WO2025090926A1 - Ultrasound-sensitive peptide particles with surface-bound gene editing tools for spatially resolved molecule delivery - Google Patents

Abstract

Provided herein are compositions comprising a plurality of peptide-based nanoparticles, each peptide-based nanoparticle comprising a perfluorocarbon liquid core, a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core, and at least one gene editing complex attached to the amphiphilic peptides. In some embodiments, each amphiphilic peptide is represented by Formula (III): HB-CL-HP-NH2 (III), wherein HB is a fluorinated hydrophobic block consisting of pentafluorinated hydrophobic amino acid residues; CL is an amino acid sequence, at least two of which are cross-linking cysteine residues; and HP is a hydrophilic amino acid sequence. In some embodiments, 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 HP extend away from the perfluorocarbon liquid core.

Description

ULTRASOUND-SENSITIVE PEPTIDE PARTICLES WITH SURFACE-BOUND GENE EDITING TOOLS FOR SPATIALLY RESOLVED MOLECULE DELIVERY

GOVERNMENT SUPPORT

[0001] This invention was made with government support under Grant No. DK.128638 awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

[0002 ] 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 “14841 l-003602_2023-5706_SequenceListing”, was created on October 24, 2024, and is 66,124 bytes in size.

REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority of US Provisional Patent Application

Serial No. 63/593,464, entitled “Ultrasound-Sensitive Peptide Particles with Surface-Bound Gene Editing Tools for Spatially Resolved Molecule Delivery,” the entire contents of which is hereby incorporated by reference herein.

FIELD OF THE INVENTION

[0004] The present invention is directed to gene delivery systems, and more specifically to peptide-based nanoparticles that permit ultrasound-triggered delivery of genetic editing materials, such as ribonucleoproteins, into tissue cultures and organoids.

BACKGROUND OF THE INVENTION

[0005] The effective delivery of therapeutic biomolecules, such as proteins, peptides, and nucleic acids, into cells poses a significant challenge in precision medicine. Protein- and nucleic acid-based therapeutics hold immense potential for the treatment of diseases such as cancer, autoimmune disorders, and genetic conditions. However, the utility of these therapies is often limited by poor tissue penetration, rapid degradation in physiological environments, and low intracellular uptake. A critical limitation of current delivery systems is their reliance on endocytic pathways, which often leads to degradation of therapeutic agertfs within the endosome, reducing their effectiveness,

[0006] The delivery of gene-editing agents to complex 3D tissue cultures and organoids presents additional challenges. Organoids, derived from pluripotent stem cells, are 3D culture systems that mimic the architecture and function of human tissues. They are essential for modeling diseases and testing therapeutics in a more biologically relevant context than traditional 2D cell cultures. However, the dense extracellular matrix of organoids limits the penetration of both viral and non-viral delivery vectors. While viral vectors can achieve high transfection efficiencies, their use is restricted by concerns over biosafety, insertional mutagenesis, and poor diffusion into the organoid core. Non-viral methods, such as electroporation and lipofection, have lower safety risks but suffer from poor tissue diffusion and low transfection efficiencies.

[0007] To overcome these limitations, researchers often resort to breaking down the organoids into smaller cell clusters, transfecting them, and re-embedding them in 3D matrices. This process compromises the organoid's structural integrity and recapitulates a 2D-like environment, undermining the benefits of 3D culture systems.

[0008] Thus, there is a need tor delivery systems that can penetrate the dense extracellular matrix of 3D organoids and bypass endocytic pathways for efficient direct delivery of gene- editing agents and therapeutic biomolecules to the cytoplasm.

SUMMARY OF THE INVENTION

[0009] Disclosed herein are compositions comprising a plurality of peptide-based nanoparticles.

[0010] In some embodiments, each peptide-based nanoparticle comprises a perfluorocarbon liquid core, a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core, and at least one gene editing complex attached to the amphiphilic peptides. In some embodiments, each amphiphilic peptide is represented by Formula (III): HB- CL-HP-NH2 (111), wherein HB is a fluorinated hydrophobic block consisting of three to five consecutively connected pentafluorinated hydrophobic amino acid residues; CL is an amino acid sequence consisting of two to 10 amino acid residues, at least two of which are cross- linking cysteine residues; and HP is a hydrophilic amino acid sequence. In some embodiments, the amphiphilic peptide consists of 8 to 30 total amino acid residues. In some embodiments, 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 HP extend away from the perfluorocarbon liquid core.

[0011] In some embodiments, the gene editing complex is selected from the group consisting of CRISPR-Cas9 ribonucleoprotein complexes, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and RNA-guided endonucleases.

[0012] In some embodiments, the CRISPR-Cas9 ribonucleoprotein complex comprises a Cas9 protein and guide RNA (gRNA) electrostatically adsorbed to the amphiphilic peptides.

[0013] In some embodiments, the gene editing complex is surface-adsorbed onto the peptide-based nanoparticles.

[0014] In some embodiments, the composition is used for targeted delivery of gene editing agents to mammalian cells.

[0015] 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

[0016] In some embodiments, the perfluorocarbon liquid core is selected from the group consisting of perfluoropentane, perfluorohexane, and perfluorobutane.

[0017] 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.

[0018] In some embodiments, HP comprises lysine, glycine, arginine, aspartic acid, or any combination thereof. In some embodiments, HP comprises the sequence KGRGD (SEQ ID NO: 35), where K is lysine, G is glycine, R is arginine, and D is aspartic acid.

[0019] In some embodiments, CL comprises GGGCCGG (SEQ ID NO: 46), where G is glycine and C is cysteine.

[0020] In some embodiments, the hydrophilic amino acid sequence of HP comprises a targeting motif. In some embodiments, the targeting motif is selected from a group consisting

[0021] In some embodiments, the targeting motif comprises a minimal targeting motif selected from the group consisting of:

[0022] In some embodiments, the targeting motif comprises KGRGD (SEQ ID NO: 35), which targets αVβ3 integrins.

[0023] In some embodiments, the amphiphilic peptide comprises an amphiphilic peptide represented by Formula (IV) or Formula (V):

wherein F_F is pentafluoro- phenylalanine, G is glycine, C is cysteine, K is lysine, G is glycine, R is arginine, and D is aspartic acid.

[0024] In some emlxxiiments, the amino acid sequence of CL consists of two to 10 amino acid residues and said hydrophilic amino acid sequence of HP consists of 3 to 15 hydrophilic amino acids, and wherein said amphiphilic peptide consists of 10 to 30 total amino acid residues.

[0025] In some embodiments, the amphiphilic peptide has a molecular weight in the range of about 2000 - 5000 daltons, wherein the amphiphilic peptide includes at least eight 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 connected consecutively by peptide bonds without any intervening amino acid residues.

[0026] Disclosed herein are methods of preparing a composition comprising a plurality of peptide-based nanoparticles loaded with a gene editing complex.

,0027] In some embod iments, the method comprises assembling a plurality of peptide- based nanoparticles by emulsifying amphiphilic peptides with a perfluorocarbon liquid; cross- linking the amphiphilic peptides; mixing the plurality of peptide-based nanoparticles with a gene editing complex solution under gentle agitation; incubating the mixture of a period sufficient to allow adsorption of the gene editing complex onto the surface of the peptide-based nanoparticles; removing unbound gene editing complex molecules by a process selected from centrifugation, filtration, or dialysis; and recovering and purifying the peptide-based nanoparticles by isolating the nanoparticles from the suspension.

[0028] Disclosed herein are methods of performing gene editing in a cellular construct

[0029] In some embodiments, the method comprises administering any composition disclosed herein to the cellular construct; and administering ultrasonic waves to the cellular construct. In some embodiments, the ultrasonic waves cause a rupture of one or more of the peptide-based nanoparticles and permeabilize the extracellular matrix of the cellular construct, thereby allowing a gene editing complex to be delivered to the cells within the cellular construct, facilitating the editing of the DNA within the cells.

[0030] In some embodiments, the administrating of ultrasonic waves comprises applying ultrasound at an intensity of less than 1 .0 watts per square centimeter. In some embodiments, the ultrasonic waves are applied at an intensity of 1 W/cm² or greater. In some embodiments, the ultrasonic waves are applied with a duty cycle ranging from about 20% to 80%.

[0031] In some embodiments. the cellular construct is an organoid. In some embodiments, the organoid is derived from pluripotent stem cells. In some embodiments, the organoid is a renal organoid comprising podocytes, proximal tubules, and distal tubules.

[0032] These and other aspects of the invention are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The foregoing and other aspects of the present invention are best understood from the following detailed description when read in connection with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments that are presently preferred, it being understood, however, that the invention is not limited to the specific instrumentalities disclosed. Included in the drawings are the following Figures: [0034] FIG. 1 is a diagram showing structure of peptide-based nanoparticles, according to an embodiment of the present disclosure.

[0035] FIG. 2 is a diagram showing target motif-mediated specific binding and ultrasound- mediated delivery of cargo into a targeted cell, according to an embodiment of the present disclosure. [0036] FIG. 3 is a graph of particle size (nanometers, nm) and count rate (kilo counts per second, kcps) versus time (days) illustrating stability of peptide-based nanoparticles (formulation B, see Table 2) during long-term storage in water at room temperature, wherein particle stability was measured via DLS over 15 days, according to an embodiment of the present disclosure.

[0037] FIG.4 is a diagram showing particle size of 1 and 2 vol% perfhioro-n-pentane (PFP) immediately after emulsion formation in the peptide-based nanoparticle pre-assembly solution (1:1 DMF:ACN, 1%TFA);

[0038] FIG. 5 is a graph showing relationship between PFP vaporization temperature

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) , according to an embodiment of the present disclosure.

[0039] FIG. 6 is a graph showing peptide-based nanoparticle cross-linking in the presence of 5% DMSO oxidizing agent used during dialysis purification, according to an embodiment of the present disclosure.

[0040] FIG. 7 is a graph of disulfide formation (percent, %) versus time (hours) illustrating percentage of disulfide cross-linking as a function of time following peptide-based nanoparticle assembly in formulation B (see Table 2) in the absence of DMSO, and showing that these peptide-based nanoparticles were not stable beyond 1 hour, according to an embodiment of the present disclosure.

[0041] FIG. 8 is a graph of particle size (nanometers, nm) versus time (hours) illustrating stability of peptide-based nanoparticles (formulation B, see Table 2) suspended in blank characterization buffer (25 mM, 150 mM NaCL, pH 7.4), or the same buffer supplemented with 5% fetal bovine serum, and incubated at 37°C for 48 hours, according to an embodiment of the present disclosure.

[0042] FIG. 9 is a graph of number percent versus particle size (nanometers, nm) illustrating particle size measurement of peptide-based nanoparticles formulated under various peptide and PFP ratios, where each letter, A, B, C, D, and E, corresponds to different formulations shown in Table 2, according to an embodiment of the present disclosure.

[0043] FIG. 10 depicts a schematic of a peptide-based nanoparticle composed of a peptide surfactant assembled at the surface of a perfluoropentane (PFP) core with a Cas9-gRNA ribonucleoprotein (RNP) complex electrostatically adsorbed to the surface of the peptide-based nanoparticle, according to an embodiment of the present disclosure.

[0044] FIG. 11 depicts a cross-sectional diagram of a renal organoid with multiple peptide- based nanoparticles distributed within the tissue, according to an embodiment of the present disclosure.

[0045] FIG. 12 illustrates the process of gene delivery at a cellular level, where a peptide- based nanoparticle is activated by ultrasound to form a microbubble and the bubble collapses, releasing the Cas9-gRNA RNP complex into the adjacent cell, according to an embodiment of the present disclosure.

[0046] FIG. 13 depicts the binding efficacy of the Cas9-gRNA RNP complex to the surface of the peptide-based nanoparticle, where the binding fraction is higher when both Cas9 and gRNA are complexed together, according to an embodiment of the present disclosure.

[0047] FIG. 14 depicts the acoustic delivery mechanism of gene editing complexes into cells, showing the interaction of a peptide-based nanoparticle with a cell, where ultrasound activation leads to the release of the gene-editing payload into the cell’s cytoplasm, according to an embodiment of the present disclosure.

[0048] FIG. 15 depicts fluorescent microscopy images showing the localization of peptide-based nanoparticles on the surface of human embryonic kidney (HEK) cells, indicating the peptide-based nanoparticles' successful binding to the cell surface after 4 hours of incubation, according to an embodiment of the present disclosure.

[0049] FIG. 16 depicts the binding fraction of peptide-based nanoparticles incubated with

GFP-expressing HEK293T cells, according to an embodiment of the present disclosure.

[0050] FIG. 17 illustrates the effect of ultrasound intensity on gene editing, as measured by the relative EGFP signal in a knockdown reporter assay, according to an embodiment of the present disclosure.

[0051] FIG. 18 shows the relationship between ultrasound duty cycle and gene editing efficiency, measured by relative EGFP signal, according to an embodiment of the present disclosure.

[0052] FIG. 19 compares the permeabilization of kidney organoids treated with

particles activated by varying US duty cycles (20%. 50%, 80%) versus CRISPRMAX and untreated controls, according to an embodiment of the present disclosure. [0053] FIG. 20 depicts the intra-organoid delivery of Cas9-GFP and its paired gRNA in kidney organoids, comparing CRISPRMAX controls, unactivated NPepRNP (-US), and US- activated (+US), according to an embodiment of the present disclosure.

[0054] FIG. 21 shows the relative expression of tdTomato fluorescence across different heights within the organoid for various transfection conditions, comparing CRISPRMAX,

without US activation, and NPepRNP activated by US as increasing intensities, according to an embodiment of the present disclosure.

[0055] FIG. 22 depicts a correlation between US intensity and duty cycle and the resulting gene editing variance and maximum signal intensity in organoids; the larger circles represent greater gene editing signal, and the shading indicates variability, with darker shades representing lower variance, according to an embodiment of the present disclosure.

[0056] FIG. 23 compares the depth of editing within kidney organoids treated with different US intensities (1 W/cm², 2 W/cm², 3 W/cm²) and duty cycles, according to an embodiment of the present disclosure.

[0057] FIG. 24 depicts confocal microscopy images showing the spatial distribution of edited cells within organoids after treatment with CRISPRMAX,

without US activation and

with US activation, according to an embodiment of the present disclosure.

[0058] FIG.25 depicts a set of graphs showing the relative fluorescence (RFU) of tdTomato, aligned with nuclei, podocyte, and tubule markers across the organoid height for CRISPRM AX- treated organoids, according to an embodiment of the present disclosure.

[0059] FIG. 26 depicts a scatter plot showing signal values for tdTomato, nuclei, podocytes, and tubules, and a comparison of the difference between means for these cell types in CRISPRMAX-treated organoids, according to an embodiment of the present disclosure.

[0060] FIG. 27 depicts a set of graphs showing the relative fluorescence (RFU) for

organoids, comparing tdTomato, nuclei, podocytes, and tubule fluorescence across the organoids height, according to an embodiment of the present disclosure.

[0061] FIG. 28 depicts a scatter plot showing signal values for tdTomato, nuclei, podocytes and tubules, and the difference between means, according to an embodiment of the present disclosure.

[0062] FIG. 29 depicts a heatmap showing p-values for the correlation between tdTomato expression and nuclei, podocytes, and tubules for both CRISPRMAX and NPepRNP treatments, according to an embodiment of the present disclosure. [0063] FIG. 30 depicts a bar chart comparing the colocalized volume of

fluorescence with podocytes and tubules in CR1SPRMAX- and

organoids, according to an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0064] 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.

[0065] 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”).

[0066] 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.

[0067] All 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 the 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.

[0068] 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 arc 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, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. [0069] 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 include

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

[0070] The opened ended term “comprising” includes the intermediate and closed terms “consisting essentially of’ and “consisting of.”

[0071] A dash (“-“) that is not between two letters or symbols is used to indicate a point of attachment for a substituent.

[0072] “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-human drugs.

[0073] A “patient” means a human or non-human animal in need of medical treatment. Medical treatment can include treatment of an existing condition, such as a disease or disorder or diagnostic treatment. In some embodiments the patient is a human patient.

[0074] “Providing” means giving, administering, selling, distributing, transferring (for profit or not), manufacturing, compounding, or dispensing.

[0075] “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 cause disease regression, to certain embodiments treatment of the disease may be commenced before the patient presents symptoms of the disease.

[0076] A “therapeutically active agent” means a compound which can be used for diagnosis or treatment of a disease. The compounds can be small molecules, peptides, proteins, or other kinds of molecules.

[0077] 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.

[0078] The present invention is directed to novel peptide-based nanoparticles prepared through the templated assembly of amphiphilic peptides.

[0079] A peptide-based particle refers to a structured entity capable of incorporating a variety of functional components. Peptide-based particles, along with, but not limited to, the terms “peptide-based nanoparticle"’, “nanoparticle”, “nanoemulsion”, “nanopeptisome”, “emulsion”, “nanofibril”, and “NPep” denote an arrangement of amiphiphilic 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 and/or cargo bound to the emulsion surface. The term peptide-based 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. [0080] Peptide-based 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 peptide-based nanoparticles and'or cargo bound to the emulsion surface into the cytoplasm directly. Thus, proteins, peptides, nucleic acids, small molecule compounds, gene editing vectors, and other materials, can be encapsulated and/or bound to the emulsion surface 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.

[0081] A typical peptide-based 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 al the surface of the perfluorocarbon (PFC) liquid core 60, which contains the cargo-fluorine-containing cargo solubilizing agent complexes 70.

[0082] 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 electrostatic interactions with oppositely charged domains on the cargo, thereby enhancing the stability and specificity of their surface attachment. FIGs. 10 and 14 depict peptide-based nanoparticles wherein the cargo is bound to the surface of the emulsion.

[0083] Peptide-based nanopanicles having a diameter of from about 250 nm to about 5 microns may be produced. An average diameter of the peptide-based 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 peptide-based 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.

[0084] The peptide-based nanoparticles contain a perfluorocarbon liquid core that allows for activation of the peptide-based 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 peptide-based nanoparticles upon application of ultrasound refers to phase transition of a perfluorocarbon liquid core into a gaseous state due to ultrasound application.

[0085] As shown diagrammatically in FIG. 2, peptide-based 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 peptide-based nanopartide 10. Application of ultrasound 85 causes acoustic vaporization of the perfluorocarbon liquid core of the peptide-based nanoparticles and leads to the formation of a gaseous core that ultimately swells and ruptures 90 the peptide-based nanoparticles. Subsequent bubble captivation 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 peptide-based nanoparticle or on the surface of the peptide-based nanoparticle into the cell cytoplasm 98. Thus, US-sensitive peptide- based 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.

[0086] Upon ultrasound activation, the perfluorocarbon liquid core undergoes a liquid-to- gas phase transition, generating microbubbles within the surrounding medium or tissue. The rapid expansion and subsequent collapse (inertial cavitation) of these microbubbles produce high-velocity fluid jets and shock waves. In the context of dense tissues or 3D structures like organoids, this cavitation process mechanically permeabilizes the extracellular matrix and creates transient pores in cell membranes. This mechanical action facilitates the penetration of peptide-based nanoparticles deeper into the tissue and permits the direct cytoplasmic delivery of cargo molecules, such as RNPs. into cells located within the tissue bulk.

[0087] Key to the assembly of these peptide-based nanoparticles is a de novo designed amphiphilic peptide, capable of assembling at the surface of a perfluorocarbon liquid. [0088] As noted above, amphiphilic peptides included in peptide-based nanoparticles according to embodiments each include a fluorinated hydrophobic block (HB), a crosslinking motif, and a hydrophilic peptide.

[0089] Thus, in one embodiment, an amphiphilic peptide represented by Formula (I) is provided:

HB-CL-HP (l) wherein HB is a fluorinated hydrophobic polymer, CL is a cross-linking motif; and HP is a hydrophilic amino acid sequence.

[0090] 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 lias 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.

[0091] 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.

[0092] According to embodiments, the amphiphilic peptide does not include lipids.

[0093] 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.

[0094] 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. [0095] 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.

[0096] 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 fluorinated hydrophobic amino acids consecutively connected by peptide bonds. Fluorinated hydrophobic amino acids include, for example, fluorinated alanine, fluorinated valine, fluorinated leucine, fluorinated isoleucine, fluorinated proline, fluorinated phenylalanine, fluorinated tryptophan, fluorinated cysteine, fluorinated methionine, fluorinated selenocysteine, and fluorinated pynolysine. 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, five, six, seven, eight, nine or ten consecutively connected pentafluoro phenylalanine residues at a terminal thereof.

[0097] Fluorinated amino acids can be obtained commercially or synthesized by standard methodologies, such as those described in detail in G. Haufe et al., Amino Acids, September 1996, Volume 11, Issue 3-4, pp 409-424.

[0098] 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.

[0099] 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-naiural and can be D or L amino acids. Examples of the naturally occurring hydrophilic amino acids include, but are not limited to, serine, threonine, asparagine, glutamine, histidine and tyrosine. Examples of the non-natural hydrophilic amino acids include amino acids having various heterocyclic groups as a part of the side chain.

[0100] 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, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen hydrophilic amino acids consecutively connected by peptide bonds.

[0101] In one embodiment, an amphiphilic peptide represented by Formula (II) is provided:

wherein HB is a fluorinated hydrophobic amino acid sequence; CL is a cross-linking motif; and HP is a hydrophilic amino acid sequence.

[0102] In one embodiment, an amphiphilic peptide represented by Formula (III) is provided:

wherein HB is a fluorinated hydrophobic amino acid sequence; CL is a cross-linking motif; ami HP is a C-terminally amidated hydrophilic amino acid sequence.

[0103] 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 peptide-based 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. [0104] 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-terminas or C terminus of the m inimal 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 acids 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-terminus of the minimal targeting motif peptide by peptide bonds can be D or L amino acids.

[0105] According to embodiments, the targeting agent includes a minimal targeting motif peptide selected from:

,

which binds to the IL-4 receptor on atherosclerotic plaques;

which binds to thrombin in blood clots; and/or

which binds to thrombin in blood clots, and optionally includes one or more hydrophilic amino acids attached to the N-terminus or C-terminus of the minimal targeting motif peptide by peptide bonds.

[0106] According to embodiments, the hydrophilic amino acid sequence including a targeting agent includes a hydrophilic amino acid sequence selected from:

[0107] 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 intcgrins and two hydrophilic amino acids. [0108] Minimal targeting motif RGD alone or with additional hydrophilic amino acids along with the binding affinity to αVβ3 integrin (IC₅₀, conc. in nM at which 50% of receptor is bound by ligand):

Thus, according to embodiments, the hydrophilic amino acid sequence HP includes,

[0109] According to embodiments, the targeting agent of the hydrophilic amino acid sequence interacts with a targeted component of a target cancer cell.

[0110] As will be appreciated by those of skill in the art, some targeting agents are recognized by both normal cells and pathological cells. In such cases, higher expression of the targeted component is typically present in tire pathological cells, such as cancer cells. For example, integrins are expressed by a wide variety of normal cells. However, inlegrins are expressed at much higher levels in cancer cells.

[0111] Examples of minimal targeting motifs suitable for inclusion in cancer taigeting agents, tumors which express targeted components which specifically bind the minimal targeting motif, and examples of hydrophilic amino acid sequences including a targeting motif which can be included in amphiphilic peptides according to embodiments, are shown in Table 1.

[0112] 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.

[0113] 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.

[0114] Naturally occurring amino acids capable of crosslinking with a corresponding amino acid residue include cysteine.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] to the peptide-based 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 liquid 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-).

[0119] 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).

[0120] 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.

[0121] In an embodiment, the amphiphilic peptide has Formula (IV):

wherein F_F is pentafluoro-phenylalanine (23 ,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 Fonnuia (IV),

is a hydrophobic block HB,

is a cross-linking motif CL, and KGRGD (SEQ ID NO:35) is a hydrophilic amino acid sequence HP, which contains targeting motif RGD.

[0122] In an embodiment, the amphiphilic peptide has Formula (V):

wherein F_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),

is a hydrophobic block HB,

is a cross-linking motif CL, and

is a C-terminally amidated hydrophilic amino acid sequence HP, which contains targeting motif ROD.

[0123] 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.

[0124] 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.

[0125] The amphiphilic peptides of all of Formulas (I). (Il), (III), (IV), and (V) are capable of assembling at the surface of a perfluorocarbon liquid and assemble into a layer to form a peptide-based nanoparticle.

[0126] Amphiphilic peptides (IV) and (V) contain three pentafluoro-phenylalanine ( F_F) residues at the N-terminus which promote interpolation and assembly of the peptide at the perfluorocarbon liquid interface. C-tenninal to this fluorous domain is a cysteine-containing crosslinking motif

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

to enable cell-surface localization of peptide-based nanoparticles including the amphiphilic peptides of Formulas (IV) and (V) mediated by binding of the targeting motif RGD with extracellular integrins.

[0127] The incorporation of the RGD sequence (SEQ ID NO:35) in the hydrophilic amino acid sequence HP permits the peptide-based nanoparticles to specifically bind to αVβ3 integrins, which are overexpressed on certain cell types such as podocytes and tubular epithelial cells within kidney organoids. This targeted binding promotes the accumulation of peptide-based nanoparticle; at the surface of these cells, enhancing the efficiency of cargo delivery upon ultrasound activation. The targeting motif can be customized based on the specific integrins or receptors expressed by different cell types, allowing for cell-specific delivery in various tissues and organoids. [0128] As noted above, the peptide-based nanopartides contain a perfluorocarbon (PFC) liquid core that allows for activation of the particle upon application of ultrasound (US). In some embodiments, the activation of the particle upon the application of US causes the delivery of a cargo associated with the peptide-based nanopanicle. In some embodiments, the cargo is present in the perfluorocarbon liquid core. In some embodiments, the cargo is associated with or bound to the surface of the peptide-based nanoparticles.

[0129] As used herein, the term '‘perfluorocarbon” refers to a hydrocarbon in which, all or a substantial portion of hydrogen atoms in C-H bonds are replaced with fluorine atoms, producing C-F bonds. The degree of replacement of hydrogen atoms with fluorine atoms may vaty, 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 embodimen t, the 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 perfluoroliexane, an 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 pcrfluorohexane, for example, perfluoro n-hexane (PFH), perfluoro-iso-hexane, or perfluoro-sec-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 peptide-based nanoparticles which leads to the formation of a gaseous core that ultimately swells and ruptures the peptide-based 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.

[0130] On the other hand, the intensity of the ultrasound should be sufficient to release the cargo from the peptide-based nanoparticles into the cells. In some embodiments, no cell damage occurs, and 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 hi 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 peptide-based nanoparticles and/or deliver a cargo to targeted cells or regions such as atherosclerotic plaques or blood clots.

[0131] In applications involving 3D tissue models such as organoids, ultrasound parameters can be adjusted to optimize the delivery of cargo while ensuring tissue viability. For instance, acoustic intensities ranging from 1 W/cm² to 3 W/cm² and duty cycles between 20% and 80% can be employed to facilitate the vaporization of peptide-based nanoparticles and enhance tissue penneabilization. These parameters are well below the FDA diagnostic limit of 190 W/cm² spatial peak pulse average intensity, ensuring that the ultrasound application is safe and does not cause collateral mechanical damage to the tissue or cells. The selection of specific ultrasound settings can be tailored based on the size of the organoid, the density of the extracellular matrix, and the desired depth of cargo delivery.

[0132] The cargo included in peptide-based nanoparticles to be delivered into a cell may include a therapeutically active agent, for example, a small molecule therapeutic agent, a protein therapeutic agent, a peptide therapeutic agent, a nucleic acid-based agent, such as RNA, DNA, an miRNA molecule, an siRNA molecule, an shRNA molecule, a dsRNA molecule, an antisense molecule, a ribozyme, a polynucleotide encoding an miRNA, siRNA, shRNA, dsRNA; or a combination of any two or more thereof, a gene editing tool (such as clustered regularly interspaced short palindromic repeals “CR1SPR”), but is not limited thereto. The therapeutically active agent may include a radioisotope.

[0133] In some embodiments, the cargo is associated with or bound to the surface of the ultrasound sensitive peptide-based nanoparticles. In some embodiments, the cargo comprises gene editing agents. In some embodiments, these gene editing agents may be ribonucleoproteins (RNPs) that consist of Cas proteins and their associated guide RNA (gRNA). In some embodiments, the RNPs may be attached to the external amphiphilic peptide 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 molecules may contain charged amino acid residues that engage in electrostatic interactions with oppositely charged domains on the RNPs, thereby enhancing the stability and specificity of their surface attachment

[0134] In some embodiments, the cargo comprises gene-editing agents such as CRISPR- Cas9 ribonucleoprolein (RNP) complexes. The RNPs, comprising Cas proteins bound to their associate guide RNA (gRNA), can be adsorbed onto the surface of the peptide-based nanoparticles via electrostatic interactions. This adsorption is facilitated by charged amino acid residues present in the amphiphilic peptide molecules, which engage in electrostatic interactions with oppositely charged domains on the RNPs. This method allows for efficient loading of RNPs onto the peptide-based nanoparticles without the need for encapsulation within the perfluorocarbon core, preserving the activity of the RNPs and facilitating their direct delivery into the cytoplasm upon ultrasound activation.

|0135[ Such therapeutically active agents include, but are not limited to, an anti- thrombotic agent, antibiotics, antivirals, antineoplastic agents, analgesics, antipyretics, antidepressants, antipsychotics, anti-cancer agents, antihistamines, anti-osteoporosis agents, anti-osteonecrosis agents, anti-inflammatory agents, non-steroidal anti-inflammatory agents, anxiolytics, chemotherapeutic agents, diuretics, growth factors, hormones, steroids and vasoactive agents.

[0136] In some embodiments, such gene-editing agents include, but are not limited to, CRISPR-Cas systems (including CRISPR-Cas9, CRISPR-Cas12, and CRISPR-Cas 13), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), base editors (such as cytidine and adenine base editors), prime editors, meganucleases, and engineered nucleases derived from homing endonucleases. Additionally, RNA-guided nucleases beyond the CRISPR-Cas systems, such as CasX and CasY variants, can be utilized. RNA editing systems, such as RNA-targeted CRISPR variants (e.g., Cas13 for RNA knockdown), as well as other ribonucleoprotein complexes that facilitate RNA-guided modification, can be employed. Transposon-based systems, integrase systems, recombinase-based tools such as phiC3l integrase and site-specific recombination systems that allow for targeted DNA or RNA modifications are also included. These gene-editing tools may be used individually or in combination, depending on the intended therapeutic or research application. The peptide-based nanoparticles may facilitate delivery of these agents into the cell cytoplasm, preserving their activity and specificity for targeted genomic modifications upon ultrasound-mediated activation.

[0137] The perfluorocarbon liquid core may further include a bioimaging agent, for example, a pholoacoustic dye (such as indocyanine green “ICG”, Cyanine 7 “Gy 7”, or dimethyl

aminonium perchlorate “IR800”), a fluorescent dye or protein (such as green fluorescent protein “GFP”. fluorescein, rhodamine, a cyanine dye), and a magnetic resonance imaging “MR1” contrast agent (such as iron oxide or gadolinium), a radiotracer, but is not limited thereto.

[0138] According to embodiments, the perfluorocarbon liquid core inchides about 1x 10³ to about 5x10⁹ molecules, such as 1 x 10⁴ to about 5x10⁸ molecules of the active agent, such as lx10⁵ to about 5x10⁷ molecules of the active agent, such as 1 x 10⁶ to about 5x 10⁶ molecules of the active agent, and may include more, or less, of the active agent.

[0139] In some embodiments, a cargo to be delivered to the interior of a cell via the peptide-based 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 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), perfluoropentanoic acid (CF₃(CF₂))COOH), peril uorononanoic 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-pentafluorophenylalanine ((CF)₅CCFl₂C(NH-Fmoc )COOH, Fmoc-pentafluoro-L-phenylalanine and/or Fmoc- pentafluoro-D-pheny lalanine, 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.

[0140] In some embodiments, the peptide-based nanoparticles are prepared with cargo molecules adsorbed onto their surface through electrostatic interactions. In some embodiments, the method comprises one or more of the following steps. The peptide-based nanoparticles are assembled by emulsifying the amphiphilic peptides with the perfluorocarbon liquid as described previously, The amphiphilic peptides are cross-linked to stabilize the peptide-based nanoparticles. A solution of the cargo molecule (e.g., RNP complexes) is prepared in an appropriate buffer. The peptide-based nanoparticle suspension is mixed with the cargo solution under gentle agitation. The mixture is incubated for a specified time (e.g., 30 minutes to 4 hours) to allow for electrostatic adsorption of the cargo onto the peptide-based nanoparticle surface. Factors such as pH, ionic strength, and temperature can be adjusted to optimize the adsorption process. Unbound cargo molecules can be removed by techniques such as centrifugation, filtration, or dialysis, if necessary. This method allows for efficient loading of sensitive biological molecules, such as RNPs, without compromising their activity. The absence of chemical conjugation steps preserves the functionality of both the peptide-based nanoparticles and the cargo.

,0141] In another embodiment, a composition including the above peptide-based 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 the pharmaceutically active agents and one that has no detrimental side effects or toxicity under the conditions of use.

[0142] The compositions may be administered as oral, sublingual, transdermal, subcutaneous, topical, absorption through epithelial or mucocutaneous linings, intravenous, intranasal, intraarterial, intramuscular, intratumoral, 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.

[0143] In still another embodiment, a method of preparing the peptide-based 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 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 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 peptide-based nanoparticles.

[0144] 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.

[0145] In some embodiments, methods of making peptide-based nanoparticles further comprise mixing together the fluorine-containing cargo solubilizing agent, the perfluorocarbon liquid, and optionally a cargo, and the amphiphilic peptides represented by any of the above Formulas (I), (II), (III), (IV), or (V), are added, forming a composition. In some embodiments, the cargo is any cargo disclosed herein. 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 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 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 peptide-based nanoparticles.

[0146] According to embodiments, the peptide-based nanoparticles have an average diameter in the range from about 1 micron to about 5 microns. According to embodiments, the peptide-based nanoparticles have an average diameter in the range from about 250 nanometers to about 1000 nanometers. According to embodiments, the peptide-based nanoparticles have an average diameter in the range from about 250 nanometers to about 750 nanometers. The size of peptide-based 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 peptide-based nanoparticles, see, for example FIG. 4 and FIG. 9. In general, the volume percent of the perfluorocarbon liquid can be increased to increase the average diameter of the peptide-based 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 peptide-based 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 peptide-based nanoparticles with a desired average diameter. [0147] 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 thiol groups of cysteine amino acids. For example, the dialysis may 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.

[0148] 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.

[0149] In another embodiment, a method of delivering an active agent to a target cell is provided. According to the method, a composition including the above peptide-based nanoparticle loaded with, for example a therapeutic or diagnostic active agent is prepared. The composition is then administered to a patient, and an ultrasound radiation is applied to release the therapeutically active entity from the peptide-based nanoparticle.

[0150] In some embodiments, peptide-based nanoparticles can be utilized for the delivery of therapeutic agents, including gene-editing tools, into three-dimensional (3D) tissue models such as organoids derived from pluripotent stem cells. Organoids are complex 3D cultures that recapitulate tire architecture and cellular organization of human tissues, serving as important models for disease study and drug testing.

[0151] For imaging, two modalities of ultrasound imaging can be used. B-mode ultrasound imaging, allows viewing of stable peptide-based nanoparticles that have cores vaporized under low intensity ultrasound to form microbubbles

but have not yet collapsed or lysed. B-mode ultrasound imaging allows a user to view and guide the peptide-based nanoparticles in space using the ultrasound pressure wave. Doppler imaging can be used and allows viewing of changes in frequency that occur when the peptide-based nanoparticles collapse due to application of ultrasound. Thus, Doppler imaging can be used to confirm carrier lysis and cargo release using higher ultrasound energies.

[0152] The term “low intensity” is used to refer to ultrasound at acoustic pressures that allow the core of the peptide-based nanoparticles to oscillate as bubbles but not collapse. The tenn “high intensity” is used to refer to ultrasound at acoustic pressures that cause bubble cavitation of the peptide-based 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 peptide-based 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 run peptide-based nanoparticles wherein the amphiphilic peptides have the sequence

stably oscillate as bubbles below 0.4 MI (mechanical index, measure of ultrasound intensity), and collapse at ultrasound pressures above this threshold.

[0153] The present disclosure is illustrated and further described in more detail with reference to the following non-limiting examples.

Enumerated Embodiments

[0154] The following examples are illustrative, but not limiting, of the compounds, compositions and methods described herein. Other suitable modifications and adaptations known to those skilled in the art are within the scope of the following embodiments.

1. A composition comprising a plurality of peptide-based nanoparticles, wherein each peptide-based nanoparticle comprises a perfluorocarbon liquid core, a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core, and at least one gene editing complex attached to the amphiphilic peptides, wherein each amphiphilic peptide is represented by Formula (III):

HB-CL-HP-NH2 (III) wherein HB is a fluorinated hydrophobic block consisting of three to five consecutively connected pentafluorinated hydrophobic amino acid residues; wherein CL is an amino acid sequence consisting of two to 10 amino acid residues, at least two of which are cross-linking cysteine residues; wherein HP is a hydrophilic amino acid sequence, wherein said amphiphilic peptide consists of 8 to 30 total amino acid residues, 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 HP extend away from the perfluorocarbon liquid core. 2. The composition of embodiment 1, wherein the gene editing complex is selected from the group consisting of CR1SPR-Cas9 ribonucleoprotein complexes, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and RNA-guided endonucleases.

3. The composition of embodiment 2, wherein the CR1SPR-Cas9 ribonucleoprotein complex comprises a Cas9 protein and guide RNA (gRNA) electrostatically adsorbed to the amphiphilic peptides.

4. The composition of any one of embodiments 1 to 3, wherein the gene editing complex is surface-adsorbed onto the peptide-based nanoparticles.

The composition of any one of embodiments 1 to 4, wherein the composition is used for targeted delivery of gene editing agents to mammalian cells.

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 perfluorocarbon liquid core is selected from the group consisting of perfluoropentane, perfluorohexane, and perfluorobutane.

8. The composition of any one of embodiments 1 to 7, 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.

9. The composition of any one of embodiments 1 to 7, wherein HP comprises lysine, glycine, arginine, aspartic acid, or any combination thereof.

10. The composition of any one of embodiments 1 to 9. wherein HP comprises the sequence KGRGD (SEQ ID NO: 35), where K is lysine, G is glycine, R is arginine, and D is aspartic acid. 11. The composition of any one of embodiments 1 to 10, wherein CL comprises GGGCCGG (SEQ ID NO: 46), where G is glycine and C is cysteine.

12. The composition of any one of embodiments 1 to 11 , wherein said hydrophilic amino acid sequence of HP comprises a targeting motif.

13. The composition of any one of embodiments 1 to 12, wherein the targeting motif is selected from a group consisting of

14. The composition of any one of embodiments 1 to 13, wherein the targeting motif comprises a minimal targeting motif selected from the group consisting of:

15. The composition of any one of embodiments 1 to 12, wherein the targeting motif comprises KGRGD (SEQ ID NO: 35), which targets αVβ3 mtegrins. 16. The composition of any one of embodiments 1 to 15, wherein said amphiphilic peptide comprises an amphiphilic peptide represented by Formula (IV) or Formula (V):

(SEQ ID NO:49), wherein F_F is pentafluoro-phenylalanine, G is glycine, C is cysteine, K is lysine, G is glycine, R is arginine, and D is aspartic acid.

17. The composition of any one of embodiments 1 to 16, wherein said amino acid sequence of CL consists of two to 10 amino acid residues and said hydrophilic amino acid sequence of HP consists of 3 to 15 hydrophilic amino acids, and wherein said amphiphilic peptide consists of 10 to 30 total amino acid residues.

18. The composition of any one of embodiments 1 to 17, wherein the amphiphilic peptide has a molecular weight in the range of about 2000 - 5000 daltons, wherein the amphiphilic peptide includes at least eight 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 connected consecutively by peptide bonds without any intervening amino acid residues.

19. A method of preparing a composition comprising a plurality of peptide-based nanoparticles loaded with a gene editing complex, the method comprising: assembling a plurality of peptide-based nanoparticles by emulsifying amphiphilic peptides with a perfluorocarbon liquid: cross-linking the amphiphilic peptides; mixing the plurality of peptide-based nanoparticles with a gene editing complex solution under gentle agitation; incubating the mixture of a period sufficient to allow adsorption of the gene editing complex onto the surface of the peptide-based nanoparticles; removing unbound gene editing complex molecules by a process selected from centrifugation, filtration, or dialysis; and recovering and purifying the peptide-based nanoparticles by isolating the nanoparticles from the suspension.

20. A method of performing gene editing in a cellular construct, the method comprising: administering a composition comprising of any one of embodiments 1 to 18 to the cellular construct; administering ultrasonic waves to the cellular construct; wherein the ultrasonic waves cause a rupture of one or more of the peptide-based nanoparticles and permeabilize the extracellular matrix of the cellular construct, thereby allowing a gene editing complex to be delivered to the cells within the cellular construct, facilitating the editing of the DNA within the cells.

21. The method of embodiment 20, wherein the administrating of ultrasonic waves comprises applying ultrasound at an intensity of less than 1.0 watts per square centimeter.

22. The method of embodiment 20, wherein the ultrasonic waves are applied at an intensity of 1 W/cm² or greater.

23. The method of embodiment 22, wherein the ultrasonic waves are applied with a duty cycle ranging from about 20% to 80%.

24. The method of embodiment 20, wherein the cellular construct is an organoid.

25. The method of embodiment 24, wherein the organoid is derived from pluripotent stem cells.

26. The method of embodiment 24, wherein the organoid is a renal oiganoid comprising podocytes, proximal tubules, and distal tubules.

EXAMPLES

Example 1: Synthesis, Characterization, and Stability of Peptide-Based Peptide-Based Nanopartides for Ultrasound-Mediated Delivery [0155] Materials And General Methods [0156] Fmoc -protected amino acids were purchased from Novabiochem. PL-Rink resin was purchased from Polymer Laboratories,

was obtained from Peptides International. Trifluoroacetic acid was obtained from Acres organics, and 1,2-elhanedi thiol was purchased from Fhika. Oregon Green 514 Phalloidin, 6 and 24 well cell culture plates, polystyrene microcuvettes, diethyl ether, dimethylformamide (DMF), acetonitrile (ACN), N- methylpyrrolidone (NMP), Slide-A LyzerTM dialysis cassettes (MWCO 3.5K) and 96-well half area high content imaging glass bottom microplates ware purchased from Fisher Scientific. Perfluorohexane (PFH) and perfluoropentane (PIT) were purchased from Oakwood Chemicals and Strem Chemicals, respectively. N,N -diisoproylcarbodiimide (DIC) was purchased from Chem Impex. Thioanisole, anisole, 3-(4,5 dimethyl-2-thiazolyl)-2,5~diphenyl-2H-teirazolium bromide (MTT), 2,2 ‘-di thiodipyridine (DTP), dimethyl sulfoxide (DMSO), 1,8- diazabicyclo[5.4.0jundec-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 Coming. Heat inactivated fetal bovine serum (FBS) and trypsin EDTA were obtained from Hyclone Laboratory Inc. HPLC solvents consisted of sol vent 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. All peptides utilized for experiments were prepared with an amidated C -terminus.

[0157] Pentide Synthesis

[0158] Fmoc-based solid-phase peptide chemistry was used to prepare the amphiphilic peptides, with HCTU activation on PL-Rink resin using an automated AB1 433A peptide synthesizer. Amphiphilic peptides were cleaved from the resin and simultaneously side-chain deprotected using a trifluoroacetic acid/thioanisok/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., followed 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. [0159] Peptide-Based Nanoparticlc Formation [0160] Peptides were weighed out as a dry fluffy solid in a round bottom flask, and dissolved in a volume of 1:1 DMF:ACN containing 1% TFA to a final concentration of 0,5 - 2.0 mg/mL. The solution was stirred at 1,000 rpm on ice for 15 min. before addition of 1% - 2% (v/v) cold PFP. After an additional 5 min. of stirring to property mix the components and create an emulsion, an equal volume of cold MilliQ water was slowly added drop wise. 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 lime the solution clarified. Unincorporated peptide was removed by dialyzing the mixture against MilliQ water containing 2.5% (v/v) DMSO to oxidize the cysteines and facilitate disulfide cross-linking of the amphiphilic peptides of the peptide-based 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 peptide-based nanoparticles were removed from the dialysis cassette, placed into a clean glass vial and used for experiments within 48 hours.

[0161] Physico-Chemical Characterization

[0162] 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 peptide-based nanoparticles in water was diluted into characterization buffer (25 mM Tris H.C1, 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 ami 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 (Rl) 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 peptide-based nanoparticles to MilliQ water to achieve a ten-fold dilution, and loading 700 pL of the sample into a disposable folded capillary cell (Malvern, DTS1070). Three independent measurements were taken at 25°C, with twenty runs each. [0163] ln separate studies, the stability of peptide-based nanoparticles during storage was evaluated via dynamic light scattering. Here, purified particles (formulation B of Table 2) 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 I 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.

[0164] Pentide-Based Nanonarticle Visualization

[0165] Differential interference contrast (DIC) microscopy was used to image the peptide- based nanoparticles in solution. Briefly, peptide-based 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 37°C, with a 15 min. sample equilibration time, using a 63x Plan-Apochromat oil objective. [0166]

[0167] A 1.5 mL solution of freshly prepared peptide-based nanoparticles in water (0.5 mg/mL peptide and 2% PFP) was placed in a round bottom 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 peptide-based nanoparticles. All values were corrected for background DTP hydrolysis. Percentage of disulfide formation was calculated by subtracting the concentration of free thiolate from the initial cysteine concentration. Studies were performed in triplicate. [0168] Results [0169] The de novo designed peptide is

capable of assembling at the surface of a perfluoro-n-pentane (PIT) droplet. The peptide sequence contains three pentafluoro-phenyialanine (Fp) 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.

[0170] It has been found that, to form peptide-based nanoparticles, a solvent-exchange procedure in which cold water is slowly added to an organic emulsion of amphiphilic peptides and PFP, ultimately leads to spontaneous assembly of the amphiphilic peptides al 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 peptide-based nanoparticles remain stable for multiple weeks when stored at room temperature in water, as depicted in FIG. 3.

[0171] It has also been found that the size of peptide-based nanoparticles could be precisely controlled between 250 nm and 1,200 nm, as a function of peptide and PFP feed ratio, as depicted in FIG. 9 and as shown in Table 2.

[0172] 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, as depicted in 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 US magnitude required for droplet cavitation.

[0173] Also evaluated was the influence of temperature on peptide-based nanoparticle size through direct visualization of particles in solution using differential interference contrast (DIC) confocal microscopy, as well as dynamic light scattering analysis. Results show that peptide-based nanoparticles with a diameter <750 nm at 258% 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=298°C) and converted the perfluorocarbon liquid cores into gaseous microbubbles at 37%, as evident by the massive increase in diameter for the purple, green and orange formulations. This influence of 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):

where

are the internal droplet pressure and atmospheric pressure, respectively, is the interfacial surface tension and 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%. The influence of vapor pressure on the temperature of the PFP solvent can be defined using the Antoine vapor Equation (2):

in which T and P represent temperature and pressure, respectively, while A, B, and C are equation parameters empirically determined for PFP (Bather 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 (Tvap), ultimately causing thermal droplet vaporization (3):

[0174] Using this equation, the relationship between T_vap and droplet size can be modeled using reported surface tension values for PFP emulsions formulated with either BSA (0.033 Nm^-1), the amphiphilic polymer PEO-PLA (0.027 Nm ^-1), or the cationic surfactant cetrimonium bromide (CTAB; 0.013 Nm ’) (Kandadai et al., 2010). Of note, the PFP-CTAB formulation most closely resembles the peptide-based nanoparticles reported here, in which the cationic amphiphilic sequence acts as the surfactant. As depicted in FIG. 5, results from the model show that, at a surface tension of 0.013 Nm"’, 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.

[0175] This suggests that the US energy required to thermally vaporize the peptide-based 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.

[0176] Next, a series of experiments to test the stability of peptide-based nanoparticles in physiologic environments was performed, and their US-mediated delivery potential was evaluated. For these studies formulation B (see Table 2) was selected, as it remains a droplet at 37^CC, and is predicted to have a core Trap slightly higher than physiologic temperature (~40°C). This should, in theory, permit low intensities of US to be used to impart the additional thermal energy necessary for particle vaporization, thereby minimizing potential physical damage to cells and cargo during insonation. The stability of peptide-based nanoparticles was first evaluated by subjecting a buffered solution of the perfluorocarbon liquid cores to repeated thermocycling, during which dynamic light scattering was used to monitor particle integrity. Results, as depicted in FIG. 9, and Table 2, show that peptide-based nanoparticles not only remained intact under these conditions, but showed little change in overall droplet size. Conversely, formulation E spontaneously vaporized at 37°C to form gaseous microbubbles, which then condensed back to their original size when cooled to room temperature. The stability of formulation B is likely imparted through the disulfide cross-links that are formed between cysteines of adjacent peptides in the peptide-based nanoparticle corona. To investigate this possibility, the propensity of the cysteine resides to undergo oxidation by measuring the rate of disulfide bond formation within freshly prepared peptide-based nanoparticles was assessed in FIG. 6. Results from three independent samples showed that approximately 60% of the available thiols were cross-linked after 1 hour, with a maximum disulfide content of 80% achieved after 24 hours. Control experiments on freshly prepared peptide-based nanoparticles suspended in pure waler, without the DMSO oxidizing agent, showed poor cross-linking ( ~20%) and loss of particle integrity I hour after their assembly, as depicted in FIG. 7.

[0177] To assess the delivery potential of peptide-based nanoparticles fluorescently-labeled phalloidin, a cell impermeable cyclic peptide that binds to intracellular filamentous actin, was loaded into the fluorous core of the particle and monitored its US-mediated transport into cells. Here, encapsulation of this model biomacromolecule was achieved simply by suspending it in the PFP solvent employed tor templated assembly of the peptide-based nanoparticle carrier. UV spectroscopy performed on peptide-based nanoparticles containing the fluorescently-labeled cargo indicated an encapsulation efficiency of 81%, and an overall loading of 2.3X 10⁶ phalloidin per particle. Particles incubated with 5% FBS showed a small increase in size to approximately 700 nm, most likely due to physical adsorption of serum proteins to the cationic particle surface. Particle count rates were 150-200 kcps for both conditions during the incubation period, suggesting peptide-based nanoparticles remained stable in physiologic milieu. Particle sizing performed on peptide-based nanoparticles also confirmed the carrier remains stable under the physiologic conditions employed for delivery studies, as shown in FIG. 8.

[0178] As described herein, a new class of peptide-based nanodroplets, peptide-based nanoparticles, capable of ultrasound-mediated delivery of membrane-impermeable cargo into cells has been developed. In this example, peptide-based nanoparticles are prepared via the de novo designed peptide

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 peptide-based nanoparticle carrier during the assembly process. Cell binding of the peptide-based nanoparticles, followed by acoustic vaporization, ultimately delivers the cargo into cells. Gaseous microbubbles generated during vaporization of peptide-based nanoparticles may also function as an US contrast agent to allow for imaging and guidance of the delivery modality in real-time. Thus, peptide-based nanoparticles of the present invention represent a potential theranostic system with broad applications in drug delivery and biomedical imaging. [0179] Example 2: Ultrasound-Activated Peptide-Based Manoparticle-Mediated Gene Editing in 3D Renal Organoid Models

[0180] Organoids derived from pluripotent stems ceils represent an integral component in the modeling spectrum of human disease and tissue development. These 3D culture systems can recapitulate the unique architecture and cellular organization of human tissues, thus establishing them as an important bridge between 2D in vitro systems and in vivo animal testing. Importantly, genetic mutations can be installed at the start of stem cell differentiation, and/or during organoid development, to mimic the various stages of disease pathology and tissue reconstruction. Yet, appropriately modeling these genetic disorders, as well as performing genomic screening and establishing reporter systems, requires methods to affect specific genetic mutations and repairs within the tissue bulk. While viral gene delivery methods demonstrate high transfection efficiencies, their deployment is limited by biosafety and mutagenesis concerns, limitations in the size of the DN A, vector and poor virion diffusion through the dense and compact organoid structure. Although non-viral approaches, including electroporation and lipofection, address some of the safety concerns inherent to viral vectors, they too suffer from poor tissue diffusion and, as a result, low transfection efficiencies (<5%-20%). To circumvent this, organoids are often sectioned into cell clusters, non-virally transfected, and then re-embedded into solid media. This compromises the spatial architecture, cellular organization, and tissue polarity of the original organoid, ultimately reverting the system back to a 2D culture. Thus, efficient in situ transfection of organoids in their native 3D state remains challenging.

[0181] A nanomaterial enabled, non-viral, acoustic transfection method was developed to improve the diffusion, penetration, and cellular accumulation of gene editing ribonucleoprotein (RNP) complexes within intact 3D tissues, relative to standard chemical transfection approaches. As an exemplary model, adult renal organoids were used, given the large variety and prevalence of genetic diseases that affect the kidneys. Intra-organoid delivery of RNPs was accomplished using a peptide-based nanoparticle vector fabricated from a cell-targeting peptide surfactant that, when emulsified with a perfluorocarbon solvent, formed an ultrasound-sensitive liquid droplet (referred to as a peptidc-based nanoparticle; NPep). NPeps are assembled via emulsification of a de novo designed peptide surfactant and perfluoropentane; ribonucleoprotein (RNP) complexes electrostatically adsorb to the emulsion surface, FIG. 10 depicts an example of this assembly.

[0182] Under ultrasound (US), NPeps arc engineered to undergo a liquid-to-gas phase transition of the particle core to generate microbubbles within the organoid interstitium. RNP- loaded NPeps diffuse within the organoid inlerstitium. where they are vaporized via ultrasound to mechanically permeabilize the tissue ECM. FIG. 11 depicts an example of this vaporization. Cavitational collapse of the bubble nuclei mechanically penneabilizes the dorse organoid extracellular matrix, and simultaneously ballistically delivers adsorbed RNPs across nearby cellular membranes to affect gene editing, as depicted in FIG. 12. NPep acoustic transfection improves both the efficiency and depth of editing compared to standard lipofection approaches in reporter kidney organoids.

[0183] Development and validation of acoustic transfection nanovector

[0184] To generate the nanoemulsion delivery vector, volatile perfluoropentane (PFP) nanodroplets were stabilized using the fluoroamphiphilic peptide emulsifier:

(F_F: pentafluorophenylalanine). The fluorinated N-terminus promotes assembly of the peptide at the PFP-water interface, while two cysteines within the central glycine-rich spacer allow for intermolecular disulfide-crosslinking between adjacent peptides to stabilize the formed particle. Finally, a C-terminal RGD motif is displayed from the surface of the emulsion to permit binding with αVβ3 integrins highly expressed on the surfaces of major nephron cell types (e.g., podocytes, tubules) to promote interstitial accumulation. [0185] Loading of Cas9 to the NPep vector was accomplished via physical mixing to adsorb the protein to the particle surface. While encapsulation and conjugation methods were considered, a simple mixing protocol was elected to form the NPep-RNP complex to match the methodology familiar to most researchers when using standard lipofection reagents. Fluorescent confocal studies using GFP-Cas9 fusion proteins demonstrated that binding of guide RNA (gRNA) to the endonuclease was a prerequisite for its adsorption to the NPep particle surface, as depicted in FIG. 12. This is likely mediated by the cationic surface RGD motif, supporting the necessity of polyanionic gRNA for RNP complexation with cationic nanovectors. Electron microcopy and dynamic light scattering studies confirmed that bound RNPs uniformly decorate the surface of the NPep carrier, as depicted in FIG. 13, and do not significantly alter particle size distribution, respectively.

[0186] Next, the ability of NPeps to acoustically deliver functional RNPs into renal cells using 2D culture models was validated. Vaporization and nonlinear expansion of peptide-based nanoparticles under US can ultimately lead to collapse of the formed microbubble; a process referred to as inertial cavitation. A high-velocity fluid jet formed during asymmetric bubble cavitation penetrates the membrane of emulsion-bound cells to form a transient pore and, in the present system, simultaneously deliver the ejected RNP payload into the cytoplasm, as shown in FIG. 14. This acoustic transfection mechanism was validated in the context of CRISPR'Cas9 gene editors using a knockdown reporter HEK293T-EGFP human embryonic kidney cell line. Importantly, during acoustic transfection, the peptide-based nanoparticle cavitation vector must be localized to the cell surface to allow the fluidic nano-jet to impinge upon the plasma membrane. Consequently, the investigation began by evaluating the time- dependent localization of fluorescently labeled NPeps to the surface of HEK293T-EGFP cells. Results in FIG. 15 show that the particles engage and uniformly decorate the kidney cell surface within a few minutes of exposure, achieving maximum surface density at 4 hours of incubation. Results in FIG. 16 also depict NPep binding properties. RGD peptide peptide-based nanoparticles were incubated with GFP-expressing HEK293T cells, then washed between 1 minute and 4 hours later. Binding fraction in FIG. 16 was normalized to the 4-hour incubation time point. Utilizing this optimized incubation time, gene editing of RNP-loaded NPep formulations

was next evaluated as a function of intensity (FIG. 17) and duty cycle (FIG. 18) of the actuating US stimulus. Results in FIG. 17 show that acoustic intensities >2 W/cm² are sufficient to deliver the RNP payload and affect statistically significant GFP knockdown. Knockdown was found to further increase in a monotonic fashion with the acoustic duty cycle, as shown in FIG. 18. Importantly, these US parameters are well below the FDA diagnostic limit of 190 W/cm² spatial peak pulse average intensity, demonstrating that this approach can permit US-guided gene editing without collateral mechanical tissue damage.

[0187] Acoustic delivery of RNPs into kidney organoids

[0188] Kidney organoids were generated from human pluripotent stem cells (hPSC). The differentiated tissues were approximately 100-400 μm in diameter and contain podocyte, proximal tubule, and distal tubule segments in nephron-like arrangements. Utilizing this renal model, the ability of NPeps to permeabilize the tissue by studying the diffusion of the nuclei staining dye DAP1 within the organoid bulk was evaluated. Signal deficient regions within the core of DAPl-stained kidney organoids was a result of poor diffusion of the dye throughout the dense tissue interstitium. Permeabilization of the organoid extracellular matrix by US-activated

particles produced more uniform staining throughout the tissue cross-section, improving total nuclei fluorescence by -25% relative to untreated and static CRISPRMAX lipofection controls, as shown in FIG. 19.

[0189] Next, the intra-organoid delivery of a Cas9-GFP fusion, and its paired gRNA , before and after US delivery from vectors was evaluated, as shown in FIG. 20. Both the

CRISPRMAX control and unactivated particles showed sequestration of Cas9

at the tissue surface, with limited penetration of the endonuclease in tire organoid core. Similarly, both conditions resulted in poor overlap of the delivered Cas9 and gRNA signals across the tissue cross-section (see fluorescent line plots in FIG. 20. right). US vaporization of NPepRNp, and acoustic ejection of the adsorbed RNP complex, led to enhanced intra-organoid penetration of Cas9, and improved Cas9-gRNA co-localization, within the tissue interstitium relative to controls.

[0190] To evaluate gene editing, a reporter organoid model derived from a human iPS cell line with Ai9 fluorescence-on reporter knocked into the AAVS1 safe harbor locus was utilized. Successful RNP-mediated knockout of the Ai9 cassette leads to production of tdTomato. Additional proximal tubule and podocyte markers were included to spatially collate gene editing events to major nephron cell types. Three-dimensional confocal microscopy was then performed to quantitate the relative expression of tdTomato as a function of depth of editing within the organoid tissue. Results in FIG. 21 show that treating organoids with RNPs complexed to CRISPRMAX, or and left unactivated by US, produced similarly poor

gene editing across the organoid height US vaporization of

however, led to a significant enhancement in gene editing efficiency that monotonically increased with US intensity. Notably, at the highest intensity tested (3 W/cm²), a 4-fold enhancement in maximum editing activity was observed for

relative to the CRISPRMAX control. All edited organoids showed a maximum of the tdTomato signal -20 μm in height from the bottom of the plate surface. This was attributed to the higher cell density at the base of the organoid, which adopts a mounded tissue architecture.

[0191] Next, the maximum tdTomato signal intensity was correlated with editing variance for each organoid transfection condition, as shown in FIG. 22. This analysis was done since, in addition to maximizing editing efficiency, low variance of edits between replicates is a key milestone for reliable transfection technologies. Variability generally decreased with increasing US intensity for treated organoids, with 3 W/cm² providing the most reproducible

editing results. No clear correlation could be elucidated from duty cycle. Ultimately, activation of at 3 W/cm² and 80% duty cycle was determined to provide optimized editing

efficiency and reproducibility. Conversely, CRISPRMAX controls showed poor editing efficiency and an intermediate level of variability.

[0192] Finally, the depth of editing in kidney organoids treated with the static (CRISPRMAX, without US activation) and active

transfection conditions was compared, as shown in FIG. 23. For this comparison, the height at which the maximum tdTomato signal was measured was plotted in a vertical confocal imaging slack for each treated organoid, collated with the overall editing efficiency (color scale), to assess how US parameters alter the depth of editing within the organoid bulk This analysis showed that, in general, increasing US intensity and duty cycle led to a higher incidence of editing in the organoid core (-15 - 20 μm height), where podocyte and proximal tubule density is the highest. Moreover, the highest US intensity (3 W/cm²) and duty cycle (80%) produced the highest editing efficiency within this focal range compared to the other conditions tested (as indicated by the darkest red hue). Conversely, low US intensities, as well as the uninsonated and CRISPRMAX controls, tended to display both low editing efficiencies and

maximum tdTomato signal that was stochastically distributed across the organoid height. Taken together, the data suggests that an increase in acoustic energy drives carriers deeper

into the organoid bulk, permitting delivery of RNP editors to internal cells that might be otherwise inaccessible to passive transfection technologies. Finally, the z-stacked images show a preference for US-activated

edited cells to cluster within specific regions of the organoid, rather than being uniformly distributed throughout the tissue bulk, as shown in FIG. 24. This suggests a cell phenotypic preference for NPep-mediated gene editing within the kidney organoids, an assertion that was next tested through immunohistochemistry analysis.

[0193] Cell Editing Specificity

[0194] To investigate cell type-specific editing in organoids treated with CRISPRMAX and

the average tdTomato signal across the tissue height was plotted to nuclei, podocyte and proximal tubule phenotypic markers, as shown in FIGs. 25-28. RNP delivery via CRISPRMAX showed a statistically significant deviation of tdTomato fluorescence from the general cell nuclei marker, as shown in FIGs. 25-26. Taken in conjunction with the confocal imaging, which showed substantial editing of the underlying stroma layer (0 - .10 pm height), suggesting that CRISPRMAX may non-specifically deliver RNPs to the supporting mesenchyma, rather than the internal podocyte and tubule cells. Conversely, US-mediated transfection showed a much sharper rise in tdTomato signal between 0 - 30 pm of

tissue height, as shown in FIG. 27, relative to CRISPRMAX, suggesting editing was directed towards intraorganoid renal cells, as opposed to the stroma. This is further supported by a lack of statistical significance between the tdTomato and general nuclei profiles of NPepRNP treated organoids, as shown in FIG. 28.

[0195] Better alignment of the tdTomato profile from

edited organoids to podocyte and proximal tubule signals, compared to CRISPRMAX, was qualitatively observed. To quantitatively evaluate this assertion, a p value heatmap was prepared to compare the degree of correlation between signals, as shown in FIG. 29. Here, higher p values (lighter colors) are indicative of greater statistical similarity between the data, and thus greater alignment between the tdTomato signal and particular phenotypic marker. This analysis showed a 5.2-fold higher correlation of tdTomato expression and podocyte and proximal tubule markers for

edited organoids relative to CR1SPRMAX. In addition to this comparative preference to edit nephron cell-types,

also showed a cumulatively greater number of podocytes and tubules edited relative to CRISPRMAX on a per organoid basis, as shown in FIG. 30. This is most likely a consequence of the superior global editing efficiency of the vector. In

sum, the data collectively suggests that ballistic ejection of RNPs from the NPep carrier leads to their preferential delivery to, and subsequent editing of, functional nephron cells within the organoid bulk, rather than the collateral mesenchymal layer, in addition to acoustically- enhanced NPep diffusion, this specificity for nephron cell types may also be due to binding of the RGD motif displayed from the surface of the emulsion to integrins overexpressed on the surfaces of podocytes and tubules. Consequently, US-actuated NPep vectors provide significantly improved efficiency, tissue depth, and precision of CRIS.PR-Cas9 gene editing in organoids relative to standard lipofection methodologies.

[0196] The elements of the figures are not exclusive. Other embodiments may be derived in accordance with the principles of the invention to accomplish the same objectives. Although this invention has been described with reference to particular embodiments, it is to be understood that the embodiments and variations shown and described herein are for illustration purposes only. Modifications to the current design may be implemented by those skilled in the art, without departing from the scope of the invention.

[0197] While various illustrative embodiments incorporating the principles of the present teachings have been disclosed, the present teachings are not limited to the disclosed embodiments. Instead, this application is intended to cover any variations, uses, or adaptations of the present teachings and use its general principles. Further, this application is intended to cover such departures from the present disclosure that are within known or customary practice in the art to which these leachings pertain.

[0198] In the above detailed description, reference is made to the accompanying drawings, which fonn a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the present disclosure are not meant to be limiting. Other embodiments may be used, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that various features of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0199] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various features. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It Ls also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0200] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and'or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

[0201] It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (for example, the term “including" should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least," the term “includes” should be interpreted as “includes but is not limited to," et cetera). While various compositions, methods, and devices are described in terms of “comprising" various components or steps (interpreted as meaning “including, but not limited to"), the compositions, methods, and devices can also “consist essentially oP or “consist oP the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups.

[0202] As used in this document, the singular forms “a," “an," and “the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Nothing in this disclosure is to be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by virtue of prior invention.

[0203] In addition, even if a specific number is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (for example, the bare recitation of “two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, et cetera" is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, and C" would include but not be limited to systems that have A done, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, et cetera). In those instances where a convention analogous to “at least one of A. B, or C, et cetera” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, or C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, et cetera). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, sample embodiments, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.” [0204] Various of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art, each of which is also intended to be encompassed.

SEQUENCES

Minimal Targeting Motif

SEQ ID NO: 1 -Minimal Target Motif 1

RGRR

SEQ ID NO: 2 -Minimal Target Motif 2

RGRRS

SEQ ID NO: 3 - Minimal Target Motif 3

YQLDV

SEQ ID NO: 4-Mtnimal Target Motif 4

VGVA

Targeting Motif

SEQ ID NO: 5 - Targeting Motif 1

GHGKHKNK

SEQ ID NO: 6 -Targeting Motif 2

CRGDKGPDC

SEQ ID NO: 7 - Targeting Motif 3

CKGAKAR

SEQ ID NO: 8 - Targeting Motif 4

CRVSRQNKC

SEQ ID NO: 9 -Targeting Motif 5 CGGERGKSC SEQ ID NO: 10 -Targeting Motif 6

CRSRKG

SEQ ID NO: 11 -Targeting Motif 7

CKAAKN

SEQ ID NO: 12 - Targeting Motif 8

CRGRRST

SEQ ID NO: 13 - Targeting Motif 9

CEYQLDVE

SEQ ID NO: 14 -Targeting Motif 10

TVRTSAD

SEQ ID NO: 15 -Targeting Motif 11

PIEDRPM

SEQ ID NO: 16 - Targeting Motif 12

ALRDRPM

SEQ ID NO: 17 -Targeting Motif 13

PEKFRPM

SEQ ID NO: 18 - Targeting Motif 14

IKVGKLQ

SEQ ID NO: 19 -Targeting Motif 15

SVSVGMKPSPRP

SEQ ID NO: 20 -Targeting Motif 16

VPEQRPM SEQ ID NO: 21 -Targeting Motif 17

CAK1DPELC

SEQ ID NO: 22 -Targeting Motif 18

CSNIDARAC

SEQ ID NO: 23 - Targeting Motif 19

RLQLKL

SEQ ID NO: 24 - Targeting Motif 20

PMMRQRPM

SEQ ID NO: 25 -Targeting Motif 21

AKATCPA

SEQ ID NO: 26 - Targeting Motif 22 QPPMEYS

SEQ ID NO: 27 - Targeting Motif 13

SISSLTD

SEQ ID NO: 28 -Targeting Motif 24

FRVGVADV

SEQ ID NO: 29 - Targeting Motif 25

CNGRCVSGCAGRC

SEQ ID NO: 30 -Targeting Motif 26

NWGDR1L

SEQ ID NO: 31 -Targeting Motif 27

CVSNPRWKC SEQ ID NO: 32 - Targeting Motif 28

CDCRGDCFC

SEQ ID NO: 33 -Targeting Motif 29

YSAYPDSVPMMS

SEQ ID NO: 34 - Targeting Motif 30

PLASRPM

SEQ ID NO: 35 - Targeting Motif 31

KGRGD

SEQ ID NO: 36 -Targeting Motif 32

RGDS

SEQ ID NO: 37 - Targeting Motif 33

GRGD

SEQ ID NO: 38 - Targeting Motif 34

GRGDS

SEQ ID NO: 39 -Targeting Motif 35

GRGDSP

SEQ ID NO: 40 - Targeting Motif 36

GRGDSPK

SEQ ID NO: 41 -Targeting Motif 37

GRGDNP

SEQ ID NO: 42 -Targeting Motif 38

GRGDTP SEQ ID NO: 43 -Targeting Motif 39

CRKRLDRNC

SEQ ID NO: 44 - Targeting Motif 40

EFEEFEIDEEEK

SEQ ID NO: 45 - Targeting Motif 41

EFEEFEIDEEEK

Cross-Linking Motif

SEQ ID NO: 46 -Cross-Linking Motif 1

GGGCCGG

Amphiphilic Peptide

SEQ ID NO: 47 - Amphiphilic Peptide (Formula IV)

Hydrophilic Amino Add Sequence HP

SEQ ID NO: 48 - C-termlnally Amidated Hydrophilic Amino Add Sequence HP

KGRGD-NH2

Amphiphilic Peptide

SEQ ID NO: 49 - Amphiphilic Peptide (Formula V)

Targeting Motif

SEQ ID NO: 50 - Targeting Motif 42

CRGRRT

Claims

CLAIMS We claim:

1. A composition comprising a plurality of peptide-based nanoparticles, wherein each peptide-based nanoparticle comprises a perfluorocarbon liquid core, a plurality of amphiphilic peptides surrounding the perfluorocarbon liquid core, and at least one gene editing complex attached to the amphiphilic peptides, wherein each amphiphilic peptide is represented by Formula (111):

HB-CL-HP-NH2 (III) wherein HB is a fluorinated hydrophobic block consisting of three to live consecutively connected pentafluorinated hydrophobic amino acid residues; wherein CL is an amino acid sequence consisting of two to 10 amino acid residues, at least two of which are cross-linking cysteine residues; wherein HP is a hydrophilic amino acid sequence, wherein said amphiphilic peptide consists of 8 to 30 total amino acid residues, 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 HP extend away from the perfluorocarbon liquid core.

2. The composition of claim 1 , wherein the gene editing complex is selected from the group consisting of CRISPR-Cas9 ribonucleoprotein complexes, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and RNA-guided endonucleases.

3. The composition of claim 2, wherein the CR1SPR.-Cas9 ribonucleoprotein complex comprises a Cas9 protein and guide RNA (gRN A) electrostatically adsorbed to the amphiphilic peptides.

4. The composition of any one of claims 1 to 3, wherein the gene editing complex is surface-adsorbed onto the peptide-based nanoparticles.

5. The composition of any one of claims 1 to 4, wherein the composition is used for targeted delivery of gene editing agents to mammalian cells.

6. The composition of any one of claims 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 claims 1 to 6, wherein the perfluorocarbon liquid core is selected from the group consisting of perfluoropentane, perfluorohexane, and perfluorobutane.

8. The composition of any one of claims 1 to 7, 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.

9. The composition of any one of claims I to 7, wherein HP comprises lysine, glycine, arginine, aspartic acid, or any combination thereof.

10. The composition of any one of claims 1 to 9, wherein HP comprises the sequence

KGRGD (SEQ ID NO: 35), where K is lysine, G is glycine, R is arginine, and D is aspartic acid.

1 1. The composition of any one of claims 1 to 10, wherein CL comprises GGGCCGG (SEQ ID NO: 46), where G is glycine and C is cysteine.

12. The composition of any one of claims 1 to 11 , wherein said hydrophilic amino acid sequence of HP comprises a targeting moti f.

13. The composition of any one of claims 1 to 12, wherein the targeting motif is selected from a group consisting of GHGKHKNK (SEQ ID NO;5), CRGDKGPDC (SEQ ID NO:6), CKGAKAR (SEQ ID NO:7), CRVSRQNKC (SEQ ID NO:8). CGGERGKSC (SEQ ID NO:9), CRSRKG (SEQ ID NO: 10), CKAAKN (SEQ ID NO: 1 1 ), CRGRRT (SEQ ID NO:50), CRGRRST (SEQ ID NO:12), CEYQLDVE (SEQ ID NO:13), CEYQLDVE (SEQ ID NO: 13), TVRTSAD (SEQ ID NO: 14), PIEDRPM (SEQ ID NO: 15), PIDERPM (SEQ ID NO: 15), ALRDRPM (SEQ ID NO: 16), PEKFRPM (SEQ ID NO: 17), 1KVGKLQ (SEQ ID NO: 18), SVSVGMKPSPRP (SEQ ID NO: 19), VPEQRPM (SEQ ID NO:20), CAKIDPELC (SEQ ID NO:21), CSNIDARAC (SEQ ID NO:22), RLQLKL (SEQ ID NO:23). PMMRQRPM (SEQ ID NO:24), AKATCPA (SEQ ID NO:25), QPPMEYS (SEQ ID NO:26), S1SSLTD (SEQ 1DNO:27), FRVGVADV (SEQ ID NO:28), CNGRCVSGCAGRC (SEQ ID NO:29), NWGDRIL (SEQ ID NO:30), CVSNPRWKC (SEQ ID NO:31), CDCRGDCFC (SEQ ID NO:32), YSAYPDSVPMMS (SEQ ID NO:33), PLASRPM (SEQ ID NO:34), KGRGD (SEQ ID NO:35), RODS (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 ), GRGDTP (SEQ ID NO:42), CRKRLDRNC (SEQ ID NO:43), EFEEFEIDEEEK (SEQ ID NO:44), and DFEE1PEEYLQ (SEQ ID NO:45).

14. The composition of any one of claims 1 to 13, wherein the targeting motif comprises a minimal targeting motif selected from the group consisting of: HGK, RGD, KAR. RSR, KAA, RGRR (SEQ ID NO:1), RGRRS (SEQ ID NO:2). YQLDV (SEQ ID NO:3), EYQ, RPM, PSP, VGVA (SEQ ID NO:4), and NGR.

15. The composition of any one of claims 1 to 12, wherein the targeting motif comprises KGRGD (SEQ ID NO: 35), which targets αVβ3 integrins.

16. The composition of any one of claims 1 to 15, wherein said amphiphilic peptide comprises an amphiphilic peptide represented by Formula (IV) or Formula (V):

wherein F_F is pentafluoro-pbenylalanine, G is glycine, C is cysteine, K is lysine, G is glycine, R is arginine, and D is aspartic acid.

17. The composition of any one of claims 1 to 16, wherein said amino acid sequence of CL consists of two to 10 amino acid residues and said hydrophilic amino acid sequence of HP consists of 3 to 15 hydrophilic amino acids, and wherein said amphiphilic peptide consists of 10 to 30 total amino acid residues.

18. The composition of any one of claims 1 to 17, wherein the amphiphilic peptide has a molecular weight in the range of about 2000 - 5000 daltons, wherein the amphiphilic peptide includes at least eight 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 connected consecutively by peptide bonds without any intervening amino acid residues.

19. A method of preparing a composition comprising a plurality of peptide-based nanoparticles loaded with a gene editing complex, the method comprising: assembling a plurality of peptide-based nanoparticles by emulsifying amphiphilic peptides with a perfluorocarbon liquid; cross-linking the amphiphilic peptides; mixing the plurality of peptide-based nanoparticles with a gene editing complex solution under gentle agitation; incubating the mixture of a period sufficient to allow adsorption of the gene editing complex onto the surface of the peptide-based nanoparticles; removing unbound gene editing complex molecules by a process selected from centrifugation, filtration, or dialysis; and recovering and purifying the peptide-based nanoparticles by isolating the nanoparticles from the suspension.

20. A method of performing gene editing in a cellular construct, the method comprising: administering a composition comprising of any one of claims 1 to 18 to the cellular construct: administering ultrasonic waves to the cellular construct; wherein the ultrasonic waves cause a rupture of one or more of the peptide-based nanoparticles and permeabilize the extracellular matrix of the cellular construct, thereby allowing a gene editing complex to be delivered to the cells within the cellular construct, facilitating the editing of the DNA within the cells.

21. The method of claim 20, wherein the administrating of ultrasonic waves comprises applying ultrasound at an intensity of less than 1 .0 watts per square centimeter.

22. The method of claim 20, wherein the ultrasonic waves are applied at an intensity of 1 W/cm² or greater.

23. The method of claim 22, wherein the ultrasonic waves are applied with a duty cycle ranging from about 20% to 80%.

24. The method of claim 20, wherein the cellular construct is an organoid.

25. The method of claim 24, wherein the organoid is derived from pluripotent stem cells.

26. The method of claim 24, wherein the organoid is a renal organoid comprising podocytes, proximal tubules, and distal tubules.