Peptide sequence and methods for atherosclerotic plaque targeting

Abstract

Provided herein are compositions comprising peptide-based nanoparticles. In some embodiments, the peptide-based nanoparticles each comprise a perfluorocarbon liquid core and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core. In some embodiments, the amphiphilic peptides comprise a peptide analogue of Apolipoprotein A1. Also provided herein are methods of preparing any of the compositions described herein, as well as methods of treating atherosclerotic plaque formation using any of the composition described herein.

Description

148411.004402 PATENTPEPTIDE SEQUENCE AND METHODS FOR ATHEROSCLEROTIC PLAQUE TARGETINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Patent Application Serial No. 63 / 670,480, filed on July 12, 2024, entitled “PEPTIDE SEQUENCE AND METHODS FOR ATHEROSCLEROTIC PLAQUE TARGETING,” the entire contents of which is hereby incorporated by reference herein.GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under Grant Nos. DK 128638 and GM 142902 awarded by the National Institutes of Health and under Grant No. DMR 1845053 awarded by the National Science Foundation. The Government has certain rights in the invention.SEQUENCE LISTING

[0003] This application contains a computer readable Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “148411004400 Sequence Listing.xinl”, was created on July 11, 2024, and is 69,005 bytes in size.FIELD OF THE INVENTION

[0004] The present invention is directed to fluorous phase-directed peptide assemblies capable of being triggered by ultrasound. It further pertains to methods for acoustically activating phase-changing nanoparticles within cells, permitting in situ imaging and targeting various cell types for specific applications.BACKGROUND OF THE INVENTION

[0005] Protein and nucleic-acid based agents are some of the most potent therapeutic tools used in precision medicine. They deliver biologically active cargo to target cells and are revolutionizing the treatment of various human diseases, including cancer, autoimmune disorders and diabetes. However, therapeutic utility of these biomacromolecules is limited by1313274296v2148411.004402 PATENTtheir ineffective distribution to diseased tissues, poor stability in physiologic environments, and toxic side effects. Importantly, many large biomolecules are unable to penetrate into cells and, therefore, existing biotherapeutics have been restricted to disease targets displayed on the cell surface. Due to the large size, traditional biocarriers rely on endocytic transport processes to enter diseased cells, ultimately leading to degradation of delivered biologies in the harsh endosomal environment. The cell-targeted biocarriers also require multi-step harsh physical and chemical formulation methods that ultimately render biological cargo inactive.

[0006] Dynamic peptide assemblies can be formed as a result of a spontaneous self-sorting and assembly process. The architecture and behavior of these self-assembled peptide structures can be controlled by modulating the physicochemical properties of the building blocks, environmental conditions, or assembly kinetics. This has resulted in a wide variety of ordered peptide arrangements including sheets, fibrils, and tubes, which are playing increasingly important roles in the formation of biomaterials and biomedical devices. Unlike traditional protein delivery vehicles that rely on endocytic pathways requiring endosomal escape – risking protein denaturation – these peptide assemblies can potentially facilitate direct intracellular delivery, bypassing the endocytic route altogether. This method is especially crucial for effective protein and nucleic acid therapy, where preserving the biological activity of the cargo is paramount.

[0007] While various peptide assemblies have been described in the literature, there have been no reports of using these peptide assemblies as drug delivery vehicles for delivering, for example, protein and nucleic acid-based therapeutic agents into cells.

[0008] Recognizing similar challenges in cellular therapies has underscored the need for advanced imaging techniques to monitor cellular behavior and response in real-time, particularly in diseases like atherosclerosis where macrophages play a pivotal role. Atherosclerosis is characterized by the build-up of lipid- and immune cell-rich plaques within arterial walls, with macrophages ingesting oxidized lipoproteins and transforming into lipid- rich foam cells. These cells exacerbate plaque growth and the progression of lesions into complex, pro-inflammatory states that can precipitate myocardial infarction or strokes.

[0009] Conventional diagnostic methods for atherosclerosis utilize intravascular ultrasound, X-ray angiography, magnetic resonance imaging, or optical coherence tomography to identify plaques based on morphologic lesion features and vessel stenosis. While these approaches provide a general anatomical assessment of lesion position, they lack the ability to distinguish stable from unstable plaques, and thus have limited resolution on disease pathology. Therefore,2313274296v2148411.004402 PATENTnovel early-detection methods that accurately identify high-risk lesions hold promise to improve patient risk stratification and enable targeted therapeutic intervention before lethal plaque rupture.SUMMARY OF THE INVENTION

[0010] Provided here are compositions comprising a plurality of peptide-based nanoparticles. In some embodiments, each peptide-based nanoparticle comprises a perfluorocarbon liquid core and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core, wherein each amphiphilic peptide is represented by Formula (X):HB-SP-APO (X)wherein HB is a fluorinated hydrophobic block consisting of three to five consecutively connected pentafluorinated hydrophobic amino acid residues; wherein SP is a spacer amino region consisting of one to five glycine residues; wherein APO is an amino acid sequence that is a peptide analogue of Apolipoprotein A1 (Apo-A1), wherein the amphiphilic peptides are oriented such that groups HB of the amphiphilic peptides are interpolated into the perfluorocarbon liquid of the perfluorocarbon liquid core and groups APO extend away from the perfluorocarbon liquid core. In some embodiments, HB consists of three, four or five consecutively connected pentafluoro-phenylalanine residues, and is located at the N-terminal end of the peptide sequence. In some embodiments, SP consists of one glycine residue. In some embodiments, APO consists of the amino acid sequence DWFKAFYDKVAEKFKEAF (SEQ ID NO: 51). In some embodiments, each amphiphilic peptide comprises the amino acid sequence FFFFFFFFFFGDWFKAFYDKVAEKFKEAF (SEQ ID NO: 52), wherein FFis pentafluoro-phenylalanine.

[0011] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient selected from the group consisting of a vehicle, an adjuvant, a carrier, and a diluent.

[0012] In some embodiments, the peptide-based nanoparticles further comprise a cargo contained within the perfluorocarbon liquid core. In some embodiments, the cargo is a statin drug. In some embodiments, the statin drug is Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, or Simvastatin.

[0013] Also provided herein are compositions comprising a plurality of cells, wherein each cell comprises at least one peptide-based nanoparticle. In some embodiments, the at least one peptide-based nanoparticle comprises a perfluorocarbon liquid core and a plurality of 3I32?429(iv2148411.004402 PATENTamphiphilic peptides surrounding the perfluorocarbon liquid core, wherein each amphiphilic peptide is represented by Formula (X):HB-SP-APO (X)wherein HB is a fluorinated hydrophobic block consisting of three to five consecutively connected pentafluorinated hydrophobic amino acid residues; wherein SP is a spacer amino region consisting of one to five glycine residues; wherein APO is an amino acid sequence that is a peptide analogue of Apolipoprotein A1 (Apo-A1), wherein the amphiphilic peptides are oriented such that groups HB of the amphiphilic peptides are interpolated into the perfluorocarbon liquid of the perfluorocarbon liquid core and groups APO extend away from the perfluorocarbon liquid core.

[0014] In some embodiments, each cell of the plurality of cells is a macrophage.

[0015] In some embodiments, HB consists of three, four or five consecutively connected pentafluoro-phenylalanine residues, and is located at the N-terminal end of the peptide sequence. In some embodiments, SP consists of one glycine residue. In some embodiments, APO consists of the amino acid sequence DWFKAFYDKVAEKFKEAF (SEQ ID NO: 51). In some embodiments, each amphiphilic peptide comprises the amino acid sequence FFFFFFFFFFGDWFKAFYDKVAEKFKEAF (SEQ ID NO: 52), wherein FFis pentafluoro-phenylalanine.

[0016] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient selected from the group consisting of a vehicle, an adjuvant, a carrier, and a diluent.

[0017] In some embodiments, the peptide-based nanoparticles further comprise a cargo contained within the perfluorocarbon liquid core. In some embodiments, the cargo is a statin drug. In some embodiments, the statin drug is Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, or Simvastatin.

[0018] In some embodiments, the composition comprises a plurality of macrophages, wherein each macrophage comprises at least one peptide-based nanoparticle, wherein the at least one peptide-based nanoparticle comprises a perfluorocarbon liquid core, a cargo contained with the perfluorocarbon liquid core, and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core, wherein each amphiphilic peptide comprises FFFFFFFFFFGDWFKAFYDKVAEKFKEAF (SEQ ID NO: 52), wherein FFis pentafluoro-phenylalanine, and wherein amphiphilic peptides are oriented such that the FFFFFFFFFFregion is interpolated into the perfluorocarbon liquid core and the DWFKAFYDKVAEKFKEAF 43! 3274296’, -2148411.004402 PATENT(SEQ ID NO: 51) region extends away from the perfluorocarbon liquid, and wherein the cargo is a statin drug.

[0019] Also provided are methods of preparing any composition comprising a plurality of cells described herein. In some embodiments, the method comprises contacting a perfluorocarbon liquid with a plurality of amphiphilic peptides to form a plurality of peptide-based nanoparticles, wherein each peptide-based nanoparticle comprises a perfluorocarbon liquid core and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core, and contacting the plurality of peptide-based nanoparticles with the plurality of cells, where in each cell of the plurality of cells internalizes at least one peptide-based nanoparticle.

[0020] In some embodiments, water is added to plurality of peptide-based nanoparticles after their formation. In some embodiments, a statin drug is added to the perfluorocarbon liquid prior to contacting the perfluorocarbon liquid with the plurality of amphiphilic peptides to form the plurality of peptide-based nanoparticles. In some embodiments, the cell is a macrophage.

[0021] Also provided are methods of detecting atherosclerotic plaques, the method comprising administering any composition described herein to a tissue, administering ultrasonic waves to the tissue, and detecting the location of atherosclerotic plaques in the tissue by locating acoustic properties of the peptide-based nanoparticles.

[0022] In some embodiments, the ultrasonic waves are administered to the tissue by a B-mode ultrasonic imaging device or Doppler ultrasonic imaging device. In some embodiments, the ultrasonic waves induce a liquid-to-gas phase transition in the peptide-based nanoparticles that generates echogenic microbubbles.

[0023] Also provided are methods of treating atherosclerotic plaques, the method comprising administering any composition described herein to a tissue with atherosclerotic plaques. In some embodiments, binding of the peptide-based nanoparticles to oxidized low-density lipoprotein (oxLDL) within lipid-rich foamy macrophages reduces the lipid burden of the foamy macrophages. In some embodiments, the method comprises administering ultrasonic waves to the tissue to induce a liquid-to-gas phase transition in the peptide-based nanoparticles. In some embodiments, the liquid-to-gas phase transition of the peptide-based nanoparticles results in mechanical fractionation of intracellular lipid droplets within lipid-rich foamy macrophages. In some embodiments, when the peptide-based nanoparticles comprise a statin drug in the perfluorocarbon liquid core, the statin drug is released into the lipid-rich foamy macrophages.148411.004402 PATENTBRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and other aspects and features of the present disclosure will become more apparent in the following detailed description when taken in conjunction with reference to the accompanying drawings, in which:

[0025] FIG. 1 is a diagram showing structure of nanoparticles according to embodiments;[0026[ F IG. 2 is a diagram showing target motif-mediated specific binding and ultrasound" mediated delivery of cargo into a targeted cell;

[0027] FIG. 3 is a graph of particle size (nanometers, nm) and count rate (kilo counts per second, kcps) versus time (days) illustrating stability of nanoparticles (formulation B, see Table 1) during long-term storage in water at room temperature, wherein particle stability was measured via DLS over 15 days;

[0028] FIG. 4 is a diagram showing particle size of 1 and 2 vol% perfluoro-n-pentane (PFP) immediately after emulsion formation in the nanoparticle pre-assembly solution (1:1 DMF:ACN, 1% TFA);

[0029] FIG. 5 is a graph showing relationship between PFP vaporization temperature (T vap, degrees Centigrade, °C) and nanodroplet size (R. H, hydrodynamic radius, nanometers, nm), modeled at three different reported surface tension values for PFP emulsions formulated with the BSA protein (open circles), PEO-PLA polymer (filled gray circles) or CTAB surfactant (filled black circles), where the dashed line indicates physiologic temperature (37°C);

[0030] FIG. 6 is a graph of number percent versus particle size (nanometers, nm) illustrating particle size measurement of nanoparticles formulated under various peptide and PFP ratios, where each letter, A, B, C, D, and E, corresponds to different formulations shown in Table 1;

[0031] FIG. 7 depicts a conceptual schematic of Apo-NPep architecture and mechanism of acoustic activation within intraplaque foam cells.

[0032] FIG. 8A depicts an optical micrograph of Apo-NPep nanoparticles; the inset shows bulk dispersion.

[0033] FIG. 8B depicts size distribution of Apo-NPep nanoparticles.

[0034] FIG. 8C depicts a transmission electron micrograph of Apo-NPep nanoparticles.[0035J FIG. 9 depicts time-dependent changes in optical density (OD600) of the nanoparticle solution at 37°C or 4!‘C.

[0036] FIG. 10A depicts confocal laser scanning micrographs of Apo-NPep binding to Dil-labeled oxLDL (left: bright field; middle: Dil-oxLDL; right merged imaged); the insets show magnified region indicated by white boxes.63l327429(iv2148411.004402 PATENT

[0037] FIG. 10B depicts confocal laser scanning micrographs of Scr-NPep binding to Dil-labeled oxLDL (left: bright field; middle: Dil-oxLDL; right merged imaged); the insets show magnified region indicated by white boxes.

[0038] FIG. 11A depicts the normalized intensity profile of Apo-NPep and Dil-oxLDL signals captured from the region depicted by the dotted line in FIG. 10A.

[0039] FIG. 11B depicts the percent co-localization of Dil-oxLDL and nanoparticle signals for Scr-Npep and Apo-Npep formulations.[0040| FIG. 12 depicts confocal laser scanning micrographs of Dil-oxLDL loaded RAW 264.7 macrophages treated with DiO-loaded Apo-NPep nanoparticles for 6 hours; DAPI indicates cell nuclei.

[0041] FIG. 13 depicts a three-dimensional fluorescent projection of Apo-NPep phagocytosis into foamy macrophages; the inset shows magnified region identified by the dashed box.

[0042] FIG. 14 depicts magnified orthographic projections identified by the white dashed box depicted in FIG. 13.

[0043] FIG. 15 depicts the fluorescent intensity profile of Apo-Npep nanoparticles and oxLDL along line in the selected confocal region shown on the left of the figure.

[0044] FIG. 16A depicts a transmission electron micrograph of untreated RAW 264.7 macrophages treated for 15 hours; electron dense oxLDL-droplets appear as dark circular / ellipsoidal structures.

[0045] FIG. 16B depicts a transmission electron micrograph of cells treated with Apo-NPep for 15 hours; electron dense oxLDL-droplets appear as dark circular / ellipsoidal structures. The triangles in the inset depict oxLDL droplets enwrapped with Apo-NPep nanoparticles.

[0046] FIG. 16C depicts a transmission electron micrograph of Scr-Npep nanoparticles treated for 15 hours; electron dense oxLDL-droplets appear as dark circular / ellipsoidal structures.

[0047] FIG. 17A depicts a representative photograph of an aortic arch isolated from mice fed a high-fat diet and perfused with Dil-loaded Apo-NPep nanoparticles.

[0048] FIG. 17B depicts a fluorescent whole tissue image of Dil-loaded Apo-NPep accumulation at atheroma sites.

[0049] FIG. 17C depicts a photograph of an atherosclerotic plaque staining in aorta tissue by Oil red O.313274296v2148411.004402 PATENT

[0050] FIG. 18 depicts the percent vaporized (activated) Apo-NPep nanoparticles at 37°C as a function of insonating US intensity.

[0051] FIG. 19A depicts a representative optical image of Apo-NPeps before US activation; the inset shows Apo-NPep solution opacity before US treatment, demonstrating formation of coalescing bubbles that confirms Apo-NPep phase-change.

[0052] FIG. 19B depicts a representative optical image of Apo-NPeps after US activation; the inset shows Apo-NPep solution opacity after US treatment, demonstrating formation of coalescing bubbles that confirms Apo-NPep phase-change.

[0053] FIG. 20 depicts the viability of Apo-NPep loaded RAW 264.7 cells following US exposure at the indicated intensity.

[0054] FIG. 21 depicts a schematic illustration of oxLDL efflux from foam cells following US activation of NPep nanoparticles.

[0055] FIG. 22 depicts the percent relative oxLDL efflux from RAW 264.7 foam cells following a 6 (left) or 24 (right) hour treatment with the indicated NPep formulation; control is untreated cells.

[0056] FIG. 23 depicts the percent relative oxLDL efflux from foam cells in the absence (left) or presence (right) of internalized Apo-Npep nanoparticles activated at the indicated US intensity.

[0057] FIG. 24 depicts the percent relative oxLDL efflux from foam cells treated with Simvastatin-loaded Apo-NPep nanoparticles and activated at the indicated US intensity after 2-hour incubation; the control is emulsion-untreated cells.

[0058] FIG. 25 depicts representative B-mode images of Apo-Npep nanoparticles in an agar phantom before (top) and after (bottom) US-mediated particle cavitation.

[0059] FIG. 26 depicts quantification of B-mode signals from the nanoparticles (+Apo-NPep) or background control (-Apo-NPep).

[0060] FIG. 27 depicts representative B-mode images of Apo-NPep loaded foam cells in agar phantoms before (top) and after (bottom) US nanoparticle vaporization using an 18 MHz transducer.

[0061] FIG. 28 depicts quantification of B-mode signals from non-labeled foam cells (-Apo-NPep) and Apo-NPep labeled foam cells (+Apo-NPep).

[0062] FIG. 29 depicts a schematic illustration of ex vivo injection of nanoparticles or Apo-NPep-foam cells to create model lesions within the porcine coronary artery.8313274296v2148411.004402 PATENT

[0063] FIG. 30 depicts representative B-mode images of injected depots containing free Apo-NPep nanoparticles; the white arrows identify the lumen of the adjacent coronary artery.

[0064] FIG. 31 depicts representative B-mode images of injected depots containing non¬ labeled foam cells; white arrows identi fy the lumen of the adjacent coronary artery, |0065] FIG. 3 IB depicts representati e B-mode images of injected depots containing Apo- NPep labeled foam cells; white arrows identify the Tumen of the adjacent coronary artery.

[0066] FIG. 32 depicts color Doppler signals, superimposed on B-mode images, from Apo-NPep nanoparticles insonated in an agar phantom; the far-left panel displays the initial B-mode image without Doppler and the remaining panels demonstrate Doppler twinkling as a function of exposure time.

[0067] FIG. 33 depicts Doppler twinkling in porcine heart tissue after delivery of free Apo-NPep nanoparticles; the white dashed region of interest (ROI) indicates the model anatomical lesion and the white arrows identify the lumen of the adjacent coronary artery.

[0068] FIG. 34 depicts Doppler twinkling in porcine heart tissue after delivery of Apo-NPep labeled foam cells; the white dashed region of interest (ROI) indicates the model anatomical lesion and the white arrows identify the lumen of the adjacent coronary artery.

[0069] FIG. 35 depicts Doppler twinkling in porcine heart tissue after delivery of non-labeled foam cells; the white dashed region of interest (ROI) indicates the model anatomical lesion and the white arrows identify the lumen of the adjacent coronary artery.

[0070] FIG. 36A depicts a spatial Doppler magnitude image corresponding to FIG. 33.

[0071] FIG. 36B depicts a spatial Doppler magnitude image corresponding to FIG. 34.[00721 FIG. 36C depicts a spatial Doppler magnitude image corresponding to FIG. 35.

[0073] FIG. 37 depicts time-dependent Doppler magnitude over a 35 second imaging window for non-labeled foamy cells (oxLDL-RAW), free Apo-NPep nanoparticles, and Apo-NPep-oxLDL-RAW cells.

[0074] FIG. 38 depicts integrated variance (area under the curve) of time-locked Doppler signals from FIG. 37.[0075| FIG. 39 depicts the persistence of twinkling features in Apo-NPep labeled foam cells over 7 days in culture.

[0076] FIG. 40 depicts a schematic of the PCD experimental setup using a 1MHz high intensity focused ultrasound (HIFU) transducer; the star (*) denotes the HIFU focal zone in the sample solution.9313274296v2148411.004402 PATENT

[0077] FIG. 41 depicts a conceptual diagram of stable and inertial cavitation of vaporized Apo-NPeps.[0078| FIG. 42A depicts frequency spectra of oscillating Apo-NPep nanoparticles at intermediate acoustic pressures.[00791 FIG. 42 B depicts frequency spectra of oscillating Apo-NPep nanoparticles at high acoustic pressures.

[0080] FIG. 42C depicts frequency spectra and cavitation activity plots from degassed control solutions insonated under similar acoustic conditions.[00811 FIG. 42D depicts frequency spectra and cavitation activity plots from degassed control solutions insonated under similar acoustic conditions.

[0082] FIG. 43 depicts cavitation activity plots of Apo-NPep over 100 pulses at the indicated acoustic pressure.[00831 FIG.44 depicts frequency spectra and cavitation activity plots from degassed control solutions insonated under similar acoustic conditions.DETA11ED DESCRIPTION OF THE INVENTION[0084| Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.[00851 The terms "a" and "an" do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. The term "or" means "and / or". The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to").[0086| Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable.[0087[ AU methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to 10313274296v2148411.004402 PATENTthe practice of the invention as used herein. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art of this disclosure.|0088] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same -base claim. Where elements are presented as lists, g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.

[0089] All compounds are understood to include all possible isotopes of atoms occurring in the compounds. Isotopes include those atoms having the same atomic number but different mass numbers and encompass heavy isotopes and radioactive isotopes. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include1’C,!'C, andi4C.. Accordingly, the compounds disclosed herein may include heavy or radioactive isotopes in the structure of the compounds or as substituents attached thereto. Examples of useful heavy or radioactive isotopes include! SF,i5N,,hO,,f>Br, -4 andi4ll.

[0090] The opened ended term "comprising" includes the intermediate and closed terms "consisting essentially of and "consisting of."[0091 | A dashthat is not between two letters or symbols is used to indicate a point of attachment for a substituent.

[0092] " Pharmaceutical compositions" means compositions including at least one active agent, such as a compound or salt of Formula 3, and at least one other substance, such as a carrier. Pharmaceutical compositions meet the U. S. FDA's GMP (good manufacturing practice) standards for human or non-huntan drugs.[00931 A "patient" means a human or nori-hurnaii animal in need of medical treatment. Medical treatment can include treatment of art existing condition, such as a disease or disorder or diagnostic treatment. In some embodiments the patient is a human patient.

[0094] " Providing" means giving, administering, selling, distributing, transferring (for profit or not), manufacturing, compounding, or dispensing.

[0095] "Treatment" or "treating" means providing an active compound to a patient in an amount sufficient to measurably reduce any disease symptom, slow disease progression or 11313274296v2148411.004402 PATENTcause disease regression. In certain embodiments treatment of the disease may he commenced before the patient presents symptoms of the disease.[0096J A "therapeutically active agent” means a compound which can be used for diagnosis or treatments of a disease. The compounds can be small molecules, peptides, proteins, or other kinds of molecules.f()097| A significant change is any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student's T-test, where p < 0.05.

[0098] The present invention is directed to novel nanoparticles prepared through the templated assembly of amphiphilic peptides at a fluorous-liquid interface containing releasable cargo-fluorine-containing cargo solubilizing agent complexes.[0099| A “nanoparticle” refers to a structured entity capable of incorporating a variety of functional components. Nanoparticles, along with, but not limited to, the terms “nanoparticle”, “nanoparticle”, “nanoemulsion”, “emulsion”, “nanofibril”, and “NPep” denote an arrangement of amphiphilic peptides forming a corona around a perfluorocarbon liquid core. This core is capable of undergoing a phase transition upon ultrasound stimulation, thus facilitating the targeted release of encapsulated cargo. The term nanoparticle encompasses a spectrum of such formulations, characterized by their molecular architecture that includes a lipid-like core surrounded by a shell of peptides, where these peptides may possess properties such as hydrophilicity, hydrophobicity, and specific binding functionalities.

[0100] Nanoparticles according to embodiments of the present invention have a perfluorocarbon liquid core that phase transitions into a gaseous state upon ultrasound application. When positioned at the cell surface, ultrasound activation serves to deliver cargo encapsulated within the nanoparticles into the cytoplasm directly. Thus, proteins, peptides, nucleic acids, small molecule compounds, and other materials, can be encapsulated and directly delivered to the cytoplasm of cells without loss of function. This ultrasound-mediated delivery is ideal for therapeutics due to its spatial and temporal precision.[01011 A typical nanoparticle is shown at 10 in FIG. 1 including a plurality of amphiphilic peptides 20, wherein the amphiphilic peptides each include a hydrophilic peptide 30, a crosslinking motif 40, and a fluorinated hydrophobic block 50. The fluorinated hydrophobic block of the amphiphilic peptides promotes peptide assembly at the surface of the perfluorocarbon (PFC) liquid core 60, which contains the cargo-fluorine-containing cargo solubilizing agent complexes 70.148411.004402 PATENT[0102| In some embodiments, the cargo is not dispersed in the perfluorocarbon liquid core 60 but is instead associated with or bound to the surface of the external amphiphilic peptide 20 molecules through various binding mechanisms, including but not limited to electrostatic interactions, ionic interactions, or other specific affinity-based interactions. In some embodiments, the amphiphilic peptide 20 molecules may contain charged amino acid residues that engage in electrosta tic interactions with oppositely charged domains on the cargo, thereby enhancing the stability and specificity of their surface attachment.

[0103] Nanoparticles having a diameter of from about 250 nm to about 5 microns may be produced. An average diameter of the nanoparticles according to embodiments may be from about 1 to about 5 microns, for example, about 1 to about 4 microns, about 1 to about 3 microns, or about 1 to about 2 microns, but is not limited thereto. In an embodiment, the nanoparticles have an average diameter in the range of about 300 nanometers to 1200 nanometers, about 250 nanometers to about 1000 nanometers, for example, 250 to about 750 nanometers, but is not limited thereto.[0104| The nanoparticles contain a perfluorocarbon liquid core that allows or activation of the nanoparticles upon application of ultrasound (US) and delivery of a cargo present in the perfluorocarbon liquid core. The term "activation" as used herein to refer to activation of nanoparticles upon application of ultrasound refers to phase transition of a perfluorocarbon liquid core into a gaseous state due to ultrasound application.[0105| As shown diagrammatically in FIG. 2, nanoparticle 10 binds to a receptor 80 disposed in or on a cell membrane 75 via specific interaction with the hydrophilic peptide 30 of the amphiphilic peptides of nanoparticle 10, Application of ultrasound 85 causes acoustic vaporization of the perfluorocarbon l iqu id core of the nanoparticles and leads to the formation of a gaseous core that ultimately swells and ruptures 90 the nanoparticles. Subsequent bubble cavitation produces a high intensity pressure wave that, when generated at the surface of a cell, transiently permeabilizes the plasma membrane 75, and simultaneously ejects cargo 95 encapsulated in the nanoparticle into the cell cytoplasm 98. Thus, US-sensitive nanoparticles represent a spatially and temporally controlled delivery modality that, as described herein, can deliver a cargo, such as biomacromolecules, directly into the cytoplasm of cells, thereby avoiding endosomal uptake and degradation of the bioactive payload.(01061 Key to the assembly of these nanoparticles is a de novo designed amphiphilic peptide, capable of assembling at the surface of a perfluorocarbon liquid.148411.004402 PATENT[0107| As noted above, amphiphilic peptides included in nanoparticles according to embodiments each include a fluorinated hydrophobic block (HB), a crosslinking motif, and a hydrophi li c peptide.|O108] Thus, in one embodiment, an amphiphilic peptide represented by Formula (I) is provided:HB— CL— HP (I)[0109| wherein HB is a fluorinated hydrophobic polymer; CL is a cross-linking motif; and HP is a hydrophilic amino acid sequence.

[0110] As used herein, the term ’’amphiphilic peptide” refers to a molecule including a fluorinated hydrophobic polymer; a cross-linking motif; and a hydrophilic amino acid sequence, wherein the amphiphilic peptide has a molecular weight in the range of about: 2000 — 5000 daltons, wherein the amphiphilic peptide includes at least five amino acid residues, and a total number of no more than 30 amino acid residues, wherein at least two of the amino acid residues are consecutively linked to each other in a chain by a peptide bond,

[0111] As used herein, the term "fluorinated hydrophobic polymer” refers to a covalently linked chain of monomer residues forming a fluorinated hydrophobic homopolymer or copolymer. The monomeric units which form the fluorinated hydrophobic polymer may each be fluorinated according to embodiments, or some, or one, of the monomeric units is fluorinated such that at least one or more of the monomer residues of the fluorinated hydrophobic polymer is fluorinated,[0112| According to embodiments, the amphiphilic peptide does not include lipids.

[0113] According to embodiments, the fluorinated hydrophobic polymer includes a hydrophobic amino acid sequence wherein the amino acids of the hydrophobic amino acid sequence have non-polar side chains, wherein the non-polar side chains do not include a group capable of forming a hydrogen bond with molecules of water; and wherein at least one of the amino acids of the hydrophobic amino acid sequence is fluorinated.

[0114] According to embodiments, the fluorinated hydrophobic polymer includes one or more synthetic non-amino acid monomeric units wherein at least one of the monomeric units is fluorinated such that at least one of the monomer residues of the fluorinated hydrophobic polymer is fluorinated. Non-limiting examples of synthetic monomeric units which can be fluorinated and reacted to form a fluorinated hydrophobic polymer include methyl methacrylate; lactic acid, glycolic acid and olefins such as ethylene, propylene, styrene.148411.004402 PATENT[0115| As used herein, the term "hydrophobic amino acid sequence" refers to a hydrophobic polymer, a sequence of hydrophobic amino acids having non-polar side chains, wherein the non-polar side chains do not include a group capable of forming a hydrogen bond with molecules of water, or a combination of a hydrophobic polymer and a sequence of hydrophobic amino acids having non-polar side chains. Hydrophobic amino acids may be naturally occurring; or non-natural (artificially produced). Examples of the naturally occurring hydrophobic amino acids include, but are not limited to, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, cysteine, and methionine. Examples of the non-natural hydrophobic amino acids may include D amino acids, as well as specific non-natural amino acids such as selenocysteine, pyrrolysine, and the like,[0U6| In the amphiphilic peptide, the fluorinated hydrophobic amino acid sequence may include one to ten fluorinated hydrophobic amino acids consecutively connected by peptide bonds, which may be unsubstituted or substituted with a substituent selected from -F, -Cl, -Br, -I, a C1-C30 alkyl group, a C2-C30 alkenyl group, a C2-C30 alkynyl group, a C3-C30 cycloalkyl group, a C3-C30 cycloalkenyl group, a C6-C30 aryl group, a C7-C30 arylalkyl group, but are not limited thereto. For example, the fluorinated hydrophobic amino acid sequence may include one, two, three, four, five, six seven, eight, nine or ten fluormated hydrophobic amino acids consecutively connected by peptide bonds. Fluorinated hydrophobic amino acids include, for example, fluorinated alanine, fluorinated saline, fluorinated leucine, fluorinated isoleucine, fluorinated proline, fluorinated phenylalanine, fluorinated tryptophan, fluorinated cysteine, fluorinated methionine, fluorinated selenocysteine, and fluorinated pyrrolysine. The fluorinated hydrophobic amino acids can be D or L amino acids and can be fluorinated at any suitable position, typically replacing a hydrogen atom. In an embodiment, the fluorinated hydrophobic amino acid sequence may include pentafluoro-phenylalanine (2,3,4,5,6-pentafluoro-L-phenylalanine and / or 2,3,4,5,6-pentafluoro-D-phenylalanine) at a terminal thereof In another embodiment, the fluorinated hydrophobic amino acid sequence may include one to ten, such as one, two, three, four, live, six, seven, eight, nine or ten consecutively connected pentafluoro-phenylalanine residues at a terminal thereof.101171 A combination of a hydrophobic polymer and a sequence of hydrophobic amino acids having non-polar side chains can be included in the fluorinated hydrophobic polymer wherein at least one of the monomer residues of the fluorinated hydrophobic polymer is fluorinated and-or at least one of the amino acid residues is fluorinated.148411.004402 PATENT[0118| As used herein, the term ‘'hydrophilic amino acid sequence" refers to a sequence of hydrophilic amino acids consecutively connected by peptide bonds, wherein the hydrophilic amino acids have a polar side chain, wherein the polar side chain includes a group capable of forming a hydrogen bond with molecules of water. Hydrophilic amino acids may be naturally occurring or non-natural and can be D or L amino acids. Examples of the naturally ’Occurring hydrophilic amino acids include, but are not limited to, senile, threonine, asparagine, glutamine, histidine and tyrosine.[0119| Examples of the non-natural hydrophilic amino acids include amino acids having various heterocyclic groups as a part of the side chain.[0120| In the amphiphilic peptide, the hydrophilic amino acid sequence HP may include three to fifteen hydrophilic amino acids consecutively connected by peptide bonds. For example, the hydrophilic amino acid sequence HP may include one, two, three, four, live, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen hydrophilic amino acids consecutively connected by peptide bonds.

[0121] In one embodiment, an amphiphilic peptide represented by Formula (II) is provided:HB— CL— HP (II)

[0122] wherein HB is a fluorinated hydrophobic amino acid sequence; CL is a cross-linking motif; and HP is a hydrophilic amino acid sequence.[0123| In one embodiment, an amphiphilic peptide represented by Formula (111) is provided:HB— CL— HP-NH2 (III)[01241 wherein HB is a fluorinated hydrophobic amino acid sequence; CL is a cross-linking motif; and HP is a C-terminally amidated hydrophilic amino acid sequence.10125] In one embodiment, the hydrophilic amino acid sequence includes a targeting agent that interacts with a targeted component of a target cell. The targeted component is at least partially external to the target cell and interaction of the targeting agent and targeted component of the target cells serves to bring nanoparticles into proximity with the target cell into which the cargo is to be delivered. The target cells can be cells of any organism, such as, but not limited to, a mammal, bird, fish, or bacterial cell. According to embodiments, the target cell is a human cell or a bacterial cell within a human body.|0126| According to embodiments, the targeting agent includes a minimal targeting motif peptide and optionally includes one or more hydrophilic amino acids attached to the N-ierminus or C-terminus of the minimal targeting motif peptide by peptide bonds. Typically, amino acids of the targeting motif peptide are L-amino acids but these may include one or more D-amino 16313274296v2148411.004402 PATENTacids so long as the targeting motif still correctly mediates binding with the targeted component. The one or more hydrophilic amino acids attached to the N-terminus or C-temrinus of the minimal targeting motif peptide by peptide bonds can be D or L amino acids.|0127] According to embodiments, the targeting agent includes a minimal targeting motif peptide selected from: HGK, RGD, EAR, RSR, KAA, RGRR (SEQ ID NO: I), RGRS (SEQ ID NO:2), YQLDV (SEQ ID NO:3), EYQ, RPM, PSP. VGVA (SEQ ID NO:4), NGR, CRKRLDRNC (SEQ ID NO 43) which binds to the 11-4 receptor on atherosclerotic plaques; EFEEFEIDEE. EK (SEQ ID NO:44) which binds to thrombin in blood clots; and / or DFEEIPEEYLQ (SEQ ID NO:45) which binds to thrombin in blood clots, and optionally incl udes one or more hydrophilic amino acids attached to the N-terminus or C-terminus of the minimal targeting motif peptide by peptide bonds.|0128] For example, the hydrophilic amino acid sequence HP may include the amino acid sequence KGRGD (SEQ ID NO:35) as a targeting agent, wherein K is lysine, G is glycine, R is arginine, and D is aspartic acid, which includes minimal targeting motif RGD capable of specific binding to integrins and two hydrophilic amino acids.|0129] Minimal targeting motif RGD alone or with additional hydrophilic amino acids along with the binding affinity to aV 3 integrin (ICjn cone, in nM at which 50% of receptor is bound by ligand): RGD (89 ± 12), RGDS (45 ± 5, SEQ ID NO:36), GRGD (55 ± 7, SEQ ID NO:37), GRGDS (28 ± 3, SEQ ID NO:38), GRGDSP (13.7 ± 0.3, SEQ ID NO:39), GRGDSPK (1.2 ± 0.1, SEQ ID NO:40), GRGDNP (45 ± 12, SEQ ID NO:41), and GRGDTP (28 ± 5, SEQ ID NO:42). Thus, according to embodiments, the hydrophilic amino acid sequence HP includes, RGD, KGRGD (SEQ ID NO:35), RGDS (SEQ ID NO:36), GRGD (SEQ ID NO:37), GRGDS (SEQ ID NO: 38). GRGDSP (SEQ ID NO:39), GRGDSPK (SEQ ID NO:40), GRGDNP (SEQ ID NO:41), or GRGDTP (SEQ ID NO:42).(0130] Various targeting agents and motifs are described in US Patent Publication No 20200197307, which is hereby incorporated by reference in its entirety.

[0131] As used herein, the phrase "cross-linking motif' refers to an amino acid sequence that Includes, at any position in the sequence, at least two amino acid residues each capable of cross-linking with a corresponding amino acid residue capable of cross-linking and present in another amphiphilic peptide.(0132] The at least two amino acid residues capable of cross-linking with a corresponding amino acid residue in the crosslinking motif of another amphiphilic peptide can be a naturally occurring amino acids and / or a non-naturally occurring amino acids.17313274296v2148411.004402 PATENT[0133| Naturally occurring amino acids capable of crosslinking with a corresponding amino acid residue include cysteine.[0134| An amino acid may be functionalized to such that it is a non-naturally occurring amino acid to provide the ability to bind to a naturally occurring or non-naturally occurring amino acid in the crosslinking motif of another amphiphilic peptide.

[0135] in exemplary embodiments, the cross-linking motif may include cross linking moieties such as sulfhydryl crosslinkers, UV cross-linkers, aza-benzenes, photosensitive crosslinkers such as azides or benzophenones, nitriles, pH sensitive cross-linkers, or enzymatic cross-linkers.10136] In an embodiment, the cross-linking motif may include cysteine, and may optionally further include glycine. For example, in one preferred embodiment, the cross-linking motif CL may include GGGCCGG (SEQ ID NO:46), wherein G is glycine and C is cysteine. The crosslinking motif CL may comprise from 1 to about 10 amino acid residues.[0137| In the nanoparticles, the amphiphilic peptide molecules are oriented in such a way that groups HB of the peptide are located at a surface of the perfluorocarbon of the perfluorocarbon liquid core, wherein the amphiphilic peptide molecules are bonded intramolecularly. For example, when the cross-linking motif of the amphiphilic peptide includes a cysteine residue, the amphiphilic peptide molecules may be cross-linked via disulfide cross-linking groups (-S-S-). When the cross-linking motif of the amphiphilic, peptide includes two or more cysteine residues, the two or more cysteine residues may be intramolecularly connected via disulfide cross-linking groups (-S-S-).[0138| A degree of cross-linking of the amphiphilic peptide molecules may be about 50% or greater, for example, about 55% or greater, about 60% or greater, about 65% or greater, about 70% or greater, about 75% or greater, about 80% or greater, about 85% or greater, about 90% or greater, or about 95% or greater. The degree of crosslinking may be measured using a colorimetric disulfide formation assay (described in detail below).[0139| The amphiphilic peptide, according to an embodiment, may include 5 to 30 amino acids and has a molecular weight in the range of about 2000 - 5000 daltons. For example, the amphiphilic peptide may include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids wherein the amphiphilic peptide has a molecular weight in the range of about 2000 - 5000 daltons.

[0140] in an embodiment, the amphiphilic peptide has Formula (IV):FFFFFFGGGCCGGKGRGD (IV) (SEQ ID NO:47)18313274296v2148411.004402 PATENT

[0141] wherein F is pentafluoro-phenylalanine (2,3,4,5,6-pentafluoro-L-phenylalanine), G is glycine, C is cysteine, K is lysine, G is glycine, R is arginine, and D is aspartic acid. In Formula (IV), FFFFFF is a hydrophobic block HB, GGGCCGG (SEQ ID NO:46) is a cross¬ linking motif CL, and KGRGD-NH2 (SEQ ID NO:48) is a C-terminally amidated hydrophilic amino acid sequence HP, which contains targeting motif RGD.[0142| in an embodiment, the amphiphilic peptide has Formula (V):FFFFFFGGGCCGGKGRGD-NH2(V) (SEQ ID NO:49)

[0143] wherein F is pentafluoro-phenylalanine, G is glycine, C is cysteine, K is lysine, G is glycine, R is arginine, and D is aspartic acid, and the amphiphilic peptide is C-terminally amidated. In Formula (IV), FFFFFF is a hydrophobic block HB, GGGCCGG (SEQ) ID NO:46) is a cross-linking motif CL, and KGRGD-NH2 (SEQ ID NO:48) is a C-terminally amidated hydrophilic amino acid sequence HP, which contains targeting motif RGD.

[0144] Amphiphilic peptides can be synthesized using techniques known to one of ordinary skill in the art, such as, but not limited to, solid-phase synthesis, recombinant methodologies, polymerization, and conjugation methods.[0145| Advantageously, amphiphilic peptides according to embodiments of the present invention may include highly fluorinated amino acid residues and such sequences may be chemically synthesized in high yield and purity using standard solid-phase techniques known to one of ordinary skill in the art.[0146 The amphiphilic peptides of all of Formulas (1), (II), ( III), (IV), and (V) are capable of assembling at the surface of a perfluorocarbon liquid and assemble into a layer to form a nanoparticle,[01471 Amphiphilic peptides (IV) and (V) contain six pentafluoro-phenylalanine (F) residues at the N-terminus which promote interpolation and assembly of the peptide at the perfluorocarbon liquid interface. C-terminal to this fluorous domain is a cysteine-con taining crosslinking motif (GGGCCGG, SEQ ID NO:46) designed to undergo disulfide cross-linking to an adjacent amphiphilic peptide in order to stabilize the peptide corona after templated assembly. Incorporation of a bioactive hydrophilic sequence at the peptide's C-terminus ultimately leads to its multivalent display at the surface of the assembled particle. The amphiphilic peptides of Formulas (IV) and (V) include the sequence KGRGD (SEQ ID NO:35) to enable, cell-surface localization of nanoparticles including the amphiphilic peptides of Formulas (IV) and (V) mediated by binding of the targeting motif RGD with extracellular integrins.19313274296v2148411.004402 PATENT

[0148] As noted above, the nanoparticles contain a perfluorocarbon (PFC) liquid core that allows for activation of the particle upon application of ultrasound (US) and delivery of a cargo present in the perfluorocarbon liquid core.J0149J As used herein, the term "perfluorocarbon" refers to a hydrocarbon in which, all or a substantial portion of hydrogen atoms in bonds are replaced with fluorine atoms, producing C-F bonds. The degree of replacement of hydrogen atoms with fluorine atoms may vary, and may be 100%, 99% or greater. 98% or greater, 97% or greater, 96% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, 75% or greater, or 70% or greater. In another embodiment, die degree of replacement of hydrogen atoms with fluorine atoms may be 100%, 99.9% or greater, 99.8% or greater, 99.7% or greater, 99.6% or greater, 99.5% or greater; 99.4% or greater, 99,3% or greater, 99.2% or greater, or 99,1% or greater. The perfluorocarbon liquid may be a perfluorobutane, a perfluoropentane, a perfluorohexane, octafluoropropane, but is not limited thereto. The perfluorocarbon liquid may be a perfluoropentane, for example, perfluoro-n-pentane (PFP) or perfluoro-iso-pentane. The perfluorocarbon liquid may be a perfluorohexane, for example, perfluoro~n~hexane (PFH), perfluoro-iso-hexane, or perfluoro- see-hexane. As used herein, the term "perfluorocarbon liquid” generally refers to a perfluorocarbon as defined above, which is present in a liquid state at ambient temperature of about 25°C. The perfluorocarbon liquid may have a boiling point of about 45°C, or lower, for example, about 40°C or lower, about 35°C or lower, or about 30°C or lower. While not wishing to be bound to any theory, it is understood that the higher the boiling point of the perfluorocarbon Liquid, the greater ultrasound intensity should be utilized to acoustically vaporize the perfluorocarbon liquid core of the nanoparticles which leads to the formation of a gaseous core that ultimately swells and ruptures the nanoparticles. Accordingly, when the boiling point of the perfluorocarbon liquid is too high, for example, greater than 45°C, cells may be damaged by the application of the ultrasound.|0150| On the other hand, the intensity of the ultrasound should be sufficient to release the cargo from the nanoparticles into the cells. To ensure, however, that no cell damage occurs, the intensity of the ultrasound should not be greater than 1.0 watts per square centimeter (W / cm2), and its mechanical index (MI) should be maintained below 1,9. Ultrasound systems for in vitro and in vivo application are commercially available, such as Toshiba Medical Systems Aplio500 and GE Healthcare Logiq E9, and these and other such systems can be used according to the manufacturers specifications to administer ultrasound to a patient, or to isolated cells to image nanoparticles to targeted cells or regions such as atherosclerotic plaques or blood clots.20313274296v2148411.004402 PATENT

[0151] The perfluorocarbon liquid core may further include a bioimaging agent, for example, a photoacoustic dye (such as indocyanine green " ICG", Cyanine 7 " Cy7", or dimethyl{4-[i.5s5-tris(4-dimethylaminopheny1)-2,4’pentadienylidene]-2.5=cyclohexadiene- 1-ylidne [ammonium perchlorate ‘’IR800”), a fluorescent dye or protein (such as green fluorescent protein " GFP", fluorescein, rhodamine, a cyanine dye), and a magnetic resonance imaging " MRI contrast agent (such as iron oxide or gadolinium), a radiotracer, but is not limited thereto.

[0152] According to embodiments, the perfluorocarbon liquid core includes about 1x103to about 5xl09molecules, such as 1x104to about 5x10smolecules of the active agent, such as 1x105to about 5x107molecules of the active agent, such as 1x106to about 5x106molecules of the active agent, and may include more, or less, of the active agent.

[0153] According to embodiments, a cargo to be delivered to the interior of a cell via the nanoparticles is contacted with is fluorine-containing cargo solubilizing agent to aid in miscibility with the perfluorocarbon liquid core. The fluorine-containing cargo solubilizing agent may be, for example, a perlluoroalkyl, a polyfluoroalkyl, a perfluorinated alkyl acid, a polyfluorinated alkyl acid, a perfluorinated aromatic compound, a polyfluorinated aromatic compound, any of which may be further substituted or unsubstituted, or a mixture of any two or more thereof. The fluorine-containing cargo solubilizing agent, may be, for example, perfluorooctane (CF3(CF2)6CF3), perfluoroteradecane (CF3(CF2)12CF3), trifluoroacetic acid (CF3COOH), pentafluoropropionic acid (CF3(CF2)COOH), perfluorotetradecanoic acid (CF3(CF2)12COOH), perfluorooctadecanoic acid (CF3(CF2)16COOH), perfluorocyclohexanecarboxylic acid ((CF2)5CFCOOH), pentafluorophenol (2,3,4,5,6-pentafluorophenol, C6F5OH), pentafluorobenzaldehyde (2,3,4,5,6-pentafluorobenzaldehyde, C6F5CHO), or Fmoc-pnetafluorophenylalanine ((CF)5CCH2C(NH-Fmoc)COOH, Fmoc-pentafluoro-L-phenylalanine and / or Fmoc-pentafluoro-D-phenylalanine, or a mixture of any two or more thereof, but is not limited thereto. In an embodiment, the fluorine-containing cargo solubilizing agent may be perfluorononanoic acid.[O154| In another embodiment, a composition including the above nanoparticle is provided that may be used for therapeutic or diagnostic use. The composition may include a pharmaceutically acceptable excipient for example, a vehicle, an adjuvant, a carrier or a diluent, that are well-known to those who are skilled in the art and are readily available to the public. Typically, the pharmaceutically acceptable carrier is one that is chemically inert to the213! 3274296’, -2148411.004402 PATENTpharmaceutically active agents and one that has no detrimental side effects or toxicity under the conditions of use.

[0155] The compositions may be administered as oral, sublingual,, transdermal, subcutaneous, topical, absorption through epithelial or mucocutaneous linings, intravenous, intranasal, intraarterial, intramuscular, mtratumoral, peritumoral, interperitoneal, intrathecal, rectal, vaginal, or aerosol formulations. In some aspects, the pharmaceutical composition is administered orally or intravenously. One preferred method of administration is through an intravenous injection.[01561 In still another embodiment, a method of preparing the nanoparticle is disclosed. According to the method, a composition including a therapeutically active agent, an amphiphilic peptide represented by any of the above Formulas (I), (11), (III), (IV'), or (V), and a perfluorocarbon liquid is provided. The composition is then contacted with water to provide an intermediate assembly including a perfluorocarbon liquid core containing the perfluorocarbon liquid and the therapeutically active agent dispersed in the perfluorocarbon liquid, and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core. In the intermediate assembly, the amphiphilic peptides are oriented in such a way that the groups I1B are interpolated into the perfluorocarbon liquid of the perfluorocarbon liquid core, while the groups MP extend away from the surface of the perfluorocarbon liquid core and away from the core of the perfluorocarbon liquid core. The amphiphilic peptide molecules of the intermediate assembly are subsequently cross-linked to form the nanoparticles.

[0157] The water may have a temperature of 10° C or lower, for example, 9° C or lower, 8° C or lower, 7° C or lower, 6° C or lower, 5° C or lower, 4° C or lower, 3° C or lower, 2° C or lower, or 1° C or lower. In an embodiment, the water may be ice-cold water, While not wishing to be bound to any theory, it is understood that when cold water is slowly added to an organic emulsion of amphiphilic peptides and perfluorocarbon liquid, spontaneous assembly of the amphiphilic peptides at the surface of the perfluorocarbon liquid core takes place. This mild procedure also eliminates the need for aggressive synthetic methods commonly used to prepare stimuli-responsive particles, which can lead to degradation of the encapsulated therapeutically active agent.

[0158] According to embodiments of methods of making nanoparticles, a cargo to be delivered to the interior of a cell via the nanoparticles is contacted with a fluorine-containing cargo solubilizing agent to aid in miscibility with the perfluorocarbon liquid core. The fluorine- containing cargo solubilizing agent may be, for example, a perfluoroalkyl, a polyfluoroalkyl, a 7313274296v2148411.004402 PATENTperfluorinated alkyl acid, a polyfluorinated alkyl acid, a perfluorinated aromatic compound, a polyfluorinated aromatic compound, any of which may be further substituted or substituted, or a mixture of any two or more thereof. The fluorine-containing cargo solubilizing agent may be, for example, perfluorooctane (CF3(CF2)6CF3, perfluoroteradecane (CF3(CF2)12CF3), trifluoroacetic acid (CF3COOH), pentafluoropropionic acid (CF3(CF2)COOH), perfluoropentanoic acid (CF3(CF2)3COOH), perfluorononanoic acid (CF3(CF2)7COOH), perfluorotetradecanoic acid (CF3(CF2)12COOH), perfluorooctadecanoic acid (CF3(CF2)16COOH), perfluorocyclohexanecarboxylic acid ((CF2)5CFCOOH), pentafluorophenol (2,3,4,5,6-pentafluorophenol, C6F5OH), pentafluorobenzaldehyde (2,3,4,5,6-pentafluorobenzaldehyde, C6F5CHO), or Fmoc-pnetafluorophenylalanine ((CF)5CCH2C(NH-Fmoc)COOH, Fmoc-pentafluoro-L-phenylalanine and / or Fmoc-pentafluoro-D-phenylalanine, or a mixture of any two or more thereof, but is not limited thereto. In an embodiment, the fluorine-containing cargo solubilizing agent may be perfluorononanoic acid.

[0159] According to embodiments of methods of making nanoparticles, the fluorine- containing cargo solubilizing agent, the perfluorocarbon liquid, and the cargo are mixed together and the amphiphilic peptides represented by any of the above Formulas (I), (IF), (111), (IV), or (V), are added, forming a composition. The composition is then contacted with water to provide an intermediate assembly including a pet-fluorocarbon core containing the perfluorocarbon liquid and the therapeutically active agent dispersed in the perfluorocarbon liquid, and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core. In the intermediate assembly, the amphiphilic peptides are oriented in such a way that the groups HB are interpolated into the perfluorocarbon liquid of the perfluorocarbon liquid core, while the groups HP extend away from the surface of the perfluorocarbon liquid cote and away from the core of the perfluorocarbon liquid core. The amphiphilic peptide molecules of the intermediate assembly are subsequently cross-linked to form the nanoparticles.

[0160] According to embodiments, the nanoparticles have an average diameter in the range from about 1 micron to about 5 microns, hi other embodiments, the nanoparticles have an average diameter in the range from about 250 nanometers to about 1000 milometers. According to embodiments, the nanoparticles have an average diameter in the range from about 250 nanometers about 750 nanometers, The size of nanoparticles can be controlled by varying the volume percent (vol%) of the perfluorocarbon liquid and / or the concentration of amphiphilic peptide in the composition when making the nanoparticles, see for example FIG. 4 and FIG. 6.23313274296v2148411.004402 PATENTIn general, the volume percent of the perfluorocarbon liquid can be increased to increase the average diameter of the nanoparticles, but this increase in average diameter is limited if the concentration of amphiphilic peptide is not also increased. According to embodiments, the volume percent of the perfluorocarbon liquid can be increased to increase the average diameter of the nanoparticles with a standard amount of amphiphilic peptides, along with simultaneous additional preparations in which the amount of amphiphilic peptides is varied to obtain a population of nanoparticles with a desired average diameter.[0161 During preparation, the cross-linking may be performed during a dialysis of the intermediate assembly. The dialysis may be conducted in an aqueous solution including dimethylsulfoxide or any other organic solvent capable of oxidizing and cross-linking filial groups of cysteine amino acids. For example, the dialysis mays be carried out in an aqueous solution of dimethylsulfoxide (DMSO) at any concentration. In an embodiment, the dialysis can be carried out in a 2.5% solution of DMSO in water. This mild cross-linking procedure also eliminates the need for aggressive synthetic methods commonly used to prepare stimuli- responsive particles, which can lead to degradation of the encapsulated cargo of the therapeutically active agent.

[0162] The degree of cross-linking of the amphiphilic peptide molecules is about 60% or greater, for example, about 65% or greater, about 70% or greater, about 75% or greater, about 80% or greater, about 85% or greater, about 90% or greater, or about 95% or greater. The degree of cross-linking of the amphiphilic peptides can be determined by a colorimetric disulfide formation assay.

[0163] According to embodiments, nanoparticles are utilized in ultrasound imaging methods, Nanoparticles having an average diameter of >1 micron provide good acoustic contrast and thus are useful as imaging agents. Such nanoparticles are too large to leave blood vessels and therefore are particularly useful in vascular applications, such as targeted imaging for diagnosis and-'or targeted, treatment of atherosclerotic plaques or blood clots,

[0164] In contrast, nanoparticles <750nm in diameter can leave blood vessels in diseased tissues and other tissues and distribute to cells, thereby allowing, for cargo delivery, including targeted cargo delivery, such as delivery of drugs and biologies including, but not limited to, small molecules, proteins and nucleic acids.

[0165] For imaging, two modalities of ultrasound imaging can be used, B-rnode ultrasound imaging allows viewing of stable nanoparticles that have cores vaporized under low intensity ultrasound to form microbubbles but have not yet collapsed or lysed. B-mode ultrasound 243! 3274296’, -2148411.004402 PATENTimaging allows a user to view and guide the nanoparticles in space using the ultrasound pressure wave. Doppler imaging can be used and allows viewing of changes in frequency that occur when the nanoparticles collapse due to application of ultrasound.

[0166] The term "low intensity" is used to refer to ultrasound at acoustic pressures that allow the core of the nanoparticles to oscillate as bubbles but not collapse. The term "high intensity" is used to refer to ultrasound at acoustic pressures that cause bubble cavitation of the nanoparticle cores. The exact threshold defining where low intensity stops, and high intensity starts will depend on the nature of the peptide shell and size of the nanoparticles. In general, application of ultrasound to a patient is at an ultrasound intensity of no higher than 1.9 Ml. For example, the 500 nm nanoparticles wherein the amphiphilic peptides have the sequence FFFFFFFFGGGCCGGKGRGD-NH2(SEQ ID NO;49), stably oscillate as bubbles below 0.4 MI (mechanical index, measure of ultrasound in tensity), and collapse at ultrasound pressures above this threshold.

[0167] In some embodiments, nanoparticles can be formed with varying acoustic properties. For example, adding water either before or after amphiphilic peptides contact the perfluorocarbon liquid, and then either cross-linking or not cross-linking the amphiphilic peptides, results in four distinct morphologies of nanoparticles. In some embodiments, when water is added to plurality of peptide-based nanoparticles after their formation and the amphiphilic peptides of each peptide-based nanoparticle are crosslinked to each other, the resulting nanoparticles comprise a crosslinked unimolecular monolayer morphology. In some embodiments, when water is added to plurality of peptide-based nanoparticles after their formation and the amphiphilic peptides of each peptide-based nanoparlicle are not crosslinked to each other, the resulting nanoparticles comprise a non-crosslinked unimolecular monolayer morphology. In some embodiments, when water is added to the plurality of amphiphilic peptides prior to their contact with the perfluorocarbon liquid and the amphiphilic peptides of each peptide-based nanoparticle are crosslinked to each other, the resulting nanoparticles comprise a 2D sheet morphology. In some embodiments, when water is added to the plurality of amphiphilic peptides prior to their contact with the perfluorocarbon liquid and the amphiphilic peptides of each peptide-based nanoparticle are crosslinked to each other, the resulting nanoparticles comprise a 1 D fibrils morphology.

[0168] In some embodiments, certain peptide-based nanoparticles can be created to specifically target atherosclerotic plaques, by combining a fluorinated hydrophobic block148411.004402 PATENTregion described herein with a peptide analogue of Apolipoprotein Al (Apo-Al). In some embodiments, the amphiphilic, peptide is represented by Formula (X):HB-SP-APO (X)wherein HB is a fluorinated hydrophobic block consisting of three to five consecutively connected pentafluorinated hydrophobic amino acid residues; wherein SP is a spacer amino region consisting of one to five glycine residues; and wherein APO is an amino acid sequence that is a peptide analogue of Apolipoprotein Al (Apo-Al ). In some embodiments, APO consists of the amino acid sequence DWFKAFYDKVAEKFKEAF (SEQ ID NO: 51). In some embodiments, the amphiphilic peptide comprises the amino acid sequence FFFFFFFFFFGDWFKAFYDKVAEKFKEAF (SEQ ID NO: 52), wherein FFis pentafluoro- phenylalanine.

[0169] In some embodiments, peptide-based nanoparticles that can target atherosclerotic plaques each comprise a perfluorocarbon liquid core and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core, as previously described. In some embodiments, the peptide-based nanoparticles further comprise a cargo contained within the perfluorocarbon liquid core, such as a statin or other appropriate medication. In some embodiments, the cargo is a statin drug selected from the group consisting of Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, and Simvastatin.

[0170] In some embodiments, any nanoparticle described herein, including nanoparticles with different morphologies and nanoparticles that can target atherosclerotic plaques can be incorporated into a cell. For example, in some embodiments, macrophages can internalize nanoparticles when brought in contact with them in vitro. The resulting composition is then a plurality of cells, each with one or more nanoparticles contained within. Thus, in some embodiments, compositions are provided that comprise a plurality of cells, wherein each cell comprises at least one nanoparticle described herein. In some embodiments, the plurality of cells is a plurality of macrophages. Additionally, in some embodiments, a method of preparing a cellular composition comprising a plurality of cells is provided, the method comprising contacting a perfluorocarbon liquid with a plurality of amphiphilic peptides to form a plurality of peptide-based nanoparticles, wherein each peptide-based nanoparticle comprises a perfluorocarbon liquid core and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core, and contacting the plurality of peptide-based nanoparticles with the plurality of cells, where in each cell of the plurality of cells internalizes at least one peptide- based nanoparticle.26313274296v2148411.004402 PATENT[0171| In some embodiments, the cellular compositions comprising the nanoparticles can be used in cellular tracking by tracking the cells with nanoparticles via ultrasound. Because the nanoparticles are internalized within the cells of the composition, as those cells move throughout a tissue, their location can be tracked with the use of an ultrasound device. Thus, in some embodiments, a method of cellular tracking is provided, the method comprising administering a cellular composition with nanoparticles described herein to a tissue, administering ultrasonic waves to the tissue, and detecting the location of the cells in the tissue by locating acoustic properties of the peptide-based nanoparticles in the plurality of cells.

[0172] For atherosclerotic plaque formation, nanoparticles comprising a peptide analogue of Apolipoprotein Al (Apo-A1) can locate within atherosclerotic plaques in a variety of ways. For example, a composition comprising nanoparticles comprising a peptide analogue of Apo-Al may be administered directly to a tissue, where the nanoparticles are taken up by lipid-rich foamy macrophages present in atherosclerotic plaques located within vasculature of the tissue. There, the nanoparticles comprising a peptide analogue of Apo- l can bind to oxidized low- density lipoprotein (oxLDL) within lipid-rich foamy macrophages. For another example, a composition comprising cells, such as macrophages, that already contain nanoparticles comprising a peptide analogue of Apo-Al may be administered to a tissue. In some embodiments, these macrophages comprising the nanoparticles locate to atherosclerotic plaques present in a tissue. Regardless of the type of composition used, once the nanoparticles are located within atherosclerotic plaques present in a tissue, those plaques can be detected by the administration of ultrasound to the tissue, which, as described herein, allows the detection of the nanoparticles, and thus the plaques, via ultrasound imaging.

[0173] Thus, in some embodiments, a method of detecting a method of detecting atherosclerotic plaque formation is provided, the method comprising administering any composition described herein that comprises peptide-based nanoparticles comprising a peptide analogue of Apo-A 1 to a tissue, administering ultrasonic waves to the tissue, and detecting the location of atherosclerotic plaque formation in the tissue by locating acoustic properties of the peptide-based nanoparticles. In some embodiments, the ultrasonic waves are administered to the tissue by a B-mode ultrasonic imaging device or a Doppler ultrasonic imaging device. In some embodiments, the ultrasonic waves induce a liquid-to-gas phase transition in the peptide- based nanoparticles that generates echogenic microbubbles.[0174| In additional to detecting atherosclerotic plaque formation, the compositions described herein that comprises peptide-based nanoparticles comprising a peptide analogue of 313274296v2148411.004402 PATENTApo- A I to a tissue may be used to treat atherosclerotic plaques. As described in the Examples herein, simply contacting the compositions described herein that comprises peptide-based nanoparticles comprising a peptide analogue of Apo-Al to atherosclerotic plaques can reduce their lipid burden, because the binding of the peptide-based nanoparticles to oxidized low- density lipoprotein (oxLDL) within lipid-rich foamy macrophages in the plaques reduced the lipid burden of the foamy macrophages. To this base technique, ultrasound can also be optionally administered to the tissue to induce a liquid-to-gas phase transition in the peptide- based nanoparticles. In some embodiments, the liquid-to-gas phase transition of the peptide- based nanoparticles results in mechanical fractionation of intracellular lipid droplets within lipid-rich foamy macrophages, further breaking down the plaques. Lastly, in some embodiments, statin drugs present within the nanoparticles as a cargo can be released into the macrophages of the plaques, both by simple diffusion and by active cavitation of the nanoparticles by ultrasonic waves.

[0175] The present disclosure is illustrated and further described in more detail with reference to the following non-limiting enumerated embodiments and examples.Enumerated Embodiments1. A composition comprising a plurality of peptide-based nanoparticles,wherein each peptide-based nanoparticle comprises a perfluorocarbon liquid core and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core, wherein each amphiphilic peptide is represented by Formula (X):HB-SP-APO (X)wherein HB is a fluorinated hydrophobic block consisting of three to five consecutively connected pentafluorinated hydrophobic amino acid residues;wherein SP is a spacer amino region consisting of one to five glycine residues; wherein APO is an amino acid sequence that is a peptide analogue of' Apolipoprotein A1 (Apo-A1),wherein the amphiphilic peptides are oriented such that groups HB of the amphiphilic peptides are interpolated into the perfluorocarbon liquid of the perfluorocarbon liquid core and groups APO extend away from the perfluorocarbon liquid core.148411.004402 PATENT2. The composition of embodiment 1, wherein FIB consists of three, four or five consecutively connected pentafluoro-phenylalanine residues, and is located at the N-terminal end of the peptide sequence.3. The composition of embodiment 1 or 2, wherein SP consists of one glycine residue.4. The composition of any one of embodiments I to 3, wherein APO consists of the amino acid sequence DWFKAFYDKVAEKFKEAF (SEQ ID NO: 51).5. The composition of any one of embodiments 1 to 4, wherein each amphiphilic peptide comprises the amino acid sequence FFFFFFFFFFGDWFKAFYDKVAEKFKEAF (SEQ ID NO: 52), wherein Fr is pentafluoro-phenylalanine.6. The composition of any one of embodiments 1 to 5, wherein the composition further comprises a pharmaceutically acceptable excipient selected from the group consisting of a vehicle, an adjuvant, a carrier, and a diluent.7. The composition of any one of embodiments 1 to 6, wherein the peptide-based nanoparticles further comprise a cargo contained within the perfluorocarbon liquid core.8. The composition of embodiment 7, wherein the cargo is a statin drug.9. The composition of embodiment 8, wherein the statin drug is Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, or Simvastatin.10. A composition comprising a plurality of cells,wherein each cell comprises at least one peptide-based nanoparticle,wherein the at least one peptide-based nanoparticle comprises a perfluorocarbon liquid core and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core. wherein each amphiphilic peptide is represented by Formula (X):HB-SP-APO (X)wherein HB is a fluorinated hydrophobic block consisting of three to five consecutively connected pentafluorinated hydrophobic amino acid residues;29313274296v2148411.004402 PATENTwherein SP is a spacer amino region consisting of one to five glycine residues; wherein APO is an amino acid sequence that is a peptide analogue of Apolipoprotein A1 (Apo-A1),wherein the amphiphilic peptides are oriented such that groups HB of the amphiphilic peptides are interpolated into the perfluorocarbon liquid of the perfluorocarbon liquid core and groups APO extend away from the perfluorocarbon liquid core.11. The composition of embodiment 10, wherein each cell of the plurality of cells is a macrophage.12. The composition of embodiment 10 or 11 wherein HB consists of three, four or five consecutively connected pentafluoro-phenylalanine residues, and is located at the N-terminal end of the peptide sequence.13. The composition of any one of embodiments 10 to 12, wherein SP consists of one glycine residue.14. The composition of any one of embodiments 10 to 13, wherein APO consists of the amino acid sequence DWFKAFYDKVAEKFKEAF (SEQ ID NO: 51).15. The composition of any one of embodiments 10 to 14, wherein each amphiphilic peptide comprises the amino acid sequence FFFFFFFFFFGDWFKAFYDKVAEKFKEAF (SEQ ID NO: 52), wherein FF is pentafluoro-phenylalanine,16. The composition of any one of embodiments 10 to 15. wherein the composition further comprises a pharmaceutically acceptable excipient selected from the group consisting of a vehicle, an adjuvant, a carrier, and a diluent.17. The composition of any one of embodiments 10 to 16, wherein the peptide-based nanoparticles further comprise a cargo contained within the perfluorocarbon liquid core.18. The composition of embodiment 17, wherein the cargo is a statin drug.30313274296v2148411.004402 PATENT19. The composition of embodiment 18, wherein the statin drug is Atorvastatin. Fluvastatin, Lovastatin. Pitavastatin, Pravastatin, Rosuvastatin, or Simvastatin.20. A composition comprising a plurality of macrophages,wherein each macrophage comprises at least one peptide-based nanoparticle, wherein the at least one peptide-based nanoparticle comprises a perfluorocarbon liquid core, a cargo contained with the perfluorocarbon liquid core, and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core,wherein each amphiphilic peptide comprises FF FFFFFFFGDWFKAFYDKV EKFK. EAF (SEQ ID NO: 52), wherein Ff is pentafluoro-pheny lalanine, andwherein amphiphilic peptides are oriented such that the FFFFFFFFFF region is interpolated into the perfluorocarbon liquid core and the DWFKAFYDKVAEKFKEAF (SEQ ID NO: 51) region extends away from the perfluorocarbon liquid, andwherein the cargo is a statin drug.21. A method of preparing the composition comprising a plurality of cells of any one of embodiments 10 to 20, the method comprising:contacting a perfluorocarbon liquid with a plurality of amphiphilic peptides to form a plurality of peptide-based nanoparticles, wherein each peptide-based nanoparticle comprises a perfluorocarbon liquid core and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core, andcontacting the plurality of peptide-based nanoparticles with the plurality of cells, where in each cell of the plurality of cells internalizes at least one peptide-based nanoparticle.22. The method of embodiment 21, wherein water is added to plurality of peptide-based nanoparticles after their formation.23. The method of embodiment 21 or 22. wherein a statin drug is added to the perfluorocarbon liquid prior to contacting the perfluorocarbon liquid with the plurality of amphiphilic peptides to form the plurality of peptide-based nanoparticles.24. The method of any one of embodiments 21 to 23, wherein the cell is a macrophage.31313274296v2148411.004402 PATENT25. A method of detecting atherosclerotic plaques, comprising:administering a composition of any one of embodiments I to 20 to a tissue, administering ultrasonic waves to the tissue, anddetecting the location of atherosclerotic plaques in the tissue by locating acoustic properties of the peptide-based nanoparticles.26. The method of embodiment 25, wherein the ultrasonic waves are administered to the tissue by a B-mode ultrasonic imaging device or a Doppler ultrasonic imaging device.27. The method of embodiment 25 or 26. wherein the ultrasonic waves induce a liquid-togas phase transition in the peptide-based nanoparticles that generates echogenic microbubbles.28. A method of treating atherosclerotic plaques, comprising administering a composition of any one of embodiments 1 to 20 to a tissue with atherosclerotic plaques.29. The method of embodiment 28, wherein binding of the peptide-based nanoparticles to oxidized low-density lipoprotein (oxLDL) within lipid-rich foamy macrophages reduces the lipid burden of the foamy macrophages.30. The method of embodiment 28 or 29, further comprising administering ultrasonic waves to the tissue to induce a liquid-to-gas phase transition in the peptide-based nanoparticles.31. The method of embodiment 30, wherein the liquid-to-gas phase transition of the peptide-based nanoparticles results in mechanical fractionation of intracellular lipid droplets within lipid-rich foamy macrophages.32. The method of any one of embodiments 28 to 31, wherein, when the peptide-based nanoparticles comprise a statin drug in the perfluorocarbon liquid core, the statin drug is released into the lipid-rich foamy macrophages.ExamplesExample 1: Nanoparticle Synthesis and Characterization Methods

[0176] Materials And General Methods32313274296v2148411.004402 PATENT

[0177] Fmoc-protected amino acids were purchased from Novabiochem. PL-Rink resin was purchased from Polymer Laboratories. 1H-Benzotriazolium 1-[bis(dimethylamino)methylene]-5chloro-hexafluorophosphate (1-),3-oxide (HCTU) was obtained from Peptides international. Tri fluoroacetic acid was obtained from Acros organics, and 1,2-ethanedithiol was purchased from Fluka. Oregon Green 514 Phalloidin, 6 arid 24 well cell culture plates, polystyrene microcuvettes, diethyl ether, dimethylformamide (DMF), acetonitrile (ACN). N- methylpyrrolidone (NMP), Slide-A-LyzerTMdialysis cassettes (MWCO 3.5K) and 96-well half area high content imaging glass bottom microplates were purchased from Fisher Scientific. Perfluorohexane (PFH) and perfluoropentane (PFP) were purchased from Oakwood Chemicals and Strem Chemicals, respectively. N,N-diisopropylcarbodiimide (DIC) was purchased from Chem Impex. Thioanisole, anisole, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2,2'-dithiodipyridine (DTP), dimethyl sulfoxide (DMSO), diazabicyclo[5.4.0]undec-7-ene (DBU) and 200 mM glutamine solution were obtained from Sigma-Aldrich. RPMI-1640 media. Hanks Balanced Salt Solution (HBSS) and Hoechst 33342 trihydrochloride dye was purchased from Invitrogen. Phosphate buffered saline (PBS) IX without calcium and magnesium, and L~ glutamine (L-Gln), were purchased from Corning. Heat inactivated fetal bovine serum (FBS) and trypsin EDTA were obtained from Hyclone Laboratory Inc. HPLC solvents consisted of solvent A (0.1% TFA in water) and solvent B (0.1% TFA in ACN) Gentamycin was purchased from VWR. RPMI-1640 without L-glutamine was purchased from Lonza. Hoechst 33342 and UltraPure™ agarose were purchased from Invitrogen. 4% paraformaldehyde in PBS was purchased, from Chem Cruz. The green fluorescent protein (GFP, 36kDa) was obtained from Dr, J. P. Schneider (Chemical Biology Laboratory, NCI) and A549 human cancer cell line was obtained from the NCI-60 repository. AU peptides utilized for experiments were prepared with an amidated C-terminus.

[0178] Peptide Synthesis

[0179] Fmoc-based solid-phase peptide chemistry was used to prepare the amphiphilic peptides, with HCTU activation on PL-Rink resin using an automated ABI 433A peptide synthesizer. Amphiphilic peptides were cleaved from the resin and simultaneously side-chain deprotected using a trifluoroacetic acid / thioanisole / 1,2-ethanedithiol / anisole (90:5:3:2) cocktail for 2 hours under argon atmosphere. The crude product was precipitated with cold diethyl ether and then lyophilized. Amphiphilic peptides were purified via reverse-phase HPLC equipped with a FluoroFlash® semi-preparative column composed of silica gel bonded with perfluorooctylethylsilyl (Si(CH2)2C8F17). A gradient of 0-50% solvent B over 25 min..33313274296v2148411.004402 PATENTfollowed by 50- 100% solvent B over an additional 50 min. was utilized. All amphiphilic peptides were lyophilized to collect the pure product, and the purity verified by analytical HPLC-MS.10180] Nanoparticle Formation(0181] Peptides were weighed out as a dry fluffy solid in a round bottom flask, and dissolved in a volume of 1:1 DM. F: ACN containing 1 % TFA to a final concentration of 0.5 - 2.0 nig / mL. The solution was stirred at 1,000 rpm on ice for 1 min. before addition of 1% - 2% (v / v) cold PFP. After an additional 5 min. of stirring to properly mix the components and create an emulsion, an equal volume of cold MilliQ water was slowly added dropwise. During this solvent exchange procedure the solution turned opaque due to self-assembly of the peptides at the interface of the water-PFP emulsion. The mixture was stirred at 1,000 rpm for 1 hour on ice, over which time the solution clarified. Unincorporated peptide was removed by dialyzing the mixture against MilliQ water containing 2.5% (v / v) DM'SO to oxidize the cysteines and facilitate disulfide cross-linking of the amphiphilic peptides of the nanoparticles. In addition, a Pasteur pipette was used to gently bubble air into the media to further promote oxidation. Dialysis was performed for 12 hours, with exchanges every four hours. Two final exchanges of the dialysis media to pure MilliQ water, for 2 hours each, removed residual DMSO. The purified nanoparticles were removed from the dialysis cassette, placed into a clean glass vial and used for experiments within 48 hours.

[0182] Physico-Chemical Characterization

[0183] Particle size and zeta potential measurements were performed via dynamic light scattering using a Zetasizer Nano-ZS instrument (Malvern, Worcestershire, UK). For size determination, a solution of nanoparticles in water was diluted times into characterization buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.4) to reach a final volume of ImL in a clean polystyrene microcuvette. The size of pure 1-2 vol% PFP emulsions prepared in 1:1 DMF: ACN containing 1% TFA were also measured as controls. Three independent measurements, ten runs each, were taken at a 175° scattering angle, a sample position of 4.65 mm and an attenuation of 11, Particle size was recorded at both 25 °C and 37!’C, with a 2 min. sample equilibration time. Material refractive index (RI) was set at 1.59 (25°C) and 1.45 (37°C) using pre-defined settings provided by the manufacturer. Dispersant Rl of 1.332 and viscosity [cP] equal to 0.9103 (25°C) and 0.7096 (37°C) were calculated using the ‘Solvent Builder’ tool in the Zetasizer software. Phase analysis light scattering (PALS) assisted zeta potential measurements were performed by adding the solution of nanoparticles to MilliQ water to313274296v2148411.004402 PATENTachieve a ten-fold dilution and loading 700 pL of the sample into a disposable folded capillary cell (Malvern, DTS 1070). Three independent measurements were taken at 25°C, with twenty runs each.

[0184] In separate studies, the stability of nanoparticles during storage was evaluated via dynamic light scattering. Here, purified particles (formulation B of Table 1) were dispersed into milliQ water and left at room temperature. At defined time points over 15 days an aliquot was removed, diluted ten times into characterization buffer, and particle size and count rate recorded at 25°C. Of note, count rate was used as a qualitative indicator of particle density and thus an estimate of stability over time. In parallel experiments, the same particles were initially diluted ten times into blank characterization buffer, or buffer supplemented with 5% fetal bovine serum, and incubated at 37°C to evaluate their stability under physiologic conditions. At defined time points over 48 hours a 1 mL aliquot was directly added to a clean polystyrene microcuvette and particle size measured at 37°C. For both experiments, three independent measurements were taken with twenty runs each.

[0185] Nanoparticle Visualization

[0186] Differential interference contrast (DIC) microscopy was used to image the nanoparticles in solution. Briefly, nanoparticles were diluted two times into characterization buffer and added to 96- well glass bottom high-content imaging microplates. The plates were then, loaded onto an LSM 710 confocal microscope (Zeiss, Thornwood, ’NY) equipped with a temperature controlled humidified chamber. Images were collected at 25 °C and 37Y", with a 15 min. sample equilibration time, using a 63x Plan- Apochromat oil objective.

[0187] Disulfide Formation Assay

[0188] A 1.5 mL solution of freshly prepared nanoparticles in water (0,5 mg / ml, peptide and 2% PFP) was placed in a round botom flask and slowly stirred with gentle bubbling of air. A 2.5% volume of DMSO was added to oxidize the thiols and initiate cross-linking. At specific time points, a 30 pL aliquot of the mixture was diluted into 200 pL of 0.1 mM DTP in characterization buffer, and allowed to react for 10 min. The solution was then transferred to a quartz cuvette (1 cm pathlength) and concentration of the free thiolate was determined via absorption at 343 nm (E₃₄₃ = 7600 cm⁻¹ M⁻¹) (Haines et al., 2005) using an Agilent 8453 UV- Vis spectrophotometer (Santa Clara, CA). In separate control experiments, the same procedure was followed without the addition of 2.5% (v / v) of DMSO to evaluate its influence on thiol oxidation and disulfide cross-linking of nanoparticles. All values were corrected for background DTP hydrolysis. Percentage of disulfide formation was calculated by subtracting 353t 3274296’, -2148411.004402 PATENTthe concentration of free thiolate from the initial cysteine concentration. Studies were performed in triplicate.

[0189] Cell Viability Assay

[0190] A549 cells were seeded onto 6 well plates at 5 x 10⁵ cells / well and allowed to adhere overnight. Cells were then washed and 3 mL of warm HBSS added to each well. Ultrasound was applied for 90 sec at a duty cycle of 10%-20% with intensity varied between 0.1-1.0 W / cm², corresponding to a peak negative pressure of 0.054–0.172 MPa, respectively. Wells not subjected to US, or cells incubated with 25% DMSO in HBSS for 1 hour, were used as negative and positive controls, respectively. Following US insonation, cells were incubated for 30 min. to recover and then washed with warm HBSS. 3mL of a 0.5 mg / mL solution of MTT reagent in HBSS was added to each well and incubated for 2 hours. The supernatant was removed and replaced with 3 mL of DMSO to dissolve the formazan product, followed by transfer of a 100 µL aliquot of the colored solution to a 96 well plate. Absorbance was then read at 540 nm using a UV plate reader (Biotek, Winooski, VT). The absorbance of negative controls was subtracted from each sample as a blank, and percent viability calculated using the equation: (Absorbance US-treated cells / Absorbance untreated cells) × 100. Results shown represent the average of three independent experiments ± standard deviation.

[0191] Results

[0192] As evidenced by the above data, the de novo designed peptide. F_FF_FGGGCCGGKGRGD-NH₂ (SEQ ID NO:49) is capable of assembling at the surface of a perfluoro-n-pentane (PFP) droplet. The peptide sequence contains three pentafluoro-phenylalanine (F_F) residues at its N-terminus, which promotes interpolation and assembly of the peptide at the PFP-liquid interface. C-terminal to this fluorous domain is a cysteine containing motif, GGGCCGG (SEQ ID NO:46), designed to undergo disulfide cross-linking to stabilize the peptide corona after templated assembly. Incorporation of a bioactive hydrophilic sequence at the peptide’s C-terminus ultimately leads to its multivalent display at the surface of the assembled particle. In this particular design, the sequence KGRGD (SEQ ID NO:35) has been included to enable cell-surface localization of the nanoparticle mediated by binding of RGD with extracellular integrins. Despite inclusion of highly fluorinated residues, this sequence was able to be chemically synthesized in high yield and purity using standard solid-phase techniques.

[0193] It has been found that, to form nanoparticles, a solvent-exchange procedure in which cold water is slowly added to an organic emulsion of amphiphilic peptides and PFP, ultimately 36313274296v2148411.004402 PATENTleads to spontaneous assembly of the amphiphilic peptides at the surface of PFP liquid core. Importantly, this mild procedure eliminates the need for aggressive synthetic methods commonly used to prepare stimuli-responsive particles, which can lead to degradation of the encapsulated cargo. Subsequent dialysis against 2.5% DMSO in water removes unincorporated peptide, and promotes disulfide cross-linking of cysteine residues in the perfluorocarbon liquid core corona. Cross-linked nanoparticles remain stable for multiple weeks when stored at room temperature in water (FIG. 3 ).

[0194] It has also been found that the size of nanoparticles could be precisely controlled between 250 nm and 1,200 nm, as a function of peptide and PFP feed ratio (FIG, 6, Table I), Table 1: Physicochemical properties of nanoparticle forma ationsDroplet size [rim 1Formulation ^Amphiphilic (PFP [vol 25°C 37°C A g, j[Peptide Conc.lgo] [mV][mg-niL] |E 11.00 [2 1.2 x 10’ 43 x 1.0336.5 3.540.2D h.oo h 863: 98 23 x HF 26.7 0.1 -.0.2C [0.75 U 739±56 7.5 x 10’ 10.1 6.8±0.5B* 4 b.?5 q 469.424 482±34 EQ _ y%o;4. _B [0.50 [2 453.4.29 528.424 1.3 8.2.40.4A*W |0.25 [2 44842“ 458423 1.0 4.640,2■A [0.50 H 3014(4 390±12 1.3 12.440.41’1 Formulations A* and B* are not shown in FIG. 6 due to their similar size to formulation B.Sbjfold change in particle size at 37°C versus 25oC.icJzetapotential.

[0195] Dynamic light scattering performed on pure PFP emulsions indicates this may be due, in part, to different sizes of PFP droplets formed in the starting emulsion (FIG. 4), At any rate, the ability to control the hydrodynamic radii of the particles is critically important for delivery applications, as this parameter is inversely correlated with passive tissue distribution, and directly proportional to the LIS magnitude required for droplet cavitation.

[0196] Also evaluated was the influence of temperature on nanoparticle size through direct visualization of particles in solution using differential interference contrast (D1C) confocal microscopy, as well as dynamic light scattering analysis. Results show that nanoparticles with a diameter <750 nm at 25°C were able to maintain their size when heated to physiologic temperature, a vital requirement for acoustic droplet vaporization in vitro and in vivo (Shpak et al., 2014). Exceeding this size threshold led to premature PFP vaporization (bp ~−29°C) and converted the perfluorocarbon liquid cores into gaseous microbubbles at 37°C, as evident by the massive increase in diameter for the purple, green and orange formulations. This influence 37313274296v2148411.004402 PATENTof particle size on the vaporization temperature of PFP is due to the inverse relationship between internal pressure and droplet dimension, as described by the Laplace pressure Equation (1):+ (D

[0197] where Pinand Patmare the internal droplet pressure and atmospheric pressure, respectively, γ is the interfacial surface tension and R_H represents the hydrodynamic droplet radii. Here, decreasing the droplet size leads to an increase in the pressure exerted on the PFP core, ultimately keeping the fluorous liquid in a superheated state well above its bulk boiling point of 29°C. The influence of vapor pressure on the temperature of the PFP solvent can be defined using the Antoine vapor equation (2):

[0198] in which T and P represent temperature and pressure, respectively, while A, B, and C are equation parameters empirically determined for PFP (Barber et al., 1956). Combining the Laplace pressure (1) and Antoine vapor (2) equations provides a single expression describing the temperature at which the vapor pressure of the core is equal to the internal droplet pressure (TSP), ultimately causing thermal droplet vaporization (3):Tvap= ... (3)B / (A-log₁₀(P_in+P_atm))-C

[0199] Using this equation, the relationship between Tvap and droplet size can be modeled using reported surface tension values for PFP emulsions formulated with either BSA (0.033 him’1), the amphiphilic polymer PEO-PLA (0.027 Nm⁻¹), or the cationic surfactant cetrimonium bromide (CTAB; 0.013 Nnf’) (FIG. 5) (Kandadai et al., 2010). Of note, the PFP- CTAB formulation most closely resembles the nanoparticles reported here, in which the cationic amphiphilic sequence acts as the surfactant. Results from the model show that, at a surface tension of 0.013 Nm⁻¹, the vaporization point of the PFP core is expected to be >378&C when particles are <800 nm in size, a finding that closely matches the experimental threshold identified for the PFP-peptide emulsions.

[0200] This suggests that the US energy required to thermally vaporize the nanoparticle core could be carefully controlled by modulating the droplet size, as well as changing the interfacial surface tension through tuning the amphiphilic character of the assembling peptide.

[0201] As described herein, a new class of peptide-based nanodroplets, nanoparticles, capable of" ultrasound-mediated delivery of membrane-impermeable cargo into cells has been 38313274296v2148411.004402 PATENTdeveloped. In this example, nanoparticles are prepared via the de novo designed peptide F_FF_FGGGCCGGKGRGD-NH₂ (SEQ ID NO:49). which efficiently assembles at the surface of organofluorine droplets, and undergoes cysteine-mediated cross-linking to stabilize the final nanostructure. Biomolecular cargo can be readily encapsulated within the nanoparticle carrier during the assembly process. Cell binding of the nanoparticles, followed by acoustic vaporization, ultimately delivers the cargo into cells. Gaseous microbubbles generated during vaporization of nanoparticles may also function as a US contrast agent to allow for imaging and guidance of the delivery modality in real-time. Thus, nanoparticles of the present invention represent a potential theranostic system with broad applications in drug delivery and biomedical imaging.Example 2: Visualizing NP_GFP tracking and vaporization by ultrasound imaging

[0202] Ultrasound waves allow' for image-guidance and temporal vaporization of NPgtp for protein delivery'.[0203| For ultrasound B-mode and Doppler imaging, NPGFPprepared as above were diluted 1:10 in degassed DI water. A 1.5% agarose phantom was degassed before gelation into mold of 50 ml beaker with 1 cm glass tube imprint (•-- 2 cm deep within gel). The agarose phantom was placed on a block of neoprene, an acoustic absorbing material, in a large bucket of degassed waler. NPGFPwere imaged in B-mode and Doppler using L7-4 (5 MHz, 3 cycles) and L22-14v (18.5 MHz, 12 cycles) transducers. Using the Verasonics Matlab script, the voltage of the transducers was incrementally increased for image-guided particle tracking in B-mode and for particle acoustic droplet vaporization with Doppler mode. Video images were captured and In-phase quadrature (IQ) Doppler data was saved throughout the entire experiment. The L7-4, 5 MHz transducer, pressures were measured using a hydrophone. The pressure threshold of acoustic droplet vaporization was performed for three replicates for average ultrasound pressure necessary' for droplet vaporization.

[0204] Real-time monitoring of NPGFPwas captured with B-mode imaging from an 18.5 MHz transducer. Three individual NPGFPwere tracked with high echogenicity over six seconds at one second time intervals. This demonstrates that NPst-> are capable of use as ultrasound image-guided particles.

[0205] Additionally, the temporal vaporization of NPGFPwas captured with Doppler mode imaging from a 5 MHz transducer.39313274296v2148411.004402 PATENTExample 3: Ultrasound Detection of Atherosclerotic Plaques

[0206] Background

[0207] Atherosclerosis is a progressive inflammatory disease characterized by accumulation of lipid- and immune cell-rich plaques in the arterial walls. Macrophages, antigen-presenting cells of the innate immune system, play a key role in all stages of atherosclerotic progression. For example, oxidized low-density lipoprotein (oxLDL) within nascent lesions creates proinflammatory complexes that promote recruitment of monocyte- derived cells into the subendothelial space. These cells differentiate into macrophages that actively ingest oxLDL, generating lipid-rich foam cells which have a limited capacity for migration. < As a result, foamy macrophages persist within plaques and promote lipid storage, plaque growth and progression of the lesion to a complex, pro-inflammatory phenotype. In this advanced stage, continued secretion of pro-inflammatory factors by foam cells, recruitment of other immune subsets and vascular cells, and eventual death of the cellular infiltrates, creates an iterative cycle that ultimately generates a pro-thrombotic necrotic core. These unstable plaques are then prone to rupture, creating high-risk pathological lesions that can potentiate lethal myocardial infarctions and ischemic stroke.

[0208] The following Example 3 describes acoustically activated, phase-changing peptide nanoparticles designed to selectively bind to oxLDL-rich foamy macrophages to provide contrast-enhanced ultrasound (US) diagnosis of vulnerable atherosclerotic plaques (FIG. 7). Preferential labeling is accomplished by exploiting the thin fibrous cap. characteristic of rupture prone lesions, to permit intraplaque diffusion of the particles into at-risk anatomical sites. Rapid engulfment by resident foam cells, and selective binding to intracellular oxLDL, leads to retention of the emulsions within the pro-atherogenic cellular components. When stimulated by US, peptide nanoparticles undergo a liquid-to-gas phase transition that produces echogenic microbubbles in situ to serve as imaging nuclei. This behavior is shown to provide contrast-enhanced B-mode imaging of lesions. Under color Doppler US, the peptide nanoparticles exhibit a unique twinkling artifact that provides enhanced diagnostic precision of intraplaque foamy macrophage location in three-dimensional tissues.

[0209] Simultaneously, US vaporization of peptide nanoparticles is shown to stimulate efflux of oxLDL from foam cells, likely due to cavitation-induced mechanical fractionation of lipid droplets that facilitates transport of oxLDL to the cellular membrane. This suggests that, in addition to transforming the diagnosis of atherosclerotic lesions, this theranostic platform may therapeutically reduce lipid burden and shrink plaques in a non-invasive fashion. By 40313274296v2148411.004402 PATENTleveraging the low-cost, portable, and non-ionizing nature of diagnostic US, this modality provides real-time, continuous, and radiation-free theranosis of atherosclerotic arterial burden.

[0210] In sum. non-invasive imaging modalities that identify rupture prone atherosclerotic plaques hold promise to improve risk stratification and advance early intervention strategies. The following Example 3 describes the development of phase-changing peptide nanoparticles as theranostic contrast agents for synchronous ultrasound diagnosis and therapy of at-risk atherosclerotic lesions. By targeting lipids within atherogenic foam cells, and exploiting characteristic features of vulnerable plaques, nanoparticles preferentially accumulate within lesions and are retained by intraplaque macrophages. The following Example 3 demonstrates that acoustic vaporization of intracellular nanoparticles both promotes lipid efflux from foam cells and generates echogenic microbubbles that provide contrast-enhanced ultrasound identification of lipid-rich anatomical sites. In Doppler mode, peptide nanoparticles generate color twinkling features due to random phased reflection of incident acoustic waves from stably oscillating bubbles. Acoustic twinkling is unique to the peptide nanoparticles, and not observed from endogenous bubble nuclei in the tissues, degenerating diagnostic features that offer spatial precision of lesion identification in tissues.

[0211] Development and Characterization of Peptide EmulsionsJ0212] Athersclerotic plaque-targeting peptide nanopailieles were prepared using the de novo designed peptide emulsifier: FFFFFFFFFFFFGDWFKAFYDKVAEKFKEAF (SEQ ID NO: 52) (Fp = pentafluoro phenylalanine). The C -terminal 18-mer represents a reported peptide analogue of the Apolipoprotein A l (Apo- I), a primary component of high-density lipoprotein, that specifically binds to oxidized lipids. This is conjugated io a fluorine-rich C- terminal tail that promotes interfacial assembly of the peptide emulsifier at the surface of a perfluorocarbon (PFC) nanodroplet to generate colloidally stable emulsions. A glycine spacer separates these two functional sequences to preserve the a-helical structure of the oxLDL- targeting motif, which is essential for its lipid-binding specificity. Mixing the peptide emulsifier with the PFC solvent perfluoropentane (PFP), and then sonicating the mixture with distilled water, generated a dispersion of oxLDL-targeting peptide nanoparticles (FIG. 8 A), hereafter referred to as Apo-NPeps, with an average diameter of approximately 500 nm (FIGs.8B-8C). This size is ideal for cardiovascular labeling as it is sufficient to promote contact of Apo-NPep emulsions with the arterial wall, which sediment in hemodynamic flow due to the density of the PEP particle core (ρPFP = 1.6 g / mL, ρblood = 0.9 g / mL), while avoiding rapid renal clearance. Optical density measurements at 4 C and 37°C demonstrated that Apo-NPep 41313274296v2148411.004402 PATENTremains colloidally stable over the first day of storage (FIG. 9) and persisted in sufficient yield for imaging studies up to 10 days (47% and 26% remaining at 4°C and 37°C, respectively).10213] Apo- Pep Targeting of Intraplaque oxLDL

[0214] Next, the oxLDL-binding specificity of the nanoparticles was assessed using confocal laser scanning microscopy (FIGs. 10A-10B and 11A-11B). These studies included nanoparticles prepared from a scrambled version of the Apo-targeting peptide (Scr-NPep) to evaluate the specificity of Apo-NPep binding to oxLDL. Micrographs in FIGs. lOA-lOB demonstrate a clear co-localization of Apo-NPep with Dil -labeled oxLDL, with no such specificity observed for Scr-NPep controls. This suggests Apo-NPeps bind to oxLDL through specific ligand-target interactions, as opposed to non-specific adsorption to the surface of the lipid droplets. Spatially collated mapping of the Apo-NPep brightfield and Dil-oxLDL signals support this assertion (FIG. 11 A), with quantification of these results demonstrating a 3.5-fold higher percentage of particle-oxLDL co-localization for Apo-NPep (98.0 ± 6.1%) relative to Scr-NPep (22.6 ± 20.8%) (FIG. 1 IB).[0215| Next, the uptake of Apo- and Scr-NPeps into oxLDL-laden foamy macrophages and their ability to bind to intracellular lipid droplets was assessed. To rule out artifacts caused by cell toxicity, the biocompatibility of the emulsions was first assessed in RAW 264.7 macrophages after a 24 h incubation with Apo-NPep and Scr-NPep formulations. No statistically significant loss of RAW 264.7 viability was observed at the imaging compatible concentration of both particles. In parallel assays, it was confirmed that treating RAW 264.7 macrophages with Dil-oxLDL (10 pg mL’1) for 4 hours was sufficient to generate lipid-rich foam cells. With these in vitro conditions optimized, fluorescently-labeled NPep nanoparticles were prepared and their time-dependent internalization into foamy macrophages via flow cytometry was tracked. Results demonstrated that maximum intracellular uptake of Apo-NPep occurred between 6 – 18 hours of incubation, and persisted within the macrophages for >24 hours. Follow up laser scanning confocal, microscopy of oxLDL-laden foam cells demonstrated significant co-localization of Apo-NPeps with intracellular lipid droplets at both 6 hours (FIG. 12) and 24 hours of incubation. Serial confocal Z-stack images (FIGs. 13-14) and fluorescence surface overlays (FIG. 15) further confirm the superposition of Apo-N Peps and oxL DL signals within foamy macrophages. TEM micrographs, shown in FIGs. 16A-16C, demonstrate that Apo-NPep nanoparticles completely envelope the oxLDL droplets in the macrophage lysosome, creating a coacervate-like internal structure (FIG. 16B). However, phagocytosed Scr-NPep controls do not show this same behavior (FIG. 16C) and are not observed localized 42313274296v2148411.004402 PATENTto the intracellular oxLDL droplets. Apo- Peps appeared to shrink the size of internal oxLDL droplets and induce a sponge-like morphology. This suggests Apo-NPeps may solubilize oxLDL to promote its removal from foamy macrophages. Conversely, intracellular oxLDL droplets in macrophages treated with Scr-NPeps appear morphologically similar to untreated controls, which corroborates later findings on the inability of Scr-NPeps to induce oxLDL efflux from foam cells. In sum. these results demonstrate the ability of Apo-NPeps to selectively target and bind oxLDL within atherogenic foamy macrophages, and reveal unique nanoarchitectures formed by the nanoparticle-functionalized intracellular lipid droplets that hints at additional therapeutic functionality.

[0216] Next, the ability of Apo-NPep nanoparticles to extravasate from the vasculature and accumulate within atherosclerotic lesions was investigated. Aortas from ApoE- / - mice fed a high-fat diet for 12—20 weeks were harvested and perfused with fluorescently labeled Apo- NPep formulations. This was then followed by a perfusion of Oil red O (ORO) to stain atherosclerotic plaques. Whole tissue imaging confirmed the presence of Apo-NPeps in the aortic arch (FIGs. 17A-17B), which is particularly prone to plaque accumulation due to the low fluid shear stress in these regions. As expected, Apo-NPep signals were highly co- localized with ORO-stained anatomical regions (FIG. 17C), indicating selective accumulation of the nanoparticles within atherosclerotic lesions. Control experiments confirmed Apo-NPep and ORO co-Iocalization was not due to non-specific hydrophobic interactions between these components, which could otherwise create artifactual staining results. The plaque-targeting specificity of Apo-NPep is likely due to both oxLDL-mediated permeabilization of the endothelial barrier, leading to preferential infiltration of nanoparticles into the subendothelial lesion space, and intraplaque retention of the nanoparticles as a result of oxLDL binding. Taken together, these results demonstrate that Apo-NPeps translocate across the fibrotic vascular surface of atherosclerotic plaques and selectively accumulate within intraplaque foamy macrophages, where they bind to intracellular oxLDL droplets to persist within the atherogenic cellular components.Acoustic Activation and oxLDL Efflux Activity of NPep Nanoparticles

[0218] Previous findings show that Apo-NPep alters the morphology of oxLDL structures within foam cells and suggests that acoustic cavitation of the bound nanoparticles may mechanically fractionate the lipid droplets io promote their efflux. To test this assertion, the acoustic sensitivity of the nanoparticles through optical density measurements was investigated (FIGs. 18 and I9A-19B). Results indicate that the population of acoustically vaporized Apo- 43313274296v2148411.004402 PATENTNPep nanoparticles increases monotonically with US intensity (FIG. 18), from 27% at 0.1 W cm-2to >70% at 2 W cm-2. This acoustic actuation of Apo-NPeps is due to a liquid-to-gas phase transition of the PFP core that occurs during the rarefaction (expansion) phase of the US waveform. Vaporization of the liquid nanoparticles to form microbubbles, a process referred to as cavitation, can be observed with the naked eye (see FIGs. 19A-19B). Under these conditions, US activation of Apo-NPeps within treated RAW 264.7 cells did not compromise their viability (FIG. 20).

[0219] Next, given the oxLDL destabilizing effects of the nanoparticles, and the anti¬ atherogenic properties of the Apo-A1 mimetic peptide, the efflux of lipids from foamy macrophages treated with NPep nanoparticles was investigated. In addition to the therapeutic Apo-NPep formulation, scrambled nanoparticles (Scr-NPep) and particles prepared from the enantiomeric, D-amino acid containing, Apo-A1 mimetic peptide emulsifier were included. The latter control formulation, referred to as D-Apo-NPep, tests the impact of emulsifier chirality on the efflux activity of NPep nanoparticles. The Apo-Al peptide interacts with ATP-binding cassette transporter ABCA1, a cholesterol efflux regulatory protein, to promote the removal of free cholesterol from cells. Therefore, inclusion of D-Apo-NPep in our experiments, which cannot bind, ABCA1 through traditional ligand-protein interactions, will test the importance of ABCA1 targeting on oxLDL efflux from NPep-treated foamy macrophages.

[0220] Time-dependent oxLDL efflux from the foamy macrophages was evaluated via flow cytometry analysis of NPep-treated foam cells with and without US exposure (see experimental design in FIG. 21). Results in FIG. 22 show that, in the absence of the US trigger, Apo-NPep nanoparticles (containing the natural L-chiral peptide) stimulate greater oxLDL efflux from macrophages compared to Scr-NPep and D-Apo-NPep. Importantly, this efflux occurred without acoustic particle cavitation, indicating that binding of intact Apo-Al mimetic nanoparticles to oxLDL is, alone, sufficient to reduce the lipid burden of foamy macrophages. This corroborates prior TEM results showing shrinkage of lipid droplets within Apo-NPep treated macrophages before the application of US (FIG. 16B), This effect also increases with time, with oxLDL release from Apo-NPep treated cells increasing 1.7-fold between the 6- and 24-hour incubation time points. As expected, Scr-NPep and D-Apo-NPep did not produce a statistically significant increase in lipid efflux relative to untreated controls, most likely due to their attenuated binding affinity to ABCA1. For D-Apo-NPep, in particular, its efflux activity may be further compromised by the altered α-helicity of the ABCA1 binding motif, which is reported to be a prerequisite for its interactions with oxLDL. US-treatment was found to further 44313274296v2148411.004402 PATENTenhance oxLDL efflux from foam cells in an intensity-dependent manner, but only in the presence of Apo-NPep nanoparticles (FIG. 23). This is presumably due to mechanical fractionation of intracellular lipid droplets by the cavitating particles. Notably, cavitation of Apo-NPep treated foam cells at the highest tested acoustic intensity (1.0 W cm-2) yielded a 3.7-fold increase in oxLDL efflux compared to non-insonated controls (0 W cm-2).(02211 Next, Simvastatin, an FDA-approved anti-atherosclerotic drug, was loaded into Apo-NPep nanoparticles to test whether US-mediated delivery of the pharmacologic cargo would further promote lipid efflux (FIG. 24). Importantly, Simvastatin demonstrates pleiotropic effects in macrophages, including promoting the efflux of cholesterol stored within lysosomes. In the absence of the US trigger. Simvastatin-loaded Apo-NPep emulsions yielded a -8% improvement in oxLDL efflux from foamy macrophages after 24 hours of treatment, relative to the non-drug loaded formulations (comparison of FIG. 24) (+Simvastatin, 0 W cm-2) to Apo-NPep results in FIG. 22 (-Simvastatin)). This suggests that Simvastatin may gradually diffuse out of the emulsions to enhance efflux activity in Apo-NPep treated foam cells. US activation of Simvastatin-loaded Apo-NPeps yielded improved results, with -32% removal of total intracellular lipid burden from Apo-NPep treated foam cells activated at the highest tested US intensity (1 W cm-2, see FIG. 24). Collectively, these findings demonstrate that Apo-NPeps can execute synergistic biochemical (ABCA1 binding and lipid solubilizing), mechanical (US cavitation) and pharmacologic (statin release) effects to stimulate rapid and efficient clearance of lipids from atherogenic foam cells.

[0222] US imaging of Apo-NPep Treated Foamy Macrophages in Tissues

[0223] The imaging potential of the nanoparticles was first evaluated by loading Apo-NPeps into tissue-mimetic agar phantoms and collecting B-mode US signals (FIG. 25). Once exposed to US (18 MHz; P+ = 1.0 MPa, P- = 0.6 MPa), Apo-NPeps generated echogenic microbubbles that could be clearly resolved from the background, producing a 5.8 signal-to-noise ratio (FIG.26). This confirms the rapid phase-shift of nanoparticles into bubble imaging nuclei in an acoustic field, further identified by movies demonstrating the buoyancy of the cavitating contrast agents. Next, the persistence of Apo-NPep signals following uptake of the nanoparticle into foam cells was investigated. B-mode imaging of Apo- Pep-loaded foamy macrophages, hereafter referred to as Apo-N Pep-foam cells, showed a further enhancement in the contrast of the nanoparticle imaging nuclei, producing a 9.2 -fold enhancement in signal-to-noise (FIGs.27-28). Control foam cells, void of intracellular Apo-NPep contrast agents, were acoustically invisible. The increase in B-mode contrast of Apo-NPep-foam cells relative to the free Apo- 45313274296v2148411.004402 PATENTNPep particles is likely due to accumulation of multiple nanoparticles within each cell. In fact, some cells had a sufficient intracellular load of the Apo-NPep nanoparticles that, following US conversion of the particles into microbubbles, the foamy macrophages became buoyant and raised to the medium surface. This hints at the potential for these nanoparticles to promote migration of foam cells out of atherosclerotic plaques, where US vaporization of phagocy tosed Apo-NPep may lead to cellular buoyancy that encourages their localization to the plaque- endothelial interface.

[0224] Next, the performance of free Apo-NPep nanoparticles and Apo- Pep-foam cells was validated in ex vivo tissues, using a porcine heart as an exemplary model (FIG. 29). To generate a facsimile of pathologic lesions, depots of free nanoparticles, or foamy macrophages loaded with Apo-NPep emulsions, were created adjacent to a coronary vessel by manually injecting the sample into the appropriate tissue anatomical site. B-mode imaging was then performed using a 5 MHz transducer, selected to enable deep tissue penetration of the acoustic signals and to match traditional frequencies of clinical cardiac ultrasonography (4 - 7 MHz), When free Apo-NPep emulsions were injected, a noticeable enhancement in B-mode greyscale contrast was observed (FIG. 30), confirming the contrast agents were active in physiologic tissues. Similar to results from our agar phantom experiments, Apo- Pep-foam cells produced the most intense imaging features, and therefore greatest contrast, while foamy macrophages without Apo-NPeps were acoustically silent (FIGs. 31A-31B). Importantly, the improved contrast of Apo-NPep-foam cells allowed for the dear resolution of the boundaries of the model, lesion and identification of the spatial localization of the plaque relative to the arterial wall.

[0225] Doppler Twinkling of Apo-NPep emulsions

[0226] While Apo-N Pep-foam cell lesions could be identified from the B-mode images, scattering of the acoustic signal in deep tissues may reduce feature resolution, increase the frequency of false positive signals, and collectively diminish the accuracy of the platform. This pitfall is shared by all current US contrast agents, and is caused by background B-mode speckling due to scattering of the signal from endogenous bubble nuclei in tissues. However, during the US imaging experiments, a unique imaging feature of Apo-NPeps that overcomes this limitation was discovered. When performing color Doppler US, rapid color changes were observed, referred to as acoustic ‘twinkling’, from the emulsions as they phase transitioned into echogenic microbubbles in agar phantoms (FIG. 32), Here, twinkling arises from random amplitude and phase changes of the US echo wave due to isotropic bubble movement, generating transient Doppler signals. The wide spectral bandwidth of Apo-NPep twinkling is 463! 3274296’, -2148411.004402 PATENTnot shared by endogenous tissue nuclei and so these twinkling features are specific to the contrast agents. This phenomenon allowed for the resolution of depots of Apo-NPep nanoparticles (FIG. 33) and Apo-NPep-foam cells (FIG. 34) more clearly in porcine tissue under Doppler imaging, relative to B-mode signals (FIGs. 36B-36C and FIG. 37), with precise spatial localization. Like B-mode imaging, foamy RAW 264.7 cells without Apo-NPep contrast agents are acoustically invisible in Doppler mode (FIG. 35). Collating the Doppler magnitude within the imaging frame further resolved the focal point of the twinkling features and improved the spatial precision of lesion identification (FIGs. 36A-36C).[02271 To quantitively compare the occurrence of twinkling among samples, the time¬ dependent variance in Doppler signals from control foam cells, free nanoparticles (Apo-NPep), and nanoparticle-loaded foam cells (Apo-NPep-foam cells) were plotted. Results in FIG. 37 demonstrate more frequent Doppler twinkling from the free nanoparticles compared to Apo- NPep-foam cells over the 35 second collection time. The extended twinkling features from the Apo-NPep in tissues compared to its shorter duration (- 10 seconds) in the agar cavity (FIG.32) may result from a high local concentration of particles within the tissues, which limits the displacement of nanoparticles in the acoustic field. Further, the reduced twinkling frequency of Apo-NPep-foam cells may be due, in part, to viscoelastic damping of cavitating microbubbles by the molecularly crowded intracellular environment However, collapsing the time-locked variance into a single integrated value demonstrates equivalent cumulative Doppler intensity from free nanoparticles and Apo-NPep-foam cells (FIG. 38). This suggests that the decreased frequency of nanoparticle twinkling inside foamy macrophages is compensated by the higher loading of particles within each cell, leading to a higher per event Doppler intensity for Apo-NPep-foam cells relative to nanoparticles alone. Apo-NPeps were observed to persist in labeled macrophages and provide Doppler twinkling features for over 7 days (FIG. 39). Finally, Apo-NPep nanoparticles stored for more than 2 months at 4l'‘C still produced twinkling features when imaged, demonstrating an extraordinary stability relative to traditional microbubble contrast agents. Collectively, these findings demonstrate that the distinct Doppler twinkling behavior of Apo-NPep can delineate atherosclerotic lesions within tissues with exceptional spatial precision, and provide stable, multi -day imaging persistence utilizing standard clinical US hardware.

[0228] T o mechanistically probe Apo-NPep Doppler twinkling, cavitation induced acoustic signals from nanoparticles loaded into an agar phantom were measured using a 1 MHz high intensity focused ultrasound (HIFU) activating transducer and passive cavitation detection 47313274296v2148411.004402 PATENT(PCD) hydrophone array (FIG. 40). By interpreting the harmonic periodicity and noise of the echo frequency spectra, stable cavitation, where microbubbles undergo pulsatile volumetric changes at their equilibrium radius, and inertial cavitation, where bubbles oscillate out of equilibrium and collapse, could be differentiated (FIG. 41 ). For this study, the cavitation activities of the nanoparticles were monitored at an intermediate (P+ ~ 1.3 MPa, P- ~ 1.0 MPa) and high (P+ ~ 2.5 MPa, P- ~ 2.0 MPa) acoustic pressure (FIGs. 42A-42D). Degassed PBS was analyzed as a control. Cavitation spectral density plots recorded at P- = 1 MPa showed harmonic echo frequencies from the activated nanoparticles at 2, 3, and 5 MHz (fundamental frequency - 1 MHz) (FIG, 42A). Increasing acoustic pressure to P- - 2.0 MPa yielded a subharmonic frequency at ~0.5 Hz (½ ω), along with an increased noise floor. (FIG. 42B), The appearance of harmonic and subharmonic frequencies, as well as lack of broadband acoustic emission, suggests vaporized Apo-NPeps stably cavitate in the acoustic field. Further, the increased noise at P- ~ 2.0 MPa suggests the bubbles enter an intermediate regime of destabilized oscillation that precedes inertial cavitation, but ultimately do not collapse. Interpreting the pulse-locked cavitation activity over 100 cycles supports this assertion, demonstrating a relatively stable cavitation signal at 11.5 mV2for Apo-NPep nanoparticles activated at P- — 1.0 MPa (FIG. 43) that was >50 times higher than the control (-0.2 mV2, FIG.44). The subtle decline in cavitation activity for Apo-NPep nanoparticles over the 100 pulse window may be due to fluidic transport of the bubbles out of the focal region in the agar cavity, which was observed via B-mode imaging. Conversely, at P- = 2.0 MPa a large fluctuation in cavitation activity was observed, which corresponded to a disappearance of the hyperechoic region in B-mode imaging captured under similar conditions (FIG. 42A). This is most likely due to acoustic movement of the deforming bubbles out of the focal region and into the agar gel. Taking the PCD results in conjunction with our Doppler twinkling imaging data, which demonstrates that twinkling features appear at intermediate acoustic amplitudes (P- = 1.6 - 1.8 MPa), suggests the origins of Apo-NPep Doppler twinkling most likely arise from stable cavitation. This indicates that Apo-NPep bubble nuclei can potentially be recondensed back to an nanoparticle after US sonication to permit reuse of the nanoparticle over multiple imaging cycles, therefore extending the persistence of the twinkling contrast agent in tissues.148411.004402 PATENTSEQUENCESSEQ ID NO: 1 - Minimal Targeting Motif IRGRRSEQ ID NO: 2 – Minimal Targeting Motif 2RGRRSSEQ ID NO: 3 - Minimal Targeting Motif 3YQLDVSEQ ID NO: 4 – Minimal Targeting Motif 4VGVASEQ ID NO: 5 – Hydrophilic amino acid sequence 1 including a targeting motif GHGKHKNKSEQ ID NO: 6 – Hydrophilic amino acid sequence 2 including a targeting motif CRGDKGPDCSEQ ID NO: 7 - Hydrophilic amino acid sequence 3 including a targeting motif CKGAKARSEQ ID NO: 8 - Hydrophilic amino acid sequence 4 including a targeting motif CRVSRQNKCSEQ ID NO: 9 – Hydrophilic amino acid sequence 5 including a targeting motif CGGERGKSCSEQ ID NO: 10 – Hydrophilic amino acid sequence 6 including a targeting motifCRSRKGSEQ ID NO: 11 - Hydrophilic amino acid sequence 7 including a targeting motif 49313274296v2148411.004402 PATENTCKAAKNSEQ ID NO: 12 - Hydrophilic amino acid sequence 8 including a targeting motif CRGRRSTSEQ ID NO: 13 – Hydrophilic amino acid sequence 9 including a targeting motif CEYQLDVESEQ ID NO: 14 - Hydrophilic amino acid sequence 10 including a targeting motif TVRTSADSEQ ID NO: 15 – Hydrophilic amino acid sequence 11 including a targeting motifPIEDRPMSEQ ID NO: 16 – Hydrophilic amino acid sequence 12 including a targeting motif ALRDRPMSEQ ID NO: 17 - Hydrophilic amino acid sequence 13 including a targeting motif PEKFRPMSEQ ID NO: 18 - Hydrophilic amino acid sequence 14 including a targeting motif IKVGKLQSEQ ID NO: 19 - Hydrophilic amino acid sequence 15 including a targeting motif SVSVGMKPSPRPSEQ ID NO: 20 - Hydrophilic amino acid sequence 16 including a targeting motif VPEQRPMSEQ ID NO: 21 - Hydrophilic amino acid sequence 17 including a targeting motif CAKIDPELCSEQ ID NO: 22 - Hydrophilic amino acid sequence 18 including a targeting motif 50313274296v2148411.004402 PATENTCSNIDARACSEQ ID NO: 23 - Hydrophilic amino acid sequence 19 including a targeting motif RLQLKLSEQ ID NO: 24 ~ Hydrophilic amino acid se uence 20 including a targeting motif PMMRQRPMSEQ ID NO: 25 - Hydrophilic amino acid sequence 2.1 including a targeting motif AKATCPASEQ ID NO: 26 - Hydrophilic amino add sequence 22 including a targeting motif QPPMEYSSEQ ID NO: 27 - Hydrophilic amino acid sequence 23 including a targeting motif SISSLTDSEQ ID NO: 28 - Hydrophilic amino add sequence 24 including a targeting motif FRVGVADVSEQ ID NO: 29 - Hydrophilic amino acid sequence 25 including a targeting motif CNGRCVSGCAGRCSEQ ID NO: 30 - Hydrophilic amino acid sequence 26 including a targeting motif NWGDRILSEQ ID NO: 31 - Hydrophilic amino acid sequence 27 including a targeting motif CVSNPRWKCSEQ ID NO: 32 - Hydrophilic amino acid sequence 28 including a targeting motif CDCRGDCFCSEQ ID NO: 33 - Hydrophilic amino acid sequence 29 including a targeting motif 51313274296v2148411.004402 PATENTYSAYPDSVPMMSSEQ ID NO: 34 - Hydrophilic amino add sequence 30 including a targeting motif PLASRPMSEQ ID NO; 35 ~ Hydrophilic amino acid sequence HP 1KGRGDSEQ ID NO: 36 - Hydrophilic amino acid sequence HP 2RGDSSEQ ID NO; 37 - Hydrophilic amino acid sequence HP 3GRGDSEQ ID NO: 38 - Hydrophilic amino add sequence HP 4GRGDSSEQ ID NO: 39 - Hydrophilic amino acid sequence HP 5GRGDSPSEQ I D NO: 40 - Hydrophilic amino acid sequence HP 6GRGDSPKSEQ ID NO: 41 - Hydrophilic amino acid sequence H P 7GRGDNPSEQ ID NO: 42 - Hydrophilic amino acid sequence HP 8GRGDTPSEQ ID NO: 43 - Targeting agent included in the hydrophilic amino acid sequence and used to target atherosclerosis or blood clotsCRKRLDRNC148411.004402 PATENTSEQ ID NO: 44 - Targeting agent included in the hydrophilic amino acid sequence and used to target thrombin in blood clotsEFEEFEIDEEEKSEQ ID NO: 45 - Targeting agent included in the hydrophilic amino acid sequence and used to bind to thrombin in blood clotsDFEEIPEEYLQSEQ ID NO: 46 - Cross-linking motif CLGGGCCGGSEQ ID NO: 47 - Formula (IV) amphiphilic peptideFFFFFFGGGCCGGKGRGDSEQ ID NO: 48 – C-terminally amidated hydrophilic amino acid sequence HP KGRGD-NH2SEQ ID NO: 49 - Formula (V) amphiphilic peptideFFFFFFGGGCCGGKGRGD-NH2SEQ ID NO: 50 - Hydrophilic amino acid sequence 31 including a targeting motif CRGRRTSEQ ID NO: 51 - peptide analogue of Apolipoprotein Al (Apo-Al) DWFKAFYDKVAEKFKEAFSEQ ID NO: 52 - Atherosclerotic plaque-targeting peptideFFFFFFFFFFFGDWFKAFYDKVAEKFKEAF

Claims

148411.004402 PATENTCLAIMSWhat is claimed is:

1. A composition comprising a plurality of peptide-based nanoparticles,wherein each peptide-based nanoparticle comprises a perfluorocarbon liquid core and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core, wherein each amphiphilic peptide is represented by Formula (X):HB-SP-APO (X)wherein HB is a fluorinated hydrophobic block consisting of three to five consecutively connected pentafluorinated hydrophobic amino acid residues;wherein SP is a spacer amino region consisting of one to five glycine residues; wherein APO is an amino acid sequence that is a peptide analogue of Apolipoprotein Al (Apo-A l),wherein the amphiphilic peptides are oriented such that groups HB of the amphiphilic peptides are interpolated into the perfluorocarbon liquid of the perfluorocarbon liquid core and groups APO extend away from the perfluorocarbon liquid core.

2. The composition of claim 1, wherein HB consists of three, four or five consecutively connected pentafluoro-phenylalanine residues, and is located at the N-terminal end of the peptide sequence.

3. The composition of claim 1, wherein SP consists of one glycine residue.

4. The composition of claim 1, wherein APO consists of the amino acid sequence DWFKAFY'DKVAEKFKEAI- (SEQ ID NO: 51).

5. The composition of claim 1, wherein each amphiphilic peptide comprises the amino acid sequence FFFFFFFFFFFGDWFKAFYDKVAEKFKEAF (SEQ ID NO: 52), wherein FFis pentafluoro-phenylalanine.

6. The composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable excipient selected from the group consisting of a vehicle, an adjuvant, a carrier, and a diluent.54313274296v2148411.004402 PATENT7. The composition of claim 1, wherein the peptide-based nanoparticles further comprise a cargo contained within the perfluorocarbon liquid core.

8. The composition of claim 7, wherein the cargo is a statin drug,9. The composition of claim 8, wherein the statin drug is Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, or Simvastatin.

10. A composition comprising a plurality of cells,wherein each cell comprises at least one peptide-based nanoparticle,wherein the at least one peptide-based nanoparticle comprises a perfluorocarbon liquid core and a plurality of' amphiphilic peptides surrounding the perfluorocarbon liquid core, wherein each amphiphilic peptide is represented by Formula (X):HB-SP-APO (X)wherein HB is a fluorinated hydrophobic block consisting of three to five consecutively connected pentafluorinated hydrophobic amino acid residues;wherein SP is a spacer amino region consisting of one to five glycine residues; wherein APO is an amino acid sequence that is a peptide analogue of Apolipoprotein A1 (Apo-A1),wherein the amphiphilic peptides are oriented such that groups HB of the amphiphilic peptides are interpolated into the perfluorocarbon liquid of the perfluorocarbon liquid core and groups APO extend away from the perfluorocarbon liquid core.

11. The composition of claim 10, wherein each cell of the plurality of cells is a macrophage.12, The composition of claim 10, wherein HB consists of three, four or five consecutively connected pentafluoro-phenylalanine residues, and is located at the N-tenninal end of the peptide sequence.13, The composition of claim 10, wherein SP consists of one glycine residue.14, The composition of claim 10, wherein APO consists of the amino acid sequence DWFKAFYDKVAEKFKEAF (SEQ ID NO: 51).55313274296v2148411.004402 PATENT15. The composition of claim 10, wherein each amphiphilic peptide comprises the amino acid sequence FFFFFFFFFFFGDWFKAFYDKVAEKFKEAF (SEQ ID NO: 52), wherein F is pentafluoro-phenylalanine.

16. The composition of claim 10, wherein the composition further comprises a pharmaceutically acceptable excipient selected from the group consisting of a vehicle, an adjuvant, a carrier, and a diluent.

17. The composition of claim.

10. wherein the peptide-based nanoparticles further comprise a cargo contained within the perfluorocarbon liquid core,18. The composition of claim 17, wherein the cargo is a statin drug.

19. The composition of claim 18, wherein the statin drug is Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, or Simvastatin.

20. A composition comprising a plurality of macrophages,wherein each macrophage comprises at least one peptide- based nanoparticle, wherein the at least one peptide-based nanoparticle comprises a perfluorocarbon liquid core, a cargo contained with the perfluorocarbon liquid core, and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core,wherein each amphiphilic peptide comprises FFFFFFFFFFFGDWFKAFYDKVAEKFKEAF (SEQ ID NO: 52), wherein FFis pentafluoro-phenylalanine, andwherein amphiphilic peptides are oriented such that the FFFFFFFFFFF region is interpolated into the perfluorocarbon liquid core and the DWFKAFYDKVAEKFKEAF (SEQ ID NO: 51) region extends away from the perfluorocarbon liquid, andwherein the cargo is a statin drug.

21. A method of preparing the composition comprising a plurality of cells of claim 10, the method comprising:contacting a perfluorocarbon liquid with a plurality of amphiphilic peptides to form a plurality of peptide-based nanoparticles, wherein each peptide-based nanoparticle comprises a 56313274296v2148411.004402 PATENTperfluorocarbon liquid core and a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core, andcontacting the plurality of peptide-based nanoparticles with the plurality of cells, where in each cell of the plurality of cells internalizes at least one peptide-based nanoparticle.

22. The method of claim 21, wherein water is added to plurality of peptide-based nanoparticles after their formation.

23. The method of claim 21, wherein a statin drug is added to the perfluorocarbon liquid prior to contacting the perfluorocarbon liquid with the plurality of amphiphilic peptides to form the plurality of peptide-based nanoparticles.

24. The method of claim 21, wherein the cell is a macrophage.

25. A method of detecting atherosclerotic plaques, comprising:administering the composition of claim 1 to a tissue,administering ultrasonic waves to the tissue, anddetecting the location of atherosclerotic plaques in the tissue by locating acoustic properties of the peptide-based nanoparticles.

26. The method of claim 25, wherein the ultrasonic waves are administered to the tissue by a B-mode ultrasonic imaging device or a Doppler ultrasonic imaging device.

27. The method of claim 25, wherein the ultrasonic waves induce a liquid-to-gas phase transition in the peptide-based nanoparticles that generates echogenic microbubbles.

28. A method of treating atherosclerotic plaques, comprising administering the composition of claim 1 to a tissue with atherosclerotic plaques.

29. The method of claim 28, wherein binding of the peptide-based nanoparticles to oxidized low-density lipoprotein (oxLDL) within lipid-rich foamy macrophages reduces the lipid burden of the foamy macrophages.57313274296v2148411.004402 PATENT30. The method of claim 28, further comprising administering ultrasonic waves to the tissue to induce a liquid-to-gas phase transition in the peptide-based nanoparticles.

31. The method of claim 30, wherein the liquid-to-gas phase transition of the peptide-based nanoparticles results in mechanical fractionation of intracellular lipid droplets within lipid-rich foamy macrophages.

32. The method of claim 28, wherein, when the peptide-based nanoparticles comprise a statin drug in the perfluorocarbon liquid core, the statin drug is released into the lipid-rich foamy macrophages.

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