WO2024254612A2

WO2024254612A2 - An ultrasound-activated ultrasound contrast agent

Info

Publication number: WO2024254612A2
Application number: PCT/US2024/033302
Authority: WO
Inventors: Travis J. Williams; Jesse Tong-Pin Yen; Van Khanh DO; Andy Yu-How CHANG
Original assignee: Childrens Hospital Los Angeles; University of Southern California USC
Current assignee: Childrens Hospital Los Angeles; University of Southern California USC
Priority date: 2023-06-09
Filing date: 2024-06-10
Publication date: 2024-12-12
Anticipated expiration: 2025-12-09
Also published as: WO2024254612A3

Abstract

An ultrasound-activated ultrasound contrast agent includes a sterilized liquid carrier; and a sterilized polymer dispersed and/or dissolved in the sterilized liquid carrier. Advantageously, the sterilized polymer includes amino groups that are at least partially functionalized with CO₂ at a plurality of the amino groups.

Description

AN ULTRASOUND-ACTIVATED ULTRASOUND CONTRAST AGENT

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application Serial No. 63/471,994 filed June 9, 2023, the disclosure of which is hereby incorporated in its entirety by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] The invention was made with Government support under Contract No. P50FD006425 awarded by The West Coast Consortium for Technology & Innovation in Pediatrics (CTIP). The Government has certain rights in the invention.

TECHNICAL FIELD

[0003] In at least one aspect, an ultrasound-activated ultrasound contrast agent is provided.

BACKGROUND

[0004] Bladder catheterization, particularly in pediatric patients, is often poorly tolerated and can be a significant source of discomfort and dissatisfaction among adults. Beyond the immediate discomfort, catheterization is associated with a considerable risk of urinary tract infections (UTls). Studies have shown infection rates range from 2% to 22% during voiding cystourethrograms (VCUGs) and from 2.1% to 21% during urodynamic studies. The invasiveness and complications associated with catheterization led the 2006 National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Strategic Plan for Pediatric Urology to prioritize the development of a catheter-free VCUG procedure as a critical national health research goal. However, despite the passage of nearly 20 years since this call to action, an effective catheter-free method has not yet been successfully developed.

[0005] Currently, the voiding cystourethrogram (VCUG) remains the gold-standard technique for detecting vesicoureteral reflux (VUR), a condition where urine flows backward from the bladder into the ureters or kidneys. This procedure is not only invasive, requiring bladder catheterization, but it also exposes patients to ionizing radiation, raising further health concerns. Although contrast- enhanced voiding urosonography offers an alternative by eliminating ionizing radiation, it still necessitates the use of a catheter, thereby not addressing the discomfort and infection risks associated with catheterization.

[0006] The ongoing reliance on such invasive techniques underscores the urgent need for innovative solutions. There is a clear demand for a method that can accurately diagnose VUR without the associated risks and discomfort of current practices. Developing a non-invasive, patient-friendly diagnostic tool would significantly enhance patient care, particularly in pediatric urology, and align with the longstanding objectives of national health research agendas.

SUMMARY

[0007] In at least one aspect, a nanoparticle is capable of the on-demand release of CO2 with ultrasound energy with visualization of released bubbles with diagnostic ultrasound for the purpose of disease diagnosis and treatment.

[0008] In another aspect, a method for visualizing bladder-to-kidney urine reflux is provided. A sterilized polymer is covalently loaded with CO2 and then administered to a subject. The CO2 is released with ultrasound once the polymer is in the bladder. This then enables the bubbling of CO2 as an ultrasound contrast agent.

[0009] In another aspect, the methods set forth herein allow the avoidance of urinary catheterization in diagnostic urologic tests.

[0010] In another aspect, the intravenous injection of covalently bound CO2 nanoparticles are excreted by the kidneys into the urine. Once in the bladder, these nanoparticles are activated by ultrasound to generate CO2 bubbles. The presence of bubbles in the ureters or kidneys, as visualized by ultrasound, would indicate vesicoureteral reflux.

[0011] In another aspect, a contrast-enhanced ultrasound agent is provided. The contrast- enhanced ultrasound agent includes a sterilized liquid carrier and a sterilized polymer dispersed and/or dissolved in the sterilized liquid carrier. Characteristically, the sterilized polymer includes amino groups that are at least partially functionalized with CO2 at a plurality of the amino groups.

[0012] In another aspect, a contrast-enhanced ultrasound agent is provided. The contrast- enhanced ultrasound agent includes a sterilized liquid carrier and a sterilized nanoparticle composed of a polyethylenimine dispersed and/or dissolved in the sterilized liquid carrier. Characteristically, the polyethylenimine includes amino groups that are at least partially functionalized with CO2 at a plurality of the amino groups. In another aspect, a method for delivering releasable, caged CO2 in vivo is provided. The method includes a step of administering a contrast-enhanced ultrasound agent to a subject. The contrast-enhanced ultrasound agent includes a polymer that includes amino groups to a subject. Advantageously, the polymer includes amino groups functionalized with CO2 at a plurality of the amino groups. Ultrasonic energy is directed into a subject’s urinary tract to release CO2 from the contrast-enhanced ultrasound agent.

[0013] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be made to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements and wherein:

[0015] FIGURE 1. Schematic of syringe holding a contrast-enhanced ultrasound agent that releases CO2.

[0016] FIGURE 2. Schematic flowchart illustrating a method for delivering releasable, caged CO2 in vivo. [0017] FIGURE 3. Schematic drawing of experimental apparatus. A rubber balloon filled with nanoparticle solution is “activated” by a sonication transducer releasing carbon dioxide bubbles. An imaging transducer allows for visualization of this process.

[0018] FIGURE 4. Representative ultrasonographic image of CO? formation in nanoparticle- filled balloon after activation.

[0019] FIGURE 5. Representative ultrasonographic image of water-filled balloon without evidence of CO2 formation after sonication.

[0020] FIGURE 6. Successful visualization of CO? bubbles after sonication in nanoparticle- filled balloon overlayed with thick pork belly.

DETAILED DESCRIPTION

[0021] Reference will now be made in detail to presently preferred compositions, embodiments and methods of the present invention, which constitute the best modes of practicing the invention presently known to the inventors. The Figures are not necessarily to scale. However, it is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for any aspect of the invention and/or as a representative basis for teaching one skilled in the art to variously employ the present invention.

[0022] Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and/or use are to be understood as modified by the word "about" in describing the broadest scope of the invention. Practice within the numerical limits stated is generally preferred. Also, unless expressly stated to the contrary: all R groups (e.g. Rj where i is an integer) include hydrogen, alkyl, lower alkyl, C1-6 alkyl, Ce-io aryl, C6-10 heteroaryl, alylaryl (e.g., C1-8 alkyl C6-10 aryl). -NO2, -NH2, -N(R’R”), -N(R’R”R”’)⁺L' , Cl, F, Br, -CF₃, -CCI3, -CN, -SO3H, -PO3H2, -COOH, -CO₂R’, -COR’, -CHO, -OH, -OR’, -O’M⁺, - SO₃-M⁺, -PO₃ M⁺, -COO-M⁺. -CF₂H, -CF₂R’. -CFH₂, and -CFR’R” where R’, R” and R'” are Ci-10 alkyl or Ce-i8 aryl groups, M is a metal ion, and L" is a negatively charged counter ion; R groups on adjacent carbon atoms can be combined as -OCH2O-; single letters (e.g., "n" or "o") are 1, 2, 3, 4, or 5; in the compounds disclosed herein a CH bond can be substituted with alkyl, lower alkyl, C i-6 alkyl, C₆-io aryl. C₆-io heteroaryl, -NO₂. -NH₂, -N(R’R”), -N(R’R”R”')⁺L; Cl, F. Br, -CF₃, -CC1₃, -CN, - SO3H, -PO3H2, -COOH, -CO2R’, -COR', -CHO, -OH, -OR’, -O’M⁺, -SO₃-M⁺, -PO₃’M⁺, -COO’M⁺, - CF₂H, -CF₂R‘, -CFH₂, and -CFR’R” where R', R” and R’” are C1-10 alkyl or C6-18 aryl groups. M⁺ is a metal ion, and L" is a negatively charged counter ion; hydrogen atoms on adjacent carbon atoms can be substituted as -OCH₂O-; when a given chemical structure includes a substituent on a chemical moiety (e.g., on an aryl, alkyl, etc.) that substituent is imputed to a more general chemical structure encompassing the given structure; percent, "parts of," and ratio values are by weight; the term "polymer" includes "oligomer," "copolymer," "terpolymer," and the like; molecular weights provided for any polymers refers to weight average molecular weight unless otherwise indicated; the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description, and does not necessarily preclude chemical interactions among the constituents of a mixture once mixed; the first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutandis to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property.

[0023] It must also be noted that, as used in the specification and the appended claims, the singular form "a," "an," and "the" comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components.

[0024] As used herein, the term “about” means that the amount or value in question may be the specific value designated or some other value in its neighborhood. Generally, the term “about” denoting a certain value is intended to denote a range within +/- 5% of the value. As one example, the phrase “about 100” denotes a range of 100 +/- 5, i.e. the range from 95 to 105. Generally, when the term “about” is used, it can be expected that similar results or effects according to the invention can be obtained within a range of +/- 5% of the indicated value. [0025] As used herein, the term ‘‘and/or” means that either all or only one of the elements of said group may be present. For example, “A and/or B” shall mean “only A, or only B. or both A and B”. In the case of “only A”, the term also covers the possibility that B is absent, i.e. “only A, but not B”.

[0026] It is also to be understood that this invention is not limited to the specific embodiments and methods described below, as specific components and/or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular embodiments of the present invention and is not intended to be limiting in any way.

[0027] The term “comprising” is synonymous with “including,” “having,” “containing,” or “characterized by.” These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps.

[0028] The phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0029] The phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

[0030] The phrase “composed of’ means “including” or “consisting of.” Typically, this phrase is used to denote that an object is formed from a material.

[0031] With respect to the terms “comprising,” “consisting of.” and “consisting essentially of,” where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.

[0032] The term “one or more” means “at least one” and the term “at least one” means “one or more.” The terms “one or more” and “at least one” include “plurality” and “multiple” as a subset. In a refinement, “one or more” includes “two or more.” [0033] The term “substantially,” “generally,” or “about” may be used herein to describe disclosed or claimed embodiments. The term “substantially” may modify a value or relative characteristic disclosed or claimed in the present disclosure. In such instances, “substantially” may signify that the value or relative characteristic it modifies is within ± 0%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10% of the value or relative characteristic.

[0034] It should also be appreciated that integer ranges explicitly include all intervening integers. For example, the integer range 1-10 explicitly includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Similarly, the range 1 to 100 includes 1, 2, 3, 4. . . . 97, 98, 99, 100. Similarly, when any range is called for, intervening numbers that are increments of the difference between the upper limit and the lower limit divided by 10 can be taken as alternative upper or lower limits. For example, if the range is 1.1. to 2.1 the following numbers 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, and 2.0 can be selected as lower or upper limits.

[0035] When referring to a numeral quantity, in a refinement, the term “less than” includes a lower non-included limit that is 5 percent of the number indicated after “less than.” For example, “less than 20” includes a lower non-included limit of 1 in a refinement. Therefore, this refinement of “less than 20” includes a range between 1 and 20. In another refinement, the term “less than” includes a lower non-included limit that is, in increasing order of preference, 20 percent, 10 percent, 5 percent, or 1 percent of the number indicated after “less than.”

[0036] In the examples set forth herein, concentrations, temperature, and reaction conditions (e.g., pressure. pH, flow rates, etc.) can be practiced with plus or minus 50 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples. In a refinement, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 30 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples. In another refinement, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 10 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples. [0037] For all compounds expressed as an empirical chemical formula with a plurality of letters and numeric subscripts (e.g.. CH2O), values of the subscripts can be plus or minus 50 percent of the values indicated rounded to or truncated to two significant figures. For example, if CH2O is indicated, a compound of formula C(o.8-i.2)H(i.6-2.4)0(o.8-i 2)- In a refinement, values of the subscripts can be plus or minus 30 percent of the values indicated rounded to or truncated to two significant figures. In still another refinement, values of the subscripts can be plus or minus 20 percent of the values indicated rounded to or truncated to two significant figures. The term '‘electrical communication” means that an electrical signal is either directly or indirectly sent from an originating electronic device to a receiving electrical device. Indirect electrical communication can involve processing of the electrical signal, including but not limited to, filtering of the signal, amplification of the signal, rectification of the signal, modulation of the signal, attenuation of the signal, adding of the signal with another signal, subtracting the signal from another signal, subtracting another signal from the signal, and the like. Electrical communication can be accomplished with wired components, wirelessly connected components, or a combination thereof.

[0038] The term “one or more” means “at least one” and the term “at least one” means “one or more.” The terms “one or more” and “at least one” include “plurality” as a subset.

[0039] The term “substantially,” “generally,” or “about” may be used herein to describe disclosed or claimed embodiments. The term “substantially” may modify a value or relative characteristic disclosed or claimed in the present disclosure. In such instances, “substantially” may signify that the value or relative characteristic it modifies is within ± 0%, 0.1%, 0.5%, 1%, 2%, 3%, 4%. 5%, or 10% of the value or relative characteristic.

[0040] Abreviations:

[0041] CEUS" means contrast-enhanced ultrasound.

[0042] CO2" means carbon dioxide.

[0043] CTIP" means The West Coast Consortium for Technology & Innovation in Pediatrics.

[0044] "D" means Dalton. [0045] "DI" means deionized.

[0046] "NIDDK" means National Institute of Diabetes and Digestive and Kidney Diseases.

[0047] "nm" means nanometer.

[0048] "PBS" means phosphate-buffered saline.

[0049] "PEG" means polyethylene glycol.

[0050] “PEI” means polyethylenimine.

[0051] "RF" means radio frequency.

[0052] "UTIs" means urinary tract infections.

[0053] "VCUG" means voiding cystourethrogram.

[0054] "VUR" means vesicoureteral reflux.

[0055] In general, a strategy to use ultrasound to modulate covalent bonding is provided. This strategy uses amine-rich materials, which are known to react reversibly with CO2 and to store CO2 as carbamic acid groups-CO will be covalently attached to the material. It is shown that this CO2 can be released with low-power ultrasound, realizing loading/unloading efficiency near 10 wt%. This observation opens the way for ultrasound-triggered CO2 effervesce from a polymeric material, which in turn serves as a contrast agent for ultrasound imaging. The CCh-amine chemistry applied herein is known in the carbon capture field, except in these applications, CO2 is usually released by pH (acidification) or heating (well above 40 °C). A key innovation is that excitation ultrasound enables CO2 release, which we demonstrated with the bladder modeled by a water balloon in a water tank.

[0056] In at least one aspect, a contrast-enhanced ultrasound agent is provided. Figure 1 proides a schematic of a syringe holding the contrast-enhanced ultrasound agent. The contrast- enhanced ultrasound agent 10 includes a liquid earner 12, and in particular, sterilized liquid polymer, and a polymer 14, and in particular, sterilized polymer 14, dispersed and/or dissolved in the sterilized liquid carrier. Characteristically, the sterilized polymer includes amino groups that are at least partially functionalized with CO2 at a plurality of the amino groups. In a refinement, the sterilized polymer 14 is water-soluble and nontoxic. It should be appreciated that the terms “polymer” and “sterilized polymer” are interchangeable. Similarly, “liquid polymer” and “sterilized liquid polymer”

[0057] In another aspect, a nanoparticle includes the sterilized polymer. In a refinement, the nanoparticles are small enough to be filtrated by the kidneys, which requires an average diameter of less than about 10 nm. In a refinement, the average diameter of the nanoparticle is less than about 5.5 nm. Typically, the nanoparticle has an average diameter greater than 0.5 nm. Alternatively, the nanoparticles have a maximum dimension of less than about 10 nm or 5.5 nm. In a refinement, the nanoparticles have a maximum dimension greater than about 0.5 nm.

[0058] In another aspect, the sterilized polymer is a poly(amine) material or a poly(amine) material that is functionalized with PEG groups.

[0059] In another aspect, the sterilized polymer is a polyethylenimine. In a refinement, the sterilized polymer is a linear polyethylenimine. In a further refinement, the sterilized polymer before carboxylation has the following formula

n is an integer from about 40 to 1000. In a further refinement, the sterilized polymer includes monomer units having following formula

where dashed line represent bonds to a polymer backbone. In another refinement, the sterilized polymer is a branched polyethylenimine. For example, the sterilized polymer before carboxylation can include a monomer unit having the following formula incorporated into the polymer backbone:

where dashed line represent bonds to a polymei' backbone. In another refinement, the sterilized polymer is a dendritic polyethylenimine.

[0060] In another aspect, the sterilized polymer has a weight average molecular weight from about 86 D to 40,000 D. In a refinement, the sterilized polymer has a weight average molecular weight from about 10,000 D to 30,000 D. In some refinements, the sterilized polymer has a weight average molecular weight greater than or equal to 86D, 100 D, 500 D, 1000 D, 5000 D, or 10,000 D. In some further refinements, the sterilized polymer has a weight average molecular weight less than or equal to 50,000 D, 45,000 D, 40,000 D, 35,000 D, 30,000 D, 25,000 D, 20,000 D, or 15000 D.

[0061] In another embodiment, a nanoparticle-containing contrast-enhanced ultrasound agent 10 is provided. Referring to Figure 1, contrast-enhanced ultrasound agent 10 includes a sterilized liquid carrier 12 and a sterilized nanoparticle 14 composed of a polyethylenimine dispersed and/or dissolved in the sterilized liquid carrier. Characteristically, the polyethylenimine includes amino groups functionalized with CO2 at a plurality of the amino groups. In a refinement, the nanoparticles are small enough to be filtrated by the kidneys, which requires an average diameter of less than about 10 nm. In a refinement, the average diameter of the nanoparticle is less than about 5.5 nm. Typically, the nanoparticle has an average diameter greater than 0.5 nm. Alternatively, the nanoparticles have a maximum dimension of less than about 10 nm or 5.5 nm. In a refinement, the nanoparticles have a maximum dimension greater than about 0.5 nm.

[0062] In another aspect of the nanoparticle-containing contrast-enhanced ultrasound agent, the polyethylenimine is functionalized with PEG groups.

[0063] In another aspect of the nanoparticle-containing contrast-enhanced ultrasound agent, the polyethylenimine is a linear polyethylenimine. In a further refinement, the polyethylenimine before carboxylation has the following formula

n is an integer from about 40 to 1000. In a further refinement, the polyethylenimine includes monomer units having following formula

thereof, where dashed line represent bonds to a polymer backbone. In another refinement, the polyethylenimine is a branched polyethylenimine. For example, the polyethylenimine before carboxylation can include a monomer unit having the following formula incorporated into the polymer backbone:

where dashed line represent bonds to a polymer backbone. In another refinement, thepolyethylenimineis a dendritic polyethylenimine.

[0064] In another aspect of the nanoparticle-containing contrast-enhanced ultrasound agent, the polyethylenimine has a weight average molecular weight from about 86 D to 40,000 D. In a refinement, the polyethylenimine has a weight average molecular weight from about 10,000 D to 30,000 D. In some refinements, the sterilized polymer has a weight average molecular weight greater than or equal to 86 D, 100 D, 500 D. 1000 D, 5000 D, or 10,000 D. In some further refinements, the sterilized polymer has a weight average molecular weight less than or equal to 50,000 D. 45,000 D, 40,000 D, 35,000 D, 30,000 D, 25,000 D, 20,000 D, or 15000 D.

[0065] In another embodiment, a method for delivering releasable, caged CO2 in vivo is provided. Referring to Figure 2, a schematic flow chart illustrating the method is provided. In step a), a contrast-enhanced ultrasound agent 10 is administered to subject 18. The contrast-enhanced ultrasound agent includes a polymer including amino groups functionalized with CO2 at a plurality of the amino groups. In step b), the ultrasonic energy from source 20 is directed into a part of a subject’s body to release CO2 from the contrast-enhanced ultrasound agent. Details of the contrast-enhanced ultrasound agent are set forth above. In a refinement, the part of a subject’s bodyis a subject's urinary tract. [0066] In another aspect, the method further includes collecting diagnostic ultrasound images from the subject. In a refinement in step c), diagnostic ultrasound images are collected from the subject with ultrasound imaging equipment 22 which is in electrical communication with the computing system 24. Ultrasound images can be displayed on a monitor 26 in computing system 24. These ultrasonic images can be used to determine if the subject has vesicoureteral reflux (VUR) from the ultrasound images. In another refinement, CO2 effervescence can be used to diagnose or surveil VUR. Advantageously, caged CO2 and ultrasound activation can be used as an alternative to a voiding cystourethrogram (VCUG), or any other methodology that involves bladder catheterization. Advantageously, it is shown for the first time that CO2 effervescence can be selectively and decisively triggered by sonication. Moreover, the level of energy required is well above that for clinical imaging and appropriate for excitation use.

[0067] In another aspect of the method, computing system 24 can have algorithms encoded therein for performing the calculations from the imaging data.

[0068] In another aspect of the method, it is shown that polymeric carbamic acid materials obtained by carboxylating the amino-containing material can serve as contrast agents for contrast- enhanced ultrasound (CEUS) imaging wherein amine-bound CO2 can be uncaged from the material in vitro by focused ultrasound.

[0069] In another aspect of the method, ultrasonic energy is low-power. Low-power ultrasound in medical imaging involves using ultrasound waves at lower intensities, typically below 720 mW/cm² Ispta. 190 W/cnr I_spPa, and a mechanical index of 1.9, to produce diagnostic images safely. Operating at frequencies between 2 to 18 MHz, it balances depth and resolution, making it ideal for various applications like obstetrics for fetal monitoring, cardiology for heart and blood vessel imaging, abdominal imaging for organs such as the liver and kidneys, musculoskeletal imaging for assessing muscles and tendons and guiding procedures like biopsies. Its modes, including B-mode for 2D images, Doppler for blood flow visualization, M-mode for motion capture, and 3D/4D for detailed imaging, enhance its versatility. Advantages include its non-invasive nature, real-time imaging capabilities, cost-effectiveness, and portability, making it an indispensable tool in diverse medical settings. [0070] In another aspect of the method, the method further includes determining if the subject has vesicoureteral reflux (VUR) from the ultrasound images.

[0071] In another aspect of the method, the polymer is a poly(amine) material or a poly(amine) material that is functionalized with PEG groups.

[0072] In another aspect of the method, the polymer is a polyethylenimine as set forth above.

[0073] In another aspect of the method, the sterilized polymer has a weight average molecular weight from about 86 D to 40,000 D. In a refinement, the sterilized polymer has a weight average molecular weight from about 10,000 D to 30,000 D. In some refinements, the sterilized polymer has a weight average molecular weight greater than or equal to 86 D, 100 D, 500 D. 1000 D, 5000 D, or 10,000 D. In some further refinements, the sterilized polymer has a weight average molecular weight less than or equal to 50,000 D, 45,000 D, 40,000 D, 35,000 D, 30.000 D, 25,000 D, 20,000 D, or 15000 D.

[0074] In another aspect of the method, the method further includes using (i.e., applying) CO2 effervescence to diagnose or surveil VUR.

[0075] In another aspect of the method, caged CO2 and ultrasound activation is applied as an alternative to a voiding cystourethrogram (VCUG), or any other methodology that involves bladder catheterization.

[0076] In another aspect of the method, the method further includes determining bladder pressure from the volume of a CO2 bubble in a subject's bladder or kidney.

[0077] In another aspect, a controlled volume of CO2 is placed in a subject's bladder or kidney to generate a gas bubble via release of caged CO2.

[0078] As an initial application of the technology set forth herein, the development of a catheter-free alternative to the standard VCUG highlights the challenges and capabilities for the innovation. Catheterization can be avoided if an imaging contrast agent were placed in the bladder through natural excretion. To achieve this, the contrast agent is injected into the body intravenously and must be small enough to pass through the kidneys. Adopting the concept of gas bubbles as ultrasound contrast agents, currently done using fluorinated materials, carbon dioxide is an excellent contrast modality once delivered to the bladder without having “active” CO2 released. CO2 is captured in an amine-rich polymer nanoparticle so that it is not detectable until it is released in the bladder, after delivery by IV, via renal excretion. Clinical experience suggests that IV administration is far better tolerated than catheterization, particularly in children. It is paramount that the contrast material not be detectable during its normal transit from the kidneys to the bladder, otherwise visualization of normal transiting contrast within the kidneys can be confused with the presence of vesicoureteral reflux (VUR).

[0079] Emphasis is placed on vesicoureteral reflux due to its commonality, with a pediatric prevalence of up to 30% and an incidence of 30% to 50% in those with urinary tract infections. When reflux coexists with urinary tract infection and intrarenal reflux, the risk of renal scarring increases significantly, potentially leading to end-stage renal disease. This technology can significantly impact clinical outcomes. Beyond catheter-free VCUG, the technology aims to surveil children and adults with neurogenic bladders. The ability to deliver CO2 gas to the bladder, where it can be released and coalesce into bubbles, could allow for bladder pressure measurements using the ideal gas law. This could enable catheter-free bladder pressure assays for urodynamic studies and catheter-free pressureflow studies for managing lower urinary tract symptoms, which have an estimated prevalence of 59.6% among US adults over 40 years of age.

[0080] The following examples illustrate the various embodiments of the present invention. Those skilled in the art will recognize many variations that are within the spirit of the present invention and scope of the claims.

[0081] Exmples 1. Selective release of CO2-loaded nanoparticles from a rubber balloon for vesicoureteral reflux imaging

[0082] Methods: Polyethylenimine (PEI, 500-940 mg) was dissolved in solvent (deionized water, ethylene glycol, or PBS; 10 mL), followed by addition of dry ice (50 g) as a CO2 source in a sealed Parr apparatus. Dry ice released CO2 and the reaction was stirred for 18 hours until it reached ambient temperature. A rubber balloon (simulating a bladder) was filled with either water only or the nanoparticle solution (Figure 3). We used a prototype 1.1 MHz spherically focused, air-backed transducer (focal depth: 55 mm) or a prototype unfocused 1.66 MHz air-backed transducer, a computer-controlled RF generator (JJ&A Instruments) to provide bubble sonication, and a Butterfly iQ imaging system to simultaneously visualize the bubbles produced. We used an electrical power of 10 W with 10% duty cycle (1 ms on, 9 ms off) for a period of 5 seconds. Following this, a 37 mm layer of pork belly was interposed between the ultrasound transducers, and the experiment was repeated.

[0083] Results: Figures 4 and 5 show the nanoparticle and water solutions upon stimulation with ultrasound, respectively in the balloon-only model. This work demonstrates the potential to produce and visualize microbubbles when applied to CCh-loaded nanoparticles. Additionally, visualization was successful with a 37 mm thick layer of pork belly interposed between the ultrasound and balloon (Figure 6).

[0084] Conclusions: The ability to selectively release covalently bound CO2 nanoparticles and utilize CO2 bubbles as an ultrasound contrast agent has been demonstrated. This represents a major technical hurdle overcome in the development of a catheter-free, radiation-free VCUG. The next steps involve refining the nanoparticles, optimizing ultrasound activation parameters, and testing this modality in an animal model to detect VUR.

[0085] Example 2. Selective release of CCh-loaded nanoparticles from a pig bladder

[0086] A young adult female swine was chosen due to its similar bladder size to humans and to ensure urinary catheter access. The pig was anesthetized and placed in the supine position for the duration of the procedure. A catheter was inserted through the urethra into the bladder, which was subsequently drained of any existing urine. For the procedure, CCh-loaded PEI solutions, which performed the best ex vivo, were fabricated, with molecular weights of either 1.8 kDa, 10 kDa, or 25 kDa, and NaaPO j buffer levels at either 0 or 7.5%. In increments of approximately 60 mL, the bladder was filled via the catheter with one of the CCh-loaded PEI solutions until full (at or above 300 mL). The stimulating transducer (1.1 MHz, spherically focused) was placed on the midline suprapubic region and coupled to the skin with ultrasound gel. For all trials, a duty cycle of 10% (1 ms on, 9 ms off) and a pulse count of 500 (for a total sonication time of 5 seconds) were used. Power levels varied between 10W and 3OW. During the active sonication period, the transducer was held with light pressure over the bladder. After sonication ceased, the imaging probe was quickly placed in the same position to capture the resulting bubbling. Between solutions, the bladder was emptied of the previous volume.

[0087] For all solutions, 10 W was not sufficient to release CO2, and 20 W only released negligible amounts. 30 W was sufficient to release significant bubble quantities in a majority of the tested solutions. Repeat stimulation was also possible- bubbles could be seen even after a solution was stimulated multiple times at 30 W. All molecular weights and buffer levels were successful in the release and visualization of bubbles, however, bubble persistence was most favorable in 10 kDa 0% NaaPCri. 10 kDa 7.5% NasPCfi, and 25 kDa 0% NasPCfi. 1.8k Da PEI produced many bubbles, but they did not linger as much as the aforementioned solutions. Conversely, 25 kDa PEI with 7.5% buffer had copious amounts of bubbles present after insertion but before sonication; their persistence made it difficult to determine if the sonication had succeeded. Notably, the stimulation did not produce any visible lesions nor cause any heating of the skin.

[0088] While exemplary embodiments are described above, it is not intended that these embodiments describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limitation, and it is understood that various changes may be made without departing from the spirit and scope of the invention. Additionally, the features of various implementing embodiments may be combined to form further embodiments of the invention.

Claims

WHAT IS CLAIMED IS:

1. A contrast-enhanced ultrasound agent comprising: a sterilized liquid carrier; and a sterilized polymer dispersed and/or dissolved in the sterilized liquid carrier, wherein the sterilized polymer includes amino groups at least partially functionalized with CO2 at a plurality of the amino groups.

2. The contrast-enhanced ultrasound agent of claim 1 , wherein a nanoparticle includes the sterilized polymer.

3. The contrast-enhanced ultrasound agent of claim 1 , wherein the sterilized polymer is a poly(amine) material or a poly(amine) material that is functionalized with PEG groups.

4. The contrast-enhanced ultrasound agent of claim 1 , wherein the sterilized polymer is a poly ethy lenimine .

5. The contrast-enhanced ultrasound agent of claim 1 , wherein the sterilized polymer is a linear polyethylenimine.

6. The contrast-enhanced ultrasound agent of claim 5, wherein the sterilized polymer before carboxylation has the following formula

n is an integer from about 40 to 1000.

7. The contrast-enhanced ultrasound agent of claim 5, wherein the sterilized polymer includes monomer units having following formula

thereof, where dashed line represent bonds to a polymer backbone.

8. The contrast-enhanced ultrasound agent of claim 5, wherein the sterilized polymer has a weight average molecular weight from about 86 D to 40,000 D.

9. The contrast-enhanced ultrasound agent of claim 5, wherein the sterilized polymer has a weight average molecular weight from about 10,000 D to 30,000 D.

10. The contrast-enhanced ultrasound agent of claim 1 , wherein the sterilized polymer is a branched polyethylenimine.

11. The contrast-enhanced ultrasound agent of claim 1, wherein the sterilized polymer before carboxylation includes a monomer unit having the following formula:

12. A contrast-enhanced ultrasound agent comprising: a sterilized liquid carrier; and a sterilized nanoparticle composed of a polyethylenimine dispersed and/or dissolved in the sterilized liquid carrier, the polyethylenimine includes amino groups at least partially functionalized with CO2 at a plurality of the amino groups.

13. The contrast-enhanced ultrasound agent of claim 12, wherein the polyethylenimine is functionalized with PEG groups.

14. The contrast-enhanced ultrasound agent of claim 12, wherein the polyethylenimine is a linear polyethylenimine.

15. The contrast-enhanced ultrasound agent of claim 12, wherein the polyethylenimine before carboxylation has the following formula

n is an integer from about 40 to 1000.

16. The contrast-enhanced ultrasound agent of claim 12, wherein the polyethylenimine includes monomer units having following formula

thereof, where dashed line represent bonds to a polymer backbone.

17. The contrast-enhanced ultrasound agent of claim 12, wherein the polyethylenimine has a weight average molecular weight from about 86 D to 40,000 D.

18. The contrast-enhanced ultrasound agent of claim 12, wherein the polyethylenimine has a weight average molecular weight from about 10,000 D to 30,000 D.

19. The contrast-enhanced ultrasound agent of claim 12, wherein the polyethylenimine is a branched polyethylenimine.

20. The contrast-enhanced ultrasound agent of claim 12, wherein the polyethylenimine includes a monomer unit having the following formula:

21. A method for delivering releasable, caged CO2 in vivo comprising: administering contrast-enhanced ultrasound agent to a subject, the contrast-enhanced ultrasound agent comprising a sterilized liquid carrier and a sterilized polymer dispersed and/or dissolved in the sterilized liquid carrier, the sterilized polymer including amino groups at least partially functionalized with CO2 at a plurality of the amino groups. a polymer that includes amino groups to a subject, the polymer including amino groups functionalized with CO2 at a plurality of the amino groups; and directing ultrasonic energy into a part of a subject’s body to release CO2 from the contrast- enhanced ultrasound agent.

22. The method of claim 21 further comprising collecting diagnostic ultrasound images from the subject.

23. The method of claim 22 further comprising determining if the subject has vesicoureteral reflux (VUR) from the ultrasound images.

24. The method of claim 21 , wherein the part of a subject’s body is a subject’s urinary tract.

25. The method of claim 21, wherein the polymer is a poly(amine) material or a poly(amine) material that is functionalized with PEG groups.

26. The method of claim 21, wherein the polymer is a polyethylenimine.

27. The method of claim 26, wherein the sterilized polymer has a weight average molecular weight from about 86 D to 40,000 D.

28. The method of claim 26, wherein the sterilized polymer has a weight average molecular weight from about 10,000 D to 30,000 D.

29. The method of claim 21 further comprising using CO2 effervescence to diagnose or surveil VUR.

30. The method of claim 21 wherein caged CO2 and ultrasound activation are used as an alternative to a voiding cystourethrogram (VCUG) or any other methodology that involves bladder catheterization.

31. The method of claim 21 further comprising determining bladder pressure from the volume of a CO2 bubble in a subject’s bladder or kidney.

32. The method of claim 21, wherein a controlled volume of CO2 is placed in a subject’s bladder or kidney to generate a gas bubble via release of caged CO2.