications, including targeted gene and drug delivery. These adaptable particles are currently being explored as protective therapeutic carriers and as cavitation nuclei to enhance payload delivery via sonoporation. Combining these functions improves payload circulation half-life and release profile, as well as tissue selectivity and cellular uptake. Regardless of the mechanism of action, it is advantageous for the bubbles to extravasate through the vasculature and reach cellular target sites to achieve the desired effect, particularly in cancer therapy. Summary of the Invention [Problem to be solved by the invention]

[0005] Commercially available UCAs available today are generally designed to function only as blood pool agents with diameters between 1 and 8 μm. While methods for reducing bubble size have previously been developed, most of these strategies involve post-formation manipulation of microbubbles, such as gradient separation by gravity or physical filtration or flotation. While effective for selecting nanosized bubbles, these methods, in addition to being labor-intensive, introduce the potential for sample contamination, reduce bubble yield and stability, and waste stock material. Furthermore, the applicability of microbubbles as carriers (e.g., in cancer therapy) is limited by their large size, which generally traps them in the vasculature. [Means for solving the problem]

[0006] Embodiments described herein relate to nanobubble-targeted therapy (TNT), which can provide a drug-free, low-toxicity method for inducing highly selective or targeted cell death in a subject. In some embodiments, the treatment or method can include administering a plurality of cell-targeted nanobubbles to a subject. Each of the cell-targeted nanobubbles can have a membrane defining at least one interior cavity containing at least one gas and a targeting moiety attached to the outer surface of the membrane. The targeting moiety can bind to a cell surface molecule of the target cell, and the nanobubbles can have a size, diameter, and / or composition that promotes internalization of the cell-targeted nanobubbles by the target cell upon binding of the targeting moiety to the cell surface molecule. Following administration of the cell-targeted nanobubbles to a subject, the cell-targeted nanobubbles internalized in the target cell can resonate with ultrasound energy effective to promote inertial cavitation of the internalized nanobubbles and apoptosis and / or necrosis of the target cell. [Effects of the Invention]

[0007] In some embodiments, the cell-targeting nanobubbles can have an average diameter of about 50 nm to about 400 nm, and the targeting moiety can include at least one of a polypeptide, a polynucleotide, a small molecule, an elemental compound, an antibody, and an antibody fragment.

[0008] In other embodiments, the targeted cells may be cancer cells of a subject, and the targeting moiety may bind to a cancer cell surface molecule. The cancer cell surface molecule may be a cancer cell antigen on the surface of the cancer cell. For example, cancer cell antigens include 5T4, α2β1 integrin, AXL receptor tyrosine kinase (AXL), B-cell maturation antigen (BCMA), c-MET (hepatocyte growth factor receptor), C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), cadherin-6, CD19, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, collagen receptor, cryptoprotein, and the like. Protein, CS1, Delta-like canonical Notch ligand 3 (DLL3), Endothelin receptor type B (EDNRB), Ephrin A4 (EFNA4), Epidermal growth factor receptor (EGFR), EGFRvIII, Ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), Fibroblast growth factor receptor 2 (FGFR2), Fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), Folate receptor 1 (FOLR1), Nonmetastatic Glycoprotein B (GPNMB), guanylate cyclase 2C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), integrin α, lysosome-associated membrane protein 1 (LAMP-1), Lewis Y, LIV-1, leucine-rich repeat-containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), sodium-dependent phosphate transport protein 2B (NaPi2b), nectin-4, NMB, NOTCH3, p-phosphorylated phosphoprotein 2B (P-phosphorylated phosphoprotein 2B), and p-phosphorylated phosphoprotein 3B (P-phosphorylated phosphoprotein 3B). The proteins may include at least one of p-CAD, prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), protein tyrosine phosphatase μ (PTPμ), solute carrier family 44 member 4 (SLC44A4), SLIT-like family member 6 (SLITRK6), STEAP family member 1 (STEAP1), tissue factor (TF), T-cell immunoglobulin and mucin protein-1 (TIM-1), or trophoblast surface antigen (TROP-2).

[0009] In one example, the target cell is a prostate cancer cell, the cell surface molecule is PSMA, and the targeting moiety is a PSMA ligand.

[0010] In some embodiments, the membrane can be a lipid membrane. The lipid membrane of the cell-targeting nanobubbles can further comprise at least one of glycerol, propylene glycol, pluronic (poloxamer), alcohol, or cholesterol in an amount effective to alter the modulus and / or interfacial tension of the nanobubble membrane.

[0011] In other embodiments, the lipid film comprises dipalmitoylphosphatidylcholine (DPPC), dibehenoylglycerophosphocholine (DBPC), distearoylphosphatidylcholine (DSPC), diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), and distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidic acid (DPPA), or a mixture of at least two of these PEG-functionalized lipids. For example, the lipid mixture can include a combination of at least about 50% by weight dibehenoylglycerophosphocholine (DBPC) and less than about 50% by weight additional phospholipids selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidic acid (DPPA), or PEG-functionalized phospholipids thereof.

[0012] In some embodiments, the gas within the interior cavity of the cell-targeting nanobubbles can include a perfluorocarbon gas, such as octafluoropropane (C3F8).

[0013] In other embodiments, the cell-targeting nanobubbles can further include at least one therapeutic agent contained within or conjugated to the membrane of each nanobubble. The therapeutic agent can include, for example, at least one chemotherapeutic agent, antiproliferative agent, biocide, biostatic agent, or antibacterial agent.

[0014] In some embodiments, resonating internalized nanobubbles induces death of targeted cells without adversely affecting normal cells and tissues in the subject.

[0015] In some embodiments, the resonance is achieved with a duty cycle of about 1% to about 50%, an ultrasound frequency of about 1 MHz to about 50 MHz (e.g., about 1 MHz to about 10 MHz), and about 0.1 W / cm 2 ~About 3W / cm 2 The pressure may be applied at an intensity of about 100 kPa to about 1 MPa, with a pressure amplitude of about 50 kPa to about 1 MPa, and for a time of about 1 minute to about 30 minutes.

[0016] In other embodiments, the resonation can include a sequence of two ultrasound pulses with pulses of different pressure amplitudes delivered to the tissue where the nanobubbles are internalized by the cells. In some embodiments, one pulse can have a greater pressure amplitude than the other pulse. For example, one pulse can have a pressure amplitude at least twice that of the other pulse.

[0017] In some embodiments, one pulse can be below the nanobubble pressure threshold for inertial cavitation, followed by another pulse above the pressure threshold for inertial cavitation. For example, for nanobubbles with a pressure threshold of 200 kPa, the first pulse can be 150 kPa, followed by another pulse at 250 kPa. In another example, for nanobubbles with a pressure threshold of 500 kPa, one pulse can be 300 kPa and the second pulse at 600 kPa.

[0018] In other embodiments, the overall pulse length may be longer (10-30 cycles) than a typical imaging pulse (3-6 cycles) to induce maximum inertial cavitation.

[0019] In some embodiments, the pulse sequence can be provided from an unfocused transducer, which differs from typical focused ultrasound transducers used for ultrasound treatments such as drug delivery and histotripsy.

[0020] In yet other embodiments, the method can be used to treat lesions containing widespread cancer micrometastases, such as in the liver or bone, which cannot be easily visualized and where focused ultrasound cannot be used.

[0021] In yet other embodiments, the methods and treatments can be used to induce the death of microbial prokaryotic cells and treat infectious diseases.

[0022] Other embodiments described herein relate to methods of treating cancer in a subject in need thereof. The method can include administering to the subject a plurality of cancer cell-targeted nanobubbles. Each of the cancer cell-targeted nanobubbles can have a membrane defining at least one interior cavity containing at least one gas and a targeting moiety attached to the outer surface of the membrane. The targeting moiety can bind to a cancer cell surface molecule of the targeted cancer cell. The cancer cell-targeted nanobubbles can have a size and / or diameter that promotes internalization of the nanobubbles by the targeted cancer cell upon binding of the targeting moiety to the cancer cell surface molecule.

[0023] After administration of cancer cell-targeted nanobubbles to a subject and internalization of the cancer cell-targeted nanobubbles into the cancer cells, the internalized nanobubbles can be resonated with ultrasonic energy effective to promote inertial cavitation of the internalized nanobubbles and apoptosis and / or necrosis of the targeted cancer cells.

[0024] In some embodiments, the cancer cell surface molecule can be a cancer cell antigen on the surface of a cancer cell. For example, the cancer cell antigen can be 5T4, α2β1 integrin, AXL receptor tyrosine kinase (AXL), B-cell maturation antigen (BCMA), c-MET (hepatocyte growth factor receptor), C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), cadherin-6, CD19, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, collagen receptor, cryptotannin, or the like. Protein, CS1, Delta-like canonical Notch ligand 3 (DLL3), Endothelin receptor type B (EDNRB), Ephrin A4 (EFNA4), Epidermal growth factor receptor (EGFR), EGFRvIII, Ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), Fibroblast growth factor receptor 2 (FGFR2), Fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), Folate receptor 1 (FOLR1), Nonmetastatic Glycoprotein B (GPNMB), guanylate cyclase 2C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), integrin α, lysosome-associated membrane protein 1 (LAMP-1), Lewis Y, LIV-1, leucine-rich repeat-containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), sodium-dependent phosphate transport protein 2B (NaPi2b), nectin-4, NMB, NOTCH3, p-phosphorylated phosphoprotein 2B (P-phosphorylated phosphoprotein 2B), and p-phosphorylated phosphoprotein 3B (P-phosphorylated phosphoprotein 3B). The proteins may include at least one of p-CAD, prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), protein tyrosine phosphatase μ (PTPμ), solute carrier family 44 member 4 (SLC44A4), SLIT-like family member 6 (SLITRK6), STEAP family member 1 (STEAP1), tissue factor (TF), T-cell immunoglobulin and mucin protein-1 (TIM-1), or trophoblast surface antigen (TROP-2).

[0025] In one example, the target cancer cells are prostate cancer cells, the cancer cell surface molecule is PSMA, and the targeting moiety is a PSMA ligand.

[0026] Yet another embodiment relates to a system for treating cancer in a subject. The system can include an ultrasound source configured to noninvasively deliver ultrasound energy to cancer cells within the subject, a plurality of cancer cell-targeted nanobubbles, and a controller coupled to the ultrasound source. Each of the cancer cell-targeted nanobubbles can have a membrane defining at least one internal cavity containing at least one gas and a targeting moiety coupled to the outer surface of the membrane. The targeting moiety can bind to a cancer cell surface molecule of the targeted cancer cell. The cancer cell-targeted nanobubbles can have a size and / or diameter that promotes internalization of the nanobubbles by the targeted cancer cell upon binding of the targeting moiety to the cancer cell surface molecule. The controller coupled to the ultrasound source can be configured to cause resonance of the cancer cell during a resonance time to promote inertial cavitation of the nanobubbles internalized by the cancer cell.

[0027] In some embodiments, the resonance is achieved with a duty cycle of about 1% to about 50%, an ultrasound frequency of about 1 MHz to about 50 MHz (e.g., about 1 MHz to about 10 MHz), and about 0.1 W / cm 2 ~About 3W / cm 2 The pressure may be applied at an intensity of about 100 kPa to about 1 MPa, with a pressure amplitude of about 50 kPa to about 1 MPa, and for a time of about 1 minute to about 30 minutes.

[0028] In other embodiments, the resonation can include a sequence of two ultrasound pulses with pulses of different pressure amplitudes delivered to the tissue where the nanobubbles are internalized by the cells. In some embodiments, one pulse can have a greater pressure amplitude than the other pulse. For example, one pulse can have a pressure amplitude at least twice that of the other pulse.

[0029] In some embodiments, one pulse can be below the nanobubble pressure threshold for inertial cavitation, followed by another pulse above the pressure threshold for inertial cavitation. For example, for nanobubbles with a pressure threshold of 200 kPa, the first pulse can be 150 kPa, followed by another pulse at 250 kPa. In another example, for nanobubbles with a pressure threshold of 500 kPa, one pulse can be 300 kPa and the second pulse at 600 kPa.

[0030] In other embodiments, the overall pulse length may be longer (10-30 cycles) than a typical imaging pulse (3-6 cycles) to induce maximum inertial cavitation.

[0031] In some embodiments, the pulse sequence can be provided from an unfocused transducer, which differs from typical focused ultrasound transducers used for ultrasound treatments such as drug delivery and histotripsy. [Brief explanation of the drawings]

[0032] [Figure 1] Figure 1 (A-E) shows A) a schematic illustrating the concept of the TNT approach, B) data showing the effect of extracellular NB+US comparable to the effect intracellularly at significantly lower NB doses, and C) feasibility data (D, E) showing the retention of acoustic activity of PSMA-NBs in prostate cancer cells at high resolution in vivo and in vitro. Intracellular NBs can be positioned to kill cells with high specificity and minimal collateral damage. [Figure 2] FIG. 2 is a flow diagram illustrating a method according to one embodiment. [Figure 3] Figure 3(B-C) shows representative ultrasound contrast images and corresponding enhancement (C) of NBs with rigid and flexible shells, showing a significant and rapid increase in signal as the driving pressure increases (B). Only pressures adjacent to the maximal signal increase are shown. [Figure 4]Figure 4 (A-E) shows images and plots illustrating in vivo nonlinear contrast scanning. A) Timeline showing bubble injection, nonlinear, and 3D US application. B) Representative tumor images showing bubble distribution at different time points in the tumor margin and tumor core for PSMA-NB. C) Plain NB. D) Lumason microbubbles. E) Mean signal intensity at the tumor margin and core plotted as a function of time for PSMA-NB (i), plain NB (ii), and Lumason MB (iii). Tumors were imaged at 18 MHz at 5 frames / s for 3 min and 1 frame / s for 16 min. [Figure 5] Figure 5 (A-C) shows a schematic diagram and plots illustrating 3D ultrasound scanning to visualize bubble distribution throughout the tumor. A) Timeline showing 3D US scan points. B) Representative 3D US images of the tumor showing PSMA-NB, NB, and Lumason at peak. C) Quantification of 3D US signal intensity at peak and at t = 25 min after baseline subtraction. N = 3, error bars represent mean ± sd. *P < 0.05. [Figure 6] Figure 6 (A-C) shows a schematic diagram and plots illustrating 3D ultrasound scanning to visualize bubble distribution throughout the tumor. A) Timeline showing 3D US scan points. B) Representative 3D US images of tumors showing PSMA-NBs, NBs, and Lumason at baseline, 25 minutes post-injection, and after cardiac puncture. C) Quantification of 3D US signal intensity of PSMA-NBs, plain NBs, and Lumason before and after cardiac puncture after baseline subtraction. N=3, error bars represent mean ± sd. *P<0.05. [Figure 7]Figure 7 (A-B) shows histological images demonstrating the accumulation and extravasation of Cy5.5-PSMA-NB in ​​resected tumors after cardiac puncture. A) Representative images of tumor tissue showing PSMA expression (cyan), vasculature (CD31 expression, red), and PSMA-NB or plain NB distribution (green). B) Signal intensity of bubbles, PSMA, and blood vessels expressed as a percentage of total cell fluorescence in tumor sections. Cy5.5-PSMA-NB signal at both the tumor edge and core was significantly higher than NB signal at both the tumor edge and core. N=3, error bars represent mean ± SD. *P<0.001. [Figure 8] Figure 8 (A-B) shows (A) representative US images of PSMA-positive PC3pip cells incubated with PSMA-NBs at different time points after the initial 1-hour NB exposure. (B) Acoustic activity of PSMA-NB-incubated PC3pip cells, NB-incubated PC3pip cells, and PSMA-negative PC3flu cells at different time points after treatment. PSMA-NB-incubated PC3pip cells show significantly higher acoustic activity from t = 0 to t = 24 h compared to all other groups. n = 3, error bars represent mean ± SD, * indicates a statistically significant difference (p < 0.05) from all other groups at each time point. [Figure 9] Figure 9 (A-E) shows (A) representative confocal images of PSMA-NBs and NB distribution in PC3pip cells; 100x magnification (blue - nuclei, red - NBs, and green - late endosomes / lysosomes). (B) Zoomed merged images of PSMA-NBs and (C) plain NB-incubated PC3pip cells. (D) Representative confocal images of PSMA-NBs distribution in PC3pip cells after 24 hours of exposure (blue - nuclei, red - NBs, green - endosomes). (E) Zoomed merged images of PSMA-NBs and (F) plain NB-incubated PC3pip cells. PSMA-NBs show high colocalization in late endosomal / lysosomal vesicles (yellow). [Figure 10]Figure 10 (A-C) shows plots showing (A) headspace GC / MS analysis of C3F8 gas generated by NB; eluting at 3.37 min, (B) calibration curve for various concentrations of bubbles versus peak area corresponding to C3F8 gas, (C) headspace GC / MS analysis of C3F8 gas generated by PSMA-NB and plain NB-internalized PC3pip cell suspensions. [Figure 11] Figure 11 (A-B) shows (A) representative US images of subcutaneous tissue obtained after injection of labeled cells (PSMA-NB-incubated cells) and unlabeled cells at different time points and at a 0.1 MI value, and (B) the mean US signal intensity at each time point. [Figure 11] Figure 11 shows tumor images showing bubble distribution at different time points in the tumor margin and tumor core for another replicate PSMA-NB. [Figure 12] Figure 12 is a schematic diagram of the tumor model and PSMA-targeted and non-targeted NBs. [Figure 13] Figure 13 (A–C) shows that PSMA-targeted NBs provide greater tumor enhancement than LUMASON. (A) Representative ultrasound images of PC3pip orthotopic tumors and livers after injection of PSMA-targeted NBs and clinically available MBs (LUMASON). Rows 1 and 2 show B-mode and CHI-mode images of the tumors and livers before UCA injection. Rows 3–5 show CHI images at different time points after UCA administration. The imaging intensity in the tumors and livers of mice administered PSMA-targeted NBs was significantly higher than that in animals administered LUMASON at different time points. Scale bars are 0.5 cm. (B1) Time-intensity curves (TICs) of PC3pip orthotopic tumors after intravenous administration of PSMA-targeted NBs (n = 11) and LUMASON (n = 3). (B2) Time-intensity curves (TICs) of livers after intravenous administration of PSMA-targeted NBs (n = 4) and LUMASON (n = 3). (C) Comparison of UCA kinetic parameters between PSMA-targeted NBs and LUMASON in tumors or livers. Data as mean ± standard deviation. *p<0.05, PSMA-targeted NB group vs. LUMASON group. [Figure 14]Figure 14 (A-C) shows images and plots demonstrating that PSMA-targeted NBs provide greater tumor enhancement compared to non-targeted NBs. (A) Representative ultrasound images of PC3pip orthotopic tumors after injection of PSMA-targeted and non-targeted NBs (n=11). Columns 1 and 2 show B-mode and CHI-mode images of the tumors before UCA injection, respectively. Columns 3 to 5 show CHI images at different time points after UCA administration. Scale bars are 0.5 cm. (B1) Time-intensity curves (TICs) of PC3pip orthotopic tumors after iv administration of PSMA-targeted and non-targeted NBs. (B2) The US signal obtained from non-targeted NB measurements was used to normalize the signal from PSMA-targeted NBs. Normalized signal enhancement means (intensity PSMA-targeted NB - intensity non-targeted NB). (C) Comparison of targeted and non-targeted NBs in tumors. Data are expressed as deviations (n=11). *p<0.05 for the comparison of targeted vs. non-targeted NBs. [Figure 15] Figure 15 (A-B) shows plots demonstrating that PSMA-targeted and non-targeted NBs provide greater tumor enhancement in small tumors (Group A) compared to large tumors (Group B). (A) Time intensity curves (TIC) of tumors after iv administration of PSMA-targeted and non-targeted NBs in Group A (n=7) and Group B (n=4). (B) TIC parameters between Group A (n=7) and Group B (n=4). Data are presented as standard deviation. *p<0.05, Group A vs. Group B. [Figure 16] Figure 16 (A-B) shows images and plots demonstrating that PSMA-targeted NBs enable prolonged imaging and greater US signal in PSMA-positive PC3pip tumors after nanobubbles are removed from the circulation. (A) The first row shows B-mode images of the tumor and liver before injection. The second row shows CHI of the tumor and liver before bubble collapse. The third row shows CHI of the tumor and liver after bubble collapse. The scale bar is 0.5 cm. (B) The average signal intensity of the bubbles in the tumor and liver before and after collapse. Data are presented as mean ± standard deviation; *p<0.05, targeted group vs. non-targeted group, n=4. [Figure 17] Figure 17 (A-B) shows histological images of Cy5.5 and CD31 signals in tumors treated with PSMA-targeted or non-targeted NBs after perfusion. (Magnification: 20x). (A) Cy5.5 and CD31 signals in tumors after perfusion. N=3 for both PSMA-1-targeted and non-targeted groups. (B1) Quantification of the fluorescence ratio (total bubble fluorescence / vascular fluorescence per field). Data are presented as mean ± standard deviation. *p<0.05, targeted group vs. non-targeted group, n=3. (B2) Quantification of the fluorescence ratio (total bubble fluorescence / cell fluorescence per field). [Figure 18] Figure 18 shows an outline of the procedure for administering and resonating PSMA-NBs to mice. [Figure 19] Figure 19 shows images of PSMA-positive and PSMA-negative tumors treated with PSMA-NB and US. [Figure 20] Figure 20 shows images of PSMA-positive and PSMA-negative tumors treated with US only (no PSMA-NB). [Figure 21] FIG. 21 shows images showing PSMA-positive tumors treated with PSMA-NBs and US. [Figure 22] FIG. 22 shows images showing PSMA-negative tumors treated with PSMA-NB and US. DETAILED DESCRIPTION OF THE INVENTION

[0033] Detailed Description All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. The definitions provided herein are intended to facilitate understanding of certain terms used frequently herein and are not intended to limit the scope of application.

[0034] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Furthermore, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The terms "comprising," "including," "having," and "consisting of" can also be used interchangeably.

[0035] The term "stable cavitation" refers to a gas cavity of nanobubbles that tends to grow in size and oscillate without exploding. When exposed to a pressure field, the gas cavity oscillates but does not explode. In stable cavitation, the collection of nanobubbles tends to behave in a relatively stable manner as long as a pressure field capable of producing rectified diffusion is present.

[0036] The term "inertial cavitation" refers to the vibration and violent collapse of nanobubbles' gas voids, typically at the resonant frequency of the gas voids, induced by an applied pressure field. When gas voids or nanobubbles implode within cells, they exert concentrated high pressure on the cells, which can disrupt organelles, cytoskeleton, and denature intracellular proteins. In addition to causing cell damage, inertial cavitation can also generate free radicals.

[0037] The term "oncologic disorder" can refer to a disease state in a subject in which abnormally proliferating cells and / or tissues are present. Oncologic disorders can include, but are not limited to, cancers, sarcomas, tumors, leukemias, lymphomas, and the like.

[0038] The term "neoplastic cells" can refer to cells that exhibit abnormal cell growth, such as increased, uncontrolled cell proliferation. Neoplastic cells can be hyperplastic cells, cells from a cell line that exhibit a lack of contact inhibition when grown in vitro, tumor cells, or cancer cells that are capable of metastasizing in vivo. Alternatively, neoplastic cells can be referred to as "cancer cells." Non-limiting examples of cancer cells can include melanoma, breast cancer, ovarian cancer, prostate cancer, sarcoma, leukemic retinoblastoma, hepatoma, myeloma, glioma, mesothelioma, carcinoma, leukemia, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, promyelocytic leukemia, lymphoblastoma, thymoma, lymphoma cells, melanoma cells, sarcoma cells, leukemia cells, retinoblastoma cells, hepatoma cells, myeloma cells, glioma cells, mesothelioma cells, and carcinoma cells.

[0039] The term "tumor" can refer to an abnormal mass or population of cells that results from excessive cell division, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0040] The terms "treating" or "treatment" of a disease (e.g., a neoplastic disorder) can refer to carrying out a therapeutic protocol to eradicate at least one neoplastic cell. As such, "treating" or "treatment" does not require complete eradication of the neoplastic cell.

[0041] The term "polymer" can refer to a molecule formed by the chemical bonding of two or more chemical units. The chemical units may be linked to each other by covalent bonds. Two or more linking units in a polymer can all be the same, in which case the polymer may be called a homopolymer. The chemical units can also be different, so that the polymer can be a combination of different units. Such a polymer may be called a copolymer.

[0042] The term "subject" can refer to any animal, including, but not limited to, humans and non-human animals (e.g., rodents, arthropods, insects, fish (e.g., zebrafish)), non-human primates, sheep, cattle, ruminants, lagomorphs, pigs, goats, horses, dogs, cats, birds, etc.) that are intended to be the recipient of a particular treatment.

[0043] Embodiments described herein relate to nanobubble-targeted therapy (TNT), which can provide a drug-free, low-toxicity approach to inducing highly selective or targeted cell death in a subject. TNT can use nanobubbles (NBs) that target cell surface molecules on target cells and then internalize into the target cells. Once inside or internalized in the target cells, the cell-targeted NBs can be resonated to promote inertial cavitation and / or destruction of the internalized NBs using nanobubble-specific ultrasound pulses. The unique combination of NBs interacting with ultrasound within the target cells can lead to highly selective or targeted cell death. TNT utilizes specific targeting of nanobubbles to only cells expressing specific cell surface molecules, followed by inertial cavitation with ultrasound.

[0044] As an example, NBs can be targeted to PSMA via highly selective ligands. PSMA levels have been reported to be associated with the aggressiveness of prostate cancer (PCa), so PSMA-targeted NBs are highly selective for tumors that express or are aggressive (Figure 1). Once PSMA-targeted NBs are internalized by PCa cells and the remaining NBs (which have zero toxicity on their own) are cleared from the bloodstream, a series of ultrasound pulses can be used to inertially cavitate or disrupt the NBs within the cancer cells. This results in highly focused cancer cell therapy while leaving normal cells untouched. Because ultrasound is frequently utilized in many cancer diagnostic and biopsy procedures, physicians already familiar with the technology can apply the same equipment and workflow, reducing costs and accelerating clinical interpretation.

[0045] 2 is a flow chart illustrating a death-inducing treatment or method 10 according to embodiments described herein. In a treatment or method 10 for inducing cell death, such as cancer cell death or microbial cell death, step 12 can involve administering to a subject a plurality of cell-targeted nanobubbles that can be internalized by the target.

[0046] Each cell-targeted nanobubble can have a membrane, such as a lipid membrane, defining at least one interior cavity containing at least one gas, and a targeting moiety linked to the outer surface of the lipid membrane. The targeting moiety can bind to a cell surface molecule of a target cell, and the cell-targeted nanobubble can be internalized by the target cell upon binding of the targeting moiety to the cell surface molecule.

[0047] Lipid membranes can exhibit selective activation and / or cavitation in response to known ultrasonic pressures. In some embodiments, lipid membranes can be specifically modified to initiate cavitation and nanobubble collapse at predictable pressures, thereby avoiding collateral damage and activation of other nanoscale gas nucleation sites. The composition of the lipid membranes used to form cell-targeted nanobubbles can also significantly lower the cavitation threshold.

[0048] In some embodiments, the lipid membrane can include, for example, multiple lipids, edge activators incorporated between the lipids of the membrane to increase the flexibility of the nanobubble, membrane stiffeners incorporated into the outer surface of the membrane to increase the membrane's resistance to tearing, and other additives such as pluronics (poloxamers), alcohols, and cholesterols to modify the modulus and / or interfacial tension of the bubble shell.

[0049] In other embodiments, each nanobubble can include a hydrophilic outer domain defined at least in part by the hydrophilic heads of the lipids and a hydrophobic inner domain defined at least in part by the hydrophobic tails of the lipids. An edge-activating agent, such as propylene glycol, can extend at least partially between the lipids from the outer domain to the inner domain. Glycerol can be provided on the outer domain of the nanobubble, extending partially between the hydrophilic heads of the lipids. Gases encapsulated by the membrane have low solubility in water (e.g., hydrophobic gases) and can include, for example, perfluorocarbons such as perfluoropropane or perfluorobutane, sulfur hexafluoride, carbon dioxide, nitrogen (N), oxygen (O), and air.

[0050] In some embodiments, each cell-targeted nanobubble can have a size that facilitates extravasation of the cell-targeted nanobubble and internalization of the cell-targeted nanobubble by the target cell upon binding of the targeting moiety to a cell surface molecule. For example, each nanobubble can have a size (diameter) of about 30 nm to about 600 nm or about 100 nm to about 500 nm (e.g., about 300 nm), depending on the particular lipids, edge activators, and membrane stiffeners and methods used to form the nanobubbles (described in more detail below).

[0051] Cell-targeted nanobubbles can have a lipid concentration that enhances the nanobubbles' in vivo circulatory stability. Higher lipid concentrations have been found to correlate with increased stability and longer circulation of the nanobubbles upon administration to a subject. In some embodiments, cell-targeted nanobubbles can have a lipid concentration of at least about 2 mg / ml, at least about 3 mg / ml, at least about 4 mg / ml, at least about 5 mg / ml, at least about 6 mg / ml, at least about 7 mg / ml, at least about 8 mg / ml, at least about 9 mg / ml, at least about 10 mg / ml, at least about 11 mg / ml, at least about 12 mg / ml, or more. In other embodiments, the lipid concentration of cell-targeted nanobubbles can be about 5 mg / ml to about 12 mg / ml, about 6 mg / ml to about 12 mg / ml, about 7 mg / ml to about 12 mg / ml, about 8 mg / ml to about 12 mg / ml, about 9 mg / ml to about 12 mg / ml, about 10 mg / ml to about 12 mg / ml, or at least about 10 mg / ml.

[0052] The lipids comprising the membrane or shell can include any natural, synthetic, or semi-synthetic (i.e., modified natural) moiety that is generally amphipathic or amphiphilic (i.e., contains a hydrophilic component and a hydrophobic component). Examples of any one or combination of lipids that can be used to form the membrane include phosphocholines such as 1-alkyl-2-acetoyl-sn-glycero-3-phosphocholines and 1-alkyl-2-hydroxy-sn-glycero-3-phosphocholines; phosphatidylcholines having both saturated and unsaturated lipids, including dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dibehenoylglycerophosphocholine (DBPC), distearoylphosphatidylcholine (DSPC), and diarachidonylphosphatidylcholine (DAPC); phosphatidylethanolamines such as dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine (DPPE), and distearoylphosphatidylethanolamine (DSPE); phosphatidylserine; distearoylphosphatidylcholine; Phosphatidylglycerols including glycerol (DSPG); phosphatidylinositol; sphingolipids such as sphingomyelin; glycolipids such as gangliosides GM1 and GM2; glucolipids; sulfatides; glycosphingolipids; phosphatidic acids such as dipalmitoylphosphatidic acid (DPPA) and distearoylphosphatidic acid (DSPA); palmitic acid; stearic acid; arachidonic acid; oleic acid; chitin, hyaluronic acid, polyvinylpyrrolidone or polyethylene lipid-containing polymers such as PEG; lipid-containing sulfonated mono-, di-, oligo-, or polysaccharides; cholesterol, cholesterol sulfate, and cholesterol hemisuccinate; tocopherol hemisuccinate; lipids with ether- and ester-linked fatty acids; polymeric lipids, a wide variety of which are well known in the art; diacetyl phosphate; dicetyl phosphate; stearylamine; cardiolipin; phospholipids with short-chain fatty acids of about 6 to about 8 carbons in length;Phospholipids having medium-chain fatty acids of about 10 to about 16 carbons in length; phospholipids having long-chain fatty acids of about 18 to about 24 carbons in length; synthetic phospholipids having asymmetric acyl chains, for example, one acyl chain of about 6 carbons and another acyl chain of about 12 carbons; ceramides; polyoxyalkylene (e.g., polyoxyethylene) fatty acid esters, polyoxyalkylene (e.g., polyoxyethylene) fatty alcohols, polyoxyalkylene (e.g., polyoxyethylene) fatty alcohol ethers, polyoxyalkylene (e.g., polyoxyethylene) sorbitan fatty acid esters (e.g., TWEEN (ICI) non-ionic liposomes, including niosomes such as glycerol polyethylene glycol oxystearate, glycerol polyethylene glycol ricinoleate, alkyloxylated (e.g., ethoxylated) soy sterols, alkyloxylated (e.g., ethoxylated) castor oil, polyoxyethylene-polyoxypropylene polymers, and polyoxyalkylene (e.g., polyoxyethylene) fatty acid stearates; sterols, including cholesterol sulfate, cholesterol butyrate, cholesterol isobutyrate, cholesterol palmitate, cholesterol stearate, lanosterol acetate, ergosterol palmitate, and phytosterol n-butyrate; fatty acid esters; sterol esters of sugar acids, including cholesterol glucuronide, lanosterol glucuronide, 7-dehydrocholesterol glucuronide, ergosterol gluconate, cholesterol gluconate, lanosterol gluconate, and ergosterol gluconate; esters of sugar acids and alcohols, including lauryl glucuronide, stearoyl glucuronide, myristoyl glucuronide, lauryl gluconic acid, myristoyl gluconic acid, and stearoyl gluconic acid; esters of sugars and fatty acids, including sucrose laurate, fructose laurate, sucrose palmitate, sucrose stearate, glucuronic acid, gluconic acid, and polyuronic acids; saponins, including sarsasapogenin, smilagenin, hederagenin, oleanolic acid, and digitoxigenin;Glycerin and glycerin esters, including glycerin dilaurate, glycerin trilaurate, glycerin dipalmitate, glycerin tripalmitate, glycerin distearate, glycerin tristearate, glycerin dimyristate, and glycerin trimyristate; long-chain alcohols, including n-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, and n-octadecyl alcohol; 6-(5-cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside; digalactosyl diglyceride; 6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside; 6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside xyl-1-thio-α-D-mannopyranoside; 12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoic acid; N-[12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoyl]-2-aminopalmitic acid; cholesteryl (4'-trimethylammonio)butanoate; N-succinyldioleoylphosphatidylethanolamine; 1,2-dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol; 1,3-dipalmitoyl-2-succinylglycerol; 1-hexadecyl-2-palmitoylglycerophosphoethanolamine and palmitoylhomocysteine; and / or any combination thereof.

[0053] In some embodiments, the lipids used to form the membrane may include a mixture of phospholipids with various acyl chain lengths. For example, the lipids may include dipalmitoylphosphatidylcholine (DPPC), dibehenoylglycerophosphocholine (DBPC), distearoylphosphatidylcholine (DSPC), diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), and distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidic acid (DPPA), or a mixture of at least two of these PEG-functionalized lipids.

[0054] In other embodiments, the mixture of phospholipids having various acyl chain lengths may include dibehenoylglycerophosphocholine (DBPC) and one or more additional phospholipids selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidic acid (DPPA), or PEG-functionalized phospholipids thereof.

[0055] In some embodiments, the mixture of phospholipids may include at least about 40%, at least about 50%, at least about 60%, at least about 70%, or about 80% by weight of dibehenoylglycerophosphocholine (DBPC); less than about 60%, less than about 50%, less than about 40%, less than about 30%, or less than about 20% by weight of an additional phospholipid selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidic acid (DPPA), or PEG-functionalized phospholipids thereof. The PEG can have a molecular weight of about 1000 to about 5000 Da, for example, about 2000 Da.

[0056] In some embodiments, the phospholipid mixture may include, by weight, about 40% to about 80%, about 50% to about 70%, or about 55% to about 65% (e.g., about 60%) dibehenoylglycerophosphocholine (DBPC); and about 20% to about 60%, about 30% to about 50%, or about 35% to about 45% (e.g., about 40%) additional phospholipids selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidic acid (DPPA), or PEG-functionalized phospholipids thereof.

[0057] In other embodiments, the one or more additional phospholipids may comprise, consist essentially of, or consist of a combination of dipalmitoyl phosphatidic acid (DPPA), dipalmitoyl phosphatidylethanolamine (DPPE), and PEG-functionalized distearoyl phosphatidylethanolamine (DSPE).

[0058] In yet other embodiments, the mixture of phospholipids can include, for example, dibehenoylglycerophosphocholine (DBPC), dipalmitoylphosphatidic acid (DPPA), dipalmitoylphosphatidylethanolamine (DPPE), and PEG-functionalized distearoylphosphatidylethanolamine (DSPE) in a ratio of about 6:1:1:1 by weight.

[0059] In some embodiments, an edge activator, incorporated between the lipids in each nanobubble's membrane and enhancing nanobubble flexibility, can include a co-surfactant, such as propylene glycol, which enhances the effectiveness of the phospholipid surfactant. The edge activator can be provided to each nanobubble in an amount effective to cause separation of the nanobubble's lipid domains and form defects that absorb excess pressure that may have caused the lipid "domains" to tear. Other edge activators that can be substituted for or used in combination with propylene glycol can include cholesterol, sodium cholate, limonene, oleic acid, and / or Span 80.

[0060] In some embodiments, the amount of propylene glycol provided in the nanobubbles can be about 0.05 ml to about 0.5 ml, about 0.06 ml to about 0.4 ml, about 0.07 ml to about 0.3 ml, about 0.08 ml to about 0.2 ml, or about 0.1 ml per ml of hydrated lipid.

[0061] In another embodiment, the membrane stiffener incorporated into the outer surface of each nanobubble membrane and enhancing membrane resistance to tearing comprises glycerol. The glycerol can be provided on each nanobubble membrane in an amount effective to stiffen the membrane and improve membrane resistance to lipid "domain" tearing. The amount of glycerol provided on the nanobubble membrane can be about 0.05 ml to about 0.5 ml, about 0.06 ml to about 0.4 ml, about 0.07 ml to about 0.3 ml, about 0.08 ml to about 0.2 ml, or about 0.1 ml per ml of hydrated lipid.

[0062] The membranes defining the nanobubbles can be concentric or otherwise and can have a monolamellar structure (i.e., composed of one monolayer or bilayer), an oligolamellar structure (i.e., composed of about two or about three monolayers or bilayers), or a multilamellar structure (i.e., composed of about four or more monolayers or bilayers). The membrane can be substantially solid (homogeneous), porous, or semi-porous.

[0063] The interior void space defined by the membrane can contain at least one gas. The gas may have low solubility in water and can be, for example, a perfluorocarbon such as perfluoropropane (e.g., octafluoropropane) or perfluorobutane. The interior void space can also contain other gases such as carbon dioxide, sulfur hexafluoride, air, nitrogen (N), oxygen (O), and helium.

[0064] In some embodiments, nanobubbles can include a linker that connects a targeting moiety and, optionally, a therapeutic agent to the membrane of each nanobubble. The linker can be of any suitable length and contain any suitable number of atoms and / or subunits. The linker can include one or a combination of chemical and / or biological moieties. Examples of chemical moieties can include alkyl groups, methylene carbon chains, ethers, polyethers, alkylamide linkers, alkenyl chains, alkynyl chains, disulfide groups, and polymers such as poly(ethylene glycol) (PEG), functionalized PEG, PEG-chelating polymers, dendritic polymers, and combinations thereof. Examples of biological moieties can include peptides, modified peptides, streptavidin-biotin or avidin-biotin, polyamino acids (e.g., polylysine), polysaccharides, glycosaminoglycans, oligonucleotides, phospholipid derivatives, and combinations thereof.

[0065] Cell-targeted nanobubbles can also contain other materials, such as liquids, oils, bioactive agents, diagnostic agents, therapeutic agents, photoacoustic agents (e.g., Sudan Black), and / or nanoparticles (e.g., iron oxide), which may be encapsulated by the membrane and / or linked or conjugated to the membrane.

[0066] The targeting moiety can bind to cell surface molecules of the target cells and / or tissues, allowing the nanobubbles to target and / or adhere to the target cells and / or tissues of interest. In some embodiments, the targeting moiety can include any molecule or complex of molecules that can interact with a cell surface or extracellular molecule or cellular biomarker. Cell surface molecules can include, for example, cellular proteases, kinases, proteins, cell surface receptors, lipids, and / or fatty acids.

[0067] In certain embodiments, the targeting moiety specifically binds to a cell surface molecule of the target cell. As used herein, the first molecule is, for example, about 10 5 M -1A first molecule "specifically binds" to a second molecule if it binds to or associates with the second molecule with an affinity or Ka (i.e., the equilibrium binding constant of a particular binding interaction in units of 1 / M) of at least about 10 6 M -1 , about 10 7 M -1 , about 10 8 M -1 , about 10 9 M -1 , about 10 10 M -1 , about 10 11 M -1 , about 10 12 M -1 , or about 10 13 M -1 "High affinity" binding is defined as binding to a second molecule with a Ka of at least 10 7 M -1 , about 10 8 M -1 , about 10 9 M -1 , about 10 10 M -1 , about 10 11 M -1 , about 10 12 M -1 , about 10 13 M -1 Alternatively, affinity can be expressed as M (e.g., 10 -5 M~10 -13 Specific binding can be defined as the equilibrium dissociation constant (KD) of a particular binding interaction in units of about 10 M or less. In certain embodiments, specific binding can be defined as the equilibrium dissociation constant (KD) of a particular binding interaction in units of about 10 M or less. -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M, 10 -10 M, 10 -11 M or 10 -12By "binding" is meant binding to a target molecule with a KD of M or less. The binding affinity of a first molecule for a target can be readily determined using conventional techniques, for example, by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, surface plasmon resonance (SPR) techniques (e.g., using a BIAcore 2000 instrument, using the general procedures outlined by the manufacturer); radioimmunoassay, etc.

[0068] In some embodiments, targeting moieties may include, but are not limited to, synthetic compounds, natural compounds or products, macromolecular entities, bioengineered molecules (e.g., polypeptides, lipids, polynucleotides, antibodies, antibody fragments), and small entities (e.g., small molecules, neurotransmitters, substrates, ligands, hormones, and elemental compounds).

[0069] In one example, targeting moieties may include antibodies such as monoclonal, polyclonal, or humanized antibodies, including but not limited to Fv fragments, single-chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent targeting moieties, including but not limited to, disulfide Fv fragments, which are generally covalently linked or otherwise stabilized (i.e., leucine zipper or helix stabilized) scFv fragments, scFv tandems ((scFv)2 fragments), monospecific or bispecific antibodies such as diabodies, tribodies, or tetrabodies; and receptor molecules that naturally interact with a desired target molecule.

[0070] Antibody preparation can be accomplished by any number of well-known methods for generating antibodies. These methods generally involve immunizing an animal, typically a mouse, with a desired immunogen (e.g., a desired target molecule or fragment thereof). Once the mouse is immunized and boosted one or more times with the desired immunogen(s), antibody-producing hybridomas can be prepared and screened according to well-known methods. For a general overview of monoclonal antibody production, see, e.g., Kuby, Janis, Immunology, 3rd Edition, pp. 131-139, W.H. Freeman & Co. (1997), portions of which are incorporated herein by reference.

[0071] Targeting moiety does not need to be derived from biological sources.Targeting moiety can be, for example, screened from a combinatorial library of synthetic peptides.One such method is described in U.S. Patent No. 5,948,635, which is incorporated herein by reference, and describes the generation of a phagemid library with random amino acid insertions in the pIII gene of M13.This phage can be clone-amplified by affinity selection.

[0072] The immunogen used to prepare a targeting moiety with the desired specificity is generally the target molecule, or a fragment or derivative thereof. Such immunogens may be isolated from their naturally occurring sources or synthesized using methods known in the art. For example, peptide chains can be synthesized by 1-ethyl-3-[dimethylaminopropyl]carbodiimide (EDC)-catalyzed condensation of amine and carboxyl groups. In certain embodiments, the immunogen can be linked to carrier beads or proteins. For example, the carrier can be functionalized beads such as SASRIN resin, commercially available from Bachem, King of Prussia, PA, or proteins such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA). The immunogen can be directly coupled to the carrier or can be linked to the carrier via a linker such as a non-immunogenic synthetic linker (e.g., polyethylene glycol (PEG) residues, aminocaproic acid or its derivatives), or a random or semi-random polypeptide.

[0073] In certain embodiments, it may be desirable to mutate the binding region of polypeptide targeting moiety and select targeting moiety with superior binding properties compared to non-mutated targeting moiety.This can be achieved by any standard mutagenesis technique, such as PCR using Taq polymerase under error-inducing conditions.In such cases, PCR primers can be used to amplify the scFv coding sequence of phagemid plasmid under mutation-inducing conditions.The PCR product can then be cloned into phagemid vector and screened for desired specificity as described above.

[0074] In other embodiments, targeting moieties can be modified to make them more resistant to cleavage by proteases. For example, the stability of targeting moieties comprising polypeptides can be increased by substituting one or more naturally occurring amino acids in the (L) configuration with D-amino acids. In various embodiments, at least 1%, 5%, 10%, 20%, 50%, 80%, 90%, or 100% of the amino acid residues of the targeting moiety can be in the D configuration. Switching from L-amino acids to D-amino acids neutralizes the digestive ability of many ubiquitous peptidases found in the gastrointestinal tract. Alternatively, enhanced stability of targeting moieties comprising peptide bonds can be achieved by introducing modifications of conventional peptide bonds. For example, introducing a cyclic ring into the polypeptide backbone can confer enhanced stability to avoid the effects of many proteolytic enzymes known to digest polypeptides in the stomach or other digestive tracts and serum. In yet other embodiments, enhanced stability of the targeting moiety can be achieved by intercalating one or more dextrorotatory amino acids (such as dextrorotatory phenylalanine or dextrorotatory tryptophan) between the amino acids of the targeting moiety. In exemplary embodiments, such modifications increase the protease resistance of the targeting moiety without affecting the activity or specificity of its interaction with the desired target molecule.

[0075] In certain embodiments, antibodies or variants thereof can be modified to reduce their immunogenicity when administered to a subject. For example, if the subject is human, the antibody can be "humanized," in which the complementarity-determining region(s) of a hybridoma-derived antibody are grafted onto a human monoclonal antibody, as described, for example, in Jones, P. et al. (1986), Nature, 321, 522-525 or Tempest et al. (1991), Biotechnology, 9, 266-273. Transgenic mice or other mammals can also be used to express humanized antibodies. Such humanization can be partial or complete.

[0076] In certain embodiments, the targeting moieties described herein may comprise a homing peptide that selectively directs nanobubbles to target cells. Homing peptides for target cells can be identified using a variety of methods known in the art. Many laboratories have identified homing peptides that are selective for cells of the brain, kidney, lung, skin, pancreas, intestine, uterus, adrenal gland, retina, muscle, prostate, or tumor vasculature. See, for example, Samoylova et al., 1999, Muscle Nerve, 22:460; Pasqualini et al., 1996, Nature, 380:364; Koivunen et al., 1995, Biotechnology, 13:265; Pasqualini et al., 1995, J. Cell Biol., 130:1189; Pasqualini et al., 1996, Mole. Psych., 1:421, 423; Rajotte et al., 1998, J. Clin. Invest., 102:430; Rajotte et al., 1999, J. Biol. Chem., 274:11593. See also U.S. Patent Nos. 5,622,6999; 6,068,829; 6,174,687; 6,180,084; 6,232,287; 6,296,832; 6,303,573; and 6,306,365.

[0077] Phage display technology provides a means for expressing diverse populations of random or selectively randomized peptides. Various methods of phage display and methods for generating diverse populations of peptides are well known in the art. For example, a method for preparing a diverse population of binding domains on the surface of phage is described in U.S. Pat. No. 5,223,409. In particular, phage vectors useful for generating phage display libraries, as well as methods for selecting potential binding domains and generating randomly or selectively mutated binding domains, are also provided in U.S. Pat. No. 5,223,409. Similarly, methods for generating phage peptide display libraries, including vectors, and methods for diversifying the population of expressed peptides are also described in Smith et al., 1993, Meth. Enzymol., 217:228-257; Scott et al., Science, 249:386-390; and two PCT publications, WO 91 / 07141 and WO 91 / 07149. Phage display technology can be particularly powerful when used in conjunction with, for example, codon-based mutagenesis methods, which can be used to generate random peptides or random or desirably biased peptides (see, e.g., U.S. Patent No. 5,264,563). Using these or other well-known methods, phage display libraries can be generated, which can be subjected to in vivo phage display to identify peptides that advance to one or a few selected tissues.

[0078] In vitro screening of phage libraries has previously been used to identify peptides that bind to antibodies or cell surface receptors (see, e.g., Smith et al., 1993, Meth. Enzymol., 217:228-257). For example, in vitro screening of phage peptide display libraries has been used to identify novel peptides that specifically bind to integrin adhesion receptors (see, e.g., Koivunen et al., 1994, J. Cell Biol. 124:373-380) and the human urokinase receptor (Goodson et al., 1994, Proc. Natl. Acad. Sci., USA 91:7129-7133).

[0079] In certain embodiments, the targeting moiety may comprise a receptor molecule, for example, a receptor that naturally recognizes a desired specific molecule on a target cell. Such receptor molecules include receptors modified to enhance the specificity of their interaction with the target molecule, receptors modified to interact with a desired target molecule that is not naturally recognized by the receptor, and fragments of such receptors (see, for example, Skerra, 2000, J. Molecular Recognition, 13:167-187). A preferred receptor is a chemokine receptor. Exemplary chemokine receptors are described, for example, in Lapidot et al., 2002, Exp Hematol, 30:973-81 and Onuffer et al., 2002, Trends Pharmacol Sci, 23:459-67.

[0080] In other embodiments, the targeting moiety may comprise a ligand molecule, including, for example, a ligand that naturally recognizes a desired specific receptor on a target cell, including ligands modified to increase the specificity of their interaction with the target receptor, ligands modified to interact with a desired receptor not naturally recognized by the ligand, and fragments of such ligands.

[0081] In yet other embodiments, the targeting moiety may comprise an aptamer. Aptamers are oligonucleotides selected to specifically bind to a desired molecular structure on a target cell. Aptamers are generally the product of an affinity selection process similar to phage display affinity selection (also known as in vitro molecular evolution). This process involves, for example, performing several tandem iterations of affinity separation using a solid support to which a disease immunogen is bound, followed by polymerase chain reaction (PCR) to amplify the nucleic acids bound to the immunogen. Each round of affinity separation thus enriches the nucleic acid population for molecules that successfully bind to the desired immunogen. In this way, a random pool of nucleic acids may be "educated" to generate aptamers that specifically bind to the target molecule. Aptamers are typically RNA, but can also be DNA or analogs or derivatives thereof, such as, but not limited to, peptide nucleic acids (PNAs) and phosphorothioate nucleic acids.

[0082] In yet other embodiments, the targeting moiety may be a peptidomimetic. For example, by employing scanning mutagenesis to map the amino acid residues of a protein involved in binding to other proteins, peptidomimetic compounds can be generated that mimic those residues and promote the interaction. Such mimetics may then be used as targeting moieties to deliver nanobubbles to target cells.For example, non-hydrolyzable peptide analogs of such residues include benzodiazepines (see, e.g., Freidinger et al., Peptides: Chemistry and Biology, GR Marshall (ed.), ESCOM Publisher: Leiden, Netherlands, 1988), azepines (see, e.g., Huffman et al., Peptides: Chemistry and Biology, GR Marshall (ed.), ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma malactam rings (Garvey et al., Peptides: Chemistry and Biology, GR Marshall (ed.), ESCOM Publisher: Leiden, Netherlands, 1988), ketomethylene pseudopeptides (Ewenson et al., 1986, J Med Chem 29:295; and Ewenson et al., Peptides: Structure and Function Function (Proceedings of the 9th American Peptide Symposium, Pierce Chemical Co., Rockland, Ill., 1985), a b-turn dipeptide core (Nagai et al., 1985, Tetrahedron Lett 26:647; and Sato et al., 1986, J Chem Soc Perkin Trans 1:1231), and a β-amino alcohol (Gordon et al., 1985, Biochem Biophys Res Cummun 126:419; and Dann et al., 1986, Biochem Biophys Res Commun 134:71).

[0083] In some embodiments, the targeting moiety binds to an antigen on a target cell. Target cells of interest include, but are not limited to, cells associated with a particular disease or condition in which it is desirable to induce cell death. According to some embodiments, the target cell may be a cancer cell, an immune cell, an endothelial cell, or a prokaryotic cell of a microorganism.

[0084] As such, in some embodiments, the target cell is a cancer cell. By "cancer cell" is meant a cell that exhibits a neoplastic cell phenotype, which may be characterized, for example, by one or more of aberrant cell growth, aberrant cell proliferation, loss of density-dependent growth inhibition, anchorage-independent growth potential, the ability to promote tumor growth and / or development in an immunocompromised non-human animal model, and / or any suitable indicator of cellular transformation. "Cancer cell" may be used interchangeably herein with "tumor cell," "malignant cell," or "carcinoma cell," and includes cancer cells of solid tumors, semi-solid tumors, primary tumors, metastatic tumors, and the like. In certain aspects, the cancer cell is a carcinoma cell.

[0085] In some embodiments, the cancer cell antigen is 5T4, alpha2beta1 integrin, AXL receptor tyrosine kinase (AXL), B-cell maturation antigen (BCMA), c-MET (hepatocyte growth factor receptor), C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), cadherin-6, CD19, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, collagen receptor, Cryptoprotein, CS1, Delta-like canonical Notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvIII, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1), Non-transferable glycoprotein B (GPNMB), guanylate cyclase 2C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), integrin α, lysosome-associated membrane protein 1 (LAMP-1), Lewis Y, LIV-1, leucine-rich repeat-containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), sodium-dependent phosphate transport protein 2B (NaPi2b), nectin-4, NMB, NOTCH3, p The protein may include at least one of p-cadherin (p-CAD), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), protein tyrosine phosphatase μ (PTPμ), solute carrier family 44 member 4 (SLC44A4), SLIT-like family member 6 (SLITRK6), STEAP family member 1 (STEAP1), tissue factor (TF), T-cell immunoglobulin and mucin protein-1 (TIM-1), or trophoblast surface antigen (TROP-2).

[0086] Non-limiting examples of antibodies that specifically bind to tumor antigens that can be used as targeting moieties include adecatumumab, asclinbacumab, ticutumumab, conatumumab, daratumumab, drozitumumab, durigotumab, durvalumab, dusigitumumab, enfortumab, enoticumab, figitumumab, ganitumumab, glembatumumab, intetumumab, ipilimumab, iratumumab, icrucumab, lexatumumab, lucatumumab, mapatumumab, narunatumumab, necitumumab, nesbacumab, ofatumumab, olaratumumab, panitumumab, patritumumab, and the like. , pritumumab, radletumumab, ramucirumab, rilotumumab, lobatumumab, seribantumumab, talectumumab, teprotumumab, tobetumab, vanticumab, besencumab, votumumab, zalutumumab, framvotumab, altumomab, anatumomab, altitumomab (Arcitumomab), bectumomab, blinatumomab, detumomab, ibritumomab, minletumomab, mitumomab, moxetumomab, naptumomab, nofetumomab, pemtumomab, pintumomab, racotumomab, satumomab, solitomab, taplitumomab, tenatumomab Mab, tositumomab, tremelimumab, abagovomab, igovomab, oregovomab, capromab, edrecolomab, nacolomab, amatuximab, bavituximab, brentuximab, cetuximab, dellotuximab, dinutuximab, ensituximab, futuximab, girentuximab, indatuximab, isatuximab, margetuximab, rituximab, siltuximab, ublituximab, ecloneximab, abituzumab, alemtuzumab, bevacizumab, bivatuzumab, brontiximab, cantuzumab, chituzumab Mab, clivatuzumab, dacetuzumab, demcizumab, dalotuzumab, denintuzumab, elotuzumab, emactuzumab, emibetuzumab, enoblitzumab, etaracizumab, farletuzumab, ficlatuzumab, gemtuzumab, imgatuzumab, inotuzumab, labetuzumab, rifastuzumab, lintuzumab, lorvotuzumab, lumuletuzumab, matuzumab, milatuzumab, nimotuzumab, obinutuzumab, ocaratuzumab, otlertuzumab, onartuzumab, oportuzumab, pulsatuzumab, pertuzumab, pinatuzumab, polatuzumab,Examples of antibodies include sibrotuzumab, simtuzumab, tacatuzumab, tigatuzumab, trastuzumab, tucotuzumab, bundurumab, vanucizumab, pertuzumab, borsetuzumab, sotituzumab, catumaxomab, ertumaxomab, depatuxizumab, ontuxizumab, brontuzumab, tamtubetumab, or tumor antigen-binding variants thereof. As used herein, "variant" refers to an antibody that specifically binds to a particular antigen (e.g., HER2 for trastuzumab) but has fewer or more amino acids than the parent antibody (e.g., is a fragment (e.g., scFv) of the parent antibody), has one or more amino acid substitutions relative to the parent antibody, or a combination thereof.

[0087] For example, if the targeted cells include ovarian cancer cells, the targeting moiety can include an antibody or peptide against human CA-125R. Overexpression of CA-125 is significant in ovarian cancer cells. Alternatively, if the targeted cells include a malignant cancer such as glioblastoma, the targeting moiety can include an antibody or peptide against extracellular growth factor receptor (EGFR), human transferrin receptor (TfR), and / or extracellularly cleaved PTPmu. Overexpression of EGFR and TfR and extracellular cleavage of PTPmu are involved in the malignant phenotype of tumor cells.

[0088] Other targeting moieties can include PSMA-targeting moieties or PSMA ligands that can selectively recognize PSMA-expressing tumors, cancer cells, and / or cancer angiogenesis in vivo. PSMA is a transmembrane protein that is highly overexpressed (100-1000-fold) in almost all prostate cancer (PC) tumors. Only 5-10% of primary PC lesions have been shown to be PSMA-negative. PSMA expression levels increase with tumor stage and grade.

[0089] Small molecule PSMA ligands bind to an active site within the extracellular domain of PSMA, are internalized, and are recycled in endosomes, resulting in enhanced tumor uptake and retention and high image quality. Examples of PSMA ligands include Afshar-Oromieh A, Malcher A, Eder M, et al., PET imaging with a [68Ga]gallium-labeled PSMA ligand for the diagnosis of prostate cancer: biodistribution in humans and first evaluation of tumor; Weineisen M, Schottelius M, Simecek J, et al., 68Ga- and 177Lu-Labeled PSMA I&T: Optimization of A PSMA-Targeted Theranostic Concept and First Proof-of-Concept Human Studies. J Nucl Med. 2015;56:1169-1176. Lesions. Eur J Nucl Med Mol Imaging.2013;40:486-495;Cho SY, Gage KL, Mease RC, et al. Biodistribution, tumor detection, and radiation dosimetry of 18F-DCFBC, a low-molecular-weight inhibitor of prostate-specific membrane antigen, in patients with metastatic prostate cancer. J Nucl Med.2012;53:1883-1891; and Rowe SP, Gage KL, Faraj SF, et al. (1)(8)F-DCFBC PET / CT for PSMA-Based Detection and Characterization of Primary Prostate Cancer ((1)(8)F-DCFBC PET / CT for PSMA-Based Detection and Characterization of Primary Prostate Cancer) J Nucl Med. 2015;56:1003-1010.

[0090] Other examples of PSMA ligands are described in U.S. Patent Nos. 6,875,886, 6,933,114, and 8,609,142, which are incorporated by reference in their entireties. Still other examples of PSMA ligands are disclosed in U.S. Patent Application Publication Nos. 2015 / 0366968, 2015 / 0366968, 2018 / 0064831, 2018 / 0369385, and U.S. Patent No. 9,889,199, all of which are incorporated by reference in their entireties.

[0091] In some embodiments, the PSMA ligand has the general formula (I): [ka] (In the formula, n and n 1 are each independently 1, 2, 3, or 4; L is an optionally substituted aliphatic or heteroaliphatic linking group; B is a linker, such as a peptide linker, that includes at least one negatively charged amino acid; Y is a lipid of the nanobubble that is directly or indirectly linked or bound to B; and Z is hydrogen or at least one of a detectable moiety or a label or a therapeutic agent linked or attached directly or indirectly to B. In other embodiments, Z can be selected from the group consisting of an imaging agent, an anti-cancer agent, or a combination thereof. In yet other embodiments, Z is a fluorescent label such as rhodamine, IRDye700, IRDye800, Cy3, Cy5, and / or Cy5.5.

[0092] Optionally, cell-targeting nanobubbles can include a therapeutic agent encapsulated by and / or linked to the membrane. Examples of therapeutic agents can include, but are not limited to, chemotherapeutic agents, biologically active ligands, small molecules, DNA fragments, DNA plasmids, interfering RNA molecules such as siRNA, oligonucleotides, and DNA encoding shRNA. A therapeutic agent can refer to any therapeutic or prophylactic agent used to treat (including prevent, diagnose, alleviate, or cure) a ailment, affliction, condition, disease, or injury in a subject. It will be understood that the membrane can additionally or optionally include proteins, carbohydrates, polymers, surfactants, and / or other membrane-stabilizing materials, any one or combination of which can be natural, synthetic, or semi-synthetic.

[0093] In some embodiments, the therapeutic agent can be at least one of a chemotherapeutic agent, an antiproliferative agent, an antibacterial agent, a biocide, and / or a biostatic agent. The therapeutic agent can be encapsulated and / or associated with the membrane of the nanobubbles.

[0094] In some embodiments, cell-targeted nanobubbles can be formed by dissolving at least one lipid and a lipid linked to a targeting moiety in propylene glycol. For example, PSMA-targeted nanobubbles can be prepared by dissolving 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC, Avanti Polar Lipids Inc., Pelham, AL), 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; DPPE (Corden Pharma, Switzerland), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](ammonium salt) (DSPE-mPEG 2000, Laysan Lipids, Arab, AL) in propylene glycol along with DSPE-PEG-PSMA-1 to produce a lipid-propylene glycol solution. It will be appreciated that other substances such as proteins, carbohydrates, polymers, surfactants, and / or other membrane stabilizing materials may be dissolved in propylene glycol.

[0095] After forming the lipid-propylene glycol solution, glycerol and phosphate buffered saline (PBS) solutions can be added to the lipid-propylene glycol solution, and the resulting solution can be mixed, for example, by sonication. The mixed solution can be transferred to a vial. Air can be removed from the sealed vial containing the hydrated lipid solution and replaced with a gas such as octafluoropropane until the pressure in the vial equalizes. The resulting solution can then be shaken or stirred for a time sufficient to form nanobubbles (e.g., about 45 seconds). In one example, a lipid-propylene glycol solution containing DBPC / DPPA / DPPE / DSPE-PEG-PSMA-1 dissolved in propylene glycol can be contacted with the hydrated PBS / glycerol solution, placed in a vial, and then placed in an incubator shaker at about 37°C and about 120 rpm for about 60 minutes. In some embodiments, the resulting solution containing nanobubbles can be lyophilized and reconstituted for storage and shipping, or frozen and thawed before use.

[0096] The cell-targeted nanobubbles so formed can be administered to a subject via any known route, such as via intravenous injection. By way of example, a composition comprising a plurality of octafluoropropane-containing nanobubbles can be administered intravenously to a subject known or suspected of having a tumor.

[0097] In some embodiments, nanobubbles are administered to a subject to treat a neoplastic disease, such as a solid tumor, e.g., a solid carcinoma, sarcoma, or lymphoma, and / or a collection of neoplastic cells. Tumors can be malignant or benign, and can contain both cancerous and pre-cancerous cells.

[0098] The composition comprising cell-targeting nanobubbles can be formulated for administration (for example, injection) to a subject diagnosed with at least one tumor disorder.For example, cell-targeting nanobubbles can be targeted to prostate cancer cells by conjugating PSMA ligand, which is specific to the PSMA antigen that is overexpressed on prostate cancer cells.Cell-targeting nanobubbles can be formulated with at least one lipid that is conjugated to PEG.Then, nanobubbles can be combined with PSMA ligand, which then becomes conjugated to the PEG of lipid.

[0099] The location(s) at which the nanobubble composition is administered to a subject can be determined based on the individual needs of the subject, such as the location of the neoplastic cells (e.g., tumor location, tumor size, and tumor location on or near a particular organ). For example, the composition can be injected intravenously into a subject. It will be understood that other injection routes can be used, including, for example, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal routes.

[0100] Cell-targeted nanobubbles administered to a subject can circulate within the subject and bind and / or complex with target cells by binding and / or complexing the targeting moiety to cell surface molecules of the target cells. Typically, cell-targeted nanobubbles can bind and / or complex with target cells within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 1 hour or less.

[0101] Once the cell-targeted nanobubbles are bound and / or complexed to the target cells, the size and / or diameter of the cell-targeted nanobubbles allows them to be internalized or enter the target cells, e.g., by endocytosis and / or phagocytosis. The cell-targeted nanobubbles accumulate within the target cells and can remain within the cells for an extended period of time, e.g., at least 1 hour, 2 hours, 3 hours, or more.

[0102] Referring again to Figures 1 and 2, following internalization of the cell-targeted nanobubbles into the target cells, in step 14 of method 10, the internalized nanobubbles are resonated with ultrasonic energy of a given frequency, acoustic pressure, and for a time effective to promote violent oscillation, vibration, and rapid volume collapse and / or inertial cavitation of the internalized nanobubbles, which can result in apoptosis and / or necrosis of the targeted cells.

[0103] Resonance of internalized nanobubbles can be achieved by using non-invasive, minimally invasive, and / or external ultrasound sources that generate ultrasound energy effective to promote inertial cavitation. The intensity and frequency of the applied ultrasound signal, as well as the duty cycle and pattern for activating the ultrasound source, can be controlled and configured for a given application. Monitoring nanobubble dynamics and correlating inertial collapse signatures with treatment parameters provides a strategy for gaining further insight into mechanisms of action and intra-treatment monitoring to improve clinical outcomes.

[0104] The ultrasound source can provide specific acoustic sequences that can promote nanobubble collapse without disrupting and / or adversely affecting normal cells and tissues. These sequences can be applied from an unfocused transducer, which differs from the typical focused ultrasound transducers used in ultrasound treatments such as drug delivery and histotripsy. The use of unfocused ultrasound makes it possible to treat lesions such as widespread cancer micrometastases in the liver or bone, for example, which cannot be easily visualized and therefore cannot be treated with focused ultrasound. It will be appreciated that focused transducers can also be used in certain applications.

[0105] In some embodiments, the resonance is at a duty cycle of about 1% to about 50%, about 1% to about 40%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, or about 5% to about 15%, an ultrasound frequency of about 1 MHz to about 50 MHz, about 1 MHz to about 40 MHz, about 1 MHz to about 30 MHz, about 1 MHz to about 20 MHz, about 1 MHz to about 15 MHz, or about 1 MHz to about 10 MHz, or about 0.1 W / cm 2 ~about 10W / cm 2 , about 0.1W / cm 2 ~about 9W / cm 2 , about 0.1W / cm 2 ~approx. 8W / cm 2 , about 0.1W / cm 2 ~about 7W / cm 2 , about 0.1W / cm 2 ~about 6W / cm 2 , about 0.1W / cm 2 ~about 5W / cm 2 , about 0.1W / cm 2 ~about 4W / cm 2 , or about 1 W / cm 2 ~about 4W / cm 2 the pressure amplitude may be about 50 kPa to about 1 MPa, about 50 kPa to about 900 KPa, about 50 kPa to about 800 KPa, about 50 kPa to about 750 KPa, about 100 kPa to about 750 KPa, or about 150 kPa to about 750 KPa, and the time may be about 1 minute to about 30 minutes, about 1 minute to about 25 minutes, about 1 minute to about 20 minutes, about 1 minute to about 15 minutes, about 1 minute to about 10 minutes, or about 1 minute to about 5 minutes.

[0106] In other embodiments, the resonation can include two ultrasound pulse sequences having pulses of different pressure amplitudes delivered to the tissue into which the nanobubbles are administered, one pulse having a greater pressure amplitude than the other pulse, for example, one pulse having a pressure amplitude at least twice that of the other pulse.

[0107] In some embodiments, one pulse is below the nanobubble pressure threshold for inertial cavitation, followed by another pulse above the pressure threshold for inertial cavitation. For example, for nanobubbles with a pressure threshold of 200 kPa, the first pulse is 150 kPa, followed by another pulse of 250 kPa. In another example, for nanobubbles with a pressure threshold of 500 kPa, one pulse is 300 kPa and the second pulse is 600 kPa.

[0108] In other embodiments, the overall pulse length may be longer (10-30 cycles) than a typical imaging pulse (3-6 cycles) to induce maximum inertial cavitation.

[0109] In some embodiments, a system including an ultrasound source may include both an ultrasound source (transmitter) and a passive cavitation detection and monitoring acoustic sensor (receiver). The acoustic sensor may be integrated into the transmitted ultrasound source as a transducer element in an array of elements, or may be implemented as a standalone sensor such as a hydrophone appropriately positioned relative to the ultrasound source and target area. Instead of simply detecting reflected ultrasound signals at the source frequency, the system may rely on detecting inertial cavitation signals resulting from the collapse of cell-targeting nanobubbles that selectively accumulate targeted cells.

[0110] In some embodiments, the treatments and / or methods described herein can be used to treat cancer in a subject in need thereof. Such methods include administering to the subject a plurality of cancer cell-targeted nanobubbles. Each of the cancer cell-targeted nanobubbles can have a lipid membrane defining at least one interior cavity containing at least one gas and a targeting moiety linked to the outer surface of the lipid membrane. The targeting moiety can bind to a cancer cell surface molecule of the targeted cancer cell. The cancer cell-targeted nanobubbles can have a size and / or diameter that promotes internalization of the nanobubbles by the targeted cancer cell upon binding of the targeting moiety to the cancer cell surface molecule. Following administration of the cancer cell-targeted nanobubbles to the subject and internalization of the cancer cell-targeted nanobubbles into the cancer cell, the internalized nanobubbles can be resonated with ultrasound energy effective to promote inertial cavitation of the internalized nanobubbles and apoptosis and / or necrosis of the targeted cancer cell.

[0111] In certain embodiments, the subject has a cancer characterized by the presence of a solid tumor, a semi-solid tumor, a primary tumor, a metastatic tumor, a liquid tumor (e.g., leukemia or lymphoma), or the like.Cancers that can be treated using the methods described herein include adult and pediatric acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, anal cancer, cancer of the appendix, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, biliary tract cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brain stem glioma, cerebellar astrocytoma, malignant glioma, glioblastoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, hypothalamic islet tumor, breast cancer, male breast cancer, bronchial adenoma, Burkitt's lymphoma, carcinoid tumor, carcinoma of unknown origin, central nervous system lymphoma, cerebellar astrocytoma, malignant neuroblastoma, and malignant neuroblastoma. Glioma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphocytic and myeloid leukemia, chronic myeloproliferative disorders, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing's family of tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumors, extracranial germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, gestational trophoblastic tumors, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and ocular Pathway glioma, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, renal cell carcinoma, laryngeal cancer, lip and oral cancer, small cell lung cancer, non-small cell lung cancer, primary central nervous system lymphoma, Waldenstrom's macroglobulinemia, malignant fibrous histiocytoma, medulloblastoma, melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous cell carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, chronic myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal gland These include, but are not limited to, blastoma, supratentorial primitive neuroectodermal tumor, pituitary carcinoma, plasma cell neoplasm, pleuropulmonary blastoma, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, squamous cell cervical carcinoma, supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, choriocarcinoma, hematological malignancies, adult T-cell leukemia, lymphoma, lymphocytic lymphoma, stromal and germ cell tumors, or Wilms' tumor.In some embodiments, the cancer is lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, brain and central nervous system cancer, skin cancer, ovarian cancer, leukemia, endometrial cancer, bone, cartilage and soft tissue sarcoma, lymphoma, neuroblastoma, nephroblastoma, retinoblastoma, or gonadal germ cell tumor.

[0112] In some embodiments, the subject has a cancer selected from breast cancer, glioblastoma, neuroblastoma, head and neck cancer, gastric cancer, ovarian cancer, skin cancer (e.g., basal cell carcinoma, melanoma, etc.), lung cancer, colorectal cancer, prostate cancer, glioma, bladder cancer, endometrial cancer, kidney cancer, leukemia (e.g., T-cell lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), etc.), liver cancer (e.g., hepatocellular carcinoma (HCC), primary or recurrent HCC, etc.), B-cell malignancies (e.g., non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, etc.), pancreatic cancer, thyroid cancer, any combination thereof, and any subtype thereof.

[0113] Pharmaceutical compositions comprising the cancer cell-targeted nanobubbles described herein can be administered to a subject in a therapeutically effective amount. In some embodiments, a therapeutically effective amount of cancer cell-targeted nanobubbles is an amount that, when administered in one or more doses alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy), is effective to reduce symptoms of a pathological condition (e.g., cancer) in an individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the symptoms in the individual in the absence of treatment with the conjugate. According to some embodiments, if a subject has cancer, the methods described herein promote apoptosis and / or necrosis of the cancer when the cancer cell-targeted nanobubbles are administered in an effective amount.

[0114] Dosage depends on the severity and responsiveness of the condition (e.g., cancer) to be treated. Optimal dosing schedules can be calculated from measurements of drug accumulation in the individual's body. The administering physician can determine the optimal dosage, administration method, and repetition rate. Optimal dosages may vary depending on the relative potency of individual drugs and can generally be estimated based on, for example, EC50s found to be effective in in vitro and in vivo animal models. Generally, dosages can be given daily, weekly, monthly, or yearly. The treating physician can estimate the dosing repetition rate based on the measured residence time and concentration of the conjugate in body fluids or tissues. After successful treatment, it may be desirable for the subject to undergo maintenance therapy to prevent recurrence of the disease state, in which cell-targeted nanobubbles can be administered at a maintenance dose once or more daily, every few months, every six months, once a year, or any other suitable frequency.

[0115] In some embodiments, therapeutic agents, such as drugs and / or chemotherapeutic agents (e.g., doxorubicin), can be loaded into nanobubbles during nanobubble formation to provide drug-loaded, cell-targeted nanobubbles. The drug-loaded, cell-targeted nanobubbles can be administered to a subject in response to ultrasound energy, which releases the therapeutic agent from the nanobubbles. Advantageously, cell-targeted nanobubbles that enable remote release of therapeutic agents, such as chemotherapeutic agents (e.g., doxorubicin), can be targeted to specific cells or tissues of a subject, such as tumors, cancers, and metastases, by systemic administration to the subject (e.g., intravenous, intravascular, or intra-arterial injection), and then targeted to the cells or tissues from which they are remotely released to specifically treat the target cells or tissues (e.g., tumors, cancers, and metastases) of the subject. Targeting, inertial cavitation of nanobubbles, and selective release of chemotherapeutic agents to malignant cancer metastases enables treatment of such metastases with chemotherapeutic agents that provide negligible effects if not targeted and remotely released using the nanobubbles described herein.

[0116] Cell-targeted nanobubbles can enable the administration of any combination of the above-mentioned therapeutic agents and treatments at low doses, i.e., lower than those traditionally used in clinical settings.

[0117] The benefits of lowering the dose of a combination therapeutic agent and therapy administered to a subject include a reduction in the occurrence of adverse effects associated with higher doses. For example, lowering the dose of a chemotherapeutic agent such as doxorubicin may result in a reduction in the frequency and severity of nausea and vomiting compared to that observed with higher doses. Similar benefits are contemplated for compounds, compositions, drugs, and therapies combined with nanobubbles.

[0118] Reducing the incidence of adverse effects is intended to improve the quality of life of patients undergoing cancer treatment. Additional benefits of reducing the incidence of adverse effects include improved patient compliance, a reduction in the number of hospitalizations required to treat adverse effects, and a reduction in the administration of analgesics required to treat pain associated with adverse effects.

[0119] It will be appreciated that the cell-targeted nanobubbles and methods described herein can be used in applications other than the diagnostic, therapeutic, and theranostic applications described above. For example, cell-targeted nanobubbles can target microbial cells, such as bacteria and fungi, and promote inertial cavitation of the nanobubbles to resonate upon cell internalization to treat infections in a subject, and particularly infections that are resistant to antimicrobial agents. Advantageously, cell-targeted nanobubbles can also deliver biocides and / or biostatic agents that kill microorganisms, as well as agents that simply inhibit their growth or accumulation.

[0120] The following examples are for illustrative purposes only and are not intended to limit the scope of the claims appended hereto.

[0121] Example 1 In this example, we investigated the dynamics of PSMA-targeted NB distribution by high-frequency ultrasound throughout the tumor volume and investigated differences in contrast agent dynamics at the tumor margin and tumor core. This example also further demonstrates the extravasation and accumulation of PSMA-NBs within the entire tumor mass using three-dimensional (3D) US imaging.

[0122] We previously demonstrated that active targeting of PSMA enhances tumor uptake of intact PSMA-NBs with prolonged retention, resulting in prolonged US signal enhancement in tumors for over 25 minutes that can be visualized by clinical nonlinear ultrasound. One hypothesis for the prolonged tumor enhancement is that PSMA-targeted NBs are internalized by targeted cancer cells, and that internalization delays octafluoropropane gas dissolution.

[0123] These examples also demonstrate the effect of receptor-mediated endocytosis of PSMA-NBs on their sonoactivity and intracellular persistence using an in vitro cellular model. Elucidating the mechanism of PSMA-NB interaction at the cellular level may provide new avenues for clinical translation of PSMA-NBs in the diagnostic and therapeutic applications of PCa.

[0124] Experimental Section / Methods NB formulation and characterization Lipid-shell-stabilized C3F8 NBs functionalized with PSMA-1 ligand were formulated as previously reported. Briefly, a lipid cocktail containing DBPC, DPPE, DPPA, mPEG-DSPE, and DSPE-PEG-PSMA-1 was dissolved in propylene glycol, glycerol, and PBS. This was followed by gas exchange with C3F8, mechanical stirring, and centrifugation, after which the NBs were isolated. PSMA-NBs and NBs were characterized as previously described.

[0125] Animal models Animals were handled according to protocols approved by the Case Western Reserve University Institutional Animal Care and Use Committee (IACUC) and all applicable protocols and guidelines for animal use were followed. Male athymic nude mice (4-6 weeks old) were anesthetized with inhalation of 3% isoflurane with 1 L / min oxygen and inoculated with 1 x 10 cells in 100 µL of Matrigel. 6 PSMA-negative PC3flu and PSMA-positive PC3pip cells were subcutaneously implanted. Animals were observed every other day until tumors reached approximately 8-10 mm in diameter.

[0126] In vivo NLC imaging for bubble dynamics analysis In vivo experiments were performed using a FUJIFILM VisualSonic Vevo 3100. A total of nine animals were used in the experiment. Animals were divided into three groups: PSMA-NB, NB, and Lumason MB. In vivo bubble distribution was imaged using 2D nonlinear contrast mode. A total volume of 200 μl of either undiluted PSMA-NB or NB was injected via the tail vein. To acquire wash-in bubble kinetics, tumors were scanned at 5 fps for approximately 3 minutes. Scanning parameters were set to an 18 MHz frequency, 4% transmit power, 30 dB contrast gain, medium beam width, and 40 dB dynamic range using an MS250 transducer. A 3D US scan was then performed at peak signal, followed by nonlinear contrast imaging to examine the washout phase kinetics at 1 fps and 1,000 frames for approximately 16 minutes, maintaining the above parameters during the imaging session.

[0127] 3D ultrasound imaging of the entire tumor Ultrasound 3D tumor imaging was performed using a Vevo 3100 (FUJIFILM, Visual Sonics) scanner. The transducer was clipped onto the 3D motor and positioned over the tumor area. The probe's XY axis position was adjusted to position it at the center of the tumor as the imaging display. The 3D setup was configured to create 0.05 mm-thick 2D slices in 383 frames. Images were acquired and assembled together to obtain a 3D volume to achieve bubble distribution throughout the tumor. Animals were divided into three groups: PSMA-NB, NB, and Lumason MB. A total volume of 200 μl of either undiluted PSMA-NB or NB was injected via the tail vein. To obtain wash-in bubble kinetics, tumors were scanned at 5 fps for approximately 3 minutes using the same parameters as above. 3D scanning was then performed to visualize bubble distribution throughout the tumor with peak contrast signals. After the bubbles were allowed to circulate freely without US scanning, 3D scanning was performed again 25 minutes after injection. To identify intact bubbles that had extravasated and accumulated in the tumor, a 3D collapse sequence was applied to the entire tumor and rescanned to obtain a 3D image.

[0128] 3D ultrasound tumor extravasation study For extravascular testing, animals were divided into three groups: PSMA-NB, NNB, and Lumason, with three animals per group (total n = 9). 200 μl of contrast agent was injected via the tail vein. Mice were subjected to 3D US scanning 25 minutes after injection, as described above. Cardiac perfusion with 50 ml of PBS was then performed via the left ventricle, and 3D US scanning was completed again to detect US signals arising from intact bubbles accumulated in the perfused tumor.

[0129] tissue analysis Animals were divided into three groups: Cy5.5-PSMA-NBs (n = 3), Cy5.5-NBs (n = 3), and non-contrast control. Cy5.5-labeled NBs were prepared by mixing DSPE-PEG-Cy5.5 (100 μl) with lipid solution. Mice were administered either 200 μl of undiluted UCA or PBS via the tail vein. At 25 min postinjection, animals were scanned using US to detect signals, followed by PBS perfusion with 50 ml of PBS via the left ventricle. The tumors were then scanned again to detect US signals originating from intact NBs. Tumors and kidneys were excised, fixed in paraformaldehyde, and embedded in optimal cutting temperature compound (OCT Sakura Finetek USA Inc., Torrance, CA). Tissues were cut into 8 μm slices, washed three times with PBS, incubated in protein blocking solution containing 0.5% Triton X-100 (Fisher Scientific, Hampton, NH), and incubated for 24 h at 4°C in a 1:250 dilution of primary CD31 (PECAM-1) monoclonal antibody (Fisher Scientific, Hampton, NH). Then, the slices were washed with PBS, incubated for 1 h with an Alexa-568-tagged secondary antibody (Fisher Scientific, Hampton, NH), and stained with DAPI (Vector Laboratories, Burlingame, CA). Fluorescent images were acquired and analyzed (by segmentation and threshold interaction functions) using Axio Vision v4.8.1.0, Carl Zeiss software (Thornwood, NY). For PSMA immunohistochemistry, tissues were washed three times with PBS, incubated with blocking solution, and then incubated with a 1:150 diluted PSMA primary antibody (Thermo Fisher Scientific, Waltham, MA) at 4°C for 24 h, following the same steps as for CD31 staining.

[0130] Preparation and characterization of contrast agents The preparation and characterization of NBs have been reported elsewhere. Briefly, a lipid cocktail containing DBPC (Avanti Polar Lipids Inc., Pelham, AL), DPPE, DPPA (Corden Pharma, Switzerland), and mPEG-DSPE2000 (Laysan Lipids, Arab, AL) was dissolved in propylene glycol (PG, Sigma-Aldrich, Milwaukee, WI), glycerol, and PBS. The gas was then exchanged with C3F8 (Electronic Fluorocarbons, LLC, PA), and the vial was subjected to mechanical agitation. NBs were isolated by centrifugation. PSMA-targeted NBs were formulated by incorporating DSPE-PEG-PSMA-1 into the lipid cocktail mixture. PSMA-NBs and NBs were characterized as previously described.

[0131] cell culture research Retroviral-transformed PSMA-positive PC3pip cells and PC3flu cells (transfection control) were originally obtained from Dr. Michel Sadelain (Memorial-Sloan Kettering Cancer Center, New York, NY). Cell lines were checked and authenticated by Western blot. Cells were grown in complete RPMI 1640 medium (Invitrogen Life Technology, Grand Island, NY) at 37°C and 5% CO2.

[0132] Cellular uptake studies Both PC3pip and PC3flu cells (2 × 10 6 Cells (1 × 10 cells / ml) were seeded onto cell culture Petri dishes (60 × 15 mm, Fisher Scientific) at approximately 70% confluence. After 24 hours, cells were incubated with PSMA-NBs or plain NBs (approximately 10,000 bubbles / cell) for 1 hour. After incubation, cells were washed three times with PBS (3 times) and maintained in RPMI at 37°C until the time of US scanning. Before trypsinization of US scanning cells, they were counted and diluted to 1 × 10 6 Cells were used.

[0133] Acoustic evaluation of PSMA-targeted NBs internalized into PC3pip cells in vitro The in vitro acoustic activity of NB-internalized cells was assessed using a clinical US scanner (AplioXG SSA-790A, Toshiba Medical, now Hitachi Healthcare America). To perform the measurements, cells (approximately 2 × 10 6 ) were washed and detached using trypsin. After detachment and resuspension in PBS, the cell suspension was placed in a custom-made 1.5% (w / v) agarose phantom.

[31] The phantoms were mounted on a 12 MHz linear array transducer, and images were acquired using contrast harmonic imaging (CHI) with a 0.1 mechanical index (MI), 65 dynamic range, 70 dB gain, and an imaging frame rate of 0.2 frames / s. Region-of-interest (ROI) analysis was performed on all specimens using the onboard software, and the mean signal intensity of each ROI was measured. Data were then exported to Microsoft Excel for further processing. Experiments were performed in triplicate.

[0134] Confocal imaging of PC3pip cells internalized into PSMA-NBs PC3pip cells were cultured in glass-bottom Petri dishes (MetTek Corporation, Ashland, MA, USA) at 10 4Cells were seeded at a density of 1000 cells / well. Rhodamine-labeled NBs were prepared by mixing 50 μL of DSPE-rhodamine with the lipid solution. After 24 h, 250 μL of rhodamine-tagged PSMA-NBs diluted 1:10 was added to the cells for 1 h. After incubation, cells were washed with PBS and then placed in RPMI for 3 and 24 h in an incubator. The 3-h time point was chosen because a low standard error and significantly higher acoustic activity were previously observed in PSMA-NB-internalized PC3pip cells at the 3-h time point. One hour before the end of the incubation, 24 μL of 5 μM Lysotracker Red (ThermoFisher Scientific), a marker for late endosomes and lysosomes, was added to the cells according to the manufacturer's instructions. After incubation, cells were washed three times with PBS and fixed with 4% paraformaldehyde for 10 min. Cells were washed with PBS and stained with DAPI mounting medium (Vecor Laboratories, Burlingame, CA). Cells were then observed using a fluorescence microscope (Leica DMI 4000B, Wetzlar, Germany) equipped with appropriate filter sets. Lysotracker Red exhibits green fluorescence (excitation: 577 nm, emission: 590 nm).

[0135] Analysis of intracellularly trapped octafluoropropane gas using GC / MS The presence of octafluoropropane (C3F8) gas within the cells was confirmed by headspace gas chromatography / mass spectrometry (GC / MS) as previously described. In these experiments, cells (1 × 10 7 cells / mL) in a cell culture flask (75 cm 2Cells were grown in 1000-well plates (size: 10000 NBs / cell) for 24 hours. Again, as described above, cells were incubated with 1 mL of either PSMA-NBs or plain NBs (approximately 10,000 NBs / cell) for 1 hour. After incubation, cells were washed with PBS and incubated in culture medium for 3 hours. After incubation, cells were trypsinized, resuspended in PBS, and centrifuged at 1,000 rpm for 4 minutes. Cells were then transferred to a GC headspace vial containing 300 μL of culture medium and 300 μL of cell lysis buffer, sealed with a PTFE / silicone septum, and capped (Thermos Fisher Scientific). The vial was sonicated for 20 minutes in an ultrasonic water bath (Branson Ultrasonics, Danbury, CT) at 50 °C to release C3F8 gas into the headspace vial. GC / MS analysis was performed as described previously using an Agilent 5977B-MSD mass spectrometer equipped with an Agilent 7890B gas chromatograph GC / MS system. A DB5-MS capillary column (30 m × 0.25 mm × 0.25 μm) was used with a helium flow of 1.5 mL / min. 1 μL of headspace sample was injected at a 1:10 split. The gas chromatography conditions used were as follows: oven temperature 60 °C, held for 1 min, ramped to 120 °C at 40 °C / min, and held for 3.5 min. Perfluoropropane was eluted in 1.2 min. Samples were analyzed in selected ion monitoring (SIM) mode using electron impact ionization (EI). An M / z of 169 (M-19) was used for all analyses. The ion dwell time was set to 10 ms. Perfluoropropane was verified against the NIST MS spectral database. A standard calibration plot was obtained by measuring the peak area of ​​the GC peak as a function of NB concentration (0–100 × 10). 8 NB / mL).

[0136] In vivo US imaging of internalized bubbles Animals were handled according to protocols approved by the Case Western Reserve University Institutional Animal Care and Use Committee (IACUC) and all applicable protocols and guidelines regarding animal use were followed. PC3pip cells were incubated with PSMA-NBs and treated as described above. Mice (n = 9, 4-6 week-old athymic (NCR nu / nu) mice weighing 20 g) were randomly divided into three groups and anesthetized with inhaled 3% isoflurane with 1 L / min oxygen. Baseline US signals were obtained on both the left and right sides of the flank region (marked with a permanent marker) using the parameters described above at mechanical indices (MI) of 0.1 and 0.5. After incubation with PSMA-NBs, PC3pip cells were suspended in a mixture of Matrigel and PBS (1:1 PBS / Matrigel), and the cell suspension (100 µL) was injected subcutaneously into the flank of nude mice. As a control, cells without NB exposure were injected adjacent to the NB-exposed cells (Figure 7A). US images of the injection site were acquired immediately after injection or 3 hours, 24 hours, or 8 days later at an MI of 0.1 using the same parameters as above. After imaging at 0.1 MI, the MI value was increased to 0.5, and the area was reimaged for all time points. Regions of interest (ROIs) were drawn around the injected cell area, excluding the skin. The mean signal intensity for each ROI was then obtained using CHIQ software. These measurements were exported to Excel, and the baseline value (pre-inoculation contrast) was subtracted from the signal enhancement in each ROI.

[0137] result Nanobubble characterization Validation of nanobubble preparation, functionalization with PSMA-1 ligand, and lipid-ligand conjugation has been reported previously. The diameters of NBs and PSMA-NBs characterized by resonance mass spectrometry (RMM) were 281×2 nm and 277×11 nm, respectively. Validation of RMM analysis and its optimization for use in NB characterization have been previously described. Importantly, the results show that the average size and concentration did not change significantly after functionalization (the concentrations of NBs and PSMA-NBs were 4×11×2.45×10 and 3.9×11×2.82×10 NB / ml, respectively).

[0138] Ultrasound signals at the tumor margins and core In vivo experiments were performed using a FUJIFILM VisualSonic Vevo 3100. A total of nine tumor-bearing mice were used in the experiment. Animals were divided into three groups: PSMA-NB, NB, and Lumason MB. A total volume of 200 μL of either undiluted PSMA-NB or NB was injected via the tail vein, after which in vivo bubble distribution was imaged in 2D nonlinear contrast mode. Figure 4A shows a schematic diagram of the ultrasound scanning process timeline. A baseline scan was performed in both nonlinear contrast mode and 3D mode before bubble injection. To acquire wash-in bubble dynamics, the tumor was scanned at 5 frames per second (fps) for approximately 3 minutes. Figures 4B, C, and D show the TIC curves corresponding to the tumor edge and core, acquired over the first 1,000 frames at 5 fps and the second 1,000 frames at 1 fps. Before changing the frame rate from 5 fps to 1 fps, a 3D US scan was performed to observe the bubble distribution throughout the tumor mass.

[0139] Rapid signal enhancement was observed in tumors imaged with either PSMA-NBs or plain NBs, reaching peak intensity 1–2 min after injection. There was no significant difference in peak enhancement (PE) across the tumor for PSMA-NBs and plain NBs, indicating similar morphology for targeted and non-targeted bubbles. The variability of bubble kinetic parameters was further investigated by analyzing the tumor margin and core separately. To distinguish them from the tumor core, loop ROIs were drawn separately at the tumor margin (Figure 4). As shown in Figure 4E, immediately after injection, PSMA-NBs and plain NBs rapidly filled the tumor margin. In contrast, PSMA-NBs showed slower washout into the tumor core compared to the tumor margin. Kinetic parameters were calculated and summarized in Table 1. The time to peak (TTP) for PSMA-NBs in the tumor core was significantly greater than that for plain NBs (2.48 □ 0.71 min vs. 1.21 □ 0.15 min, P < 0.05). However, the time to peak (TTP) for tumor margins relative to PSMA-NB and NB was not significantly different. There was no significant difference in wash-in area under the curve (WiAUC) for both groups. Furthermore, the peak enhancement (PE) for tumor margins with both NB and PSMA-NB was not significantly different. The PE for tumor cores with both NB and PSMA-NB was also not significantly different. The comparable WiAUC and PE for both bubbles showed similar kinetics. However, the PE for tumor margins and cores was significantly different for both NB groups (Table 1).

[0140] Table 1 - Summary of tumor margin and core kinetic parameters obtained from time intensity curves (TIC) [Table 1] All values ​​are expressed as mean s.d. * , # , $ ,Statistical significance in each group; p<0.05.

[0141] Furthermore, NB accumulation was compared with that of the commercially available MB contrast agent, Lumason. Contrast enhancement occurred rapidly with Lumason (TTP: 0.84 for the margin, 0.06 min; TTP: 0.77 for the core, 0.11 min), significantly different from that of both types of NB. Furthermore, the PE and WAUC of Lumason were significantly lower compared with the two NB groups. After peak enhancement, the US signal decayed over time in both the PSMA-NB and NB groups (Figure 4E). The washout AUC of the tumor margin with PSMA-NB was significantly higher than that of the NB group (Table 1). Similarly, the washout AUCs for the tumor core with PSMA-NB and NB were also significantly different. Furthermore, the washout AUCs for the tumor margin and core were significantly different for both groups. Importantly, the nonlinear contrast imaging parameters used in the bubble study were kept constant, and the transmit frequency used to image Lumason was higher than that typically used for this agent. This may result in reduced detection sensitivity and subsequent signal enhancement, but the relative contrast agent kinetics should be relatively unaffected.

[0142] The significant differences observed in NB dynamics between the tumor edge and core may be due to the discrepancy in vascularity at the tumor edge and core caused by angiogenesis. Angiogenesis is a key process in tumor development and progression, contributing to the formation of new capillaries from pre-existing blood vessels and promoting tumor growth and metastasis by supplying tumors with essential nutrients and oxygen. The distribution of PSMA biomarkers in the tumor mass also plays an important role in target bubble accumulation in tumors. Furthermore, PSMA expression has been reported in endothelial cells of the neovasculature. One hypothesis for the difference in TTP is the presence of a biomarker that controls the flow of contrast agents. PSMA-targeted NBs tend to bind to the biomarker within the tumor and slow its flow rate. Therefore, the time required to fill the tumor with PSMA-NBs is slower than that of free-flowing NBs. After reaching peak signal, the contrast of both types of NBs begins to decrease at both the tumor edge and core. However, the washout AUC of PSMA-NBs was significantly higher than that of NBs, indicating high retention of targeted NBs in the tumor. The decrease in NB signal was likely due to the loss of NBs from the tumor margin and tumor matrix due to tumor interstitial pressure (TIP). In addition to abnormal angiogenesis, tumor tissue exhibits poor lymphatic drainage compared with normal tissue, which impedes drug and nanoparticle delivery. Due to the binding of PSMA-targeted NBs to the PSMA biomarker, TIP-induced removal may be minimized for PSMA-NBs.

[0143] 3D ultrasound imaging of the entire tumor To better understand the bubble distribution throughout the tumor mass, 3D nonlinear contrast US was performed after administration of PSMA-NBs, NBs, and Lumason MBs as an extension of 2D imaging. 0.05 mm-sized 2D US image slices of the tumor were constructed together to obtain the bubble distribution throughout the tumor. After the nonlinear contrast scan, 3D US was performed to obtain the contrast signal at the peak throughout the tumor mass (Figure 5). 3D analysis calculates the percentage of voxels within the imaging volume that exhibit nonlinear signal. Consistent with the 2D scan, 3D analysis showed similar signal at the peak for both PSMA-NBs and NBs (Figure 5B and C). At 3 minutes, both PSMA-NBs and NBs covered approximately 90% of the tumor margin (86.9 ± 0.8% and 87.7 ± 6.6%, respectively, p = 0.6). Similarly, PSMA-NBs and NBs were detected in approximately 60% of the tumor core (64.9 ± 14.5% and 62.4 ± 28.1%, respectively). The percentage of Lumason drug detected within the tumor margin was significantly lower compared to PSMA-NB and NB. At 3 minutes, Lumason was detected in 10% (5.7±2.1%) of the tumor margins.

[0144] Interestingly, when tumors were imaged with continuous nonlinear ultrasound (1 fps for 16 minutes after 3D acquisition), no significant difference in drug coverage was observed between the PSMA-NB and NB groups. Furthermore, the ratio of PSMA-NB to NB signal throughout the tumor was lower compared to previous observations using 2D scans. We speculate that continuous exposure of NBs immobilized within tumor tissue to US may have resulted in rapid bubble lysis. To test this hypothesis, we performed another series of experiments in which mice were injected with NBs, PSMA-NBs, or Lumason, but then allowed to circulate for 30 minutes without US exposure. In these experiments, PSMA-NBs demonstrated a significantly higher proportion of nonlinear signal in the tumor core compared to both NBs and Lumason (25.2 ± 1.5%, 13.9 ± 5.1%, and 0.4 ± 0.4%, respectively, p < 0.05). The signal fraction significantly decreased to approximately 12% after applying the collapse sequence, confirming the destruction of intact bubbles accumulated within the tumor (data not shown). 3D tumor analysis also showed that PSMA-NBs also accumulated more at the tumor margin compared to NBs, although the difference was not statistically significant (54.2 ± 4.8% vs. 38.7 ± 14.4%, respectively).

[0145] 3D ultrasound tumor extravasation study To examine the extravasation and accumulation of NBs throughout the intact tumor volume, whole blood perfusion via cardiac puncture was performed at 25 min postinjection, and 3D US scans were completed before and after cardiac puncture (Figure 6A). Whole blood perfusion evacuates blood from the vasculature and removes any material, including freely moving nanobubbles, from the tumor vasculature. After perfusion, the 3D US signal within the tumor corresponds to material remaining throughout the tumor parenchyma or in the intracellular space. Before perfusion, at 25 min postinjection, the proportion of PSMA-NBs at the tumor margin was 1.4-fold higher than that of NBs (Figure 6B,C; 46.8□21.3% vs. 33.2□25.4%, p=0.2). The proportion of PSMA-NBs in the tumor core was 4.1-fold higher than that of NBs (37.7□20.1% vs. 18.9□18.7%). After perfusion, the percentage of US signal decreased in both groups. The percentages of PSMA-NBs and NBs were reduced by 67% and 92%, respectively, at the tumor margin compared to their peak values ​​(15.0□7.21% vs. 2.45□3.4%, respectively). Furthermore, after perfusion, PSMA-NBs were significantly reduced in the tumor core compared to that of NBs (12.2□2.3% and 3.2□2.2%, respectively) (p<0.05). The significantly higher (approximately fourfold) 3D US signal of PSMA-NBs in the tumor core compared to NBs indicates a relatively high accumulation and extravasation of PSMA-NBs in the tumor core environment. The presence of US contrast after cardiac perfusion provided evidence of the accumulation and extravasation of intact NBs in the tumor parenchyma. As expected, the percentage of Lumason became negligible in both the tumor margin and core at 25 min postinjection and after perfusion.

[0146] Histological analysis To confirm the US data, we performed histological analysis after injection of Cy5.5-PSMA-NBs or CY5.5-NBs into tumor tissue. PSMA expression, CD31 expression, and PSMA-NB and NB distribution were assessed separately in the tumor margin and core. Prior to injection, bubbles were tagged with a fluorescent dye, Cy5.5. As described in the extravasation study, 3D US scans were performed 25 min postinjection, and the animals were then perfused with PBS via cardiac puncture. After perfusion, tumors were rescanned in 3D as described above, and the tumors were excised for histological analysis. The tumor core and margin were imaged and analyzed separately. CD31, representing the vasculature, showed a higher percentage in the tumor margin compared to the tumor core. PSMA expression was also higher in the tumor margin compared to the tumor core, but the difference was not significant. Cy5.5-PSMA-NB signal was more evenly distributed within the tumor, providing evidence that targeted NBs had migrated from the vasculature and accumulated in the tumor matrix. Quantification of the histological signal reveals that the Cy5.5-PSMA-NB signal in both the tumor core and margins was significantly higher (3-fold) compared to the signal in plain NBs (p<0.001) (Figure 7). Enhanced interaction of the contrast agent with the tumor matrix explains the high accumulation of PSMA-NBs after extravasation.

[0147] Persistence of PSMA-targeted NBs in PC3pip cells in vitro In this study, we investigated the effect of cellular internalization of nanobubble ultrasound contrast agents on the persistence of acoustic activity. Specifically, we compared the effects of passive cellular uptake of PSMA-targeted NBs versus receptor-mediated endocytosis in PSMA-expressing human prostate cancer cells. We first examined the dynamics of nonlinear acoustic properties in both PSMA-positive PC3pip and PSMA-negative PC3flu cells after 1 hour of incubation with PSMA-targeted or non-targeted NBs. As shown in Figure 8, after 1 hour of incubation, PC3pip cells incubated with PSMA-NBs exhibited 3.25-fold higher acoustic activity compared to plain NB-incubated PC3pip cells (Figure 8; P<0.05; 9.69 ± 1.78 dB vs. 2.19 ± 1.22 dB, respectively), demonstrating higher signal intensity compared to all other groups (NBs in PC3pip cells, PSMA-NBs in PC3flu cells, and plain NBs in PC3flu cells) and the negative control (cells alone). Furthermore, the significantly higher acoustic activity of internalized PSMA-NBs persisted for all time points tested except for the 48-h time point (Figure 8). After 24 h, PC3pip cells exposed to PSMA-NBs exhibited significantly higher US signal intensity (4.11 ± 0.68 dB; P < 0.005) compared with all other groups. PSMA-NBs incubated under the same conditions but without cells exhibited initial signal intensity comparable to that of PSMA-NBs incubated with PC3pip cells. However, from the 3-h time point onward, the signal intensity of the PSMA-NB-only group was significantly lower than that of PSMA-NBs internalized by PC3pip cells, indicating the greater stability of internalized PSMA-NBs in the cellular environment compared with free PSMA-NBs.

[0148] Previous studies have investigated the targeting of microbubbles (MBs) to genetically engineered cell surface markers on endothelial progenitor cells (EPCs), demonstrating selective binding to EPCs in vitro and enabling imaging with CEU. We have also previously shown that either internalized or membrane-bound MBs are protected by much greater viscous attenuation by cells compared to free MBs. Consistent with these findings, we observed that internalized NBs exhibited significantly higher backscattering over time compared to free NBs under the same conditions. Furthermore, at later time points, PSMA-NBs in PC3pip cells exhibited higher contrast than plain NBs incubated with either PC3pip or PC3flu cells and PSMA-NBs incubated with PSMA-NB-negative PC3flu cells. Nonspecific uptake of NBs by cells slightly reduces the rate of signal decay from NBs. However, much slower decay was observed for PSMA-NBs localized within endosomes. Therefore, we hypothesize that the long-term survival of PSMA-NBs in cells may be due to stabilization by endosomal / lysosomal entrapment.

[0149] Confocal imaging of PSMA-NB internalization in PC3pip cells PSMA acts as a cell membrane receptor and internalizes the PSMA-targeting ligand along with the payload attached to the targeting agent. When the PSMA ligand binds to the biomarker on the cell membrane, the cell membrane invaginates and the entire particle is engulfed by the cell. The localization of internalized PSMA-NBs within the cell was investigated by confocal microscopy imaging using the fluorescent dye Lysotracker Red. Lysotracker Red staining stains late endosomes and lysosomal structures.

[0150] Our previous fluorescence imaging data demonstrated that PSMA-targeted NBs are selectively internalized by PC3pip cells. Our confocal imaging results here demonstrate the internalization of PSMA-NBs by PC3pip cells, more specifically, receptor-mediated endocytosis (Figure 9, 100x; Figure 18, 40x). Substantial colocalization of rhodamine-labeled PSMA-NBs (red) with Lysotracker Red (green)-stained late endosomes / lysosomes was observed in all PSMA-expressing cells. As shown in Figure 9A, plain NBs exhibited some nonspecific uptake by PC3pip cells, but colocalization with late endosomes / lysosomes was limited. Uptake of nontargeted NBs by PC3pip cells was substantially lower compared to uptake of PSMA-NBs. Furthermore, a higher degree of colocalization of PSMA-NBs with late endosomes / lysosomes is shown in Figures 9B and C.

[0151] Imaging of PC3pip cells at 24 h after bubble exposure revealed a lower amount of fluorescent signal compared to earlier time points (Figure 8D). However, these images also showed a high degree of colocalization of PSMA-NBs with late endosomal / lysosomal vesicles. After 24 h, very low or no fluorescent signal was observed in the cytoplasm or late endosomal / lysosomal compartments of NB-incubated cells. Furthermore, images showed yellow staining near the nucleus, suggesting that the majority of PSMA-NBs colocalized with either late endosomes or lysosomes and were transported to the cytoplasm.

[0152] When PC3pip cells were incubated with targeted NBs, greater Lysotracker staining was observed compared to non-targeted NBs, correlating with a receptor-mediated internalization mechanism that enters the late endosomal / lysosomal pathway. PSMA possesses a unique internalization motif and has been reported to have a robust baseline internalization rate of 60% of its surface PSMA at 2 hours. Its transmembrane location and internalization make PSMA an ideal target for imaging and therapy. Overall, PSMA-mediated endocytosis appears to be the primary pathway for internalization of PSMA-targeted nanobubbles in PSMA-expressing prostate cancer PC3pip cells.

[0153] Intracellular C using headspace GC / MS 3 F 8 Analysis of To confirm that intact gas-containing nanobubbles were internalized and remained within PC3pip cells, cells were collected 3 hours after exposure to PSMA-NBs or NBs and analyzed for the presence of C3F8 within the cells using headspace GC / MS. The use of headspace GC / MS to quantify C3F8 gas concentrations in NBs has been previously reported and validated. Analysis was performed using the relative abundance of a peak observed at a mass-to-charge ratio (m / z) of 169, corresponding to C3F8. Calibration plots were generated by GC / MS using different concentrations of NBs (Figures 10A and 10B). A linear relationship was observed between peak area and bubble number. GC / MS data showed that the peak area obtained from PSMA-NB-incubated PC3pip cells was 3.5-fold higher than that of plain NB-incubated PC3pip cell suspensions (Figure 9C; the peak areas for PSMA-NBs, NBs, and cells were 16,778 (au), 6,274 (au), and 2,172 (au), respectively. Based on the calibration curve, this corresponds to approximately 500 average-sized PSMA-NBs per cell versus 138 average-sized plain NBs. To our knowledge, this is the absolute most direct method for confirming the presence of intracellular gas vesicles. The ratio of peak areas is consistent with the difference in acoustic activity from cells, as shown in Figure 10. It is also strikingly similar to the difference in ultrasound signal seen from PSMA-NB versus plain NB accumulation in PC3pip tumors after clearance of circulating NBs, supporting the data indicating that target NBs extravasate and are retained in tumors in vivo in an intact form.

[0154] In vivo application of PSMA-NB-internalized cells To confirm that the extended intracellular retention could also be visualized in vivo, we studied the acoustic activity of internalized bubbles within cells upon injection into mice. PC3PIP cells incubated with PSMA-NBs were injected subcutaneously into the flank region of nude mice and imaged at 12 MHz. As a control, cells without NB exposure were injected adjacent to the area of ​​labeled cell injection. Figure 11A shows US images obtained at 0-24 h and 8 days after cell injection. NB-incubated cells showed significantly higher contrast compared to unlabeled cells immediately after injection (9.7 ± 2.9, 5.2 ± 1.5: P < 0.05). The initial signal seen from control cells is most likely the result of air bubbles trapped in the Matrigel. After 3 and 24 h, contrast in the area with NB-incubated cells remained significantly higher compared to control cells (Figure 11B).

[0155] Internalized PSMA-NBs in PC3pip cells primarily colocalized with intracellular vesicles and exhibited substantial backscattering activity for 48 hours after incubation in vitro and for 1 week in vivo. To our knowledge, this study provides the first direct evidence of PSMA-targeted NB uptake and long-term retention in cancer cells and demonstrates the critical role of endosomes / lysosomes in stabilizing NB acoustic activity.

[0156] This example demonstrated that active targeting of NBs to PSMA increased extravasation and accumulation within PSMA-expressing tumors in both 2D nonlinear contrast and 3D US modes. The data showed that both tumor wash-in and retention of PSMA-NBs were delayed due to biomarker interaction and binding. The longer retention of PSMA-NB signal in the tumor core also further supports targeting-enhanced bubble extravasation. Furthermore, in vitro studies suggested that active targeting of NBs to PSMA selectively enhanced cellular internalization in PSMA-positive PC3pip cells. US was able to detect internalized PSMA-NBs in PC3pip cells, and internalized PSMA-NBs demonstrated long-term stability in the cellular environment, likely due to encapsulation in endosomal vesicles. GC / MS analysis further confirmed the persistence of intact NBs in cells after internalization. This finding supports previous studies demonstrating long-term acoustic activity of targeted nanobubbles in biomarker-expressing tumors and opens the door to new molecular imaging and targeted therapy approaches using ultrasound.

[0157] Example 2 In this example, we investigate PSMA-targeted NBs for in vivo US imaging of PCa using a more clinically relevant orthotopic prostate tumor model in nude mice (Figure 12). Given the robust nature of NB-enhanced ultrasound, we also used this technique to examine the effect of PSMA targeting efficiency on tumor progression and size in the same model. This may provide a method for related studies on targeted ultrasound NBs.

[0158] Materials and Methods Preparation of PSMA-targeted and non-targeted NBs PSMA-targeted NBs (10 mg / mL) were prepared by first dissolving a mixture of lipids containing 1,2-dibehenoyl-sn-glycero-3-phosphocholine (C22, Avanti Polar Lipids Inc., Pelham, AL), 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA, Corden Pharma, Switzerland), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE, Corden Pharma, Switzerland), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-mPEG 2000, Laysan Lipids, Arabidopsis) in propylene glycol (0.1 mL, Sigma-Aldrich). Lipids were prepared as previously reported by dissolving lipids in glycerol (Aldrich, Milwaukee, WI) and heating and sonicating at 80°C until all lipids were dissolved. A mixture of glycerol (0.1 mL, Acros Organics) and phosphate-buffered saline (0.8 mL, Gibco, pH 7.4) preheated to 80°C was added to the lipid solution. The resulting solution was sonicated at room temperature for 10 minutes. DSPE-mPEG-PSMA (25 μL of 1 mg / mL in PBS) was added. The solution was transferred to a 3 mL headspace vial, capped with a rubber septum and aluminum seal, and sealed with a vial crimper. Air was manually removed using a 30 mL syringe and replaced by injecting octafluoropropane (C3F8, Electronic Fluorocarbons, LLC, PA) gas. The phospholipid solution was then activated by mechanical shaking for 45 seconds using a VialMix shaker (Bristol-Myers Squibb Medical Imaging Inc., N. Billerica, MA). PSMA-targeted NBs were separated from the foam and microbubble mixture by centrifugation at 50 rcf for 5 minutes with the headspace vial inverted. 200 μL of PSMA-targeted NB solution was then withdrawn from a fixed distance of 5 mm from the bottom using a 21G needle. A similar preparation was performed for non-targeted NBs, without the addition of DSPE-mPEG-PSMA.

[0159] NB size, concentration, and surface charge The size distribution and concentration of PSMA-targeted and non-targeted NBs were characterized by nanosensor-equipped resonance mass spectrometry (ARCHIMEDES, Malvern Panalytical) capable of measuring particle sizes between 50 and 2000 nm. To obtain acceptable detection limits (<0.01 Hz), the NB solution was diluted with PBS (500x), loaded at 2 psi for 120 s, analyzed at (500x), and measured on an Anton Paar Litesizer 500.

[0160] Animal models Animals were handled according to protocols approved by the Case Western Reserve University Institutional Animal Care and Use Committee (IACUC), and all applicable protocols and guidelines for animal use were followed. Male athymic Balb / c nude mice, 4-6 weeks old, were purchased from the Case Western Reserve University Animal Research Center and housed in the approved Small Animal Imaging Center of the Animal Resource Center. All animals received standard care: free access to food and water; a 12 / 12 light / dark cycle; species-appropriate temperature and humidity; environmental enrichment and group housing whenever possible; standard cage disinfection; and solid-bottom cages. Mice were anesthetized with inhalation of 1-2% isoflurane with 0.5-1 L / min of oxygen. A 28 1 / 2-gauge insulin needle was inserted into the ventral prostate to deliver 10 μL of PSMA(+) PC3Pip cells suspended in PBS (phosphate-buffered saline). A well-localized bleb within the injected prostate lobe was a sign of technically satisfactory injection. Animals were observed every other day until tumors reached approximately 3-5 mm in diameter and then used for imaging studies.

[0161] Pharmacokinetic studies Animals were used for the study 10 days after inoculation, when tumor diameters reached 3–5 mm. The pharmacokinetics of NB was monitored using an APLIXG SSA-790A Toshiba Medical Imaging Systems (Otawara-Shi, Japan) ultrasound probe PLT-1204BT. After anesthetizing mice with 1–2% isoflurane with 0.5–1 L / min of oxygen, each mouse was positioned supine, and the ultrasound probe (PLT-1204BT) was positioned longitudinally along the animal's body axis to visualize ultrasound images of the PC3pip orthotopic tumor. To compare contrast-enhanced ultrasound images with the same tumor in the same mouse (n=11), 200 μL of either PSMA-targeted NB (3.9 ± 0.282 × 10 11 / mL) or non-targeted NB (4.0 ± 0.245 × 10 11 / mL) was administered via the tail vein. Images were acquired in raw data format for 5 seconds before NB injection. To image changes in tissue contrast density after NB injection, contrast harmonic imaging (CHI) was used (CHI, frequency 12.0 MHz; MI 0.1; dynamic range 65 dB; gain 70 dB; imaging frame rate 0.2 frames / s). Mice were continuously imaged for 30 min. Remaining NBs were collapsed by repeated flushing, and then 30 min later, the same mice were administered non-targeted or PSMA-targeted NBs. LUMASON (200 μL, 1–5 × 10⁻⁶ / mL, sulfur hexafluoride lipid type A microspheres, Bracco Diagnostics Inc.) was tested in three other mice. LUMASON was prepared according to the protocol provided by the manufacturer. Raw data were processed using software provided by the scanner manufacturer. Acquired linear raw data images were processed using CHI-Q quantification software (Toshiba Medical Imaging Systems, Otawara-Shi, Japan). Regions of interest (ROIs) were outlined for tumor and liver regions. The signal intensity of each ROI as a function of time (time-intensity curve - TIC) was calculated and exported to Excel. To analyze the ultrasound contrast decay, the baseline was subtracted from the TIC.

[0162] Research on the collapse of the bubble economy Mice were administered 200 μL of NB (3.9 ± 0.282 × 10 11 / mL) via tail vein injection. Five minutes after contrast injection, images were taken at four different planes, including the tumor and liver, within the same field of view. Then, 25 flashes were performed at different locations, from the liver plane to the cardiac plane, to disintegrate any remaining NB in ​​the circulation. Subsequently, images were taken again at four different planes, including the tumor and liver, within the same field of view, using contrast-mode imaging. Mean intensities were analyzed using Image J. This experiment was repeated in four nude mice bearing PC3pip orthotopic tumors.

[0163] Histological analysis Animals were divided into three groups: PSMA-NB (n = 3), plain-NB (n = 3), and non-contrast control (n = 3). The method was the same as in our previous study. Mice were administered 200 μL of contrast agent or PBS alone via the tail vein. Ten minutes after contrast agent injection, PBS perfusion was performed with 50 mL of PBS via the left ventricle. After perfusion, tumors and livers were excised and embedded in optimal cutting temperature compound (OCT Sakura Finetek USA Inc., Torrance, CA). Tissues were cut into 9 μm slices and then stained for CD31 to visualize tumor vasculature. Briefly, tissues were washed three times with PBS and incubated with a protein blocking solution containing 0.5% Triton X-100 (Fisher Scientific, Hampton, NH). The tissues were then incubated in a 1:250 dilution of CD31 primary antibody (Fisher Scientific, Hampton, NH) for 24 hours at 4°C. After washing with PBS, the tissues were incubated with an Alexa 568-tagged secondary antibody (Fisher Scientific, Hampton, NH) for 1 hour and stained with DAPI (Vecor Laboratories, Burlingame, CA) using standard techniques. Fluorescent images were then viewed with a Leica DM4000B fluorescent microscope (Leica Microsystems Inc, Buffalo Grove, IL) and analyzed using Image J.

[0164] result Contrast-enhanced ultrasound imaging of orthotopic prostate tumors using PSMA-targeted NB and LUMASON After tail vein injection of PSMA-targeted NBs (200 µL of 3.9 ± 0.282 × 10⁻¹⁻¹ / mL PSMA-targeted NBs) (n = 11) or LUMASON (200 µL of 1–5 × 10⁻¹ / mL LUMASON MBs) (n = 3), contrast harmonic imaging (CHI) images were acquired sequentially (receive frequency 12 MHz) to determine bubble dynamics in the tumor and liver. The LUMASON dose, PSMA-targeted NB dose, and imaging parameters used were optimized in previous studies. It is noteworthy that the nonlinear contrast imaging parameters in these studies utilized higher frequencies than those commonly used clinically for LUMASON (3 MHz). These do not affect the kinetic parameters of LUMASON, but may affect overall image quality. In CHI mode, the tumor and liver were invisible before injection of either PSMA-targeted NBs or LUMASON (Figure 13A). Rapid enhancement began approximately 15–30 seconds after NB injection and was observed first in the liver, followed by the tumor. UCA kinetic parameters (Figure 13C) were obtained from time-intensity curves (TICs) (Figures 13B1 and 13B2). These included time to peak, peak intensity, half-time, washout area, and area under the curve (AUC). These parameters were compared between PSMA-targeted NBs and LUMASON in both the tumor and liver. Although the group size of the LUMASON group was relatively small, the differences between the LUMASON and PSMA-targeted NB groups in the imaging parameters used in this study were large, and statistically significant differences were observed between the two groups. This is also consistent with previously published literature. The results showed that time to peak, peak intensity, half-time, washout area, and AUC were significantly different (p<0.05) between PSMA-targeted NBs and LUMASON in the tumor, and the last four parameters were significantly different (p<0.05) between PSMA-targeted NBs and LUMASON in the liver. All of the above indicated greater stability and longer circulation time of our PSMA-targeted NBs than LUMASON MBs in the bloodstream.

[0165] The tumor size in the LUMASON group was 280–520 mm 3 The tumor size in the NB group was 90–1100 mm 3 To ensure that the difference between LUMASON and PSMA-targeted NB was not a result of tumor size, we divided the PSMA-targeted NB group into two groups based on tumor size: those with tumor volumes between 90 and 670 mm 3 Group A (small tumors) and Group B (tumor volumes 670–1100 mm 3 We divided the patients into group A (large tumors) and group B (large tumors), and compared the LUMASON group with these two groups separately. Nevertheless, the parameters of both groups A and B were significantly different from those of the LUMASON group. Our results confirmed that the lower peak enhancement of LUMASON was not related to tumor size.

[0166] Comparison of contrast agent kinetics and orthotopic prostate tumors using PSMA-targeted and non-targeted NBs To evaluate the selective imaging ability of PSMA-targeted NBs for prostate tumors, non-targeted NBs were used as a comparison. US scans using both bubble formulations were performed under identical conditions, and the average results for 11 nude mice bearing PC3pip orthotopic tumors are reported. First, PC3pip tumors were localized in B-mode and then switched to contrast mode. The tumors were not visible in contrast mode before bubble injection (Figure 14A). Continuous contrast mode US was performed to monitor bubble dynamics in tumors after intravenous injection of PSMA-targeted or non-targeted NBs. Bubble dynamics obtained from time-intensity curves (TICs), including time to peak, peak intensity, half-time, washout area, and area under the curve (AUC) (Figure 14B1), were compared between PSMA-targeted and non-targeted NBs in PSMA(+) PC3pip orthotopic tumors (Figure 14C). Significant differences were measured between PSMA-targeted and non-targeted NBs in peak intensity (p = 0.0001), half-time (p = 0.0056), washout area (p = 0.0092), and area under the curve (p < 0.0001). The US signal from non-targeted NB measurements was used to normalize the signal from PSMA-targeted NBs. Normalized TICs showed that the mean intensity from PSMA-targeted NBs was consistently higher than that of non-targeted NBs at different time points (Figure 14B2). The tumor sizes used in this study ranged from 90 to 1100 mm. 3 Because tumor size varied, the inventors also divided the animals into two cohorts: Group A (small tumors, 90–670 mm); 3 , n=7) and Group B (large tumors, 670-1100 3We compared parameters between PSMA-targeted and non-targeted NBs in groups of 10-110 (n = 4 mm). In group A, which had small tumors, significant differences were observed in peak intensity and area under the curve. In group B, which had large tumors, significant differences were observed in peak intensity, area under the curve, and half-time. Individual mouse TICs also showed differences between PSMA-targeted and non-targeted NBs in 10 of 11 mice. While interanimal survival rates were observed, the overall results between the two groups were similar. Overall, our data demonstrated greater stability and longer circulation time of PSMA-targeted NBs than non-targeted NBs in the bloodstream.

[0167] Comparison of contrast agent kinetics based on different tumor sizes Because there is clear variability in the kinetics of PSMA-targeted and non-targeted NBs depending on tumor size, tumors were divided into two groups: those with tumor volumes between 90 and 670 mm 3 Group A (n = 7), tumor volume 670–1100 mm 3 The tumors were divided into two groups: Group A (n = 4) and Group B (n = 4). UCA kinetic parameters (Figure 15B) were obtained from the time-intensity curves (TIC) (Figure 15A). As tumor size increased, the peak intensity, washout area, and total area under the curve were significantly different between Group A and Group B (Figure 15B). As shown in Figure 14A, in Group B, some tumors were not fully filled after bubble injection, and the signal was heterogeneous in B-mode. In Group A, the signal was relatively homogeneous in both B-mode and contrast mode (Figure 13A).

[0168] PSMA-targeted NBs are retained in orthotopic prostate tumors after bubble clearance from the circulation Research on the collapse of the bubble economy, 300-800mm 3The study was performed in four additional mice bearing orthotopic PC3pip tumors measuring 100 μm in size, and the average results are reported in Figure 16. The series in Figure 16A shows CHI images before and after circulating bubble collapse by repeated high-intensity pulses applied to the liver. Quantitative analysis of enhancement (Figure 16B) showed a 47.9 ± 18.6% reduction in signal after clearance in PC3pip tumors with targeted NBs, compared with 74.8 ± 8.9% for untargeted NBs in the tumor and 92.2 ± 2.4% for those in the liver. These data demonstrated significantly higher peak enhancement in tumors enhanced with PSMA-targeted NBs compared with untargeted NBs. Most importantly, after clearance of circulating NBs via the associated high-intensity pulses, signal intensity in tumors enhanced with PSMA-targeted NBs remained significantly higher than that in untargeted NBs. This suggests significant NB retention in PSMA-expressing PCa cells. In contrast to the tumor, signal intensity in the liver was similar for both PSMA-targeted and untargeted NBs before decay and was almost completely eliminated after clearance of both agents. It is important to note that the tumor regions in these studies were not exposed to constant resonance (in contrast to the tumors used to generate TICs) and retained bubble echogenicity for a longer period of time. This likely contributed to the larger difference between targeted and untargeted NBs seen in this experiment. These data demonstrate for the first time significant extravascular retention of PSMA-targeted NBs within the PSMA-positive PC3pip tumor parenchyma (presumably within tumor cells) after clearance of NBs from the circulation in live mice.

[0169] Immunohistochemical analysis To further verify that PSMA-targeted NBs can extravasate into the tumor matrix, bubbles were labeled with the fluorescent dye Cy5.5 and injected into a new set of animals bearing orthotopic PC3pip tumors. Ten minutes after injection, mice underwent a cardiac flush perfusion procedure with cold PBS to remove circulating bubbles and tumors, and tumors were excised for histological analysis. CD31 staining was used to visualize tumor blood vessels. Fluorescence in blood vessels and cells was used to normalize the bubble signal per field. Histological images showed that Cy5.5 signals in the PSMA-targeted NB group were found outside tumor capillaries and deep within the parenchyma (Figure 17A), providing strong evidence of bubble extravasation and subsequent interstitial penetration. The NB fluorescence ratios (quantification of the fluorescence ratios from total bubble fluorescence / vascular fluorescence and total bubble fluorescence / cellular fluorescence per field) in the PSMA-targeted NB group were significantly higher than those in the non-targeted NB group (Figure 24B1, B2), confirming that PSMA-targeted NBs can not only extravasate into tumors but also become trapped within them.

[0170] The goal of this example was to formulate a novel targeted nanoscale ultrasound contrast agent for detecting PSMA(+) PCa in a clinically relevant orthotopic model. Our previous study in a flank tumor model already examined the dynamics of PSMA-targeted and non-targeted NBs, and histological findings confirmed that PSMA-targeted NBs can specifically recognize tumors with PSMA expression. In this example, significant differences were observed in peak intensity, half-time, washout area, and area under the curve between PSMA-targeted and non-targeted NBs in orthotopic tumors (Figure 14). Comparing the results from the orthotopic tumor model with previous studies in a flank tumor model, the signal difference between PSMA-targeted and non-targeted NBs in orthotopic PC3pip tumors was less pronounced than in flank PC3pip tumors. More specifically, while the total AUC for PSMA-NBs between the two models was comparable, the non-targeted NB AUC and, particularly, the washout AUC increased in the orthotopic model. Western blot studies showed that flank PC3pip tumors and orthotopic PC3pip tumors had similar levels of PSMA expression, so the difference is not due to different levels of PSMA expression. We hypothesize that this difference may be the result of three factors: 1) variations in vascular density and vascular permeability in the two tumor models, 2) differences in overall tumor burden, and 3) differences in cell density and central necrosis. Differences in the tumor microenvironment between flank and orthotopic tumors are known to affect these factors. Specifically, orthotopic PC3 tumors have been reported to have higher vascular volume and permeability than flank PC3 tumors. The higher vascular permeability of orthotopic tumors may allow greater extravasation of all nanobubbles through the enhanced permeability and retention (EPR) effect. Therefore, enhanced permeability may lead to increased uptake of both PSMA-targeted and non-targeted NBs. Therefore, the difference in NB accumulation in orthotopic tumors between PSMA-targeted and non-targeted NBs is smaller than that in the flank tumor model. This is also due to the higher overall area under the time-intensity curve seen in this model (193.7 ± 64.38 dB in the orthotopic model) compared with the contralateral flank tumors. * 130.4±50.11dB for the flank model* min) and washout AUC of non-targeted NB (149.5 ± 52.19 dB* min in the orthotopic model vs. 116.51 ± 25.61 dB* min in the flank model). * This is reflected by the time to delivery (min). Higher necrosis and cell density also result in greater retention of all bubbles, thus reducing washout of non-targeted bubbles. In general, there are many potential differences between these models, and specific changes in TIC may occur. This is, in part, why using NB contrast-enhanced ultrasound may provide some insight into nanoparticle transport in tumors. The average tumor size of flank tumors is approximately 125 mm. 3 However, the average size of orthotopic tumors is approximately 500 mm 3 Stratification of tumors into large and small cohorts showed significant differences in peak intensity, washout area, and area under the curve (Figure 15), indicating that tumor burden is also a factor influencing kinetics. Animal-to-animal variability was also observed within animals. When normalized to individual animals (as shown in Figure 14B2), the differences in enhancement become even larger. Orthotopic tumors are likely to be more heterogeneous than flank tumors, reducing the differences on average.

[0171] In this example, we used bubble collapse studies to detect intratumoral signal after circulating bubble collapse, demonstrating that PSMA-targeted NBs are retained in tumors to a greater extent than non-targeted NBs (Figure 16). In addition, we found that the dynamics and tumor distribution of NBs differed depending on tumor size / stage. Histological studies of small and large tumors revealed that the centers of large tumors were more necrotic than the centers of small tumors.

[0172] The targeting ligand, PSMA-1, is a peptide-based, highly negatively charged PSMA ligand that is available for clinical studies and can be easily synthesized. The average diameter of PSMA-targeted NBs was 277 ± 11 nm. Our smaller NB size should achieve better tumor penetration than larger bubbles. Smaller particle size has been shown to improve nanoparticle biodistribution and enhance permeability and retention in mouse xenograft tumor models. Overall, the current data suggest that 1) echogenic nanobubbles labeled with a high-affinity ligand for PSMA are fairly stable in vivo and exhibit significant differences in kinetics between clinical MBs and untargeted NBs; and 2) NBs appear to have distinct kinetics and retention in tumors of different sizes. This may be a promising area of ​​future research as a means to stage and potentially grade tumors using the same drug.

[0173] Example 3 This example demonstrates the precise tuning of membrane and / or shell composition, nanobubble size, and acoustic pulse sequence to elicit superior nanobubble behavior at a given ultrasonic frequency and pressure. It was found that the acoustic response of nanobubbles with a narrow size distribution range can be altered by their membrane shell structure (Figure 2). The membrane composition was adjusted by the inclusion of glycerol and propylene glycol (PG). PG has been shown to increase lipid membrane fluidity, while glycerol leads to shell stiffening. To perform this experiment, NBs with a CF gas core were formulated as previously described. Briefly, a lipid cocktail containing DBPC, DSPE-PEG2000, and glycerol was dissolved in PG and PBS, followed by gas exchange and activation via mechanical agitation. NBs with low polydispersity were prepared via separation by centrifugation and filtration through a 400 nm membrane. The shell and pressure dependence of the onset of the detectable nonlinear response of NB solutions in PBS was determined using US in contrast harmonic mode (Toshiba Aplio XG, 12 MHz) at pressures ranging from 74 kPa to 857 kPa (MI = 0.03 to 0.35). As shown in Figure 2, bubbles with a softer shell composed of PG and a cocktail of phospholipids exhibited significantly lower pressures required for the onset of activity (as measured by signal intensity within the imaging field of view). These bubbles exhibited the onset of a detectable nonlinear response at lower pressures (123 kPa to 245 kPa). US images of stiffer NB dispersions showed a minimal 6% signal increase with increasing pressure from 343 kPa to 465 kPa, and a significant 146% increase from 465 kPa to 710 kPa.

[0174] A controllable pressure threshold has potential advantages over contrast enhancement methods based on the nonlinear response of bubbles. One such technique is amplitude modulation, in which two pulses with different pressure amplitudes are sent to the tissue. One pulse typically has twice the amplitude of the other pulse. The signals are scaled and subtracted upon reception. Due to the linear tissue response, the signal from the tissue is canceled, and the only remaining signal is from the bubbles. This increases the contrast (CTR) to the tissue. Sending pulses below the pressure threshold and above the enhancement threshold significantly increases the CTR. Applying a flexible shell results in a smaller pressure for enhancement, resulting in a higher scattering cross-section and therefore better results for imaging purposes. Applying a stiffer shell distorts the pressure to higher values, making it more suitable for therapeutic purposes such as enhanced heating applications, where higher pressures are required. Furthermore, the vibration amplitude of the prefocal bubble is negligible, and by utilizing the steep pressure gradient of some ultrasound transducers, the attenuation of the prefocal bubble can be significantly reduced. Therefore, delivering sufficient energy to the target's resonant NB contributes more efficiently to the enhanced heating effect. Furthermore, unwanted heating in off-target regions is minimized due to the off-resonance bubbles. Finally, this type of approach is also expected to be more effective in eliciting anti-tumor-induced immunity via the so-called "abscopal effect," which has been reported for high-intensity focused ultrasound and histotripsy.

[0175] In summary, TNT-applied bubbles with specific shell compositions 1) reduce the typical activation pressure, 2) exhibit the necessary tunable and predictable pressure-sensitive behavior, and 3) enable cell-mediated endocytosis and long-term residence within intracellular vesicles, making this technology unique compared to conventional microbubble-mediated cell destruction.

[0176] Example 4 This example presents the results of in vitro cellular uptake studies and in vivo experiments demonstrating that targeting NBs with PSMA-1 ligands selectively increased binding to PSMA-expressing PC3pip cells and high accumulation in PC3pip tumors. We hypothesize that combining accumulated PSMA-targeted NBs with therapeutic US selectively damages PSMA-positive PC3pip tumor tissue via intracellular detonation.

[0177] Preparation and characterization of NBs A lipid solution (10 mg / mL) for nanobubbles was prepared by dissolving 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC, Avanti Polar Lipids Inc., Pelham, AL), 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; DPPE, Corden Pharma, Switzerland), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-mPEG 2000, Laysan Lipids, Arabidopsis, AL) in propylene glycol (PG, Sigma Aldrich, Milwaukee, WI) in a 6:1:2:1 ratio, followed by heating at 80 °C and sonication. A mixture of glycerol (Gly, Acros Organics) and phosphate buffer (0.8 mL, Gibco, pH 7.4) preheated to 80°C was added and sonicated for 10 minutes at room temperature. The solution (1 mL) was transferred to a 3 mL headspace vial and capped with a rubber septum and aluminum seal. The air was replaced with octafluoropropane (C3F8, Electronic Fluorocarbons, LLC, PA) gas and activated by mechanical shaking for 45 seconds using a VialMix shaker (Bristol-Myers Squibb Medical Imaging Inc., N. Billerica, MA). Nanobubbles were separated from microbubbles by centrifugation at 50 rcf for 5 minutes with the headspace vial inverted, and 100 μL of NB solution was withdrawn from the vial using a 21G needle at a fixed distance of 5 mm from the bottom.

[0178] PSMA-NBs were prepared by adding DSPE-PEG-PSMA-1 (25 μg / ml) to the initial lipid solution and following the protocol described above. To prepare DSPE-PEG-PSMA-1, PSMA-1 (from Professor James Basilion's laboratory) was mixed with DSPE-PEG-MAL (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000-maleimide], Laysan Bio, Arab, AL) in a 1:2 ratio in PBS at pH 8.0. After combining, the mixture was thoroughly vortexed and allowed to react for 4 hours on a vial rotating wheel at 4°C. The product was lyophilized, and the resulting powder was dissolved in PBS to obtain the DSPE-PEG-PSMA-1 stock solution. The conjugation of DSPE-PEG-PSMA-1 was confirmed by high-performance liquid chromatography (HPLC) and MALDI-TOF. HPLC was performed on a Shimadzu HPLC system equipped with an SPD-20A Prominence UV / Visible detector and monitored at a wavelength of 220 nm. Analytical HPLC was performed using an analytical Luna HPLC system at a flow rate of 1.0 mL / min. The run was performed using a 5μ C18(2) 100A column (250 mm × 4.6 mm × 5 μm, Phenomenex). The gradient used was 10% to 40% acetonitrile with 0.1% TFA over 20 minutes.

[0179] The size distribution and concentration of NBs were characterized by resonant mass spectrometry (Archimedes®, Malvern Panalytical) as described above in 1-2. Measurements were terminated after measuring 1000 particles. Data were exported from Archimedes software (version 1.2) and analyzed for positive and negative values. The surface charge of diluted NB solutions (500X) was measured with an Anton Paar Litesizer 500.

[0180] procedure Figure 18 outlines the procedure for administering and resonating PSMA-NBs to mice, including the following:

[0181] 200 μL of targeted PSMA-NBs are injected via the tail vein into dual tumor-bearing mice (M1, M2 and M3, M4).

[0182] Thirty minutes later, TUS is applied to the PC3pip tumors of M1 and M3 mice.

[0183] Thirty minutes later, TUS is applied to PC3flu tumors in M2 and M4 mice.

[0184] Control mice were injected with PBS, and 30 minutes later, TUS was administered to both PC3pip and PC3flu tumors. Parameters: TUS treatment: 5 minutes, 3 MHz, 2.2 W / cm 2 / 10 DC (small probe).

[0185] After 24 hours of treatment, the tumors are excised and subjected to histological examination.

[0186] Apoptosis is analyzed using the TUNEL assay.

[0187] Group 1 - PSMA-NB injection and 30 minutes later, TUS is applied to PC3pip tumors (M1 and M3).

[0188] Group 2 - PSMA-NB injection and 30 minutes later, TUS is applied to PC3flu tumors (M2 and M4).

[0189] Group 3 - PBS injection and 30 minutes later, TUS was applied to PC3pip and PC3flu tumors (M5).

[0190] Figure 19 shows the results of a PSMA-expressing tumor treated with a 10% duty cycle, 3 MHz, 2.2 W / cm 2We show that treatment with PSMA-targeted nanobubbles in combination with 5 minutes of ultrasound at 100 rpm resulted in significant apoptosis 24 hours after treatment. When the same treatment was applied to PSMA-negative tumors, little or no apoptosis was observed. This suggests that only nanobubbles internalized by tumor cells via receptor-mediated endocytosis had a significant toxic effect.

[0191] Figure 20 shows that little to no apoptosis was observed when PSMA-positive or PSMA-negative tumors were treated with 10% duty cycle, 3 MHz, 5 minutes of ultrasound alone (no bubbles). This suggests that only nanobubbles in combination with ultrasound have a toxic effect. Bubbles alone (no ultrasound) had no effect. Data not shown.

[0192] Figures 21 and 22 show PSMA-expressing tumors treated with PSMA-NBs. TUS showed significant apoptosis in cell death compared to all other groups. Apoptosis was approximately 33 (33.55 ± 1.10)% of the DAPI used for ROI selection.

[0193] From the above description of the invention, those skilled in the art will recognize improvements, changes, and modifications. Such improvements, changes, and modifications by those skilled in the art are intended to be covered by the appended claims. All references, publications, and patents cited in this application are incorporated herein by reference in their entirety.

Claims

1. A method of inducing cell death in a subject, comprising administering to the subject a plurality of nanobubbles, each nanobubble having a membrane defining at least one interior cavity containing at least one gas and a targeting moiety coupled to an exterior surface of the membrane, wherein the targeting moiety binds to a cell surface molecule of a target cell, and the nanobubbles have a size and / or diameter that promotes internalization of the nanobubbles by the target cell upon binding of the targeting moiety to the cell surface molecule; and vibrating the internalized nanobubbles in the target cells with ultrasonic energy effective to promote inertial cavitation of the internalized nanobubbles and apoptosis and / or necrosis of the target cells; A method comprising:

2. 10. The method of claim 1, wherein the nanobubbles have an average diameter of about 50 nm to about 400 nm.

3. 10. The method of claim 1, wherein the cell is a cancer cell and the targeting moiety binds to a cancer cell surface molecule.

4. 10. The method of claim 1, wherein the targeting moiety is selected from the group consisting of a polypeptide, a polynucleotide, a small molecule, an elemental compound, an antibody, and an antibody fragment.

5. The method of claim 1 , wherein the cancer cell surface molecule is a cancer cell antigen on the surface of the cancer cell.

6. The cancer cell antigen is 5T4, α2β1 integrin, AXL receptor tyrosine kinase (AXL), B cell maturation antigen (BCMA), c-MET (hepatocyte growth factor receptor), C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), cadherin-6, CD19, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, collagen receptor, or cryptoprotein. , CS1, Delta-like canonical Notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvIII, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1), non-transferable glycoprotein Protein B (GPNMB), guanylate cyclase 2C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), integrin α, lysosome-associated membrane protein 1 (LAMP-1), Lewis Y, LIV-1, leucine-rich repeat-containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), sodium-dependent phosphate transport protein 2B (NaPi2b), nectin-4, NMB, NOTCH3, p-cadherin 5. The method of claim 4, wherein the target protein comprises at least one of prostate cancer cells expressing a leukemia antigen (LCC11A), prostate-specific membrane antigen (p-CAD), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), protein tyrosine phosphatase μ (PTPμ) solute carrier family 44 member 4 (SLC44A4), SLIT-like family member 6 (SLITRK6), STEAP family member 1 (STEAP1), tissue factor (TF), T-cell immunoglobulin and mucin protein-1 (TIM-1), or trophoblast surface antigen (TROP-2).

7. 2. The method of claim 1, wherein the target cells are prostate cancer cells and the cell surface molecule is PSMA.

8. The method of claim 1 , wherein the membrane is a lipid membrane.

9. 9. The method of claim 8, wherein the lipid membrane further comprises at least one of glycerol, propylene glycol, pluronic (poloxamer), alcohol, or cholesterol, which alter the modulus and / or interfacial tension of the bubble membrane.

10. 9. The method of claim 8, wherein the nanobubbles have a lipid concentration of at least about 5 mg / ml.

11. 9. The method of claim 8, wherein the lipid membrane comprises dipalmitoylphosphatidylcholine (DPPC), dibehenoylglycerophosphocholine (DBPC), distearoylphosphatidylcholine (DSPC), diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), and distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidic acid (DPPA), or a mixture of at least two of these PEG-functionalized lipids.

12. 12. The method of claim 11, wherein the lipid mixture comprises a combination of at least about 50% by weight of dibehenoylglycerophosphocholine (DBPC) and less than about 50% by weight of an additional phospholipid selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidic acid (DPPA), or a PEG-functionalized phospholipid thereof.

13. The method of claim 1 , wherein the gas comprises a perfluorocarbon gas.

14. 10. The method of claim 1, wherein the resonance induces cell death without adversely affecting normal cells and tissues.

15. The resonance has a duty cycle of about 1% to about 50%, an ultrasonic frequency of about 1 MHz to about 12 MHz, and a frequency of about 0.1 W / cm ２ ~Approx. 3W / cm ２ 2. The method of claim 1, wherein the pressure is applied at an intensity of about 100 kPa to about 100 kPa, a pressure amplitude of about 50 kPa to about 1 MPa, and a time period of about 1 minute to about 10 minutes.

16. 2. The method of claim 1, wherein the resonance comprises two ultrasound pulse sequences having pulses of different pressure amplitudes delivered to the tissue into which the nanobubbles are administered, one pulse having a greater pressure amplitude than the other pulse.

17. 17. The method of claim 16, wherein one pulse has a pressure amplitude at least twice that of the other pulse.

18. 17. The method of claim 16, wherein one pulse is below the nanobubble pressure threshold for inertial cavitation, followed by another pulse above the threshold pressure threshold for inertial cavitation.

19. The method of claim 1 , wherein the ultrasonic energy is provided by an unfocused ultrasonic transducer.

20. The method of claim 1 , wherein the target cells comprise widespread cancer micrometastases in the subject.

21. The method of claim 1 , wherein the cells comprise prokaryotic cells of a microorganism.

22. 22. The method of any of claims 1-21, wherein the nanobubbles further comprise at least one therapeutic agent contained within or conjugated to the membrane of each nanobubble.

23. 23. The method of claim 22, wherein the therapeutic agent further comprises at least one chemotherapeutic agent, antiproliferative agent, biocide, biostatic agent, or antibacterial agent.

24. 1. A method of treating cancer in a subject in need thereof, comprising administering to the subject a plurality of nanobubbles, each nanobubble having a membrane defining at least one interior cavity containing at least one gas and a targeting moiety attached to an exterior surface of the membrane, wherein the targeting moiety binds to a cell surface molecule of a target cancer cell, the nanobubble having a size and / or diameter that promotes internalization of the nanobubble by the target cancer cell upon binding of the targeting moiety to the cell surface molecule; and resonating the internalized nanobubbles in the target cancer cells with ultrasound energy effective to promote inertial cavitation of the internalized nanobubbles and apoptosis and / or necrosis of the target cancer cells; A method comprising:

25. 25. The method of claim 24, wherein the nanobubbles have an average diameter of about 50 nm to about 400 nm.

26. 25. The method of claim 24, wherein the targeting moiety is selected from the group consisting of a polypeptide, a polynucleotide, a small molecule, an elemental compound, an antibody, and an antibody fragment.

27. 25. The method of claim 24, wherein the cancer cell surface molecule is a cancer cell antigen on the surface of the cancer cell.

28. The cancer cell antigen is 5T4, α2β1 integrin, AXL receptor tyrosine kinase (AXL), B cell maturation antigen (BCMA), c-MET (hepatocyte growth factor receptor), C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), cadherin-6, CD19, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, collagen receptor, or cryptoprotein. , CS1, Delta-like canonical Notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvIII, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1), non-transferable glycoprotein Protein B (GPNMB), guanylate cyclase 2C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), integrin α, lysosome-associated membrane protein 1 (LAMP-1), Lewis Y, LIV-1, leucine-rich repeat-containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), sodium-dependent phosphate transport protein 2B (NaPi2b), nectin-4, NMB, NOTCH3, p-cadherin ( 28. The method of claim 27, wherein the target polypeptide comprises at least one of the following: p-CAD), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), protein tyrosine phosphatase μ (PTPμ) solute carrier family 44 member 4 (SLC44A4), SLIT-like family member 6 (SLITRK6), STEAP family member 1 (STEAP1), tissue factor (TF), T-cell immunoglobulin and mucin protein-1 (TIM-1), or trophoblast surface antigen (TROP-2).

29. 25. The method of claim 24, wherein the target cell is a prostate cancer cell and the cell surface molecule is PSMA.

30. 25. The method of claim 24, wherein the membrane is a lipid membrane.

31. 31. The method of claim 30, wherein the lipid membrane further comprises at least one of glycerol, propylene glycol, pluronic (poloxamer), alcohol, or cholesterol, which alter the modulus and / or interfacial tension of the bubble membrane.

32. 31. The method of claim 30, wherein the nanobubbles have a lipid concentration of at least about 5 mg / ml.

33. 33. The method of claim 32, wherein the lipid membrane comprises dipalmitoylphosphatidylcholine (DPPC), dibehenoylglycerophosphocholine (DBPC), distearoylphosphatidylcholine (DSPC), diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), and distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidic acid (DPPA), or a mixture of at least two of these PEG-functionalized lipids.

34. 34. The method of claim 33, wherein the lipid mixture comprises a combination of at least about 50% by weight of dibehenoylglycerophosphocholine (DBPC) and less than about 50% by weight of an additional phospholipid selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidic acid (DPPA), or a PEG-functionalized phospholipid thereof.

35. 25. The method of claim 24, wherein the gas comprises a perfluorocarbon gas.

36. 25. The method of claim 24, wherein the resonance induces cell death without adversely affecting normal cells and tissues.

37. The resonance has a duty cycle of about 1% to about 50%, an ultrasonic frequency of about 1 MHz to about 12 MHz, and a frequency of about 0.1 W / cm ２ ~Approx. 3W / cm ２ 25. The method of claim 24, wherein the pressure is applied at an intensity of about 100 kPa to about 100 kPa, a pressure amplitude of about 50 kPa to about 1 MPa, and a time period of about 1 minute to about 10 minutes.

38. 25. The method of claim 24, wherein the resonance comprises two ultrasound pulse sequences having pulses of different pressure amplitudes delivered to the tissue into which the nanobubbles are administered, one pulse having a greater pressure amplitude than the other pulse.

39. 39. The method of claim 38, wherein one pulse has a pressure amplitude at least twice that of the other pulse.

40. 39. The method of claim 38, wherein one pulse is below the nanobubble pressure threshold for inertial cavitation, followed by another pulse above the threshold pressure threshold for inertial cavitation.

41. 25. The method of claim 24, wherein the ultrasonic energy is provided by an unfocused ultrasonic transducer.

42. 25. The method of claim 24, wherein the target cells comprise widespread cancer micrometastases in the subject.

43. 25. The method of claim 24, wherein the cells comprise microbial prokaryotic cells.

44. 44. The method of any of claims 24-43, wherein the nanobubbles further comprise at least one therapeutic agent contained within or conjugated to the membrane of each nanobubble.

45. 45. The method of claim 44, wherein the therapeutic agent further comprises at least one chemotherapeutic agent, antiproliferative agent, biocide, biostatic agent, or antibacterial agent.

46. 1. A system for treating cancer in a subject, comprising: an ultrasound source configured to non-invasively deliver ultrasound energy to cancer cells within the subject; a plurality of nanobubbles, each nanobubble having a membrane defining at least one interior cavity containing at least one gas, and a targeting moiety coupled to an exterior surface of the membrane, wherein the targeting moiety binds to a cell surface molecule of a target cancer cell, the nanobubble having a size and / or diameter that promotes internalization of the nanobubble by the target cancer cell upon binding of the targeting moiety to the cell surface molecule; and a controller coupled to the ultrasound source configured to induce resonance in cancer cells during a resonance time and promote inertial cavitation of nanobubbles internalized by the cancer cells. A system including:

47. The resonance has a duty cycle of about 1% to about 50%, an ultrasonic frequency of about 1 MHz to about 12 MHz, and a frequency of about 0.1 W / cm ２ ~Approx. 3W / cm ２ 47. The system of claim 46, wherein the pressure is applied at an intensity of about 100 kPa to about 100 kPa, a pressure amplitude of about 50 kPa to about 1 MPa, and a duration of about 1 minute to about 10 minutes.

48. 47. The system of claim 46, wherein the resonance comprises two ultrasound pulse sequences having pulses of different pressure amplitudes delivered to the tissue into which the nanobubbles are administered, one pulse having a greater pressure amplitude than the other pulse.

49. 49. The system of claim 48, wherein one pulse has a pressure amplitude at least twice that of the other pulse.

50. 49. The system of claim 48, wherein one pulse is below the nanobubble pressure threshold for inertial cavitation, followed by another pulse above the nanobubble pressure threshold for inertial cavitation.

51. 47. The system of claim 46, wherein the ultrasonic energy is provided by an unfocused ultrasonic transducer.

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