Compositions and methods for detecting and treating thrombosis and vascular plaques

Abstract

The present invention provides nanodroplets labeled with targeting ligands that can be used to detect and treat vascular thrombosis (e.g., fibrin clots) and vascular plaques, or related diseases and disorders, as well as methods of making and using the same.

Description

[0001] Priority and related patent applications This application claims priority to U.S. Provisional Application No. 62 / 857,766, filed June 5, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to a pharmaceutical composition and its preparation method, as well as its diagnostic or therapeutic uses. More specifically, this invention relates to targeted microbubbles and / or nanodroplets labeled with diagnostic and / or therapeutic ligands and their emulsions, which can be used to detect and disrupt vascular thrombosis (e.g., fibrin clots) and vascular plaques, and methods for their preparation and use. Background of the Invention Cardiovascular disease is a leading cause of death and disability worldwide. Thrombosis is a potential cause of many types of cardiovascular disease, including venous thromboembolism (VTE), ischemic heart disease, and ischemic stroke. The outcomes of methods for removing occlusive thrombi, including angioplasty / stenting, thrombectomy, mechanical rupture, and / or biochemical dissolution, are complex. These methods are often time-consuming and costly, and frequently carry the risk of bleeding complications.

[0003] Microbubbles have been used to enhance coronary ultrasound thrombolysis in the treatment of acute myocardial infarction and acute ischemic stroke. In myocardial infarction and ischemic stroke, thrombi cause arterial blockage, loss of tissue downstream of the bloodstream, leading to ischemia and potential cell death. Thrombi are composed of fibrin and platelets, and may contain abundant red blood cells.

[0004] Fibrin, also known as factor Ia, is a fibrous, non-globulin involved in blood clotting. Fibrin is present in high concentrations in venous and arterial thrombi, and fibrin-targeted therapies exhibit high sensitivity. Conversely, fibrin is absent in circulating blood, making these therapies potentially highly specific. In addition to protein-based approaches, this application also describes small cyclic peptides with high affinity for fibrin and high selectivity for fibrinogen. The potential benefits of small peptides compared to antibodies include faster blood flow clearance and the ability to penetrate the fibrin network, both of which improve the target-to-background ratio.

[0005] Inflammation and endothelial dysfunction are key thresholds for the development of atherosclerosis. The expression of endothelial cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), has been shown to play a crucial role in leukocyte recruitment and is typically increased at sites of pathological inflammation. Persistent expression of VCAM-1 in dysfunctional endothelial cells mediates the adhesion, rolling, and binding of monocytes and promotes their migration to forming atherosclerotic plaques. Therefore, VCAM-1 is not only a target for early detection via imaging but also for therapeutic drug delivery.

[0006] Ultrasound can be used to break up blood clots; however, a trade-off must be made between time / efficiency and damage to healthy tissue. Agents that can locally amplify the sound within the cavity, such as microbubbles, can accelerate disruption while maintaining low energy delivery. When using bubbles, care must be taken regarding their size (1-5 micrometers), as this may hinder entry into the blood clot. Blood clots have a porous matrix, but the voids within the clot typically prevent micrometer-sized structures from entering.

[0007] Therefore, improvements are still needed in the methods used to detect and treat thrombosis and related diseases and disorders. Efforts to improve the safety, effectiveness, and efficiency of thrombus removal have significant potential clinical implications. Summary of the Invention

[0008] This invention is partly based on novel microbubbles and nanodroplets with targeting capabilities to select biomarkers and their emulsions that can be used for the diagnosis and treatment of certain diseases and disorders, particularly thrombosis. These carriers are capable of targeting various protein targets, such as fibrin and VCAM-1, to improve the detection or destruction of thrombi, platelets, and vascular plaques in cardiovascular diseases. This invention also relates to a pharmaceutical composition and methods of its preparation and use.

[0009] In one respect, the present invention generally relates to microbubbles and / or nanodroplet aqueous emulsions or suspensions having one or more fibroin-binding ligands connected thereto.

[0010] In another aspect, the present invention generally relates to aqueous emulsions or suspensions of microbubbles and / or nanodroplets having one or more VCAM-1 binding ligands connected thereto.

[0011] In another aspect, the present invention generally relates to an aqueous emulsion or suspension comprising microbubbles and / or nanodroplets thereof with one or more fibrin-binding ligands attached, as disclosed in this application, and microbubbles and / or nanodroplets thereof with one or more VCAM-1 binding ligands attached, as disclosed in this application.

[0012] In another aspect, the present invention generally relates to a method for detecting vascular thrombosis or plaque. The method includes: administering the aqueous emulsion or suspension disclosed in this application to a subject in need; and imaging a subset of the subject to detect the presence of vascular thrombosis or plaque.

[0013] In another aspect, the present invention generally relates to a method for diagnosing or assessing thrombosis. The method includes: administering the aqueous emulsion or suspension disclosed in this application to a subject in need; and imaging a subset of the subject to diagnose or assess thrombosis in the subject.

[0014] In another aspect, the present invention generally relates to a method for disintegrating or destroying vascular thrombi or plaques. The method includes: administering the aqueous emulsion or suspension disclosed in this application to a subject in need; and applying ultrasound to a target area of ​​the subject's organ with vascular thrombi or plaques, thereby destroying or reducing the vascular thrombi or plaques.

[0015] In another aspect, the present invention generally relates to a method for treating thrombosis or arterial plaque. The method includes: administering the aqueous emulsion or suspension disclosed in this application to a subject in need; and applying ultrasound to a target area of ​​the subject.

[0016] In another aspect, the present invention generally relates to a method for performing ultrasonic thrombolysis. The method includes: administering the aqueous emulsion or suspension disclosed in this application to a subject in need; and applying ultrasound to a target area of ​​the subject. Attached Figure Description

[0017] Figure 1 A fibroin-binding peptide (FBP) with an azide functional group bound to DSPE-PEG5000-DBCO is shown to prepare a product with a dibenzotriazole linker.

[0018] Figure 2 The combination of FBP with amine functional groups with DSPE-PEG5000-NHS ester was shown to generate a product with an amide linker.

[0019] Figure 3 The experiment demonstrates the oxidation of perfluorobiphenyl sulfides to generate more reactive sulfone derivatives, which then react with DSPE-PEG5000-amine to form DSPE-PEG5000-PFPhSO2. Finally, DSPE-PEG5000-PFPhSO2 reacts with FBP containing an amino group to yield the conjugated final product.

[0020] Figure 4 Mass spectrometry data are shown, confirming the binding of FBP to DSPE-PEG5000-DBCO (A), DSPE-PEG5000-NHS ester (B), and DSPE-PEG5000-PFPhSO2 (C).

[0021] Figure 5 The images show FBP labeled with 5(6)-carboxytetramethylrhodamine N-succinimide to generate FBP-Rh (MW=2100.75 Da (top), and DK-12 labeled with 5(6)-carboxytetramethylrhodamine N-succinimide to generate DK-12-Rh (MW=2182.49 Da) (bottom).

[0022] Figure 6The in vitro affinity binding analysis of the control peptide (DK12) fluorescence (rhodamine labeling) and the fibroin-binding peptide fluorescence (rhodamine labeling) is shown.

[0023] Figure 7 A general representation of the target MBs is shown. Various combinations of phospholipids form a spherical shell, while the interior is filled with perfluorocarbon gas, preferably octafluoropropane. Target-binding ligands, including VCAM-1 ligands or FBP ligands (shown as green stars), are attached to the surface shell of the bubble via PEG connectors.

[0024] Figure 8 The size distribution of various types of MBs with different FBP conjugated phospholipids and MPEG controls is shown in (A), and the number-weighted average of all samples is shown in (B).

[0025] Figure 9 The gas content of MBs is shown. The gas content of all four samples was determined by gas chromatography.

[0026] Figure 10 The images show (A) TEM micrographs of fibrin-binding peptides targeting microbubbles; and (B) fibrin-binding peptides targeting nanodroplets.

[0027] Figure 11 The images show (A) TEM micrographs of fibrin-binding peptide-targeted microbubbles permeating fibrin clots; and (B) fibrin-binding peptide-targeted nanodroplets permeating fibrin clots.

[0028] Figure 12 The conjugation of the VCAM-1 ligand to the DSS linker via the N-terminal amine group is shown. The DSPE-PEG2K-amine is conjugated to the other head of the DSS linker to obtain the VCAM-1_DSPE-PEG2K conjugate.

[0029] Figure 13 Exemplary fluorescence data of fibrin clot rupture are shown. Detailed Implementation

[0030] This invention provides novel microbubbles and nanodroplets with targeted capabilities for selective biomarkers, and their emulsions, which can be used as diagnostic probes and therapeutic agents for certain diseases and disorders, particularly thrombosis and arterial plaque. These microbubbles and / or nanodroplets can target various protein targets, such as fibrin and VCAM-1, to improve the detection and / or disruption of blood clots (e.g., thrombi, platelets, and vascular plaques) that occur in many cardiovascular diseases. The targeted microbubbles and / or nanodroplets can be acoustically activated in situ to induce clot rupture. This invention also provides a pharmaceutical composition and a method for its preparation and use.

[0031] A key feature of this invention is the use of nanoscale acoustically active nanodroplets, for example, in the range of about 100 nanometers to 300 nanometers, which is a fraction of the size of typical microbubbles. This smaller size allows the droplets to penetrate thrombi more easily, thereby significantly improving the efficiency of ultrasound thrombolysis and clinical efficacy.

[0032] Another key feature of this invention is the use of low temperature and high pressure to condense fluorocarbon microbubbles (e.g., octafluoropropane microbubbles) into nanodroplets (e.g., octafluoropropane nanodroplets). Even though the boiling point of octafluoropropane (-34°C) is substantially below body temperature, the nanodroplets remain condensed after intravenous (IV) injection and then reform into microbubbles upon entering an acoustic field.

[0033] Another key feature of this invention is that nanodroplets carrying one or more targeting ligands can be locally activated in situ using acoustic methods. High specificity can be achieved because fibrin is absent in circulating blood. The small peptides used as targeting ligands in this application exhibit high affinity for fibrin and high selectivity for fibrinogen. These small peptides provide faster blood flow clearance and the ability to penetrate the fibrin network, thereby improving the target-to-background ratio.

[0034] Another key feature of this invention is the unique formulation disclosed in this application, which provides the enhanced and sufficient stability required for handling and processing nanodroplets during preparation, storage, and processing.

[0035] The disclosures of U.S. Patents No. 9,801,959 B2 and No. PCT / US19 / 24713, filed March 28, 2019, are incorporated herein by reference in their entirety.

[0036] In one respect, the present invention generally relates to microbubbles and / or nanodroplet aqueous emulsions or suspensions having one or more fibroin-binding ligands connected thereto.

[0037] In some embodiments, each microbubble and / or nanodroplet is conjugated with multiple fibrin-binding ligands.

[0038] In some embodiments, the one or more fibrin-binding ligands comprise a fibrin-binding peptide having about 11 to 16 amino acids.

[0039] In some embodiments, the fibrin-binding peptide is selected from the Tn6, Tn7, or Tn10 series (Table 1).

[0040] Table 1. Examples of fibrin-specific peptides Oliveira et al.2017 Dalton Trans.46(42):14488–14508. Kolodziej, et al. 2012 Bioconj. Chem. 23:548–556. In some embodiments, the fibrin-binding ligand is conjugated to microbubbles and / or nanodroplets via a bifunctional spacer, preferably a polyethylene glycol (PEG) group, with a preferred number average molecular weight (MW) of about 1,000 to 10,000 Daltons (e.g., about 2,000 to 10,000, about 3,000 to 10,000 Daltons, about 4,000 to 10,000 Daltons, about 1,000 to 8,000 Daltons, about 1,000 to 6,000 Daltons, about 3,000 to 7,000 Daltons, about 4,000 to 6,000 Daltons), more preferably about 5,000 Daltons. The PEG group is covalently bound to a lipid anchor, preferably a phospholipid.

[0041] In some embodiments, the phospholipid composition comprises dipalmitoylphosphatidylcholine (“DPPC”). DPPC is an amphoteric compound and is essentially a neutral phospholipid. In some embodiments, the composition comprises PEGylated lipids.

[0042] Examples of lipids include: ethanolamine phosphate-N-[methoxy(polyethylene glycol)-2000] (ammonium salt), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-3000] (ammonium salt), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-3000] (ammonium salt), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-3000] (ammonium salt), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-3000], 1,2-distearyl ... [Ammonium salt of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-3000]], 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-5000]], 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-5000], 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-5000]], and 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-5000]] (ammonium salt). Dipalmitoylphosphatidylethanolamine (“DPPE”) is a preferred lipid, preferably present in the formulation along with other lipids at a concentration of 5-20 mol%, most preferably 10 mol%.

[0043] In some embodiments, the microbubbles and / or nanodroplets are filled with gaseous material.

[0044] In some embodiments, the gaseous material comprises a fluorinated gas. The term "fluorinated gas" as used herein refers to hydrofluorocarbons containing hydrogen, fluorine, and carbon, or compounds containing only carbon and fluorine atoms (also known as perfluorinated carbon), or compounds containing sulfur and fluorine. In this invention, the term may refer to materials whose molecular structure consists of carbon and fluorine or sulfur and fluorine, and which are gaseous at room temperature and pressure.

[0045] In some embodiments, the fluorinated gas is selected from perfluoromethane, perfluoroethane, perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoron-pentane, perfluorocyclopentane, perfluorohexane, perfluorocyclohexane, and mixtures of two or more thereof.

[0046] In some embodiments, the fluorinated gas is selected from perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoron-pentane, perfluorocyclopentane, and mixtures of two or more thereof.

[0047] In some embodiments, the fluorinated gas includes octafluoropropane.

[0048] In some embodiments, the aqueous emulsion or suspension also includes a stabilizer.

[0049] In some embodiments, the stabilizer is selected from D(+)-trehalose dihydrate, propylene glycol, glycerol, polyethylene glycol, glucose, and sucrose.

[0050] In some embodiments, the gaseous material further comprises an appropriate percentage of a nonfluorinated gas or gas mixture, such as about 2% to 20% air or nitrogen (e.g., about 5% to 20%, about 10% to 20%, about 15% to 20%, about 2% to 15%, about 2% to 10%, about 2% to 5% air or nitrogen).

[0051] In some embodiments, the fluorocarbons within the microbubbles and / or nanodroplets are present in a concentrated (i.e., liquid) state.

[0052] In another aspect, the present invention generally relates to aqueous emulsions or suspensions of microbubbles and / or nanodroplets having one or more VCAM-1 binding ligands connected thereto.

[0053] In some embodiments, each microbubble and / or nanodroplet is conjugated with multiple VCAM-1 binding ligands.

[0054] In some embodiments, the one or more VCAM-1 binding ligands comprise a VCAM-1 binding peptide having about 8 to 16 amino acids.

[0055] In some embodiments, the VCAM-1 binding peptide is selected from: B2702p1-20 peptide (Table 2).

[0056] Table 2. Exemplary VCAM-1 binding peptides Dimastromatteo, et al. 2013 J Nucl Med.54(8):1442-9. In some embodiments, the VCAM-1 binding ligand is conjugated to the microbubbles and / or nanodroplets via the PEG connector disclosed in this application.

[0057] In some embodiments, the microbubbles and / or nanodroplets are filled with gaseous material.

[0058] In some embodiments, the gaseous material comprises fluorinated gas.

[0059] In some embodiments, the fluorinated gas is selected from perfluoromethane, perfluoroethane, perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoron-pentane, perfluorocyclopentane, perfluorohexane, perfluorocyclohexane, and mixtures of two or more thereof.

[0060] In some embodiments, the fluorinated gas is selected from perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoron-pentane, perfluorocyclopentane, and mixtures of two or more thereof.

[0061] In some embodiments, the fluorinated gas includes octafluoropropane.

[0062] In some embodiments, the aqueous emulsion or suspension also includes a stabilizer.

[0063] In some embodiments, the stabilizer is selected from D(+)-trehalose dihydrate, propylene glycol, glycerol, polyethylene glycol, glucose, and sucrose.

[0064] In another aspect, the present invention generally relates to an aqueous emulsion or suspension comprising microbubbles and / or nanodroplets thereof with one or more fibrin-binding ligands attached, as disclosed in this application, and microbubbles and / or nanodroplets thereof with one or more VCAM-1 binding ligands attached, as disclosed in this application.

[0065] In some embodiments of the aqueous emulsions or suspensions disclosed in this application, the microbubbles and / or nanodroplets are coated with a film-forming material.

[0066] In some embodiments, the film-forming material comprises one or more lipids.

[0067] In some embodiments, the lipid comprises phospholipids or mixtures of phospholipids.

[0068] Any suitable lipid can be used. The lipid chain length can vary from about 10 to 24 (e.g., about 10 to 20, about 10 to 18, about 12 to 20, about 14 to 20, about 16 to 20, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) carbons. More preferably, its chain length is about 16 to 18 carbons.

[0069] In some embodiments, the diameter of the micron or nano-sized bubbles is in the range of about 10 nm to 10 m (e.g., about 10 nm to 5 m, about 10 nm to 1 m, about 10 nm to 500 nm, about 10 nm to 100 nm, about 50 nm to 10 m, about 100 nm to 10 m, about 1 m to 10 m). In some embodiments, the diameter of the micron or nano-sized particles or bubbles is about 10 nm to 100 nm. In some embodiments, the diameter of the micron or nano-sized particles or bubbles is about 100 nm to 1 m. In some embodiments, the diameter of the micron or nano-sized particles or bubbles is about 1 m to 10 m.

[0070] In some embodiments, the microbubbles and / or nanodroplets are microbubbles having a microscale size of about 0.5 to 10 micrometers (e.g., about 1 µm to 10 µm, about 2 µm to 10 µm, about 5 µm to 10 µm, about 0.5 µm to 5 µm, about 0.5 µm to 2 µm, about 1 µm to 5 µm).

[0071] In some embodiments, the microbubbles and / or nanodroplets are nanodroplets with a nanoscale size of about 100 nanometers to about 800 nanometers (e.g., about 100 nm to 500 nm, about 100 nm to 300 nm, about 120 nm to 280 nm). In some embodiments, the microbubbles and / or nanodroplets are nanodroplets with a nanoscale size of about 120 nanometers to 280 nanometers.

[0072] In some embodiments, the microbubbles and / or nanodroplets do not include microbubbles and / or nanodroplets with sizes outside of about 120 nanometers to 280 nanometers (i.e., essentially all microbubbles and / or nanodroplets are nanodroplets with nanoscale sizes in the range of about 120 nanometers to 280 nanometers).

[0073] In some embodiments, the aqueous emulsion or suspension is in a homogeneous form.

[0074] In some embodiments, the aqueous emulsion or suspension may also include pharmaceutically acceptable excipients, carriers, or diluents.

[0075] In another aspect, the present invention generally relates to a method for detecting vascular thrombosis or plaque. The method includes: administering the aqueous emulsion or suspension disclosed in this application to a subject in need; and imaging a subset of the subject to detect the presence of vascular thrombosis or plaque.

[0076] In another aspect, the present invention generally relates to a method for diagnosing or assessing thrombosis or atherosclerosis. The method includes: administering the aqueous emulsion or suspension disclosed in this application to a subject in need; and imaging a subset of the subject to diagnose or assess thrombosis in the subject.

[0077] In another aspect, the present invention generally relates to a method for disintegrating or destroying vascular thrombi or plaques. The method includes: administering the aqueous emulsion or suspension disclosed in this application to a subject in need; and applying ultrasound to a target area of ​​the subject's organ with vascular thrombi or plaques, thereby destroying or reducing the vascular thrombi or plaques.

[0078] On the other hand, the present invention generally relates to a method for treating thrombosis, atherosclerosis, or arterial plaque. The method includes: administering the aqueous emulsion or suspension disclosed in this application to a subject in need; and applying ultrasound to a target area of ​​the subject.

[0079] In another aspect, the present invention generally relates to a method for performing ultrasonic thrombolysis. The method includes: administering the aqueous emulsion or suspension disclosed in this application to a subject in need; and applying ultrasound to a target area of ​​the subject.

[0080] In some method embodiments, the fluorinated gas is selected from perfluoromethane, perfluoroethane, perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoron-pentane, perfluorocyclopentane, perfluorohexane, perfluorocyclohexane, and mixtures of two or more thereof.

[0081] In some method embodiments, the fluorinated gas includes octafluoropropane.

[0082] In some method embodiments, the microbubbles and / or nanodroplets are microbubbles with a microscopic size of about 0.5 to 10 micrometers.

[0083] In some embodiments of the method, the microbubbles and / or nanodroplets are nanodroplets with a nanoscale size of about 120 nanometers to 280 nanometers.

[0084] In some method embodiments, the microbubbles and / or nanodroplets do not include microbubbles and / or nanodroplets with sizes outside of about 120 nanometers to 280 nanometers (i.e., essentially all microbubbles and / or nanodroplets are nanodroplets with nanoscale sizes in the range of about 120 nanometers to 280 nanometers).

[0085] As used herein, an "emulsion" refers to a heterogeneous system consisting of at least one immiscible liquid dispersed in droplets in another liquid, the size of which can range from nanometers to micrometers. The stability of emulsions varies considerably, and emulsion separation times can range from seconds to years. Suspensions can consist of solid particles or droplets in the bulk liquid phase. For example, dodecyl fluoropentane emulsions can be prepared using phospholipids or fluorinated surfactants and conjugates incorporated into the emulsion at a ratio of about 0.1 mol% to about 1 mol% or even up to 5 mol%, relative to the surfactants used to stabilize the emulsion.

[0086] In some embodiments, the emulsion or suspension may also include pharmaceutically acceptable excipients, carriers, or diluents. Each excipient, carrier, or diluent must be "acceptable," meaning it is compatible with the other components of the emulsion or suspension and is harmless to the patient. Some examples of materials that can be used as pharmaceutically acceptable excipients, carriers, or diluents include, but are not limited to, physiological saline, phosphate-buffered saline, propylene glycol, glycerin, and polyethylene glycol, such as PEG 400 or PEG 3350 MW.

[0087] In this application, the terms "subject" and "patient" are used interchangeably to refer to a living animal (human or non-human). A subject can be a mammal. The term "mammal" refers to any animal in the mammalian class. Mammals can be human or non-human mammals, such as dogs, cats, pigs, cattle, sheep, goats, horses, rats, and mice. The term "subject" does not exclude individuals who are completely normal in terms of disease or physical condition, or individuals who are normal in all respects.

[0088] As used herein, a “treatment” or “curative action” for a disease or disorder refers to a method of reducing, delaying, or improving the condition before or after the onset of the disease or disorder. Treatment can be one or more effects or symptoms of the disease and / or underlying lesions. Treatment can be a reduction, and can be, but is not limited to, the complete disappearance of the disease or its symptoms. This reduction or prevention is measured by various standard techniques to be at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to an equivalent untreated control group.

[0089] Example Example 1. Preparation of fibrin-targeting bioconjugates The following three binding methods were used to generate peptide-phospholipid binding molecules with different linkers. (1) A fibroin-binding peptide (FBP) with a micro PEG linker and an azide functional group was directly conjugated to N-[dibenzocyclooctyl(polyethylene glycol-5000)]carbamoyl-distearylphosphatidyl-ethanolamine (ammonium salt) (DSPE-PEG5000-DBCO) to generate a product with a dibenzotriazole linker (Scheme 1). (2) An FBP with a micro PEG linker and an amine functional group was conjugated to [(succinimideoxyglutarate)aminopropyl,polyethylene glycol-5000]-carbamoyl-distearylphosphatidyl-ethanolamine (sodium salt) (DSPE-PEG5000-NHS ester) to synthesize a product with an amide linker (Scheme 2). (3) The third method includes a first reaction in which N-[aminopropyl(polyethylene glycol-5000)]-carbamoyl-distearatephosphatidylethanolamine (sodium salt) (DSPE-PEG5000-amine) and 6,6'-tetrabromobisphenol S (1,2,3,4,5-pentafluorobenzene) (PFPhSO2) are reacted to generate DSPE-PEG5000-PFPhSO2. Then, a product with a perfluorobenzene linker is prepared using a micro FBP linker and an FBP conjugated with the amine to DSPE-PEG5000-PFPhSO2 (Scheme 3).

[0090] Figure 1 The combination of FBP with azide functional groups with DSPE-PEG5000-DBCO yielded a product with a dibenzotriazole linker.

[0091] Figure 2 The combination of FBP with amine functional groups with DSPE-PEG5000-NHS esters was shown to generate a product with an amide linker.

[0092] Figure 3 The experiment demonstrates the oxidation of perfluorobiphenyl sulfides to generate more reactive sulfone derivatives, which then react with DSPE-PEG5000-amine to form DSPE-PEG5000-PFPhSO2. Finally, DSPE-PEG5000-PFPhSO2 reacts with FBP containing an amino group to yield the conjugated final product.

[0093] All products were purified by high-performance liquid chromatography (HPLC) and characterized by mass spectrometry (MS). Figure 1 ). Figure 4 Mass spectrometry data are shown, confirming the binding of DSPE-PEG5000-DBCO (A), DSPE-PEG5000-NHS Ester (B), and DSPE-PEG5000-PFPhSO2 (C).

[0094] Example 2. Fibrin-targeted and non-targeted microbubble formulations A mixture of dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), N-(carbonyl-methoxy polyethylene glycol 5000)-1,2-distearyl-sn-glycerol-3-phosphoethanolamine sodium salt (DPPE-MPEG5000), and DSPE-PEG5000-FBP conjugates was used in the formulation of targeted microbubbles (MBs) (Figure 2). In the non-targeted microbubble formulation, DSPE-PEG5000-FBP was replaced by N-(carbonyl-methoxy polyethylene glycol 5000)-carbamoyldistearylphosphatidyl-ethanolamine (sodium salt) (DSPE-MPEG5000). Vials containing conjugated phospholipids with amide, dibenzotriazole, and perfluorobenzene linkages were named ester and DBCO, respectively. The control sample containing DSPE-MPEG5000 in PFPhSO2 was named MPEG and used in the experiments.

[0095] Figure 5 This is a schematic diagram of targeted MBs, where a combination of various phospholipids forms a spherical shell filled with perfluorocarbon gas, preferably octafluoropropane. FBP (shown as green stars) is attached to the surface of the bubble via a PEG connector.

[0096] All vials containing the phospholipid mixture solution were filled with octafluoropropane gas (OFP). Two to four samples from each series of vials were dimensionally measured using a NiComp Acusazie 780 instrument. Figure 3 The results showed that all ester, DBCO, PFPhSO2, and MPEG samples formed molecular bubbles (MBs); however, the size distribution of MBs composed of different FBP conjugates differed. Compared to ester and MPEG vials, vials containing DBCO and PFPhSO2 samples had approximately 10% fewer bubbles with diameters of 0.56–1.06 µm. In contrast, compared to ester and MPEG vials, DBCO and PFPhSO2 samples had approximately 7% more bubbles with diameters of 1.06–2.03 µm and approximately 2% more bubbles with diameters of 2.03–5.99 µm. No significant differences were observed in the number-weighted averages among the different samples.

[0097] Figure 6 The size distribution of various types of MBs with different FBP conjugated phospholipids and MPEG controls is shown in (A), along with the numerically weighted average of all samples (B). Two to four samples were drawn from each group, and the gas content in each series of vials was analyzed using a GC instrument. Figure 4 ).

[0098] Figure 7 The gas content of all four samples is shown as measured by gas chromatography.

[0099] In this experiment, the ester sample exhibited the highest gas content, while the PFPHSO2 and MPEG vials showed the lowest OFP gas content. However, GC results confirmed that gas filling resulted in a gas content >80%, which is highly beneficial for MB formation.

[0100] Example 3. Preparation of VCAM-1 targeted bioconjugates The bioconjugate was prepared using an activated VCAM-1 ligand in diisopropylamine and dimethylformamide. The activated peptide was then reacted with DSPE-PEG5000-NH2 to generate the final product, which was purified by high-performance liquid chromatography.

[0101] Figure 8 The preparation of DSPE-PEG2000-VCAM ligand bioconjugates is shown.

[0102] Example 4. VCAM-1 Targeted Microbubble Formulation The targeted microbubble formulation contains dipalmitoylphosphatidylcholine (DPPC), dipalmitoyl-tin-glycerophosphatidylethanolamine-polyethylene glycol-2000-OMe (MPEGMPEG-2000), and a lipid-ligand bioconjugate composed of DPPE-PEG2000-NH linked to the ligand via a subalkyl linker (Sub) or DPPE-PEG2000-C(=O)-linked via an amide bond. The conjugate is used at approximately 1 mol% of the total phospholipids. Microbubbles are prepared by adding DPPC (90 mol%), DPPE-PEG2000 (9 mol%), and the targeted phospholipid-PEG2000-linker-peptide conjugate (1%) to stirred propylene glycol (50-65°C) until the solid is completely dissolved. Then, while stirring at 50-65°C, add several portions of the warm phospholipid solution in propylene glycol to a phosphate buffer solution containing 5% glycerol by volume; stir the solution for 5-10 minutes. Transfer the solution to serum vials, immediately stopper and cap. Allow the solution to reach ambient temperature, then store at 4°C. For the frozen phospholipid solution, fill 1.5 mL aliquots into a batch of 25-50 mL nominal volume serum vials of 2 mL each, apply a slight vacuum and purge with perfluorobutane gas, then quickly stopper and cap the vials. Store the vials at 4°C until use, then heat to ambient temperature and agitate at 75 Hz (4500 rpm) for 45 seconds on a Bristol Myers Squibb vial mixer to create microbubbles.

[0103] Example 5. Preparation of nanodroplets Lipid suspensions were prepared from DPPC (82%), DPPE (10%), DPPE-MPEG5000 (7%), and a DSPE-MPEG5000-FBP bioconjugate (1%) with a total lipid concentration of 0.75 mg / mL in propylene glycol (10.35 mg / mL), and heated at 75 °C for 1 hour. The lipid suspension was then mixed with aqueous solutions of sodium chloride (4.78 mg / mL), sodium dihydrogen phosphate (2.34 mg / mL), disodium hydrogen phosphate (2.16 mg / mL), and glycerol (12.62 mg / mL) to prepare the final solution. This final solution was used to fill vials (1.5 mL / vial), with perfluoropropane gas added before sealing and capping. The vials were incubated in an ice bath at -15 to -18 °C for 3 minutes. In addition to the excipients mentioned above, 3% w / v glucose, 0.25% w / v, 0.5% w / v, and 1.0% w / v D(+)trehalose dihydrate were added as excipients. The vials were stirred for 45 seconds using an amalgam shaker (Vialmix, BMS Medical Imaging, 4500 rpm) to create a milky white appearance, indicating microbubble (MB) formation. The vials were incubated in an ice bath at -15 to -18°C for 3 minutes. The vials were then pressurized with N2 at 40–80 psi to create a more transparent appearance, indicating nanodroplet formation. The vials were then incubated in an ice bath at -15 to -18°C for 10 minutes. The vials were left at room temperature for 1 hour and then stored under different conditions.

[0104] Microbubbles, designated MVT-100, served as a control group. All samples underwent particle size analysis using an AccuSizer 780 (PSS.NiComp particle size analyzer) and a Nanobrook 90 Plus (Brookhaven) micrometer to measure the size of both microbubbles and nanodroplets. The average size of MVT-100 microbubbles and fibrin-targeting microbubbles ranged from 1 to 3 micrometers. The results are shown in the table below. The average size of the MVT-100-derived nanodroplets rapidly increased and then decreased as perfluoropropane gas escaped from the nanodroplets. 3% glucose provided protection, but not as effectively as D(+) trehalose dihydrate. 1% D(+) trehalose dihydrate was preferred because it produced nanodroplets stable for 24 hours.

[0105] Example 6. Disruption of fibrin clots by FTMB Fibrin was used to coat all wells of a 24-well plate by adding fibrinogen and thrombin and allowing the plate to stand overnight. In short, 160 μL of fibrinogen (1.75 M in PBS) was added to each well in 30 M thioflavin. Then, 40 µL of thrombin (7.5 units / mL in PBS) was added to each well. The plate was incubated overnight in the dark at room temperature. Fibrin clots were visible under a phase-contrast microscope.

[0106] Table 3. Stability C of different nanodroplet formulations incubated at 37℃ (n=3) Table 4. Size distribution, gas content, and zeta potential of FBP-targeted and untargeted microbubbles and nanodroplets (n=3) MB was activated (vial mixing, 45 seconds). A final stock solution of each MB formulation was prepared using 500 µL of 5.2 mL PBS. The fibrin-coated wells were washed with PBS (1.0 mL x 1) before adding MB to the wells. MB was incubated in the fibrin-coated wells for 3 minutes.

[0107] Ultrasonic waves were delivered to each hole for 30 seconds (parameters: 2000mW, PRF 10, 10ms burst length, frequency 590Hz).

[0108] Collect the supernatant and rotate it at 10,000 rpm for 15 minutes at room temperature. Measure the emitted fluorescence in a dark 96-well plate. Measure the fluorescence of thioflavin at 485 nm (excit = 450 nm; emis = 485 nm).

[0109] In one embodiment, the power level reading on the amplifier is 2,000 mW, but the power reading on the wattmeter aligned with the transducer is approximately 100 mW. The estimated mechanical properties of the ultrasound are approximately 0.28 MPa. Figure 9 ).

[0110] In another embodiment, an ultrasound MI greater than 0.40 MPa is used for ultrasonic thrombolysis of ND.

[0111] Example 8 A patient with acute STEMI received nanodroplet-enhanced ultrasound-guided thrombolysis. The nanodroplet formulation consisted of MVT-100 + 1% D(+) trehalose dihydrate, formed into nanodroplets via a proprietary cooling / pressurization process. The patient received intravenous infusion of the nanodroplets (4 mL over a 30-minute infusion period during simultaneous ultrasound). The ultrasound protocol used was as described by Mathias (Mathias, Wilson, et al. 2016 J. Am. Coll. Cardiol. 67.21.2506-2515). Image-guided high-mechanical-index ultrasound (1.8 MHz; mechanical index 1.1 to 1.3; 3-ms pulse duration) was used, with pulses applied to apical four-chamber, two-chamber, and three-chamber views encompassing areas of myocardial risk. Following ultrasound-guided thrombolysis, the patient underwent routine angioplasty and stent placement. At 30 days post-treatment, myocardial flow and left ventricular ejection fraction improved.

[0112] Example 9 Using similar ultrasound parameters as described in Example 1, another patient with acute STEMI was treated with fibrin-targeted nanodroplets. It can be seen that the targeted nanodroplets achieve coronary revascularization faster than the non-targeted nanodroplets.

[0113] Example 10 In a patient with acute ischemic stroke, three vials of fibrin-targeting nanodroplets (6 mL total) were infused intravenously over 60 minutes during simultaneous intravenous infusion of t-PA. Ultrasound was applied within the time window using a 1 MHz probe (MU = 1.0) for the same duration as the simultaneous infusion of t-PA and nanodroplets. Blood flow in the middle cerebral artery was rapidly restored.

[0114] Example 11 A patient had extensive plaque buildup in the left anterior descending coronary artery, resulting in 90% occlusion of the artery. The patient received an intravenous infusion of 6 mL of VCAM-1 targeted nanodroplets, simultaneously using the ultrasound described in Example 1. This resulted in plaque reduction and improved coronary blood flow.

[0115] Example 12 A patient presented with acute peripheral arterial occlusion in the lower extremity. A blood clot was confined to the femoral artery, resulting in reduced blood flow to the leg. Intravenous infusion of fibrin-targeted nanodroplets was administered. Using a 3D ultrasound transducer with a center frequency of 2 MHz, ultrasound was percutaneously applied to the occluded area of ​​the artery with ultrasound pulses at a power of 1.6 MPa (2 seconds on, 2 seconds off), while the nanodroplets were infused intravenously at a rate of 2.0 cc per hour for 2 hours. This cleared the arterial blockage and restored blood flow to the lower extremity.

[0116] The applicant has described the disclosure herein with reference to the accompanying drawings, in which the same numerals denote the same or similar elements. References to “an embodiment,” “embodiment,” or similar language in this specification indicate that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the use of the terms “in one embodiment,” “in an embodiment,” and similar terms throughout this specification may, but not necessarily, refer to the same embodiment.

[0117] In one or more embodiments, the features, structures, or characteristics disclosed by the applicant may be combined in any suitable manner. Numerous specific details are set forth in this description to provide a thorough understanding of embodiments of the invention. However, those skilled in the art will recognize that the applicant's compositions and / or methods may be implemented without one or more specific details, or with other methods, ingredients, materials, etc. In other instances, well-known structures, materials, or operations have not been shown or described in detail to avoid obscuring aspects of this disclosure.

[0118] In this specification and the appended claims, the singular forms “a,” “the,” and “the” include plural references unless the context clearly indicates otherwise. Unless otherwise specified or apparent from the context, the term "about" in this application should be understood as being within the normal tolerances in the art, such as within two standard deviations of the average. "Approximately" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context otherwise requires, all numerical values ​​provided in this application may be modified with "about".

[0119] Unless otherwise specified or obvious in the context, the word “or” used here should be understood as inclusive.

[0120] The term "comprising," when used to define compositions and methods, is intended to mean that the composition and method include the listed elements, but does not exclude other elements. The term "consistently composed of," when used to define compositions and methods, means that the composition and method include the listed elements and excludes other elements that are of any significant importance to the composition and method. For example, "consistently composed of" refers to the administration of a pharmacologically active agent that is explicitly listed, but does not include pharmacologically active agents that are not explicitly listed. The term "consistently composed of" does not exclude pharmacologically inactive or inert agents, such as pharmaceutically acceptable excipients, carriers, or diluents. The term "composed of," when used to define compositions and methods, refers to the exclusion of trace elements and substantial method steps that are other components. Examples defined by each of these transitional terms are within the scope of this invention.

[0121] Unless otherwise defined, all technical and scientific terms used in this application have the meanings commonly understood by one of ordinary skill in the art. While any methods and materials similar to or equivalent to those described and used herein may also be used in practice or testing, only preferred methods and materials are described herein. Except for the specific order disclosed, the methods described herein may be operated in any logically possible order.

[0122] By incorporating via reference This application references and cites other literature, such as patents, patent applications, patent publications, journals, books, papers, and online content, and incorporates the entire contents of such materials through citation. Any material or part thereof, incorporated herein by reference, that conflicts with existing definitions, statements, or other disclosed materials expressly set forth herein, shall be incorporated only if there is no conflict between the incorporated material and this disclosure. In the event of a conflict, the conflict shall be resolved in a manner favorable to this application, and the disclosure favorable to this application shall be adopted as the preferred embodiment.

[0123] equivalent The above-described representative embodiments are intended to help explain the present invention and are not intended to limit the scope of the invention, nor should they be construed as limiting the scope of the invention. In fact, apart from those shown and described in the application, the entire contents of this application, including examples included in the application and references to scientific and patent literature, will make various modifications to the invention and many further embodiments apparent to those skilled in the art. These embodiments contain important additional information, examples, and guidance applicable to the practice of various embodiments of the invention and their equivalents.

Claims

1. A nano-droplet aqueous emulsion or suspension, characterized in that, The fibroin-binding ligand has one or more ligands conjugated via polyethylene glycol (PEG) linkers; the number-average molecular weight (MW) of the PEG linkers is in the range of 1,000 to 10,000 Daltons, wherein The nanodroplets are coated with a film-forming material, which includes a phospholipid mixture. The size range of the nanodroplets is 100 nanometers to 300 nanometers; The nanodroplets are filled with condensed octafluoropropane; The aqueous emulsion or suspension includes D(+)-trehalose dihydrate as a stabilizer.

2. The aqueous emulsion or suspension according to claim 1, characterized in that, Each nanodroplet is conjugated with multiple fibroin-binding ligands.

3. The aqueous emulsion or suspension according to claim 1 or 2, characterized in that, The fibroin-binding peptides are selected from Table 1.

4. A nano-droplet aqueous emulsion or suspension, characterized in that, VCAM-1 binding ligands having one or more conjugated via polyethylene glycol (PEG) links; wherein the number-average molecular weight (MW) of the PEG links is in the range of 1,000 to 10,000 Daltons, wherein The nanodroplets are coated with a film-forming material, which includes a phospholipid mixture. The size range of the nanodroplets is 100 nanometers to 300 nanometers; The nanodroplets are filled with condensed octafluoropropane; The aqueous emulsion or suspension includes D(+)-trehalose dihydrate as a stabilizer.

5. The aqueous emulsion or suspension according to claim 4, characterized in that, Each nanodroplet is conjugated with multiple VCAM-1 binding ligands.

6. The aqueous emulsion or suspension according to claim 4 or 5, characterized in that, The VCAM-1 binding peptides are selected from Table 2.

7. The aqueous emulsion or suspension according to claim 1 or 4, characterized in that, This includes nanodroplets with one or more fibrin-binding ligands attached thereto, and nanodroplets with one or more VCAM-1 binding ligands attached thereto.

8. The aqueous emulsion or suspension according to claim 1 or 4, characterized in that, The nanodroplets include nanodroplets with a nanoscale size of 120 nanometers to 280 nanometers.

9. The aqueous emulsion or suspension according to claim 8, characterized in that, It is homogeneous.

10. The aqueous emulsion or suspension according to claim 9, characterized in that, It also includes pharmaceutically acceptable excipients, carriers, or diluents.

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