Patent Application
20240423904
The present invention generally relates to systems and methods for controlling chemical reactions using ultrasound.
Ultrasound is sound with frequencies greater than 20 kilohertz (kHz). This frequency is the approximate upper audible limit of human hearing. Ultrasonic devices operate with frequencies from 20 kHz up to several gigahertz. Ultrasound can be used in many different fields. Ultrasonic devices are used to detect objects and measure distances. Ultrasound imaging or sonography is often used in medicine. In the nondestructive testing of products and structures, ultrasound is used to detect invisible flaws. Industrially, ultrasound is used for cleaning, mixing, and accelerating chemical processes. Animals such as bats and porpoises use ultrasound for locating prey and obstacles.
Focused ultrasound uses an acoustic lens to concentrate multiple intersecting beams of ultrasound on a target deep in the body with high precision and accuracy. Depending on the design of the lens and the ultrasound parameters, the target can have dimensions in the millimeter ranges and centimeter ranges.
Many embodiments are directed to systems and methods for activating mechanochemical reactions using biocompatible ultrasound. In some embodiments, the biocompatible ultrasound can control mechanochemical reactions under physiological conditions remotely with spatial and temporal precision. Several embodiments implement synergistic systems that can cause the collapse of gas-filled structures such as (but not limited to) gas vesicles and/or microbubbles using biocompatible ultrasound. In turn, collapse of the gas-filled structures can mechanically activate the chemical reaction of mechanophore-functionalized polymers in solution. The gas-filled structures function as seeds for gas-filled structures formation and cavitation upon treatment with biocompatible ultrasound, which is effectively coupled to the mechanochemical activation of mechanophore-functionalized polymers.
In many embodiments, mechanophores can be any stress-sensitive molecules that exhibit selective reactivity under mechanical forces. Several embodiments can activate various types of mechanophores using ultrasound via gas-filled structures. Ultrasound in accordance with some embodiments applies mechanical forces to activate chemical transformations of mechanophores to produce a wide range of functional responses. Examples of various types of mechanophores include (but are not limited to) mechanophores with covalently attached cargo molecules and/or payloads, mechanochromic mechanophores, derivatives of cyclobutane, benzocyclobutene, dihalocyclopropranes, and/or Diels-Alder adducts. In some embodiments, ultrasound can activate mechanochemical reactions by triggering the release of cargo molecules attached to the mechanophores. The cargo molecules can have various functions and can be released by ultrasound with spatial and temporal control. In some embodiments, ultrasound can activate mechanochromic mechanophores (also known as color-changing mechanophores). The mechanochromic mechanophores can change colors or luminescence when a mechanical force is applied to the mechanophores such that it enables applications such as (but not limited to) force sensing. Examples of mechanochromic mechanophores include (but are not limited to) spiropyran, derivatives of spiropyran, naphthopyran, benzopyran, rhodamine, oxazine, triarylmethane, and/or diarylbibenzofuranone. In some embodiments, ultrasound can activate mechanophores such as (but not limited to) ladderenes to change and/or modulate their electrical conductivity. In some embodiments, ultrasound can activate mechanophores such as (but not limited to) benzocyclobutene, beta-lactams to reveal reactive groups. As can be readily appreciated, any of a variety of mechanophores can be activated via ultrasound as appropriate to the requirements of specific applications in accordance with various embodiments.
In many embodiments, the mechanochemical activation of mechanophore-containing polymers under physiological conditions can trigger the release and/or deliver a variety of cargos. The cargo molecules can include (but are not limited to) small molecules, proteins, polypeptides, nucleic acids, pharmaceutical molecules, catalysts, fluorescent molecules, luminescent probes, molecules that can change color upon release, and any combinations thereof. The remote-controlled and targeted release of desired molecules under biocompatible conditions in accordance with many embodiments enables diverse biological and biomedical applications such as (but not limited to) diagnostics, detection, therapeutics, imaging, and/or pharmaceuticals. The applications can be in vitro and/or in vivo for human and non-human subjects.
Some embodiments include a method for activating a mechanochemical reaction comprising: applying ultrasound to a solution at least containing a concentration of at least one polymer and a plurality of gas-filled structures; wherein each of the at least one polymer comprises a polymer chain functionalized with at least one mechanophore; and wherein the ultrasound is applied such that the plurality of gas-filled structures collapses thereby transducing the ultrasound to a mechanical force, such that the mechanical force activates the mechanochemical reaction to produce a functional response from the at least one mechanophore.
In some embodiments, the at least one mechanophore is selected from the group consisting of: a mechanochromic mechanophore, a mechanophore with a cargo molecule, a mechanophore that changes electrical conductivity, and a mechanophore that reveals at least one reactive group.
In some embodiments, the function response from the at least one mechanophore is selected from the group consisting of: changing color, changing luminescence, releasing the cargo molecule, changing electrical conductivity, and revealing the at least one reactive group.
In some embodiments, the at least one mechanophore is selected from the group consisting of: spiropyran, a derivative of spiropyran, naphthopyran, benzopyran, rhodamine, oxazine, triarylmethane, diarylbibenzofuranone, a derivative of cyclobutane, benzocyclobutene, dihalocycloproprane, a Diels-Alder adduct, a beta-lactam, ladderane, and masked 2-furylcarbinol.
In some embodiments, the at least one mechanophore is attached to the polymer chain via: a covalent bond, bioconjugation, or click chemistry.
In some embodiments, the at least one mechanophore comprises at least one cargo molecule selected from the group consisting of: a macromolecule, a small molecule, a micromolecule, an organic molecule, an inorganic molecule, an amino acid, a polypeptide, a protein, a nucleic acid, a DNA, an RNA, a monosaccharide, a polysaccharide, and any combinations thereof.
In some embodiments, the at least one mechanophore comprises at least one cargo molecule selected from the group consisting of: a drug, a chemotherapeutic drug, a catalyst, a fluorescent molecule, a fluorescent probe, a fluorophore, and a luminescent molecule.
In some embodiments, the ultrasound is a focused ultrasound.
In some embodiments, the polymer chain is poly(2-(methylsulfinyl)ethyl acrylate), the mechanophore is a masked 2-furylcarbinol mechanophore, and an anticancer drug camptothecin is covalently attached to the mechanophore.
In some embodiments, the solution is selected from the group consisting of: an aqueous solution, an organic solution, a buffer solution, an intercellular environment, an intracellular environment, an in situ environment, an in vitro environment, an in vivo environment, a physiological environment, and a clinically relevant environment.
In some embodiments, each of the plurality of gas-filled structures is selected from the group consisting of: a gas vesicle, a natural gas vesicle, a synthetic gas vesicle, a microbubble, and any combinations thereof.
In some embodiments, each of the plurality of gas-filled structures comprises a gas selected from the group consisting of: a non-reactive gas, an inert gas, air, nitrogen, carbon dioxide, helium, argon, neon, xenon, and any combinations thereof.
In some embodiments, each of the plurality of gas-filled structures has an average diameter from 50 nm to 10 microns.
In some embodiments, each of the plurality of gas-filled structures has an average length from 50 nm to 10 microns.
In some embodiments, the plurality of gas-filled structures comprises a plurality of gas vesicles, and the plurality of gas vesicles has an average diameter from 45 nm to 250 nm and an average length from 100 nm to 600 nm.
In some embodiments, the ultrasound causes a temperature increase of the solution less than or equal to 5° C.
In some embodiments, the ultrasound has an acoustic intensity Isppa (spatial-peak-pulse-average) less than or equal to 1000 W/cm2.
In some embodiments, the ultrasound has an acoustic intensity Ispta (spatial-peak-time-average) less than or equal to 50 W/cm2.
In some embodiments, the ultrasound has an acoustic intensity Isppa less than or equal to 80 W/cm2 and Ispta less than or equal to 4 W/cm2.
Some embodiments include a method for delivering one or more cargo molecules comprising: applying focused ultrasound to an aqueous environment at least containing a concentration of at least one polymer and a plurality of gas-filled structures; wherein each of the at least one polymer comprises a polymer chain functionalized with at least one mechanophore, and wherein the at least one mechanophore comprises at least one cargo molecule; and wherein the focused ultrasound is applied such that the plurality of gas-filled structures collapses thereby transducing the focused ultrasound to a mechanical force, such that the mechanical force activates the at least one mechanophore to release the at least one cargo molecule into the aqueous environment.
In some embodiments, the one or more cargo molecules is delivered with temporal and spatial control.
Some embodiments include a method for drug delivery comprising: applying focused ultrasound to an aqueous environment at least containing a concentration of at least one polymer and a plurality of gas-filled structures; wherein each of the at least one polymer comprises a polymer chain functionalized with at least one mechanophore, and wherein the at least one mechanophore comprises at least one drug molecule; and wherein the focused ultrasound is applied such that the plurality of gas-filled structures collapses thereby transducing the focused ultrasound to a mechanical force, such that the mechanical force activates the at least one mechanophore to release the at least one drug molecule into the aqueous environment.
In some embodiments, the at least one drug molecule is selected from the group consisting of: an anticancer drug, a chemotherapeutic drug, a small molecule drug, a biologic drug, a macromolecule drug, and a micromolecule drug.
Additional embodiments and features are set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the specification or may be learned by the practice of the disclosure. A further understanding of the nature and advantages of the present disclosure may be realized by reference to the remaining portions of the specification and the drawings, which forms a part of this disclosure.
The description will be more fully understood with reference to the following figures, which are presented as exemplary embodiments of the invention and should not be construed as a complete recitation of the scope of the invention. It should be noted that the patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
External control of specific chemical reactions with spatial and temporal precision can have great benefits for biomedical applications. The targeted delivery of therapeutic compounds to precise locations in the body can maximize accumulation of drugs at the sites of disease and reduce undesirable side effects to healthy tissues. Spatially triggered reactions can establish patterned engineered tissues and functional living materials. Among various ways to drive chemical transformations remotely, light is a prototypical external stimulus for achieving high spatiotemporal resolution, but its limited tissue penetration depth may hamper biological utility. (See, e.g., Y. Tao, et al., Adv. Funct. Mater. 30, 2005029 (2020); T. L. Rapp, et al., Adv. Drug Deliv. Rev. 171, 94-107 (2021); H. Kobayashi, et al., Chem. Rev. 110, 2620-2640 (2010); the disclosures of which are incorporated by reference.) Research in polymer mechanochemistry has focused on the development of force sensitive molecules termed mechanophores, including those that release covalently bound small molecules upon mechanochemical activation. (See, e.g., J. Li, C. Nagamani, et al., Acc. Chem. Res. 48, 2181-2190 (2015); B. A. Versaw, et al., J. Am. Chem. Soc. 143, 21461-21473 (2021); M. A. Ghanem, et al., Nat. Rev. Mater. 6, 84-98 (2021); the disclosures of which are incorporated by reference.) Mechanical force, which can be applied with spatial and temporal precision, is transduced to the mechanophore through covalently linked polymer chains to elicit a chemoselective response. Mechanophores have been developed to release carbon monoxide, acid, catalysts, and a variety of other payloads. (See, e.g., Y. Sun, et al., J. Am. Chem. Soc. 144, 1125-1129 (2022); S. Nijem, et al., Polym. Chem. 13, 3986-3990 (2022); C. E. Diesendruck, et al., J. Am. Chem. Soc. 134, 12446-12449 (2012); Y. Lin, et al., J. Am. Chem. Soc. 142, 99-103 (2020); H. Shen, et al., Angew. Chem. Int. Ed. 60, 13559-13563 (2021); the disclosures of which are incorporated by reference.) Previous research has shown general and modular mechanophore platforms enabling the mechanically triggered release of functionally diverse molecules that are especially promising for biomedical applications. (See, e.g., Z. Shi, et al., Chem. Sci. 12, 1668-1674 (2021); S. Huo, et al., Nat. Chem. 13, 131-139 (2021); X. Hu, et al., J. Am. Chem. Soc. 141, 15018-15023 (2019); X. Hu, et al., ACS Cent. Sci. 7, 1216-1224 (2021); T. Zeng, et al., Chem. Commun. 57, 11173-11176 (2021); U.S. patent application Ser. No. 17/469,728 to Robb et al., filed Sep. 8, 2021; the disclosures of which are incorporated by reference.)
Ultrasonication is an effective technique for achieving mechanophore activation in the laboratory. High intensity low frequency sonication (typically 20 kHz) using an immersion probe is commonly employed to exert mechanical forces on polymers in solution via acoustic cavitation, a process in which the nucleation, growth, and collapse of gas bubbles produces solvodynamic shear and results in the rapid elongation of polymer chains. (See, e.g., J. Li, et al., Acc. Chem. Res. 48, 2181-2190 (2015); P. A. May, et al., Chem. Soc. Rev 42, 7497-7506 (2013); the disclosures of which are incorporated by reference). However, the cavitation of dissolved gases under these conditions is highly destructive to tissues, making it incompatible with most biological applications. (See, e.g., B. A. Versaw, et al., J. Am. Chem. Soc. 143, 21461-21473 (2021); S. Mitragotri, et al., Nat. Rev. Drug Discov. 4, 255-260 (2005); the disclosures of which are incorporated by reference). Researches have used clinically relevant focused ultrasound (FUS) operating at higher frequencies for mechanophore activation in polymer networks and hydrogels. (See, e.g., G. Kim, et al., Proc. Natl. Acad. Sci. USA 116, 10214-10222 (2019); G. Kim, et al., Proc. Natl. Acad. Sci. USA 119, e2109791119 (2022); the disclosures of which are incorporated by reference.) Focused ultrasound is an external stimulus that can be applied with sub-millimeter spatial resolution and is highly penetrant to biological tissues. (See, e.g., W. J. Elias, et al., N. Engl. J. Med. 375, 730-739 (2016); the disclosure of which is incorporated by reference.) Nevertheless, while these earlier studies provide a promising direction, the high acoustic intensities employed can cause unsafe heating, which represents a major obstacle for biomedical applications. (See, e.g., B. Fowlkes, et al., Acoust. Today (2012) https:/doi.org/10.1121/1.4788649 (Jun. 6, 2023); the disclosure of which is incorporated by reference.) Moreover, coupling biocompatible ultrasound with the remote activation of mechanochemical reactions in solution remains an unsolved challenge. New methodologies are therefore required to realize the translational potential of polymer mechanochemistry.
Many embodiments provide systems and methods for remote control of mechanochemical reactions in aqueous environments using biocompatible focused ultrasound. Several embodiments implement gas-filled structures as acousto-mechanical transducers that can be selectively collapsed with biocompatible focused ultrasound to activate various types of mechanophores to achieve the desired functional responses. In some embodiments, the gas-filled structures can selectively trigger the release of a variety of cargo molecules from mechanophore-functionalized polymers. Some embodiments implement gas vesicles as acousto-mechanical transducers that can be triggered by biocompatible focused ultrasound to facilitate the selective release of fluorogenic and therapeutic cargo molecules from mechanophore-functionalized polymers. Many embodiments can achieve remote control of the desired mechanochemical reactions with spatial and temporal precision in biologically relevant environments.
Many embodiments activate various types of mechanophores using ultrasound and gas-filled structures. In several embodiments, mechanophores can functionalize various types of polymer chains. The mechanophores can be attached to the polymer chains via chemical bonds such as (but not limited to) covalent bonds, bonds formed via bioconjugation, bonds formed via click chemistry. Ultrasound can apply mechanical forces that are needed to activate the chemical transformations of the mechanophores to achieve the desired applications. Some embodiments use ultrasound to activate mechanochromic mechanophores such as (but not limited to) spiropyran, derivatives of spiropyran, naphthopyran, benzopyran, rhodamine, oxazine, triarylmethane, and/or diarylbibenzofuranone to initiate the color changes and/or luminescence changes of the mechanophores. Some embodiments use ultrasound to activate mechanophores that can change electrical conductivity and/or reveal reactive groups. Examples of the mechanophores include (but are not limited to) ladderenes, derivatives of cyclobutane, benzocyclobutene, beta-lactams, dihalocyclopropranes, and/or Diels-Alder adducts.
Many embodiments implement mechanophore functionalized polymers to carry the desired cargo molecules. Mechanophores are chemical moieties that are sensitive to mechanical forces. In several embodiments, a variety of cargo molecules can be attached to the mechanophores. When triggered by mechanical forces, mechanophores can react selectively and release the cargo molecules. The cargo molecules can be attached to the mechanophore via chemical bonds such as (but not limited to) covalent bonds, bonds formed via bioconjugation, bonds formed via click chemistry. As can be readily appreciated, any of a variety of chemical bonds can be utilized as appropriate to the requirements of specific applications in accordance with various embodiments of the invention. In some embodiments, the polymer chains can be functionalized with different types of mechanophores. Some embodiments use mechanophores derived from 2-furylcarbinol derivatives for mechanically triggered molecular release. As can be readily appreciated, any of a variety of mechanophores can be utilized as appropriate to the requirements of specific applications in accordance with various embodiments of the invention.
In some embodiments, various types of cargo molecules can be attached to the mechanophores. The cargo molecules can be selected in accordance with the desired applications. The triggered release of the cargo molecules in accordance with many embodiments enables diverse biomedical applications such as (but not limited to) diagnostics, detection, sensing, therapeutics, imaging, and/or pharmaceuticals. The applications can be in situ, in vitro and/or in vivo for human and non-human subjects. Examples of the cargo molecules include (but are not limited to) macromolecules, small molecules, biomolecules, organic compounds, inorganic compounds, organometallic compounds, amino acids, polypeptides, proteins, monosaccharides, polysaccharides, nucleic acids, DNAs, RNAs, and any combinations thereof. The cargo molecules can have various functions such as (but not limited to) pharmaceutical molecules, drugs, chemotherapeutic drugs, biological drugs, catalysts, fluorescent molecules, fluorescent probes, fluorogenic probes, fluorophores, luminophores, molecules that can change color upon release, and any combinations thereof. In certain embodiments, the cargo molecules can be anticancer drugs such as (but not limited to) camptothecin. As can be readily appreciated, any of a variety of cargo molecules can be utilized as appropriate to the requirements of specific applications in accordance with various embodiments of the invention.
Many embodiments provide methods of delivery for in vivo applications. Some embodiments provide administration routes that involve systemic co-injection of the gas-filled structures and functionalized polymer, relying on these synergistic components to accumulate at sufficient concentration. In some embodiments, the gas-filled structures and polymers can be colocalized through non-covalent interactions or covalent anchoring, which may further enhance the local concentration of the two components necessary to achieve mechanochemical transduction. In some embodiments, the gas-filled structures and functionalized polymer can be confined within a semipermeable implant, enabling temporal control of mechanophore activation using ultrasound.
In many embodiments, various types and sizes of polymer chains can be used. In several embodiments, the polymer chains are soluble in aqueous solutions. In some embodiments, the polymer chains functionalized with the mechanophores and the cargo molecules are soluble in aqueous solutions. In several embodiments, the polymer chains, the mechanophores, and/or the cargo molecules are biocompatible and non-toxic to the intended subjects. The polymer chains can have various chain lengths and molar mass. A longer polymer chain may enable a faster mechanophore reaction upon mechanical activation. Examples of types of polymer chains include (but are not limited to) polyacrylates including poly(2-(methylsulfinyl)ethyl acrylate) (PMSEA), poly(oligoethylene glycol acrylate)s, and poly(2-hydroxyethyl acrylate), polymethacrylates, polyacrylamides such as poly(N-isopropyl acrylamide), polyoxazolines, poly(ethylene glycol) (PEG), polypeptides, polyesters, and polysaccharides. In some embodiments, the mechanophores can be attached to a suitable position of the polymer chain such as (but not limited to) in the middle of a polymer chain, at an end of a polymer chain, or any position along a polymer chain.
Many embodiments apply a mechanical force using biocompatible ultrasound. In many embodiments, the mechanochemical activation reactions can take place at a temperature from about 5° C. to about 10° C.; or from about 10° C. to about 15° C.; or from about 15° C. to about 20° C.; from about 20° C. to about 25° C.; or from about 25° C. to about 30° C.; or from about 30° C. to about 35° C.; or from about 35° C. to about 40° C.; or from about 40° C. to about 45° C.; or from about 45° C. to about 50° C.; or greater than or equal to about 50° C. In some embodiment, the mechanical activation reactions can take place at room temperature (from about 20° C. to about 25° C.). In some embodiments, the mechanical activation reactions can take place at body temperature (from about 36° C. to about 38° C.). In some embodiments, the mechanical activation reactions can take place at an elevated temperature greater than or equal to about 25° C.
In many embodiments, the chemical reactions take place in solution. In some embodiments, the solution is an organic solution. In some embodiments, the solution is an aqueous environment. In several embodiments, the aqueous environment is the desired physiological environment for the mechanophores, the mechanophore functionalized polymers, and/or the intended subjects. In some embodiments, the aqueous environment is in water or a water-based solution. In some embodiments, the aqueous environment is in a buffer solution. In some embodiments, the aqueous environment is a cellular environment such as (but not limited to) an intercellular environment, and/or an intracellular environment. In some embodiments, the aqueous environment is an in situ environment, an in vitro environment, or an in vivo environment. In some embodiments, the aqueous environment is a physiological environment. In some embodiments, the aqueous environment is a clinically relevant environment.
In many embodiments, molecular release occurs selectively in the presence of gas-filled structures upon exposure to ultrasound. The gas-filled structures are pressure sensitive. In many embodiments, the gas-filled structures can function as acousto-mechanical transducers to effectively couple ultrasound operating under physiologically relevant conditions with the mechanochemical activation of mechanophore-containing polymers. The acousto-mechanical transducer, or acoustosensitizer, can transduce acoustic waves to mechanical energy. Mechanical energy activates the mechanophore functionalized polymers such that the mechanophores can achieve various functional responses such as (but not limited to) color changes, modulating electrical conductivity, sensing forces, revealing reactive groups, and/or releasing attached cargo molecules.
In many embodiments, gas-filled structures refer to gas-filled microstructures, gas-filled nanostructures, gas vesicles, natural gas vesicles, synthetic gas vesicles, and/or microbubbles, unless specified otherwise. Gas vesicles (GVs) are a type of genetically encodable, air-filled protein nanostructure that can be used as contrast agents for non-invasive biomedical imaging, including high-frequency diagnostic ultrasound. Microbubbles are small, gas-filled bubbles that can be used as contrast agents in medical imaging. In several embodiments, the gas-filled structures have a stable structure before being activated by the mechanical forces. In some embodiments, the gas-filled structures can have spherical shapes; or ellipsoid shapes; or cylindrical shapes; or polygon shapes; or cubical shapes; or cuboid shapes; or triangular prism shapes; or triangular pyramid shapes; or square pyramid shapes; or cone shapes; or tetrahedron shapes; or hexagon shapes; or any combinations thereof. In several embodiments, the gas-filled structures can enclose a non-reactive gas such as (but not limited to) air, nitrogen, carbon dioxide, an inert gas, helium, argon, neon, xenon, and a combination of any of the non-reactive gases. In several embodiments, the gas-filled structures can have an average diameter ranging from about 50 nm to about 10 microns; or from about 50 nm to about 100 nm; or from about 100 nm to about 200 nm; or from about 200 nm to about 300 nm; or from about 300 nm to about 400 nm; or from about 400 nm to about 500 nm; or from about 500 nm to about 600 nm; or from about 600 nm to about 700 nm; or from about 700 nm to about 800 nm; or from about 800 nm to about 900 nm; or from about 900 nm to about 1 micron; or from about 1 micron to about 5 microns; or from about 5 microns to about 10 microns. In several embodiments, the gas-filled structures can have an average length ranging from about 50 nm to about 10 microns; or from about 50 nm to about 100 nm; or from about 100 nm to about 200 nm; or from about 200 nm to about 300 nm; or from about 300 nm to about 400 nm; or from about 400 nm to about 500 nm; or from about 500 nm to about 600 nm; or from about 600 nm to about 700 nm; or from about 700 nm to about 800 nm; or from about 800 nm to about 900 nm; or from about 900 nm to about 1 micron; or from about 1 micron to about 5 microns; or from about 5 microns to about 10 microns. In some embodiments, the gas vesicles can have an average diameter from about 45 nm to about 250 nm and an average length from about 100 nm to about 600 nm. In certain embodiments, the gas vesicles can have amphiphilic protein shells that are gas-permeable but exclude liquid water from their hydrophobic interior. Gas vesicles can act as seeds for bubble formation and cavitation upon exposure to biocompatible ultrasound. Several embodiments apply biocompatible ultrasound pulses to the gas-filled structures. Under sustained ultrasound pulses with relatively low-pressure levels, large bubbles liberated from the rupture of the gas-filled structures can undergo rapid growth followed by intense collapse in an inertial cavitation process with pronounced mechanical effects. With the biocompatible ultrasound parameters in accordance with several embodiments, which are otherwise benign to tissues and/or cells, these mechanical effects take place only in the vicinity of gas-filled structures.
In many embodiments, biocompatible focused ultrasound conditions are applied to trigger the activation of mechanochemical reactions. Insonation can result in unsafe heating and tissue damage. Non-specific acoustic cavitation can cause undesirable thermal and/or mechanical effects and disrupt tissues. The biocompatible focused ultrasound conditions applied in conjunction with gas vesicles in accordance with several embodiments, are selected to minimize and/or eliminate overheating and tissue damage. In many embodiments, the biocompatible ultrasound conditions can cause an increase in the temperature of the medium of less than or equal to about 5° C. In several embodiments, an upper boundary of biocompatible ultrasound conditions can be the threshold at which acoustic cavitation can be activated selectively in the presence of gas vesicles. The upper boundary conditions in accordance with several embodiments do not include the conditions without the gas vesicles.
In some embodiments, the biocompatible ultrasound conditions can have an acoustic intensity Isppa (spatial-peak-pulse-average) less than or equal to about 1000 W/cm2. The acoustic intensity Isppa can prevent mechanical damage to tissues. In some embodiments, the biocompatible ultrasound conditions can have an acoustic intensity Ispta (spatial-peak-time-average) less than or equal to about 50 W/cm2. The acoustic intensity Ispta can effectively suppress thermal damage to tissues. In certain embodiments, Isppa is less than or equal to about 1000 W/cm2 and Ispta is less than or equal to about 50 W/cm2. In certain embodiments, Isppa is less than or equal to about 900 W/cm2 and Ispta is less than or equal to about 45 W/cm2. In certain embodiments, Isppa is less than or equal to about 800 W/cm2 and Ispta is less than or equal to about 40 W/cm2. In certain embodiments, Isppa is less than or equal to about 700 W/cm2 and Ispta is less than or equal to about 35 W/cm2. In certain embodiments, Isppa is less than or equal to about 600 W/cm2 and Ispta is less than or equal to about 30 W/cm2. In certain embodiments, Isppa is less than or equal to about 500 W/cm2 and Ispta is less than or equal to about 25 W/cm2. In certain embodiments, Isppa is less than or equal to about 400 W/cm2 and Ispta is less than or equal to about 20 W/cm2. In certain embodiments, Isppa is less than or equal to about 300 W/cm2 and Ispta is less than or equal to about 15 W/cm2. In certain embodiments, Isppa is less than or equal to about 200 W/cm2 and Ispta is less than or equal to about 10 W/cm2. In certain embodiments, Isppa is less than or equal to about 100 W/cm2 and Ispta is less than or equal to about 5 W/cm2. In certain embodiments, Isppa is less than or equal to about 90 W/cm2 and Ispta is less than or equal to about 5 W/cm2. In certain embodiments, Isppa is less than or equal to about 80 W/cm2 and Ispta is less than or equal to about 4 W/cm2.
Although specific embodiments of systems and apparatuses are discussed in the following sections, it will be understood that these embodiments are provided as exemplary and are not intended to be limiting.
Several embodiments identify focused ultrasound parameters for safe operation under physiological conditions with a particular emphasis on maintaining low-to-moderate pressure levels and avoiding temperature increases greater than about 6° C. to stay within a biomedically relevant regime. Focused ultrasound applied at high acoustic intensities can induce coagulative necrosis in tissues resulting from thermal disruption. Some embodiments use about 330 kHz focused ultrasound on either an 800 μL water sample in a sealed plastic microcentrifuge tube or a tissue-mimicking agarose gel inside a water tank and monitor peak negative pressure (PNP) and sample temperature using a hydrophone and internal thermocouple, respectively. An upper limit for PNP of 1.47 MPa can be established with a 4.5% duty cycle (about 3000 cycles per pulse with about 5 Hz pulse repetition frequency), which can result in a maximum temperature increase of about 3.6° C. in the agarose phantom (about 7.3° C. inside the plastic tube) that equilibrates in less than about 2 min. This biocompatible upper limit results in a spatial peak-temporal average acoustic intensity (Ispta) of about 3.6 W/cm2 that is within the safety limits established for the use of ultrasound in therapeutic applications. Similar ultrasound parameters have been successfully applied for therapeutic purposes to delicate tissues like the brain in both live animal studies and human clinical trials. Using similar focused ultrasound parameters as those previously employed for mechanophore activation at about 330 kHz and about 916 kHz result in an unsafe temperature increase exceeding about 27° C. above baseline due to the significantly higher acoustic intensity.
In several embodiments, mechanophore activation can be achieved under physiologically relevant conditions. In some embodiments, a masked 2-furylcarbinol mechanophore for mechanically triggered molecular release can be used.
Some embodiments use a PMSEA polymer containing a chain-centered mechanophore loaded with a fluorogenic aminocoumarin payload (PMSEA-CoumNH2, Mn=260 kg/mol; Ð=1.47). Release of the aminocoumarin small molecule results in a pronounced increase in fluorescence that is easily detected and quantified spectroscopically. Exposing a dilute solution of the polymer (2 mg/mL) in water to 330 kHz focused ultrasound under the conditions identified above in the presence of gas vesicles (GVs, 1.4 nM) results in a strong fluorogenic response indicating the successful release of aminocoumarin. Approximately 15% release can be observed after 10 min of focused ultrasound exposure. Given their relative concentrations, about 800 equivalents of PMSEA-CoumNH2 can be activated per GV within this timeframe. Extended exposure to focused ultrasound results in additional aminocoumarin release. Importantly, a fluorogenic response is not observed in the absence of GVs under the same conditions, confirming the GVs function as essential acousto-mechanical transducers, enabling mechanophore activation and selective cargo release under biocompatible conditions. Additional control experiments are performed on an analogous polymer with the mechanophore located at the chain end, which is not subjected to mechanical force. Minimal aminocoumarin release is observed under otherwise identical experimental conditions, confirming the mechanochemical origin of molecular release from PMSEA-CoumNH2 upon exposure to focused ultrasound in the presence of GVs.
Several embodiments investigate the mechanically triggered release of a small molecule therapeutic to demonstrate the capability of this system for drug delivery applications. Camptothecin can be used as a chemotherapeutic anticancer agent. Following a similar procedure as above, camptothecin can be conjugated to the mechanophore bis-initiator through a carbonate linkage followed by polymerization to afford PMSEA-CPT (Mn=319 kg/mol; Ð=1.47). Preliminary experiments performed on an isolated small molecule furfuryl carbonate model compound resembling the intermediate that is unmasked upon mechanophore activation confirm that camptothecin is successfully released in aqueous media, as evidenced by 1H NMR spectroscopy and high-performance liquid chromatography (HPLC) measurements. Importantly, PMSEA-CPT is stable in aqueous solution, with negligible release of camptothecin detected over a period of 2 months under ambient conditions.
The triggered release of camptothecin from PMSEA-CPT is then investigated using biocompatible focused ultrasound in the presence of GVs under the physiologically relevant conditions established above. Approximately 8% release of camptothecin can be achieved after 10 min exposure to focused ultrasound in the presence of GVs, as evidenced by quantitative HPLC measurements. Similar to the results above for the release of aminocoumarin, no camptothecin release can be detected upon exposure of PMSEA-CPT to focused ultrasound in the absence of GVs. To assess the feasibility of this synergistic platform for targeted chemotherapy, cytotoxicity is investigated in vitro on the viability of Raji cells, a diffuse large B-cell lymphoma model of non-Hodgkin lymphomas, using an MTT colorimetric assay. Cells are treated with samples of PMSEA-CPT before and after mechanochemical activation with focused ultrasound at various concentrations and incubated for 2 days at 37° C. Cells incubated with PMSEA-CPT that was previously exposed to focused ultrasound in the presence of GVs exhibit a significant decrease in viability with a half-maximal effective concentration (EC50) of about 250 nM. This EC50 is approximately one order of magnitude higher than cells treated with isolated camptothecin small molecule, fully consistent with the extent of cargo release after 10 min of insonation characterized above. In contrast, no significant cytotoxicity is observed across all polymer concentrations in control experiments performed with PMSEA-CPT that is not exposed to focused ultrasound, with PMSEA-CPT that is exposed to ultrasound in the absence of GVs, or with a chain-end functional control polymer (PMSEA-Control) subjected to identical focused ultrasound conditions in the presence of GVs.
Several embodiments provide a synergistic system that couples biocompatible focused ultrasound with the solution-phase activation of mechanochemical reactions under physiologically relevant conditions. Some embodiments use pressure-sensitive air-filled protein nanostructures called gas vesicles that function as acousto-mechanical transducers, effectively converting focused ultrasound energy into a coordinated mechanical stimulus for the activation of mechanophore-containing polymers. The mechanically triggered release of molecular payloads aminocoumarin and camptothecin from polymers in aqueous media illustrates the power of this approach for noninvasive bioimaging and therapeutic applications of polymer mechanochemistry. Several embodiments provide methods for remote control of specific chemical reactions under biomedically relevant conditions with the spatiotemporal precision and tissue penetration afforded by focused ultrasound.
Some embodiments provide the preparation methods of PMSEA-CoumNH2 and PMSEA-CPT. The masked 2-furylcarbinol mechanophore with an aminocoumarin payload can be synthesized according to the literature. (See, e.g., X. Hu, et al., ACS Cent. Sci. 7, 1216-1224, 2021; the disclosure of which is incorporated by reference.) A similar procedure was used to synthesize the mechanophore incorporating camptothecin as the payload. The monomer 2-methylsulfinyl ethyl acrylate (MSEA) was prepared according to the literature. (See, e.g., S. Li, et al., Biomacromolecules 18, 475-482, 2017; the disclosure of which is incorporated by reference.) Water soluble linear PMSEA polymers incorporating the mechanophores were synthesized by controlled radical polymerization from the mechanophore initiator. Polymers were purified by precipitation into diethyl ether three times and subsequently dialyzed against deionized water for at least five cycles prior to use.
Some embodiments provide general protocols for FUS experiments. GVs were produced and purified from cyanobacteria Anabaena flos-aquae. Polymer solutions and GV suspensions were prepared using MilliQ water and separately placed under vacuum at 0.5 atm overnight to remove transiently trapped air bubbles prior to treatment with FUS. The GV suspension and polymer solution were gently mixed in a 2 mL microcentrifuge tube to achieve the desired concentrations of each component without introducing observable air bubbles. The total sample volume was 800 μL. The microcentrifuge tube was stabilized and exposed to FUS on a custom stage that positions the center of the sample at the focus of the FUS transducer operating at 330 kHz (fundamental frequency) or 916 kHz (third harmonic). After FUS exposure, samples were subsequently analyzed by fluorescence spectroscopy or high performance liquid chromatography, or evaluated for cytotoxicity.
Some embodiments provide cell viability assays procedures. Raji cells were incubated in RPMI 1640 media with 10% fetal bovine serum and 1% antibacterial Penicillin/Streptomycin (PenStrep) at 37° C. and 5% CO2. An MTT colorimetric assay was used to quantify cell viability.
NMR spectra were recorded using a 400 MHz Bruker Avance III HD with Prodigy Cryoprobe. All 1H NMR spectra are reported in δ units, parts per million (ppm), and were measured relative to the signals for residual chloroform (7.26 ppm) or toluene (2.08 ppm) in deuterated solvent. All 13C NMR spectra were measured in deuterated solvents and are reported in ppm relative to the signals for chloroform (77.16 ppm). Multiplicity and qualifier abbreviations are as follows: s=singlet, d=doublet, t=triplet, q=quartet, dd=doublet of doublets, dq=doublet of quartets, ABq=AB quartet, m=multiplet, br=broad.
High resolution mass spectra (HRMS) were obtained via direct injection on an Agilent 1260 Infinity II Series HPLC coupled to a 6230 LC/TOF system in electrospray ionization (ESI+) mode.
Polymer molar mass and dispersity was determined on a gel permeation chromatography (GPC) system with an Agilent pump (G7110B), equipped with an autosampler (G7129A), a Wyatt DAWN 8 multi-angle laser light scattering detector (λ=658.9 nm), a Wyatt Optilab refractive index detector (RI) (λ=658 nm), and an Agilent PL Aquagel-OH MIXED-H column. Aqueous buffer was prepared containing 0.2 M NaNO3 with 200 ppm NaN3. Filtered aqueous buffer was used as the eluent at a flow rate of 0.3 mL/min at 25° C. The data were analyzed using Wyatt Astra 7 with dn/dc=0.153 mL/g to obtain average molar masses (Mw and Mn) for each PMSEA polymer reported.
Photoluminescence spectra were recorded on a Shimadzu RF-6000 spectrofluorophotometer using a quartz microcuvette (Starna Cells 18F-Q-10-GL14-C, 10×2 mm). Excitation and emission slit widths were 5 nm and 3 nm, respectively. A Tecan Spark microplate reader was also used to acquire photoluminescence spectra in some experiments with 96-well microplate. Excitation and emission slit widths were both 5 nm.
High-Performance Liquid Chromatography (HPLC) measurements were performed with an Agilent Eclipse Plus C18 column or a C8 column using an in-line UV-vis detector and acetonitrile/water as the eluent.
Sonication experiments with an immersion probe were performed using a 500 Watt Vibra Cell 505 liquid processor (20 kHz) equipped with a 0.5-inch diameter solid probe, sonochemical adapter, and a Suslick reaction vessel.
LCMS measurements were performed with an Agilent 6140 Series Quadrupole LCMS Spectrometer System equipped with an Agilent Eclipse Plus C18 column using an in-line UV-vis detector and acetonitrile/water as the eluent.
Compounds DA-1, DA-2, 1-CoumNH2, and 2-CoumNH2 were synthesized as reported previously (1). Internal standard 4-methyl-2-oxo-2H-chromen-7-yl acetate (IS) was prepared according to the literature (2). Monomer 2-methylsulfinyl ethyl acrylate (MSEA) was prepared according to the literature (3).
Polymer solutions were dialyzed against deionized water for at least 5 cycles to remove any residual small molecule in the reaction mixture to prevent any aminocoumarin (CoumNH2) or camptothecin (CPT) from affecting functional assays.
GVs were produced and purified from cyanobacteria Anabaena flos-aquae. GvpC was removed from the GVs by urea treatment followed by dialysis process. As a quality assurance step to confirm successful GvpC removal, pressure-dependent optical density (OD) measurements were performed for each sample at 0-7 bar to match previously reported measurements using an echoVis Vis-NIR light source coupled with an STS-VIS spectrometer and a 176.700-QS sample chamber. GV concentrations were measured as OD at 500 nm using a spectrophotometer. OD500=1 corresponds to 114 pM of GV particles (4, 5).
Both polymer solution and GV suspension were placed in a vacuum environment at 0.5 atm overnight to remove transiently trapped air bubbles before FUS treatment. GV suspension and polymer solution were gently mixed to desired concentration without introducing observable air bubbles in a 2 mL microcentrifuge tube. The total volume of sample was 800 μL. The microcentrifuge tube was stabilized on a homemade holder which positions the center of the sample to be at the focus of a focused ultrasound transducer operating at 330 kHz (fundamental frequency) or 916 kHz (third harmonic).
Synthesis of ((3aR,4R,7S,7aS)-2-(2-((2-bromo-2-methylpropanoyl)oxy)ethyl)-7-(1-(((((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)oxy)ethyl)-1,3-dioxo-6-phenoxy-1,2,3,3a,7,7a-hexahydro-4H-4,7-epoxyisoindol-4-yl)methyl 2-bromo-2-methylpropanoatebromo-2-methylpropanoate (1-CPT). A flame-dried 25 mL round bottom flask was charged with 4-dimethylaminopyridine (DMAP) (544 mg, 4.43 mmol) and camptothecin (302 mg, 0.867 mmol). The flask was evacuated and refilled with N2 three times, after which 5 mL of dichloromethane was added. Phosgene (15 wt % in toluene, 700 μL, 0.981 mmol) was added to the off-yellow suspension and the reaction was allowed to stir at room temperature. After 1 h the reaction had become homogenous and dark red, and DA-1 was added as a solution in 5 mL dichloromethane. The reaction continued to stir for 20 h, after which it was quenched with sat. NH4Cl (8 mL). The products were extracted into dichloromethane (3×5 mL) and the combined organic layers were washed with brine (5 mL), dried with sodium sulfate, and filtered. The crude mixture was purified via silica gel chromatography (0-10% methanol/dichloromethane), followed by separation of the diastereomers by reverse-phase HPLC (70% acetonitrile/water). The title product (single diastereomer) was isolated as a light yellow solid (84 mg, 27%).
TLC (5% methanol/dichloromethane): Rf=0.55
1H NMR (400 MHz, CDCl3) δ: 8.38 (s, 1H), 8.07 (d, J=8.5 Hz, 1H), 7.91 (d, J=8.1 Hz, 1H), 7.84-7.76 (m, 1H), 7.68-7.59 (m, 1H), 7.36 (s, 1H), 7.25-7.18 (m, 2H), 7.13-7.05 (m, 1H), 6.90-6.81 (m, 2H), 5.69 (d, J=17.3 Hz, 1H), 5.49 (q, J=6.5 Hz, 1H), 5.39 (d, J=17.2 Hz, 1H), 5.26 (s, 2H), 4.98 (s, 1H), 4.66 (Abq, ΔVAB=104 Hz, JAB=12.5 Hz, 2H), 3.91-3.79 (m, 2H), 3.76-3.72 (m, 2H), 3.25-3.14 (m, 1H), 3.05-2.94 (m, 1H), 2.33 (dq, J=14.9, 7.4 Hz, 1H), 2.18 (dq, J=14.7 Hz, 7.5 Hz, 1H), 1.94 (s, 3H), 1.91 (s, 3H), 1.70 (s, 3H), 1.68 (s, 3H), 1.53 (d, J=6.6 Hz, 3H), 1.01 (t, J=7.4 Hz, 3H).
13C{1H} NMR (101 MHz, CDCl3) δ: 173.5, 172.6, 171.2, 171.1, 167.3, 162.2, 157.3, 154.4, 152.9, 152.4, 148.6, 146.5, 145.5, 131.4, 131.0, 130.0, 129.3, 128.7, 128.4, 128.23, 128.19, 125.9, 120.6, 119.4, 101.2, 95.8, 90.2, 88.3, 78.2, 71.8, 67.3, 63.6, 62.2, 55.6, 55.5, 51.4, 50.1, 47.8, 36.9, 32.0, 30.75, 30.71, 30.5, 30.4, 15.7, 7.7.
HRMS (ESI, m/z): calcd for [C48H46Br2N3O14+] (M+H)+1046.1341, found 1046.1355.
Synthesis of ((3aR,4R,7S,7aS)-2-(2-((2-bromo-2-methylpropanoyl)oxy)ethyl)-7-(1-(((((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)oxy)ethyl)-1,3-dioxo-6-phenoxy-1,2,3,3a,7,7a-hexahydro-4H-4,7-epoxyisoindol-4-yl)methyl pivalate (2-CPT). A flame-dried 25 mL round bottom flask was charged with DMAP (336 mg, 2.75 mmol) and camptothecin (192 mg, 0.552 mmol). The flask was evacuated and refilled with N2 three times, after which 5 mL of dichloromethane was added. Phosgene (15 wt % in toluene, 430 μL, 0.602 mmol) was added to the off-yellow suspension and the reaction was allowed to stir at room temperature. After 1 h the reaction had become homogenous and dark red, and DA-2 was added as a solution in 5 mL of dichloromethane. The reaction continued to stir for 18 h, after which it was quenched with sat. NH4Cl (8 mL). The products were extracted into dichloromethane (3×5 mL) and the combined organic layers were washed with brine (5 mL), dried with Na2SO4, and filtered. The crude mixture was purified via silica gel chromatography (0-10% methanol/dichloromethane), followed by separation of the diastereomers by reverse-phase HPLC (70% acetonitrile/water). The title product (single diastereomer) was isolated as a light yellow solid (50 mg, 28%).
TLC (5% methanol/dichloromethane): Rf=0.57
1H NMR (400 MHz, CDCl3) δ: 8.39 (s, 1H), 8.07 (d, J=8.5 Hz, 1H), 7.92 (dd, J=8.3, 1.3 Hz, 1H), 7.81 (ddd, J=8.4, 6.9, 1.2 Hz, 1H), 7.69-7.62 (m, 1H), 7.35 (s, 1H), 7.25-7.19 (m, 2H), 7.13-7.07 (m, 1H), 6.85 (dd, J=7.6, 1.6 Hz, 2H), 5.70 (d, J=17.2 Hz, 1H), 5.50 (q, J=6.5 Hz, 1H), 5.41 (d, J=17.3 Hz, 1H), 5.28 (s, 2H), 4.93 (s, 1H), 4.56 (Abq, ΔVAB=120 Hz, JAB=12.7 Hz, 2H), 3.90-3.79 (m, 2H), 3.73-3.63 (m, 2H), 3.26-3.13 (m, 1H), 3.04-2.92 (m, 1H), 2.35 (dq, J=14.9, 7.4 Hz, 1H), 2.19 (dq, J=14.8, 7.5 Hz), 1.71 (s, 3H), 1.69 (s, 3H), 1.54 (d, J=6.6 Hz, 3H), 1.21 (s, 9H), 1.01 (t, J=7.5 Hz, 3H).
13C{1H} NMR (101 MHz, CDCl3) δ: 177.8, 173.5, 172.6, 171.2, 167.3, 162.1, 157.3, 154.5, 153.0, 152.5, 148.8, 146.6, 145.4, 131.4, 131.0, 130.1, 129.4, 128.7, 128.4, 128.24, 128.22, 125.9, 120.8, 119.4, 101.3, 95.7, 90.2, 88.6, 78.2, 71.8, 67.4, 62.3, 62.2, 55.5, 51.4, 50.1, 47.9, 39.0, 36.9, 32.1, 30.53, 30.46, 27.2, 15.7, 7.8.
HRMS (ESI, m/z): calcd for [C49H49BrN3O14+] (M+H)+982.2392, found 982.2408.
Representative procedure for the synthesis of poly(2-(methylsulfinyl)ethyl acrylate) (PMSEA) polymers. PMSEA polymers were synthesized by controlled radical polymerization following an adaptation of the procedure by Nguyen et aL. (7). A flame-dried Schlenk flask was charged with freshly cut 20 G copper wire (2 cm), initiator 1-CoumNH2 (3.5 mg, 0.0040 mmol), dimethyl sulfoxide (DMSO) (5 mL), and 2-methylsulfinyl ethyl acrylate (2.46 g, 15.2 mmol). The flask was sealed and the solution was degassed via three freeze-pump-thaw cycles, then backfilled with nitrogen and warmed to room temperature. Me6TREN (5.0 μL, 0.019 mmol) was added via microsyringe and the reaction was stirred at room temperature. Upon completion of the polymerization after 6 h, the flask was opened to atmosphere and diluted with a minimal amount of methanol. The polymer was precipitated 3× into room temperature diethyl ether, dried under vacuum, dialyzed in water (10 mL in 4 L, 5×) and finally lyophilized to afford PMSEA-CoumNH2 as a foamy white solid (822 mg, 33%). Mn=260 kg/mol, Ð=1.47.
An oven-dried sonication vessel was fitted with rubber septa, placed onto the sonication probe, and allowed to cool under a stream of dry nitrogen. The vessel was charged with a solution of the polymer in either MilliQ water or 3:1 acetonitrile/water (2 mg/mL, 20 mL total volume) and submerged in an ice bath. The solution was sparged with nitrogen for 1 h prior to sonication, and a nitrogen headspace was maintained throughout the experiment. Pulsed ultrasound (1 s on/1 s off, 20% amplitude, 20 kHz, 9.04 W/cm2) was then applied to the system. “Sonication time” is defined as the duration of exposure to sonication, which is 50% of experimental or “clock” time under 1 s on/1 s off pulsation. The solution temperature during sonication was measured to be ˜10° C. by an internal temperature probe. Aliquots taken from the sonicated solution were filtered through a 0.45 μm PTFE syringe filter prior to analysis.
FUS setup. A 2-mL microcentrifuge tube was placed on a 3D-printed sample stage, and the center position of the tube was determined using an L22-14vX 128-element Verasonics imaging probe. The tube was removed and exchanged with a needle hydrophone (Onda, HNR-0500) situated in the exact position as the previously registered center of the tube. A 330 kHz focused ultrasound transducer (Precision Acoustics, H115) was mounted on a computer-controlled 3D translatable stage (Velmex) orthogonally to the Verasonics probe. A previously reported MATLAB program was used to automatically scan and align the transducer's focus at the center of the microcentrifuge tube, according to the feedback from the needle hydrophone, which reports the acoustic pressure (5). To calibrate pressure, the needle hydrophone was placed inside and at the center of a plastic microcentrifuge tube to account for the attenuation of ultrasound wave through the plastic tube.
Spatial peak-temporal average acoustic intensity (Ispta) is calculated with eq.
where p is the peak negative pressure (PNP) of ultrasound, ρ is the density of aqueous media, c is the speed of sound in aqueous media, DC is the duty cycle applied in ultrasound treatment.
Temperature calibration. Temperature change inside the microcentrifuge tube was monitored by a temperature probe, which is inserted inside the tube. The volume of sample inside the microcentrifuge tube was maintained at 800 μL in all experiments. Once 330 or 916 kHz FUS was turned on, the temperature inside the tube was recorded until it reached equilibrium. The measured temperature remained relatively constant when the temperature probe was moved within the tube while the ultrasound remained on. Different ultrasound conditions, including pressure, duty cycle, cycle number in each emitting pulse were systematically investigated to find the upper limit of ultrasound conditions for maintaining biocompatible temperatures. Temperature increment in different sample container (plastic microcentrifuge tubes vs tissue-mimicking 1% agarose phantom) was also investigated with the same ultrasound conditions. As demonstrated with the biocompatible FUS conditions, the temperature increase is higher for the sample in the microcentrifuge tube because the plastic walls absorb more ultrasound than agarose gel or water, resulting in increased heat dissipation at the interface.
Aliquots from either the sonication (immersion probe) or FUS experiments were filtered through 0.45 μm syringe filters and added to either a quartz microcuvette or a 96-well microplate for photoluminescence measurements. The samples were then analyzed by fluorescence spectroscopy on either a Shimadzu fluorimeter or a Tecan Spark microplate reader at room temperature. Emission spectra were recorded using an excitation wavelength of 365 nm. The concentration of released aminocoumarin (CoumNH2) was determined from calibration curves constructed on both the Shimadzu fluorimeter and the Tecan Spark microplate reader.
Aminocoumarin Release with Immersion Probe Sonication. As expected, efficiency of CoumNH2 release from PMSEA-CoumNH2 (2 mg/mL in water) increases as a function of exposure time to sonication. Maximum release of CoumNH2 was observed after 120 min sonication in water (44%). In contrast to the significant release triggered from PMSEA-CoumNH2 containing a chain-centered mechanophore, sonication of chain-end functional polymer Control-CoumNH2 (2 mg/mL in water) does not result in significant release of CoumNH2, supporting that release occurs via a mechanochemical process.
Aminocoumarin Release with Biocompatible FUS. While typical biocompatible FUS experiments were performed for only 10 min, CoumNH2 release efficiency was observed to increase with additional insonation of PMSEA-CoumNH2 (2 mg/mL in water, 1.4 nM GVs). In contrast, exposure of chain-end functional control polymer Control-CoumNH2 (2 mg/mL in water) to biocompatible FUS resulted in insignificant release of CoumNH2. Likewise, no significant release of CoumNH2 was observed from either PMSEA-CoumNH2 or Control-CoumNH2 over 17 h upon heating to 50° C., further supporting that release under FUS is mechanochemically triggered. Insignificant release of CoumNH2 was also observed from PMSEA-CoumNH2 when peak negative pressure (PNP) was <1.3 MPa, despite collapse of GVs occurring under these conditions. The extent of CoumNH2 release was also dependent on the concentration of GVs, whereby a higher concentration of GVs resulted in greater percent release of CoumNH2, which can be attributed to an increased number of cavitation events and consequently a greater extent of mechanochemical activation. Furthermore, insonation of PMSEA-CoumNH2 in the presence of pre-collapsed GVs (1.4 nM), wherein gas content inside the nanostructures and surrounding fluid is substantially depleted, resulted in insignificant CoumNH2 release, demonstrating that the gas content within intact GVs is crucial for efficient mechanochemical activation. Finally, the extent of CoumNH2 release varied insignificantly with polymer concentration between 2 and 8 mg/mL.
Several embodiments provide confirmation of CPT release via 1H NMR spectroscopy and LCMS. Prior to mechanochemical activation studies, we first confirmed that camptothecin was released in polar protic solvent upon unmasking of the expected unstable furfuryl carbonate. To enable characterization, the furfuryl carbonate species was generated thermally from small molecule initiator 1-CPT. Masked furfuryl carbonate 1-CPT was dissolved in toluene-d8 and analyzed by 1H NMR spectroscopy before and after heating for 24 h at 100° C. Comparison to spectrum of the expected maleimide fragment confirmed significant thermal retro-Diels-Alder reaction occurred under these conditions. A drop of this solution was then diluted into 3:1 acetonitrile/methanol and analyzed by LCMS (gradient of 0-50% acetonitrile/water over 4 minutes). Comparison of the newly generated peak to a standard sample of pure camptothecin (CPT) reveals identical retention times, absorption spectra, and mass-to-charge ratios, supporting that CPT is released from the unmasked furfuryl carbonate upon exposure to polar protic solvent.
Calculation of HPLC Relative Response Factors (RRF). Three standard solutions with known concentrations of 4-methyl-2-oxo-2H-chromen-7-yl acetate internal standard (IS) and CPT were prepared and analyzed by HPLC equipped with a UV detector. The RRF is calculated from the HPLC results (C8 column, isocratic 30% acetonitrile/water) of the standard solution using eq. S2:
RRF at 254 nm was determined to be 6.71±0.23 for CPT:IS.
Determination of the concentration of released CPT from polymers after FUS exposure. A solution of IS of known concentration was added to analyte solutions in a volumetric ratio of 1:3 (IS:analyte solution). The solution was then kept at room temperature and analyzed by HPLC (50 μL injection, C8 column, isocratic 30% acetonitrile/water) at various time intervals. The total concentration of released CPT was calculated using the relationship in eq. S3:
No release of CPT is observed from aqueous solutions of PMSEA-CPT stored under ambient conditions for 2 months. Applying FUS (330 kHz, 3.6 W/cm2, 1.47 MPa PNP, 4.5% duty cycle, 10 min) to PMSEA-CPT (4 mg/mL in water) triggers release of CPT (7.732±0.006%, average of three trials) in the presence of GVs (1.4 nM). No release is observed with FUS in the absence of GVs. Release of CPT is also not observed upon insonation of chain-end functional polymer Control-CPT in the presence of GVs (1.4 nM), supporting that mechanical force is responsible for activation. Finally, free CPT was confirmed to be stable upon exposure to FUS (10 min) in either the presence or absence of GVs (1.4 nM).
Several embodiments use MTT-based cell viability assay. Raji cells were used to evaluate the effectiveness of CPT release for potential chemotherapy. Raji cells were incubated in RPMI 1640 media, with 10% fetal bovine serum and 1% antibacterial Penicillin/Streptomycin (PenStrep) at 37° C. and 5% CO2. The instructions provided by the manufacturer (Abcam) were followed to perform the MTT assay. Approximately 10,000 Raji cells suspended in 150 μL of media were seeded into each well of a 96-well plate. Subsequently, 50 μL of polymer sample (or pure CPT) at varying concentrations was added to corresponding wells. For calibration purposes, wells without cells and wells with only cells and media were also prepared, representing 0% and 100% cell viability, respectively. After 48 hours of incubation, cells were spun down inside the plate and media were carefully aspirated. Then 50 μL of serum free media and 50 μL of MTT solution were added to each well. Following a 3 h incubation, 150 μL MTT solution was added to each well. The plate was thoroughly mixed and shaken on an orbital shaker at 37° C. for 15 min. The absorbance at 590 nm was measured by a microplate reader (TECAN Spark) to quantify the cell viability in each well. Cell viability is calculated using eq. S4:
where A, Amedia only, and Acell only represent absorbance of the sample, control with media only, and control with only cell and media but no analyte.
As can be inferred from the above discussion, the above-mentioned concepts can be implemented in a variety of arrangements in accordance with embodiments of the invention. Accordingly, although the present invention has been described in certain specific aspects, many additional modifications and variations would be apparent to those skilled in the art. It is therefore to be understood that the present invention may be practiced otherwise than specifically described. Thus, embodiments of the present invention should be considered in all respects as illustrative and not restrictive.
As used herein, the singular terms “a,” “an,” and “the,” may include plural referents unless the context clearly dictates otherwise. Reference to an object in the singular is not intended to mean “one and only one” unless explicitly so stated, but rather “one or more.”
As used herein, the terms “approximately,” and “about” are used to describe and account for small variations. When used in conjunction with an event or circumstance, the terms can refer to instances in which the event or circumstance occurs precisely as well as instances in which the event or circumstance occurs to a close approximation. When used in conjunction with a numerical value, the terms can refer to a range of variation of less than or equal to ±10% of that numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to +0.1%, or less than or equal to ±0.05%.
Additionally, amounts, ratios, and other numerical values may sometimes be presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.
The current application claims the benefit of and priority under 35 U.S.C. § 119 (e) to U.S. Provisional Patent Application No. 63/472,767 entitled “Method For Controlling Chemical Reactions Using Ultrasound” filed Jun. 13, 2023. The disclosure of U.S. Provisional Patent Application No. 63/472,767 is hereby incorporated by reference in its entirety for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 63472767 | Jun 2023 | US |
