Nanobubbles for ultrasound mediated nucleic acid delivery

Abstract

Gas-filled nanobubbles for negatively charged genetic material delivery each includes a lipid membrane defining a gas containing internal void, wherein the lipid membrane includes a plurality of cationic lipids for complexing the negatively charged genetic material, an edge-activator incorporated between lipids of the membrane that enhances the flexibility of the membrane, and a membrane stiffener incorporated on an outer surface of the membrane that enhances the membrane's resistance to tearing.

Description

NANOBUBBLES FOR ULTRASOUND MEDIATED NUCLEIC ACID DELIVERYRELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application No. 63 / 587,839, filed October 4, 2023, the subject matter of which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] This application relates to therapeutic compositions, and more particularly to gas-filled nanoparticles or nanobubbles for therapeutic applications.BACKGROUND

[0003] Ultrasound contrast agents (UCA) are small gas-filled bubbles with a stabilizing shell made from a variety of materials, such as polymer, protein or lipid. Other than the traditional applications of these agents in diagnostic ultrasound imaging, UCA have found relevance in therapeutic applications including targeted gene and drug delivery. These adaptable particles are currently being explored as protective therapeutic carriers and as cavitation nuclei to enhance delivery of their payload by sonoporation. Together these functions improve payload circulation half-life and release profiles as well as tissue selectivity and cell uptake.

[0004] Commercial UCA available today are typically designed to serve only as blood pool agents with diameters of 1-8 pm. Although previous methodologies have been developed to reduce bubble size, most of these strategies involve manipulations of microbubbles post formation, such as gradient separation by gravitational forces or by physical filtration or floatation. While effective for selecting nanosized bubbles, these methods introduce potential for sample contamination, reduce bubble yield and stability, and waste stock materials in addition to being labor intensive. Additionally, the applicability of microbubbles as carriers has been limited by a large size, which typically confines them to the vasculature.SUMMARY

[0005] Embodiments described herein relate to cationic gas-filled nanobubbles (also referred to as sub-micron bubbles, gas-core nanoparticles or ultrafine bubbles) and their use in nucleic acid cell delivery, transfection, and / or gene therapy. The cationic nanobubbles can readily complex or be loaded with at least one negatively charged genetic material or nucleicacid, protect the complexed or loaded nucleic acids from degradation when administered systemically to a subject, be targeted to and readily internalized by targeted cells, and release the complexed or loaded nucleic acids within the cells upon insonation with ultrasound energy. In contrast to commercial microbubbles, the nanobubbles described herein are small enough, e.g., less than about 600 nm in diameter (e.g., average diameter approximately 300 nm with a 10% polydispersity), to extravasate through endothelial barriers and readily enter and cross cell membranes.

[0006] Advantageously, in contrast to traditional transfection methods, ultrasound stimulated delivery of nucleic acids to cells using nanobubbles described herein can provide contrast agents to allow real-time visualization of nanobubble delivery. Further, nanobubble stimulation increases cell permeability and enhances tumor, cell, and nucleus uptake and has been shown to promote endosomal escape and cytosolic release upon cellular uptake.Moreover, the nanobubbles can be readily functionalized with receptors and ligands to target tissues or cells of interest. Additionally, focusing ultrasound at a target site will facilitate transfection or nucleic acid release form the nanobubbles only at that site, leaving other cells and / or unaffected.

[0007] In some embodiments, each nanobubble can include a lipid membrane that defines a gas containing internal void or space. The membrane can include a plurality of cationic lipids that can provide the nanobubble with a cationic charge effective to complex at least one negatively charged genetic material, an edge- activator incorporated between lipids of the membrane that enhances the flexibility of the membranes, and a membrane stiffener incorporated on an outer surface of the membrane that enhances the membrane's resistance to tearing.

[0008] In some embodiments, the nanobubbles can be complexed and / or loaded with at least one negatively charged genetic material or nucleic acid. The negatively charged genetic material or nucleic acid can include at least one of DNA, pDNA, mRNA, microRNA, siRNA, lincRNA, plasmids, nanoplasmids, or other negatively charged genetic material.

[0009] In some embodiments, the plurality of cationic lipids can include cationic lipids having saturated C16-C22 hydrophobic tails and optionally cationic lipids having unsaturated C16-C22 hydrophobic tails. Cationic lipids having saturated C16-C22 hydrophobic tails can include, for example, l,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1,2- Dipalmitoyl-3-trimethylammonium propane (DPTAP), l,2-distearoyl-3-dimethylammonium-propane (DSDAP), N-[l-(2,3-dilauroyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DLTAP), N-[l-(2,3-dimyristoyloxy)propyl]-N,N,N-tri methylammonium methylsulfate (DMTAP), N-[l-(2- palmitoyloxy-3-lauroyloxy)propyl]-N,N,N- trimethylammonium methylsulfate (LPTAP), or a combination thereof.

[0010] In some embodiments, the lipid membrane further includes a plurality of ionizable lipids, anionic or negatively charged lipids, and / or neutral lipids. The ionizable lipids, anionic lipids, and / or neutral lipids can include phospholipids.

[0011] In some embodiments, the phospholipids can each include a hydrophilic head containing a phosphate group and two C16-C22 hydrophobic tails. Preferably, the C16-C22 hydrophobic tails are saturated. Examples of phospholipids including a hydrophilic head containing a phosphate group and two saturated C16-C22 hydrophobic tails are selected from dibehenoylglycerophosphocoline (DBPC), dipalmitoylphosphatidic acid (DPP A), dipalmitoylphosphatidylethanolamine (DPPE), PEG functionalized, distearoylphosphatidylethanolamine (DSPE-PEG), or combinations thereof. Preferably, the phospholipids include a combination of dibehenoylglycerophosphocoline (DBPC), dipalmitoylphosphatidylethanolamine (DPPE), and PEG functionalized, distearoylphosphatidylethanolamine (DSPE-PEG).

[0012] In some embodiments, the lipid membrane includes a mixture of dibehenoylglycerophosphocoline (DBPC), dipalmitoylphosphatidylethanolamine (DPPE), PEG functionalized distearoylphosphatidylethanolamine (DSPE-PEG), and 1 ,2-Dipalmitoyl- 3-trimethylammonium propane (DPTAP).

[0013] In some embodiments, the ratio of DBPC, DPPE, DSPE-PEG, and DPTAP in lipid membrane is about 6:2: 1 :0.25-1.

[0014] In some embodiments, the stabilized nanobubbles can have a lipid concentration of at least about 5 mg / ml, preferably, about 5 mg / ml to about 30 mg / ml.

[0015] In some embodiments, the edge activator consists of propylene glycol and the membrane stiffener consists of glycerol.

[0016] In some embodiments, the gas includes at least one perfluorocarbon gas, preferably C3F8 or C4F10.

[0017] In some embodiments, the lipid membrane optionally includes at least one of a surfactant, alcohol, or cholesterol, that change the shape, modulus, and / or interfacial tension of the bubble membrane. For example, the cationic nanobubble can include additionalsurfactants, such as erucyl bis-(hydroxyethyl)methylammonium chloride (EH AC), cocamidopropyl betaine, or other phospholipids, to adjust the shape of the nanobubbles and provide nanobubbles having a filament, vermiform, or worm-like shape.

[0018] In some embodiments, the nanobubbles can have an average diameter of about 30 nm to about 600 nm, preferably, about 50 nm to about 500 nm.

[0019] In some embodiments, the nanobubbles can further include at least one targeting moiety that is linked to the membranes of the nanobubbles. The targeting moiety can be selected from the group consisting of polypeptides, polynucleotides, small molecules, elemental compounds, antibodies, and antibody fragments.

[0020] In other embodiments, the nanobubbles can further include a polymer that coats at least a portion of an outer surface of the lipid membrane. The polymer coating can inhibit degradation of the at least one negatively charged genetic material or nucleic acid loaded on and / or complexed the nanobubbles. In one example, the polymer coating can include a layer of polydopamine coated on at least the portion of the outer surface.

[0021] Other embodiments described herein relate to a method of delivering a genetic material to a cell or tissue of interest. The method includes providing a plurality of nanobubbles as described herein loaded with at least one negatively charged genetic material or nucleic acid. The plurality of nanobubbles loaded with the negatively charged genetic material are administered to the cell or tissue. Optionally the nanobubbles internalized by the cell or tissue are insonated with ultrasound energy effective to release the negatively charged genetic material.

[0022] In some embodiment, the plurality of nanobubbles are insonated at a duty cycle of about 1% to about 100%, an ultrasound frequency of about 0.2 kHz to about 50 MHz (e.g., about 0.1 MHz to about 50 MHz or about 0.5 MHz to about 18 MHz), an intensity of about 0.1 W / cm2to about 5 W / cm2, a pressure amplitude of about 1 kPa to about 10 MPa (e.g., 50 kPa to about 5 MPa), and a time of about 5 seconds to about 30 minutes.

[0023] In some embodiments, the ultrasound energy is provided by a non-focused ultrasound transducer, a diagnostic imaging ultrasound transducer, a focused ultrasound transducer, or high-intensity focused ultrasound (HIFU) transducer.

[0024] In some embodiments, the ultrasound energy can be provided from a nonfocused transducer, which is distinct from typical focused ultrasound transducers used fordrug delivery and ultrasound therapy, such as high intensity focused ultrasound (HIFU) or histotripsy.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Fig. 1 illustrates a schematic image of a nucleic acid loaded nanobubble in accordance with an embodiment.

[0026] Fig. 2 illustrates a schematic of a lipid solution production protocol for use in forming nanobubbles in accordance with an embodiment.

[0027] Fig. 3 illustrates a schematic of nanobubble activation and pDNA loading protocol.

[0028] Figs. 4(A-B) illustrate graphs comparing the (A) size and (B) surface charge (zeta potential) of unloaded anionic nanobubbles and cationic nanobubbles prepared using a DOTAP lipid solution and DPTAP lipid solution.

[0029] Fig. 5 illustrates an image showing concentrations of nanobubbles prepared using a DOTAP lipid solution and a DPTAP lipid solution.

[0030] Fig. 6 illustrates a graph comparing concentration of buoyant and non-buoyant nanobubbles prepared using DOTAP lipid solution and DPTAP solution.

[0031] Figs. 7(A-B) illustrate plots comparing ultrasound stabilities of nanobubbles prepared using DOTAP lipid solution and DPTAP solution.

[0032] Fig. 8 illustrates ultrasound contrast images of nanobubbles prepared using a DOTAP lipid solution and a DPTAP lipid solution of Figs. 7(A-B).

[0033] Fig. 9 illustrates images showing co-localization of a plasmid vector loaded on nanobubble prepared using a DPTAP lipid solution.

[0034] Fig. 10 illustrates images showing loading capacity of pmaxGFP plasmid and cmvGFP nano-plasmid of nanobubbles prepared using a DPTAP lipid solution by electrophoresis.

[0035] Fig. 11 illustrates zeta potential of pmaxGFP plasmid and cmvGFP nanoplasmid of nanobubbles prepared using a DPTAP lipid solution.

[0036] Figs. 12(A-B) illustrate a plot and graph showing the effect of pDNA loading on concentration and ultrasound contrast stability of nanobubbles prepared using a DPTAP lipid solution.

[0037] Figs. 13(A-B) illustrate an image and graph showing PC3-pip cell uptake of nanobubbles prepared using a DPTAP lipid solution.

[0038] Fig. 14 illustrates a graph showing PC3-pip cell uptake of nanobubbles prepared using anionic nanobubbles.

[0039] Figs. 15(A-D) illustrate plots, images, and graphs showing ultrasound contrast of nanobubbles prepared using a DPTAP solution in PC3-pip cells.

[0040] Fig. 16 illustrates images showing transfection efficiency of nanobubbles prepared using a DPTAP solution loaded with pmaxGFP in PC3-pip cells without and with administration of therapeutic ultrasound.

[0041] Figs. 17(A-B) illustrates a plot and images showing stability of nanobubbles prepared using a DPTAP lipid solution loaded with cmvGFP nano-plasmid in mouse liver.

[0042] Fig. 18 illustrates images showing transfection efficiency of nanobubbles prepared using a DPTAP lipid solution loaded with pmaxGFP in prostate cancer cells without and with administration of therapeutic ultrasound.

[0043] Fig. 19 illustrates images showing transfection efficiency of nanobubbles prepared using a DPTAP solution loaded with pmaxGFP in livers of mice administered the nanobubbles after therapeutic ultrasound.DETAILED DESCRIPTION

[0044] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the application.

[0045] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Furthermore, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. The terms "comprising", "including", "having" and "constructed from" can also be used interchangeably.

[0046] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0047] The term "antibody" as used herein, refers to an immunoglobulin molecule, which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1988; Houston et al., 1988; Bird et al., 1988).

[0048] The term "antigen" or "Ag" as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0049] The term “genetic material” includes within its meaning any nucleic acid (or nucleotide sequence) which can be used either as such or for expressing a corresponding amino acid, peptide or protein once delivered into a cell. The genetic material includes in particular nucleic acids, RNA, and DNA, of either natural or synthetic origin including recombinant RNA and DNA, antisense or antigen RNA. The term refers both to the nucleotide sequence (gene) as such or preferably to the gene incorporated into a suitable vehicle. Suitable vehicles for carrying the genes include expression vectors such as plasmids, phagemids, cosmids, yeast artificial chromosomes (YAC), bacterial artificial chromosomes(BAC), human artificial chromosomes (HAC), chimeric oligonucleotide comprising RNA and DNA residues in a duplex conformation, hairpin or hammerhead ribozyme, small interfering RNA (siRNA), siRNA expression cassettes, DNA ribonucleases, transposon, both single and double strand RNA and DNA and analog thereof, such as phosphorothioate, phosphorodithioate oligodesoxynucleotide.

[0050] The terms “inhibit,” “silencing,” and “attenuating” can refer to a measurable reduction in expression of a target mRNA (or the corresponding polypeptide or protein) as compared with the expression of the target mRNA (or the corresponding polypeptide or protein) in the absence of an interfering RNA molecule of the present invention. The reduction in expression of the target mRNA (or the corresponding polypeptide or protein) is commonly referred to as “knock-down” and is reported relative to levels present following administration or expression of a non-targeting control RNA.

[0051] The term "nucleic acids," "nucleic acid molecules," "nucleotides," "nucleotide(s) sequence," and "polynucleotide" are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxy guanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Single stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or singlestranded RNA (ssRNA). Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences can be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (z.c., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" of the disclosure comprises one or more nucleic acids as described herein.

[0052] As used herein, a "coding region" or "coding sequence" is a portion of polynucleotide which consists of codons translatable into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows, then, that a single vector can contain just a single coding region, or comprise two or more coding regions.

[0053] The term "downstream" refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0054] The term "upstream" refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that are located on the 5' side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0055] The term "expression" as used herein refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes without limitation transcription of the polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, associationwith other protein subunits, or proteolytic cleavage. The term "yield," as used herein, refers to the amount of a polypeptide produced by the expression of a gene.

[0056] A "vector" refers to any vehicle for the cloning of and / or transfer of a nucleic acid into a host cell. A vector can be a replicon to which another nucleic acid segment can be attached so as to bring about the replication of the attached segment. A "replicon" refers to any genetic element e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "vector" includes vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini.

[0057] Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and / or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), P-galactosidase (LacZ), ^-glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters.

[0058] The term "heterologous" means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide). Similarly, a cellular sequence e.g., a gene or portion thereof) that is incorporated into a viral vector is a heterologous nucleotide sequence with respect to the vector.

[0059] The term "heterologous gene" or “heterologous nucleic acid” are used interchangeably and refers to a gene or nucleic acid that does not naturally occur as part of a viral genome. For instance, a heterologous gene can be a mammalian gene, e.g., a therapeutic gene, e.g., a mammalian gene that encodes a therapeutic protein. In some embodiments, a heterologous gene encodes a protein or portion thereof that is defective or absent in the target cell and / or subject. In some embodiments, the heterologous gene contains one or more exons encoding a protein that is defective or absent in the target cell and / or subject. For example, in some embodiments, the heterologous gene includes one or more trans-splicing molecules, e.g., as described in WO 2017 / 087900, which is incorporated herein by reference in its entirety. In some embodiments, a heterologous gene includes a therapeutic nucleic acid, such as a therapeutic RNA e.g., microRNA).

[0060] The term "promoter" refers to a sequence that regulates transcription of a heterologous gene operably linked to the promoter. Promoters provide the sequence sufficient to direct transcription and / or recognition sites for RNA polymerase and other transcription factors required for efficient transcription and can direct cell-specific expression. In addition to the sequence sufficient to direct transcription, a promoter sequence can also include sequences of other regulatory elements that are involved in modulating transcription e.g., enhancers, kozak sequences, and introns).

[0061] The term "target cell" refers to any cell to which the nanobubbles described herein can be targeted. Target cells can include cells that reside in a subject (in situ) or cells in culture. In some embodiments, target cells are post-mitotic cells. Target cells include both vertebrate and invertebrate animal cells (and cell lines of animal origin). Representative examples of vertebrate cells include mammalian cells, such as humans, rodents (e.g., rats and mice), and ungulates (e.g., cows, goats, sheep and swine). Target cells include ocular cells, such as retinal cells. Alternatively, target cells can be stem cells (e.g., pluripotent cells (i.e., a cell whose descendants can differentiate into several restricted cell types, such as hematopoietic stem cells or other stem cells) or totipotent cells (i.e., a cell whose descendants can become any cell type in an organism, e.g., embryonic stem cells, and somatic stem cells e.g., hematopoietic cells)). In yet other embodiments, target cells include oocytes, eggs, cells of an embryo, zygotes, sperm cells, and somatic (non-stem) mature cells from a variety of organs or tissues, such as hepatocytes, neural cells, muscle cells and blood cells (e.g., lymphocytes).

[0062] The term "host cell" as used herein refers to, for example microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of ssDNA or vectors. The term includes the progeny of the original cell which has been transduced. Thus, a "host cell" as used herein generally refers to a cell which has been transduced with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to natural, accidental, or deliberate mutation. In some embodiments, the host cell can be an in vitro host cell.

[0063] The term “target tissues” refers to any tissue that is affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease- associated pathology, symptom, or feature.

[0064] The terms "disorder associated with a mutation" or "mutation associated with a disorder" refer to a correlation between a disorder and a mutation. In some embodiments, a disorder associated with a mutation is known or suspected to be wholly or partially, or directly or indirectly, caused by the mutation. For example, a subject having the mutation may be at risk of developing the disorder, and the risk may additionally depend on other factors, such as other (e.g., independent) mutations (e.g., in the same or a different gene), or environmental factors.

[0065] The term “subject” can refer to any animal, including, but not limited to, humans and non-human animals (e.g., rodents, arthropods, insects, fish e.g., zebrafish)), non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.), which is to be the recipient of a particular treatment. Typically, the terms “patient” and “subject” are used interchangeably herein in reference to a human subject.

[0066] The term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0067] The term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduceseverity of and / or reduce incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and / or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0068] Throughout this disclosure, various aspects of this invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual and partial numbers within that range, for example, 1, 2, 3, 4, 5, 5.5 and 6. This applies regardless of the breadth of the range.

[0069] Embodiments described herein relate to cationic gas-filled nanobubbles (also referred to as sub-micron bubbles, gas-core nanoparticles or ultrafine bubbles) and their use in nucleic acid cell delivery, transfection, and / or gene therapy. The cationic nanobubbles can readily complex or be loaded with at least one negatively charged genetic material or nucleic acid, protect the complexed or loaded nucleic acids from degradation when administered systemically to a subject, be targeted to and readily internalized by targeted cells, and release the complexed or loaded nucleic acids within the cells upon insonation with ultrasound energy. In contrast to commercial microbubbles, the nanobubbles described herein are small enough, e.g., less than about 600 nm in diameter (e.g., average diameter approximately 300 nm with a 10% polydispersity), to extravasate through endothelial barriers and readily enter and cross cell membranes.

[0070] Advantageously, in contrast to traditional transfection methods, ultrasound stimulated delivery of nucleic acids to cells using nanobubbles described herein can provide contrast agents to allow real-time visualization of nanobubble delivery. Further, nanobubble stimulation increases cell permeability and enhances tumor, cell, and nucleus uptake and has been shown to promote endosomal escape and cytosolic release upon cellular uptake. Moreover, the nanobubbles can be readily functionalized with receptors and ligands to target tissues or cells of interest. Additionally, focusing ultrasound at a target site will facilitatetransfection or nucleic acid release form the nanobubbles only at that site, leaving other cells and / or tissues unaffected.

[0071] Fig. 1 illustrates an example of a cationic nanobubble loaded or complexed with at least one negatively charged genetic material or nucleic acid. The cationic nanobubble can include a lipid membrane that defines a gas containing internal void or space. The lipid membrane can have a hydrophilic outer domain at least partially defined by hydrophilic heads of lipids and a hydrophobic inner domain at least partially defined by hydrophobic tails of the lipids. The hydrophilic outer domain can have a cationic charge effective to complex the at least one negatively charged genetic material or nucleic acid to the outer domain. The cationic charge can be provided by a plurality of cationic lipids of the lipid domain. An edge activator, such as propylene glycol, can at least partially extend between the lipids from the outer domain to the inner domain. The glycerol can be provided on the outer domain of the nanobubbles and extend partially between hydrophilic heads of the lipids.

[0072] In some embodiments, each of the cationic nanobubbles can have a size that facilitates extravasation of the cationic nanobubbles and internalization of the cationic nanobubbles by a cell or tissue. For example, each of the cationic nanobubbles can have a size (diameter) of about 30 nm to about 600 nm or about 100 nm to about 500 nm (e.g., about 300 nm), depending upon the particular lipids, edge activator, and membrane stiffener as well as the method used to form the nanobubble (described in greater detail below).

[0073] Examples of cationic lipids that can be used to form the lipid membrane can include derivatives of ethylphosphatidylcholine, in particular esters of ethylphosphatidylcholine with fatty acids, such as l,2-Distearoyl-sn-glycero-3- Ethylphosphocholine (Ethyl-DSPC or DSEPC), l,2-Dipalmitoyl-sn-glycero-3- Ethylphosphocholine (Ethyl-DPPC or DPEPC). Examples of positively charged lipids are alkylammonium salts with a halogen counter ion (e.g., chlorine or bromine) comprising at least one (C10-C22), preferably (C14-C22), alkyl chain, such as, for instance stearylammonium chloride, hexadecylammonium chloride, dimethyldioctadecyl ammonium bromide (DDAB), hexadecyltrimethylammonium bromide (CTAB). Further examples of positively charged lipids are tertiary or quaternary ammonium salts with a halogen counter ion (e.g., chlorine or bromine) comprising one or two (C10-C22), preferably (C16-C22), acyl chain linked to the N- atom through a (Cs-Ce) alkylene bridge, such as, for instance, l,2-distearoyl-3- trimethylammonium-propane (DSTAP), 1 ,2-dipalmitoyl-3-trimethylammonium-propane(DPTAP), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), l,2-distearoyl-3- dimethylammonium-propane (DSDAP).

[0074] In some embodiments, the cationic lipids can include saturated C16-C22 hydrophobic tails and / or a mixture of cationic lipids with saturated and / or unsaturated C16-C22 hydrophobic tails. Advantageously, it was found that cationic nanobubbles formulated with cationic lipids that include saturated C16-C22 hydrophobic tails (e.g., l,2-dipalmitoyl-3- trimethylammonium-propane (DPTAP)) with resistant to coalescence or aggregation in contrast to cationic nanobubbles formulated with unsaturated C16-C22 hydrophobic tails (e.g., l,2-dioleoyl-3-trimethylammonium-propane (DOTAP)). Cationic lipids having saturated C16-C22 hydrophobic tails can include, for example, l,2-distearoyl-3- trimethylammonium-propane (DSTAP), l,2-Dipalmitoyl-3-trimethylammonium propane (DPTAP), l,2-distearoyl-3-dimethylammonium-propane (DSDAP), N-[l-(2,3- dilauroyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DLTAP), N-[l-(2,3- dimyristoyloxy)propyl|-N,N,N-tri methylammonium methylsulfate (DMTAP), N-| l-(2- palmitoyloxy-3-lauroyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (LPTAP) or a combination thereof.

[0075] Synthetic cationic lipids can also be used, such as those disclosed in U.S. Pat. No. 5,830,430, herein incorporated by reference in its entirety. These include, for example, N,N'-bis(dodecyaminocarbonylmethylene)-N,N'-bis(P-N,N,N-trimethylammoniumethyl- aminocarbonylmethyleneethylenediamine tetraiodide; N,N"-bis hexadecylaminocarbonylmethylene)-N,N',N"-tris(P-N,N,N-trimethyl-ammonium- ethylaminocarbonylmethylenediethylenetriamine hexaiodide; N,N'- Bis(dodecylaminocarbonylmethylene)-N,N”-bis(P-N,N,N-trimethylammonium- ethylaminocarbonylmethylene)cyclohexylene- 1 ,4-diamine tetraiodide; 1 , 1 ,7,7-tetra-(P-3- N,N,N,N-tetramethylammoniumethylaminocarbonylmethylene)-3-hexadecylaminocarbonyl- methylene-l,3,7-triaazaheptane heptaiodide; and N,N,N'N' -tetra phosphoethanolaminocarbonylmethylene)diethylenetriamine tetraiodide, dioctadecylamidoglycyl spermine (DOGS); 2-3-dioleyloxy-N- (2(sperminecarboxyamido)ethyl)-N,N-dimethyl- 1 -propanaminium trifluoroacetate (DOSPA) ; dipalmitoyl phosphatidyl-ethanolamidospermine (DPPES); glycine betaine derivatives such as GB12; cationic lipids bearing unsaturated double chain based on 3,4-dihydroxybenzoic acid (MVL5); guanidine-containing lipids such as bis-guanidiniumtren-cholesterol (BGTC),cholesterol derivatives such as cholesteryl-3Pcarboxyamidoethylene dimethyl amine (DC- Chol).

[0076] The cationic lipids may also be formulated in combination with neutral, anionic or negatively charged lipids, and / or ionizable lipids. The neutral, anionic or negatively charged lipids, and / or ionizable lipids including, but not limited to phosphatidylcholines, such as dilauroyl-phosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), dibehenoylglycerophosphocoline (DBPC), diarachidoyl-phosphatidylcholine (DAPC), distearoylphosphatidylcholine (DSPC) or dioleoyl-phosphatidylcholine (DOPC); phosphatidylethanolamines such as dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl phosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine (DOPE), diarachidoylphosphatidyl-ethanolamine (DAPE) or dilinoleylphosphatidylethanolamine (DLPE); phosphatidylserines, such as dimyristoylphosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS), dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), di oleoylphosphatidylserine (DOPS); phosphatidic acid derivatives, such as dipalmitoyl phosphatidic acid (DPP A), dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatidic acid (DSPA), diarachidoylphosphatidic acid (DAPA) and their alkali metal salts; phosphatidylglycerols such as dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts, distearoylphosphatidylglycerol (DSPG) and its alkali metal salts, dioleoylphosphatidy Iglycerol (DOPG); phosphatidylinositols, such as dilauroyl-phosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoylphosphatidylinositol (DOPI); PEG-modified phosphatidylethanolamines, such as DMPE- PEG1000, DMPE-PEG2000, DMPE-PEG3000, DMPE-PEG4000, DMPE-PEG5000, DPPE- PEG1000, DPPE-PEG2000, DPPE-PEG3000, DPPE-PEG4000, DPPE-PEG5000, DSPE- PEG1000, DSPE-PEG2000, DSPE-PEG3000, DSPE-PEG4000, DSPE-PEG5000, DAPE- PEG1000, DAPE-PEG2000, DAPE-PEG3000, DAPE-PEG4000 or DAPE-PEG5000; bile acid salts such as cholic acid salts, deoxycholic acid salts or glycocholic acid salts; and (C12- C24), preferably (C14-C22) fatty acid salts such as, palmitic acid salt, stearic acid salt, 1,2- dipalmitoyl-sn-3-succinylglycerol salt or l,3-dipalmitoyl-2-succinylglycerol salt; cholesterol,or polymerized lipids (such as lipids comprising for example cysteine / or thiol moieties to enable oxidative polycondensation of lipids).

[0077] In some embodiments, the neutral, ionizable, and / or negatively charged lipids can include a mixture of neutral, ionizable, and / or negatively charged phospholipids. The phospholipids can each including a hydrophilic head containing a phosphate group and two C16-C22 hydrophobic tails. Preferably, the C16-C22 hydrophobic tails are saturated. Examples of phospholipids including a hydrophilic head containing a phosphate group and two saturated C16-C22 hydrophobic tails are selected from are selected from dibehenoylglycerophosphocoline (DBPC), dipalmitoylphosphatidic acid (DPP A), dipalmitoylphosphatidylethanolamine (DPPE), PEG functionalized, distearoylphosphatidylethanolamine (DSPE-PEG), or combinations thereof. Preferably, the phospholipids include a combination of dibehenoylglycerophosphocoline (DBPC), dipalmitoylphosphatidylethanolamine (DPPE), and PEG functionalized, distearoylphosphatidylethanolamine (DSPE-PEG).

[0078] In some embodiments, the plurality of lipids used to form the liquid membrane can include a mixture of cationic lipids and anionic and / or neutral phospholipids having varying acyl chain lengths. For example, the plurality of lipids can include a mixture of 1,2- distearoyl-3-trimethylammonium-propane (DSTAP), 1 ,2-Dipalmitoyl-3-trimethylammonium propane (DPTAP), l ,2-distearoyl-3-dimethylammonium-propane (DSDAP), N-[l-(2,3- dilauroyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DLTAP), N-[l-(2,3- dimyristoyloxy)propyl]-N,N,N-tri methylammonium methylsulfate (DMTAP), and / or N-[l- (2- palmitoyloxy-3-lauroyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (LPTAP) and at least two of dibehenoylglycerophosphocoline (DBPC), distearoylphosphatidylcholine (DSPC), diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE); dipalmitoylphosphatidic acid (DPP A), or PEG functionalized lipids thereof.

[0079] In other embodiments, the mixture of cationic lipids and anionic, ionizable, and / or neutral phospholipids having varying acyl chain lengths can include 1 ,2-Dipalmitoyl- 3-trimethylammonium propane (DPTAP) or N-[l-(2- palmitoyloxy-3-lauroyloxy)propyl]- N,N,N-trimethylammonium methylsulfate (LPTAP), dibehenoylglycerophosphocoline (DBPC), and one or more additional phospholipids selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC),diarachidonylphosphatidylcholine (DAPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidic acid (DPPA), or PEG functionalized phospholipids thereof.

[0080] In some embodiments, the mixture of lipids can include at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at about 80%, by weight of dibehenoylglycerophosphocoline (DBPC); and less than about 60%, less than about 50%, less than about 40%, less than about 30%, or less than about 20%, by weight, of a combination of additional lipids selected from dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), 1 ,2-Dipalmitoyl-3-trimethylammonium propane (DPTAP) or N-[l-(2- palmitoyloxy-3-lauroyloxy)propyl]-N,N,N- trimethylammonium methylsulfate (LPTAP), or PEG functionalized phospholipids thereof. The PEG can have a molecular weight of about 1000 to about 5000 Da, for example, about 2000 Da.

[0081] In some embodiments, the mixture of lipids can include about 40% to about 80%, about 50% to about 70%, or about 55% to about 65% e.g., about 60%) by weight dibehenoylglycerophosphocoline (DBPC); and about 20% to about 60%, about 30% to about 50%, or about 35% to about 45% (e.g., about 40%) by weight of a combination of additional phospholipids selected from dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), 1 ,2-Dipalmitoyl-3-trimethylammonium propane (DPTAP) or N-[l-(2- palmitoyloxy-3-lauroyloxy)propyl]-N,N,N- trimethylammonium methylsulfate (LPTAP), or PEG functionalized phospholipids thereof.

[0082] In some embodiments, the lipid membrane includes a mixture of dibehenoylglycerophosphocoline (DBPC), dipalmitoylphosphatidylethanolamine (DPPE), PEG functionalized distearoylphosphatidylethanolamine (DSPE-PEG), and 1 ,2-Dipalmitoyl- 3-trimethylammonium propane (DPTAP).

[0083] In still other embodiments, the lipid membrane includes a mixture of dibehenoylglycerophosphocoline (DBPC), dipalmitoylphosphatidylethanolamine (DPPE), PEG functionalized distearoylphosphatidylethanolamine (DSPE-PEG), and 1 ,2-Dipalmitoyl- 3-trimethylammonium propane (DPTAP) at a ratio of, for example, about 6:2:1:0.25-1 by weight.

[0084] The cationic nanobubbles can have a lipid concentration that enhances the in vivo circulation stability of the nanobubbles. It was found that a higher lipid concentrationcorrelated with an increase in stability and longer circulation of the nanobubbles upon administration to a subject. In some embodiments, the cationic nanobubbles can have a lipid concentration of at least about 2 mg / ml, at least about 3 mg / ml, at least about 4 mg / ml, at least about 5 mg / ml, about 6 mg / ml, at least about 7 mg / ml, at least about 8 mg / ml, at least about 9 mg / ml, at least about 10 mg / ml, at least about 11 mg / ml, at least about 12 mg / ml or more. In other embodiments, the lipid concentration of the cationic nanobubbles can be about 5 mg / ml to about 12 mg / ml, about 6 mg / ml to about 12 mg / ml, about 7 mg / ml to about 12 mg / ml, about 8 mg / ml to about 12 mg / ml, about 9 mg / ml to about 12 mg / ml, about 10 mg / ml to about 12 mg / ml, or at least about 10 mg / ml.

[0085] In some embodiments, the edge-activator, which is incorporated between lipids of the membrane of each nanobubble and enhances the flexibility of the nanobubbles can include a co- surfactant, such as propylene glycol, which enhances the effectiveness of phospholipid surfactants. The edge activator can be provided in each of the nanobubbles at an amount effective to cause separation of lipid domains of the nanobubble and form defects that absorb excessive pressure, which could have caused lipid “domain” tearing. Other edge activators, which can be substituted for propylene glycol or used in combination with propylene glycol, can include cholesterol, sodium cholate, limonene, oleic acid, and / or span 80.

[0086] In some embodiments, the amount of propylene glycol provided in the nanobubbles can be about 0.05 ml to about 0.5 ml, about 0.06 ml to about 0.4 ml, about 0.07 ml to about 0.3 ml, about 0.08 ml to about 0.2 ml, or about 0. 1 ml, per 1 ml of hydrated lipids.

[0087] In other embodiments, a membrane stiffener, which is incorporated on the outer surface of the membrane of each nanobubble and enhances the membranes resistance to tearing, includes glycerol. Glycerol can be provided on the membrane of each of the nanobubbles at an amount effective to stiffen the membrane and improve the membrane’s resistance to lipid “domain” tearing. The amount of glycerol provided on the membranes of the nanobubbles and microbubbles can be about 0.05 ml to about 0.5 ml, about 0.06 ml to about 0.4 ml, about 0.07 ml to about 0.3 ml, about 0.08 ml to about 0.2 ml, or about 0.1 ml, per 1 ml of hydrated lipids.

[0088] The membranes defining the nanobubbles can be concentric or otherwise and have a unilamellar configuration (i.e., comprised of one monolayer or bilayer), anoligolamellar configuration i.e., comprised of about two or about three monolayers or bilayers), or a multilamellar configuration (i.e., comprised of more than about three monolayers or bilayers). The membrane can be substantially solid (uniform), porous, or semi-porous.

[0089] The internal void space defined by the membrane can include at least one gas. The gas can have a low solubility in water (e.g., hydrophobic) and be, for example, hexafluoro acetone; isopropyl acetylene; allene; tetrafluoroallene; boron trifluoride; 1,2- butadiene; 1,3 -butadiene; 1,3-butadiene; 1,2,3-trichloro, 2-fluoro- 1,3-butadiene; 2-methyl,l,3 butadiene; hexafluoro- 1,3-butadiene; butadiyne; 1 -fluoro-butane; 2-methyl-butane; decafluoro butane; 1 -butene; 2-butene; 2-methy- 1 -butene; 3-methyl-l -butene; perfluoro- 1- butene; perfluoro- 1 -butene; perfluoro-2-butene; l,4-phenyl-3-butene-2-one; 2-methyl-l- butene-3-yne; butyl nitrate; 1-butyne; 2-butyne; 2-chloro-l,l,l,4,4,4-hexafluoro-butyne; 3- methyl-1 -butyne; perfluoro-2-butyne; 2-bromo-butyraldehyde; carbonyl sulfide; crotononitrile; cyclobutane; methyl-cyclobutane; octafluoro-cyclobutane; perfluorocyclobutene; 3-chloro-cyclopentene; perfluoro ethane; perfluoro propane; perfluoro butane; perfluoro pentane; perfluoro hexane; cyclopropane; 1,2-dimethyl-cyclopropane; 1,1 -dimethyl cyclopropane; 1,2-dimethyl cyclopropane; ethyl cyclopropane; methyl cyclopropane; diacetylene; 3 -ethyl-3 -methyl diaziridine; 1 , 1 , 1 -trifluorodiazoethane; dimethyl amine; hexafluorodimethyl amine; dimethylethylamine; -bis-(Dimethyl phosphine)amine; 2,3- dimethyl-2-norbornane; perfluorodimethylamine; dimethyloxonium chloride; 1,3-dioxolane- 2-one; perfluorocarbons such as and not limited to 4-methy 1,1, 1,1, 2-tetrafluoro ethane; 1,1,1- trifluoroethane; 1,1,2,2-tetrafluoroethane; l,l,2-trichloro-l,2,2-trifhioroethane; 1,1 dichloroethane; 1, 1 -dichloro- 1,2, 2, 2-tetrafluoro ethane; 1 ,2-difhioro ethane; 1-chloro- 1,1 ,2,2,2-pentafluoro ethane; 2-chloro, 1 , 1 -difluoroethane; 1 -chloro- 1,1, 2, 2-tetrafluoro ethane; 2-chloro, 1,1 -difluoroethane; chloroethane; chloropentafluoro ethane; dichlorotrifluoroethane; fluoro-ethane; hexafluoro-ethane; nitro-pentafluoro ethane; nitrosopentafluoro ethane; perfluoro ethane; perfluoro ethylamine; ethyl vinyl ether; 1,1 -dichloro ethylene; 1,1 -dichloro- 1,2-difluoro ethylene; 1,2-difluoro ethylene; Methane; Methanesulfonyl chloride- trifluoro; Methanesulfonyl fluoride-trifluoro; Methane- (pentafluorothio)trifluoro; Methane-bromo difluoro nitroso; Methane-bromo fluoro; Methane-bromo chloro-fluoro; Methanebromo-trifluoro; Methane-chloro difluoro nitro;Methane-chloro dinitro; Methanechloro fluoro; Methane-chloro trifluoro; Methane-chloro-difluoro; Methane dibromo difluoro; Methane-dichloro difluoro; Methane-dichloro-fluoro; Methanedifluoro; Methane-difluoro-iodo; Methane-disilano; Methane-fluoro; Methaneiodo; Methane-iodo-trifhioro; Methane-nitro-trifluoro; Methane-nitroso-trifluoro; Methane- tetrafluoro; Methane- trichlorofluoro; Methane- trifluoro; Methanes ulfenylchloride-trifluoro; 2-Methyl butane; Methyl ether; Methyl isopropyl ether; Methyl lactate; Methyl nitrite; Methyl sulfide; Methyl vinyl ether; Neon; Neopentane; Nitrogen (N.sub.2); Nitrous oxide; 1,2,3-Nonadecane tricarboxylic acid-2-hydroxytrimethylester; 1 -Nonene-3 -yne; Oxygen (0.sub.2); 1, 4-Pentadiene; n-Pentane; Pentane-perfluoro; 2-Pentanone-4-amino-4-methyl; 1- Pentene; 2-Pentene [cis]; 2-Pentene (trans); 1 -Pentene- 3 -bromo; 1-Pentene-perfluoro;Phthalic acid-tetrachloro; Piperidine-2,3,6-trimethyl; Propane, Propane- 1,1, 1,2, 2, 3- hexafluoro; Propane- 1,2-epoxy; Propane-2,2 difluoro; Propane 2-amino, Propane-2-chloro; Propane-heptafluoro-1 -nitro; Propane-heptafluoro- 1 -nitroso; Propane-perfluoro; Propene; Propyl-l,l,l,2,3,3-hexafluoro-2,3 dichloro; Propylene- 1 -chloro; Propylenechloro- (trans); Propylene-2-chloro; Propylene-3-fluoro; Propylene-perfluoro; Propyne; Propyne-3,3,3- trifluoro; Styrene-3-fluoro; Sulfur hexafluoride; Sulfur (di)-decafluoro(S2F10); Toluene-2,4- diamino; Trifluoroacetonitrile; Trifluoromethyl peroxide; Trifluoromethyl sulfide; Tungsten hexafluoride; Vinyl acetylene; Vinyl ether; Xenon; Nitrogen; air; and other ambient gases.

[0090] In some embodiments, the gas is a perfluorocarbon. Perfluorocarbons can include, for example, fluorine gas, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, or combinations thereof.

[0091] In some embodiments, the lipid membrane optionally includes at least one of a surfactant, alcohol, or cholesterol, that change the shape, modulus, and / or interfacial tension of the nanobubble membrane. For example, the cationic nanobubble can include additional surfactants, such as erucyl bis-(hydroxyethyl)methylammonium chloride (EH AC), cocamidopropyl betaine, or other phospholipids, to adjust the shape of the nanobubbles and provide nanobubbles having a filament, vermiform, or worm-like shape.

[0092] The negatively charged genetic material or nucleic acid that is complexed to hydrophilic outer domain of the lipid membrane includes within its meaning any nucleic acid (or nucleotide sequence) which can be used either as such or for expressing a corresponding amino acid, peptide or protein once delivered into a cell. The genetic material includes in particular nucleic acids, RNA, and DNA, of either natural or synthetic origin including recombinant RNA and DNA, antisense or antigen RNA, pDNA, mRNA, microRNA,interfering RNA molecules, such as siRNAs, DNA encoding for shRNA, lincRNA, plasmids, or other negatively charged genetic material. The term refers both to the nucleotide sequence (gene) as such or preferably to the gene incorporated into a suitable vehicle. Suitable vehicles for carrying the genes include expression vectors such as plasmids, phagemids, cosmids, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), human artificial chromosomes (HAC), chimeric oligonucleotide comprising RNA and DNA residues in a duplex conformation, hairpin or hammerhead ribozyme, small interfering RNA (siRNA), siRNA expression cassettes, DNA ribonucleases, transposon, both single and double strand RNA and DNA and analog thereof, such as phosphorothioate, phosphorodithioate oligodesoxynucleotide.

[0093] The genetic material can be used, for instance, for replacing missing or defective genes, for catalyzing the destruction of cancer cells or causing cancer cells to revert back to normal tissue, for radio sensitization promotion, for enhancing or stimulating an immune response, for promoting the growth of new tissue or stimulating regeneration of damaged tissue, for enabling repair of mutated or damaged genes, for catalyzing endoribonucleolytic cleavage, for blocking gene expression by triplex formation or for blocking mRNA translation.

[0094] In some embodiments, the nucleic acid can be an oligonucleotide, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or peptide nucleic acid (PNA). The nucleic acid can be a nucleic acid from any source, such as a nucleic acid obtained from cells in which it occurs in nature, recombinantly produced nucleic acid, or chemically synthesized nucleic acid. For example, the nucleic acid can be cDNA or genomic DNA or DNA synthesized to have the nucleotide sequence corresponding to that of naturally-occurring DNA. The nucleic acid can also be a mutated or altered form of nucleic acid (e.g., DNA that differs from a naturally occurring DNA by an alteration, deletion, substitution or addition of at least one nucleic acid residue) or nucleic acid that does not occur in nature.

[0095] In one aspect, the nucleic acid can be a functional nucleic acid. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction. Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting. For example, functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, siRNA, miRNA, shRNA and external guide sequences. The functional nucleic acidmolecules can act as affectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.

[0096] Functional nucleic acids can be a small gene fragment that encodes dominantacting synthetic genetic elements (SGEs), e.g., molecules that interfere with the function of genes from which they are derived (antagonists) or that are dominant constitutively active fragments (agonists) of such genes. SGEs can include, but are not limited to, polypeptides, inhibitory antisense RNA molecules, ribozymes, nucleic acid decoys, and small peptides. The small gene fragments and SGE libraries disclosed in U.S. Patent Publication No. 2003 / 0228601, which is incorporated by reference, can be used herein.

[0097] The functional nucleic acids can function to inhibit the function of an endogenous gene at the level of nucleic acids, e.g., by an antisense, RNAi or decoy mechanism. Alternatively, certain functional nucleic acids can function to potentiate (including mimicking) the function of an endogenous gene by encoding a polypeptide that retains at least a portion of the bioactivity of the corresponding endogenous gene, and may in particular instances be constitutively active.

[0098] Other therapeutically important nucleic acids include antisense polynucleotide sequences useful in eliminating or reducing the production of a gene product, as described by Tso, P. et al Annals New York Acad. Sci. 570:220-241 (1987). Also contemplated is the delivery of ribozymes. These antisense nucleic acids or ribozymes can be expressed (replicated) in the transfected cells. Therapeutic nucleic acids or polynucleotides useful herein can also code for immunity -conferring polypeptides, which can act as endogenous immunogens to provoke a humoral or cellular response, or both. The nucleic acids or polynucleotides employed can also code for an antibody. In this regard, the term "antibody" encompasses whole immunoglobulin of any class, chimeric antibodies and hybrid antibodies with dual or multiple antigen or epitope specificities, and fragments, such as F(ab)2, Fab2, Fab and the like, including hybrid fragments. Also included within the meaning of "antibody" are conjugates of such fragments, and so-called antigen binding proteins (single chain antibodies) as described, for example, in U.S. Pat. No. 4,704,692, the contents of which are hereby incorporated by reference.

[0099] In some embodiments, the nucleic acid is siRNA. siRNAs are double stranded RNA molecules (dsRNAs) with approximately 20 to 25 nucleotides, which are generated bythe cytoplasmic cleavage of long RNA with the RNase III enzyme Dicer. siRNAs specifically incorporate into the RNA-induced silencing complex (RISC) and then guide the RNAi machinery to destroy the target mRNA containing the complementary sequences. Since RNAi is based on nucleotide base-pairing interactions, it can be tailored to target any gene of interest, rendering siRNA an ideal tool for treating diseases with gene silencing. Gene silencing with siRNAs has a great potential for the treatment of human diseases as a new therapeutic modality. Numerous siRNAs have been designed and reported for various therapeutic purposes and some of the siRNAs have demonstrated specific and effective silencing of genes related to human diseases. Therapeutic applications of siRNAs include, but are not limited to, inhibition of viral gene expression and replication in antiviral therapy, anti-angiogenic therapy of ocular diseases, treatment of autoimmune diseases and neurological disorders, and anticancer therapy. Therapeutic gene silencing has been demonstrated in mammals, which bodes well for the clinical application of siRNA. It is believed that siRNA can target every gene in human genome and has unlimited potential to treat human disease with RNAi.

[0100] In certain embodiments, the nucleic acid can be mRNA. The mRNA can be a therapeutic and / or prophylactic mRNA. An mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide encoded by an mRNA may be of any size and may have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.

[0101] In some embodiments, the mRNA has an mRNA sequence encoding a protein or a peptide. In some embodiments, the mRNA sequence is codon optimized for efficient expression human cells. In some embodiments, the mRNA sequence is naturally-occurring or a wild-type sequence. In some embodiments, the mRNA sequence encodes a protein or a peptide that contains one or mutations in amino acid sequence.

[0102] In some embodiments, the mRNA can encode a peptide or polypeptide for use in the delivery of or treatment with a vaccine for a subject or a cell of a subject. For example, the mRNA can encode an antigen from an infectious agent, such as a virus, coronavirus, CO VID virus, influenza virus, respiratory syncytial virus, rabies virus, cytomegalovirus, rotavirus, hepatitis A virus, hepatitis B virus, or hepatitis C virus, human papillomavirus herpes simplex virus, such as herpes simplex virus 1 or herpes simplex virus 2. In certainembodiments, the mRNA can encode an antigen from a human immunodeficiency virus, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2, human metapneumovirus, human parainfluenza virus, human parainfluenza virus type 1, human parainfluenza virus type 2, human parainfluenza virus type 3, malaria virus, zika virus, or chikungunya virus.

[0103] In other embodiments, the negatively charged genetic material or nucleic acid can be plasmid vector. The plasmid vector can be a circular plasmid vector. The circular plasmid vector may be monomeric, dimeric, trimeric, tetrameric, pentameric, hexameric, etc. Preferably, the circular plasmid vector is monomeric or dimeric. In other embodiments, the circular plasmid vector is a monomeric, supercoiled circular molecule. In some embodiments, the plasmid vector can be nicked. In some embodiments, the plasmid vector can be open circular. In some embodiments, the plasmid vector can be double-stranded circular.

[0104] The plasmid vectors described herein can be used to insert or express a heterologous gene into a target cell. As disclosed herein, a broad range of heterologous genes may be delivered to target cells by way of the present vectors. In some embodiments, the heterologous gene is configured to transfect a target cell having a mutation associated with a disease which can be treated by expression of the heterologous gene, e.g., a gene encoding a therapeutic protein, e.g., a protein that is defective or absent in the target cell and / or subject.

[0105] In some embodiments, the heterologous gene or nucleic acid may encode for a viable therapeutic protein so as to replace the defective protein which is naturally expressed in the targeted tissue. Examples of therapeutic proteins include one or more polypeptides selected from growth factors, interleukins, interferons, anti-apoptosis factors, cytokines, antidiabetic factors, anti-apoptosis agents, coagulation factors, anti-tumor factors. Various retina-derived neurotrophic factors have the potential to rescue degenerating photoreceptor cells, and may be expressed using a vector as described herein. Preferred biologically active agents may be selected from VEGF, Angiogenin, Angiopoietin- 1 , DeM, acidic or basic Fibroblast Growth Factors (aFGF and bFGF), FGF-2, Follistatin, Granulocyte Colony- Stimulating factor (G-CSF), Hepatocyte Growth Factor (HGF), Scatter Factor (SF), Leptin, Midkine, Placental Growth Factor (PGF), Platelet-Derived Endothelial Cell Growth Factor (PD-ECGF), Platelet-Derived Growth Factor-BB (PDGF-BB), Pleiotrophin (PTN), RdCVF (Rod-derived Cone Viability Factor), Progranulin, Proliferin, Transforming Growth Factor-alpha (TGF-alpha), PEDF, Transforming Growth Factor-beta (TGF-beta), Tumor Necrosis Factor-alpha (TNF-alpha), Vascular Endothelial Growth Factor (VEGF), Vascular Permeability Factor (VPF), CNTF, BDNF, GDNF, PEDF, NT3, BFGF, angiopoietin, ephrin, EPO, NGF, IGF, GMF, aFGF, NT5, Gax, a growth hormone, [alpha] -1 -antitrypsin, calcitonin, leptin, an apolipoprotein, an enzyme for the biosynthesis of vitamins, hormones or neuromediators, chemokines, cytokines such as IL-1 , IL-8, IL-10, IL-12, IL-13, a receptor thereof, an antibody blocking any one of said receptors, TIMP such as TIMP-1, TIMP-2, TIMP-3, TIMP-4, angioarrestin, endostatin such as endostatin XVIII and endostatin XV, ATF, angiostatin, a fusion protein of endostatin and angiostatin, the C-terminal hemopexin domain of matrix metalloproteinase-2, the kringle 5 domain of human plasminogen, a fusion protein of endostatin and the kringle 5 domain of human plasminogen, the placental ribonuclease inhibitor, the plasminogen activator inhibitor, the Platelet Factor-4 (PF4), a prolactin fragment, the Proliferin-Related Protein (PRP), the antiangiogenic antithrombin III, the Cartilage-Derived Inhibitor (CDI), a CD59 complement fragment, C3a and C5a inhibitors, complex attack membrane inhibitors, Factor H, ICAM, VCAM, caveolin, PKC zeta, junction proteins, JAMs, CD36, MERTK vasculostatin, vasostatin (calreticulin fragment), thrombospondin, fibronectin, in particular fibronectin fragment gro-beta, an heparinase, human chorionic gonadotropin (hCG), interferon alpha / beta / gamma, interferon inducible protein (IP- 10), the monokine-induced by interferon-gamma (Mig), the interferonalpha inducible protein 10 (IP10), a fusion protein of Mig and IP10, soluble Fms-Like Tyrosine kinase 1 (FLT-1) receptor, Kinase insert Domain Receptor (KDR), regulators of apoptosis such as Bcl-2, Bad, Bak, Bax, Bik, Bcl-X short isoform and Gax, fragments or derivatives thereof and the like.

[0106] In other embodiments, the heterologous gene can encode a site-specific endonuclease that provides for site-specific knock-down of gene function, e.g., where the endonuclease knocks out an allele associated with a disease. For example, where a dominant allele encodes a defective copy of a gene that, when wild-type, is a structural protein and / or provides for normal function, a site-specific endonuclease (such as TALEnuc leases, meganucleases or Zinc finger nucleases) can be targeted to the defective allele and knock out the defective allele. In addition to knocking out a defective allele, a site-specific nuclease can also be used to stimulate homologous recombination with a donor DNA that encodes a functional copy of the protein encoded by the defective allele. Thus, e.g., the cationicnanobubbles described herein can be used to deliver both a site-specific endonuclease that knocks out a defective allele, and can be used to deliver a functional copy of the defective allele, resulting in repair of the defective allele, thereby providing for production of a functional protein.

[0107] In some embodiments, the vector comprises a polynucleotide that encodes a site-specific endonuclease; and a polynucleotide that encodes a functional copy of a defective allele, where the functional copy encodes a functional protein. Site-specific endonucleases that are suitable for use include, e.g., zinc finger nucleases (ZFNs); and transcription activator-like effector nucleases (TALENs), where such site-specific endonucleases are non- naturally occurring and are modified to target a specific gene. Such site-specific nucleases can be engineered to cut specific locations within a genome, and non-homologous end joining can then repair the break while inserting or deleting several nucleotides. Such site-specific endonucleases (also referred to as "INDELs") then throw the protein out of frame and effectively knock out the gene. See, e.g., U.S. Patent Publication No. 2011 / 0301073.

[0108] In some embodiments, the plasmid vector or heterologous gene includes a reporter sequence, which can be useful in verifying heterologous gene expression, for example, in specific cells and tissues. Reporter sequences that may be provided in a transgene include, without limitation, DNA sequences encoding 0-lactamase, P-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art. When associated with regulatory elements which drive their expression, the reporter sequences provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry. For example, where the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for P-galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer.

[0109] In some embodiments, the plasmid vectors can be engineered to encode selectable markers. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin,bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Selectable markers can also be considered to be reporters.

[0110] In some embodiments, the heterologous gene does not include a coding sequence. Non-coding sequences such as shRNA, promoters, enhancers, sequences to mark DNA e.g., for antibody recognition), PCR amplification sites, sequences that define restriction enzyme sites, site-specific recombinase recognition sites, sequences that are recognized by a protein that binds to and / or modifies nucleic acids, and linkers, may be included in the vector. In instances in which a heterologous gene is a trans-splicing molecule, non-coding sequences include binding domains that bind a target intron.

[0111] In some embodiments, the heterologous gene is from 0.1 Kb to 100 Kb in length (e.g., the heterologous gene is from 0.2 Kb to 90 Kb, from 0.5 Kb to 80 Kb, from 1.0 Kb to 70 Kb, from 1.5 Kb to 60 Kb, from 2.0 Kb to 50 Kb, from 2.5 Kb to 45 Kb, from 3.0 Kb to40 Kb, from 3.5 Kb to 35 Kb, from 4.0 Kb to 30 Kb, from 4.5 Kb to 25 Kb, from 4.6 Kb to24 Kb, from 4.7 Kb to 23 Kb, from 4.8 Kb to 22 Kb, from 4.9 Kb to 21 Kb, from 5.0 Kb to20 Kb, from 5.5 Kb to 18 Kb, from 6.0 Kb to 17 Kb, from 6.5 Kb to 16 Kb, from 7.0 Kb to15 Kb, from 7.5 Kb to 14 Kb, from 8.0 Kb to 13 Kb, from 8.5 Kb to 12.5 Kb, from 9.0 Kb to 12.0 Kb, from 9.5 Kb to 11.5 Kb, or from 10.0 Kb to 11.0 Kb in length, e.g., from 0.1 Kb to 0.5 Kb, from 0.5 Kb to 1.0 Kb, from 1.0 Kb to 2.5 Kb, from 2.5 Kb to 4.5 Kb, from 4.5 Kb to 8 Kb, from 8 Kb to 10 Kb, from 10 Kb to 15 Kb, from 15 Kb to 20 Kb in length, or greater, e.g., from 0.1 Kb to 0.25 Kb, from 0.25 Kb to 0.5 Kb, from 0.5 Kb to 1.0 Kb, from 1.0 Kb to1.5 Kb, from 1.5 Kb to 2.0 Kb, from 2.0 Kb to 2.5 Kb, from 2.5 Kb to 3.0 Kb, from 3.0 Kb to3.5 Kb, from 3.5 Kb to 4.0 Kb, from 4.0 Kb to 4.5 Kb, from 4.5 Kb to 5.0 Kb, from 5.0 Kb to5.5 Kb, from 5.5 Kb to 6.0 Kb, from 6.0 Kb to 6.5 Kb, from 6.5 Kb to 7.0 Kb, from 7.0 Kb to7.5 Kb, from 7.5 Kb to 8.0 Kb, from 8.0 Kb to 8.5 Kb, from 8.5 Kb to 9.0 Kb, from 9.0 Kb to9.5 Kb, from 9.5 Kb to 10 Kb, from 10 Kb to 10.5 Kb, from 10.5 Kb to 11 Kb, from 11 Kb to11.5 Kb, from 11.5 Kb to 12 Kb, from 12 Kb to 12.5 Kb, from 12.5 Kb to 13 Kb, from 13 Kb to 13.5 Kb, from 13.5 Kb to 14 Kb, from 14 Kb to 14.5 Kb, from 14.5 Kb to 15 Kb, from 15 Kb to 15.5 Kb, from 15.5 Kb to 16 Kb, from 16 Kb to 16.5 Kb, from 16.5 Kb to 17 Kb, from 17 Kb to 17.5 Kb, from 17.5 Kb to 18 Kb, from 18 Kb to 18.5 Kb, from 18.5 Kb to 19 Kb, from 19 Kb to 19.5 Kb, from 19.5 Kb to 20 Kb, from 20 Kb to 21 Kb, from 21 Kb to 22 Kb, from 22 Kb to 23 Kb, from 23 Kb to 24 Kb, from 24 Kb to 25 Kb in length, or greater,e.g., about 4.5 Kb, about 5.0 Kb, about 5.5 Kb, about 6.0 Kb, about 6.5 Kb, about 7.0 Kb, about 7.5 Kb, about 8.0 Kb, about 8.5 Kb, about 9.0 Kb, about 9.5 Kb, about 10 Kb, about 11 Kb, about 12 Kb, about 13 Kb, about 14 Kb, about 15 Kb, about 16 Kb, about 17 Kb, about 18 Kb, about 19 Kb, about 20 Kb in length, or greater).

[0112] In addition to the heterologous gene, the plasmid vectors described herein can include conventional control elements which are operably linked to the heterologous gene in a manner which permits transcription, translation, and / or expression in a target cell.

[0113] Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product. Various expression control sequences, including promoters which are native, constitutive, inducible, and / or tissue-specific, are known in the art and may be utilized. A promoter region is operably linked to a heterologous gene if the promoter region is capable of effecting transcription of that gene such that the resulting transcript might be translated into the desired protein or polypeptide. Promoters useful as part of the vectors described herein include constitutive and inducible promoters.

[0114] In some embodiments, the cationic nanobubbles can include a linker to link a targeting moiety to the membrane of each nanobubble. The linker can be of any suitable length and contain any suitable number of atoms and / or subunits. The linker can include one or combination of chemical and / or biological moieties. Examples of chemical moieties can include alkyl groups, methylene carbon chains, ether, polyether, alkyl amide linkers, alkenyl chains, alkynyl chains, disulfide groups, and polymers, such as poly(ethylene glycol) (PEG), functionalized PEG, PEG-chelant polymers, dendritic polymers, and combinations thereof. Examples of biological moieties can include peptides, modified peptides, streptavidin-biotin or avidin-biotin, polyaminoacids e.g., polylysine), polysaccharides, glycosaminoglycans, oligonucleotides, phospholipid derivatives, and combinations thereof.

[0115] The targeting moiety can bind to a cell surface molecule of a target cell and / or tissue and is capable of targeting and / or adhering the cationic nanobubbles to the targeted cell and / or tissue of interest and / or facilitate uptake of the cationic nanobubble by the cell and / or tissue of interest. In some embodiments, the targeting moiety can comprise any molecule, orcomplex of molecules, which is / are capable of interacting with a cell surface or extracellular molecule or biomarker of the cell. The cell surface molecule can include, for example, a cellular protease, a kinase, a protein, a cell surface receptor, a lipid, and / or fatty acid.

[0116] In certain embodiments, the targeting moiety includes a first molecule specifically binds the cell surface second molecule of the target cell. As used herein, a first molecule "specifically binds" to a second molecule if it binds to or associates with the second molecule with an affinity or Ka (that is, an equilibrium association constant of a particular binding interaction with units of 1 / M) of, for example, greater than or equal to about 105M1. In certain embodiments, the first molecule binds to the second molecule with a Ka greater than or equal to about 106M1, 107M’1, 108M’1, 109M'1, lO^ M’1, 1011M'1, 1012M’^ or 1013M1. "High affinity" binding refers to binding with a Ka of at least 107M1, at least 108M1, at least 109M’1, at least IO10M’1, at least 1011M1, at least 1012M1, at least 1013M1, or greater. Alternatively, affinity may be defined as an equilibrium dissociation constant (KD) of a particular binding interaction with units of M (e.g., 10'5M to 1013M, or less). In certain aspects, specific binding means binding to the target molecule with a KD of less than or equal to about 10’5M, less than or equal to about 10'6M, less than or equal to about 10'7M, less than or equal to about 10’8M, or less than or equal to about 10"9M, IO10M, 10’1 1M, or 10’12M or less. The binding affinity of the first molecule for the target can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like.

[0117] In some embodiments, the targeting moiety can include, but is not limited to, synthetic compounds, natural compounds or products, macromolecular entities, bioengineered molecules e.g., polypeptides, lipids, polynucleotides, antibodies, antibody fragments), and small entities (e.g., small molecules, neurotransmitters, substrates, ligands, hormones and elemental compounds).

[0118] In one example, the targeting moiety can comprise an antibody, such as a monoclonal antibody, a polyclonal antibody, or a humanized antibody, including without limitation: Fv fragments, single chain Fv (scFv) fragments, Fab’ fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing;multivalent targeting moieties including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((scFv)2 fragments), diabodies, tribodies or tetrabodies, which typically are covalently linked or otherwise stabilized (i.e., leucine zipper or helix stabilized) scFv fragments; and receptor molecules, which naturally interact with a desired target molecule.

[0119] Preparation of antibodies may be accomplished by any number of well-known methods for generating antibodies. These methods typically include the step of immunization of animals, typically mice, with a desired immunogen (e.g., a desired target molecule or fragment thereof). Once the mice have been immunized and boosted one or more times with the desired immunogen(s), antibody-producing hybridomas may be prepared and screened according to well-known methods. See, for example, Kuby, Janis, Immunology, Third Edition, pp. 131-139, W.H. Freeman & Co. (1997), for a general overview of monoclonal antibody production, that portion of which is incorporated herein by reference.

[0120] The targeting moiety need not originate from a biological source. The targeting moiety may, for example, be screened from a combinatorial library of synthetic peptides. One such method is described in U.S. Pat. No. 5,948,635, incorporated herein by reference, which describes the production of phagemid libraries having random amino acid insertions in the pill gene of Ml 3. This phage may be clonally amplified by affinity selection.

[0121] The immunogens used to prepare targeting moieties having a desired specificity will generally be the target molecule, or a fragment or derivative thereof. Such immunogens may be isolated from a source where they are naturally occurring or may be synthesized using methods known in the art. For example, peptide chains may be synthesized by 1-ethyl- 3-[dimethylaminoproply]carbodiimide (EDC)-catalyzed condensation of amine and carboxyl groups. In certain embodiments, the immunogen may be linked to a carrier bead or protein. For example, the carrier may be a functionalized bead such as SASRIN resin commercially available from Bachem, King of Prussia, PA. or a protein such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA). The immunogen may be attached directly to the carrier or may be associated with the carrier via a linker, such as a non-immunogenic synthetic linker (for example, a polyethylene glycol (PEG) residue, amino caproic acid or derivatives thereof) or a random, or semi-random polypeptide.

[0122] In certain embodiments, it may be desirable to mutate a binding region of the polypeptide targeting moiety and select for a targeting moiety with superior bindingcharacteristics as compared to the un-mutated targeting moiety. This may be accomplished by any standard mutagenesis technique, such as by PCR with Taq polymerase under conditions that cause errors. In such a case, the PCR primers could be used to amplify scFv- encoding sequences of phagemid plasmids under conditions that would cause mutations. The PCR product may then be cloned into a phagemid vector and screened for the desired specificity, as described above.

[0123] In other embodiments, the targeting moiety may be modified to make them more resistant to cleavage by proteases. For example, the stability of a targeting moiety comprising a polypeptide may be increased by substituting one or more of the naturally occurring amino acids in the (L) configuration with D-amino acids. In various embodiments, at least 1%, 5%, 10%, 20%, 50%, 80%, 90% or 100% of the amino acid residues of targeting moiety may be of the D configuration. The switch from L to D amino acids neutralizes the digestion capabilities of many of the ubiquitous peptidases found in the digestive tract. Alternatively, enhanced stability of a targeting moiety comprising a peptide bond may be achieved by the introduction of modifications of the traditional peptide linkages. For example, the introduction of a cyclic ring within the polypeptide backbone may confer enhanced stability in order to circumvent the effect of many proteolytic enzymes known to digest polypeptides in the stomach or other digestive organs and in serum. In still other embodiments, enhanced stability of a targeting moiety may be achieved by intercalating one or more dextrorotatory amino acids (such as, dextrorotatory phenylalanine or dextrorotatory tryptophan) between the amino acids of targeting moiety. In exemplary embodiments, such modifications increase the protease resistance of a targeting moiety without affecting the activity or specificity of the interaction with a desired target molecule.

[0124] In certain embodiments, antibodies or variants thereof may be modified to make them less immunogenic when administered to a subject. For example, if the subject is human, the antibody may be "humanized"; where the complimentarily determining region(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones, P. et al. (1986), Nature, 321, 522-525 or Tempest et al. (1991), Biotechnology, 9, 266-273. Also, transgenic mice, or other mammals, may be used to express humanized antibodies. Such humanization may be partial or complete.

[0125] In certain embodiments, a targeting moiety as described herein may comprise a homing peptide, which selectively directs the nanobubble to a targeted cell. Homing peptidesfor a targeted cell can be identified using various methods well known in the art. Many laboratories have identified the homing peptides that are selective for cells of the vasculature of brain, kidney, lung, skin, pancreas, intestine, uterus, adrenal gland, retina, muscle, prostate, or tumors. See, for example, Samoylova et al., 1999, Muscle Nerve, 22:460; Pasqualini et al., 1996 Nature, 380:364; Koivunen et al., 1995, Biotechnology, 13:265; Pasqualini et al., 1995, J. Cell Biol., 130:1 189; Pasqualini et al., 1996, Mole. Psych., 1 :421 , 423; Rajotte et al., 1998, J. Clin. Invest., 102:430; Rajotte et al., 1999, J. Biol. Chem., 274:11593. See, also, U.S. Pat. Nos. 5,622,6999; 6,068,829; 6,174,687; 6,180,084; 6,232,287; 6,296,832; 6,303,573; and 6,306,365.

[0126] Phage display technology provides a means for expressing a diverse population of random or selectively randomized peptides. Various methods of phage display and methods for producing diverse populations of peptides are well known in the art. For example, methods for preparing diverse populations of binding domains on the surface of a phage have been described in U.S. Pat. No. 5,223,409. In particular, phage vectors useful for producing a phage display library as well as methods for selecting potential binding domains and producing randomly or selectively mutated binding domains are also provided in U.S. Pat. No. 5,223,409. Similarly, methods of producing phage peptide display libraries, including vectors and methods of diversifying the population of peptides that are expressed, are also described in Smith et al., 1993, Meth. Enzymol., 217:228-257, Scott et al., Science, 249:386-390, and two PCT publications WO 91 / 07141 and WO 91 / 07149. Phage display technology can be particularly powerful when used, for example, with a codon based mutagenesis method, which can be used to produce random peptides or randomly or desirably biased peptides (see, e.g., U.S. Pat. No. 5,264,563). These or other well-known methods can be used to produce a phage display library, which can be subjected to the in vivo phage display method in order to identify a peptide that homes to one or a few selected tissues.

[0127] In vitro screening of phage libraries has previously been used to identify peptides that bind to antibodies or cell surface receptors (see, e.g., Smith, et al., 1993, Meth. Enzymol., 217:228-257). For example, in vitro screening of phage peptide display libraries has been used to identify novel peptides that specifically bind to integrin adhesion receptors (see, e.g., Koivunen et al., 1994, J. Cell Biol. 124:373-380), and to the human urokinase receptor (Goodson, et al., 1994, Proc. Natl. Acad. Sci., USA 91:7129-7133).

[0128] In certain embodiments, the targeting moiety may comprise a receptor molecule, including, for example, receptors, which naturally recognize a specific desired molecule of a target cell. Such receptor molecules include receptors that have been modified to increase their specificity of interaction with a target molecule, receptors that have been modified to interact with a desired target molecule not naturally recognized by the receptor, and fragments of such receptors (see, e.g., Skerra, 2000, J. Molecular Recognition, 13:167-187). A preferred receptor is a chemokine receptor. Exemplary chemokine receptors have been described in, for example, Lapidot et al, 2002, Exp Hematol, 30:973-81 and Onuffer et al, 2002, Trends Pharmacol Sci, 23:459-67.

[0129] In other embodiments, the targeting moiety may comprise a ligand molecule, including, for example, ligands which naturally recognize a specific desired receptor of a target cell. Such ligand molecules include ligands that have been modified to increase their specificity of interaction with a target receptor, ligands that have been modified to interact with a desired receptor not naturally recognized by the ligand, and fragments of such ligands.

[0130] In still other embodiments, the targeting moiety may comprise an aptamer. Aptamers are oligonucleotides that are selected to bind specifically to a desired molecular structure of the target cell. Aptamers typically are the products of an affinity selection process similar to the affinity selection of phage display (also known as in vitro molecular evolution). The process involves performing several tandem iterations of affinity separation, e.g., using a solid support to which the diseased immunogen is bound, followed by polymerase chain reaction (PCR) to amplify nucleic acids that bound to the immunogens. Each round of affinity separation thus enriches the nucleic acid population for molecules that successfully bind the desired immunogen. In this manner, a random pool of nucleic acids may be "educated" to yield aptamers that specifically bind target molecules. Aptamers typically are RNA, but may be DNA or analogs or derivatives thereof, such as, without limitation, peptide nucleic acids (PNAs) and phosphorothioate nucleic acids.

[0131] In yet other embodiments, the targeting moiety may be a peptidomimetic. By employing, for example, scanning mutagenesis to map the amino acid residues of a protein, which is involved in binding other proteins, peptidomimetic compounds can be generated that mimic those residues, which facilitate the interaction. Such mimetics may then be used as a targeting moiety to deliver the nanobubble to a target cell. For instance, non-hydrolyzable peptide analogs of such resides can be generated using benzodiazepine (e.g., see Freidinger etal. in Peptides: Chemisty and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al., 1986, J Med Chem 29:295; and Ewenson et al., in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), b-turn dipeptide cores (Nagai et al., 1985, Tetrahedron Lett 26:647; and Sato et al., 1986, J Chem Soc Perkin Trans 1: 1231), and P-aminoalcohols (Gordon et al., 1985, Biochem Biophys Res Cummun 126:419; and Dann et al., 1986, Biochem Biophys Res Commun 134:71).

[0132] In some embodiments, the targeting moiety binds to an antigen on a target cells. Target cells of interest include, but are not limited to, cells that are relevant to a particular disease or condition, for example, where it is desirable to induce cell death or treat a cell or tissue. According to some embodiments, the target cell can be a cancer cell, an immune cell, an endothelial cell, or a prokaryotic cell of a microorganism.

[0133] As such, in some embodiments, the target cells are cancer cells. By "cancer cell" it is meant a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage-independent growth potential, ability to promote tumor growth and / or development in an immunocompromised non-human animal model, and / or any appropriate indicator of cellular transformation. "Cancer cell" may be used interchangeably herein with "tumor cell", "malignant cell" or "cancerous cell", and encompasses cancer cells of a solid tumor, a semi-solid tumor, a primary tumor, a metastatic tumor, and the like. In certain aspects, the cancer cell is a carcinoma cell.

[0134] Optionally, the nanobubbles can further include a polymer that coats at least a portion of an outer surface of the lipid membrane complexed with the negatively charged genetic material. The polymer coating can inhibit degradation of the at least one negatively charged genetic material or nucleic acid, such as mRNA, loaded on and / or within the nanobubbles. For example, messenger RNAs, which are single stranded, are more structurally liable and unstable than the double stranded DNA or siRNA. It was estimated that the half-life of the naked double-stranded DNA in the cytosol of mammalian cells is between about 50 and 90 minutes, while the half-life of naked mRNA is reported to be onlyseconds or less than a second; based on the half-life, mRNA is about 5,500 times more unstable than DNA.

[0135] In one example, the polymer coating can include a layer of polydopamine (PDA) coated on at least the portion of the outer surface of the lipid membrane of the nanobubble complexed with the negatively charged genetic material. PDA is a melanin-like mimic of mussel adhesion proteins, exhibiting excellent photothermal stability, biocompatibility and biodegradability. PDA for coating the surface of the cationic nanobubbles complexed with the negatively charged genetic material described herein can be produced by in situ self-polymerization of dopamine in a mild reaction environment with the nanobubbles.

[0136] In some embodiments, the PDA covers just a portion of the outer surface of the lipid membrane and not the entire outer surface of the lipid membrane, or it may cover a substantial portion of the outer surface of the lipid membrane. For example, the biocompatible PDA coating may cover at least about 10%, 20, 30%, 40% 50%, 60%, 70%, 80%, or at least about 90% of the surface of the outer surface of the lipid membrane of the nanobubbles but less than 100% of the outer surface of the lipid membrane. In other embodiments, the amount of PDA applied to the outer surface of the lipid membrane is an amount effective to inhibit degradation of complexed negatively charged genetic material.

[0100] In some embodiments, the cationic nanobubbles can be formed by dissolving at the lipids in propylene glycol. For example, cationic nanobubble can be prepared by dissolving l,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC, Avanti Polar Lipids Inc., Pelham, AL), l,2-Dipalmitoyl-3-trimethylammonium propane (DPTAP), 1 ,2-dipalmitoyl-sn- glycero- 3 -phosphor ethanolamine; DPPE (Corden Pharma, Switzerland), and 1,2- distearoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (ammonium salt) (DSPE-mPEG 2000, Laysan Lipids, Arab, AL) in propylene glycol to produce a lipidpropylene glycol solution. It will be appreciated that other materials can be dissolved in the propylene glycol, such as proteins, carbohydrates, polymers, surfactants, and / or other membrane stabilizing materials.

[0101] After producing the lipid-propylene glycol solution, a glycerol and phosphate buffered solution (PBS) solution can be added to lipid-propylene glycol solution and the resulting solution can be mixed by, for example, sonication. The mixed solution can be transferred to a vial. The air can be removed from the sealed vial containing the hydratedlipid solution and replaced with a gas, such as octafluoropropane, until the vial pressure equalized. The resultant solution can then be shaken or stirred for a time (e.g., about 45 seconds) sufficient to form the nanobubbles. In one example, a lipid-propylene glycol solution comprising DBPC / DPTAP / DPPE / DSPE-PEG-PSMA-1 dissolved in propylene glycol can be contacted with a hydration PBS / glycerol solution, placed in a vial, and then placed in an incubator-shaker at about 37°C and at about 120 rpm for about 60 minutes. In some embodiments, the resultant solution containing the nanobubbles can be freeze dried and reconstituted for storage and shipping or frozen and thawed complexing with the negatively charged genetic material.

[0102] In some embodiments, the sizes of nanobubbles can be adjusted, if desired, by a variety of procedures including extrusion, filtration, sonication, homogenization, employing a laminar stream of a core of liquid introduced into an immiscible sheath of liquid, extrusion under pressure through pores of defined size, and similar methods. The foregoing techniques, as well as others, are discussed, for example, in U.S. Pat. No. 4,728,578; U.K. Patent Application GB 2193095 A; U.S. Pat. No. 4,728,575; U.S. Pat. No. 4,737,323; International Application PCT / US85 / 01161 ; Mayer et al., Biochimica et Biophysica Acta, Vol. 858, pp. 161-168 (1986); Hope et al., Biochimica et Biophysica Acta, Vol. 812, pp. 55-65 (1985);U.S. Pat. No. 4,533,254; Mayhew et al., Methods in Enzymology, Vol. 149, pp. 64-77 (1987); Mayhew et al., Biochimica et Biophysica Acta, Vol 755, pp. 169-74 (1984); Cheng et al, Investigative Radiology, Vol. 22, pp. 47-55 (1987); PCT / US89 / 05040, U.S. Pat. No. 4,162,282; U.S. Pat. No. 4,310,505; U.S. Pat. No. 4,921,706; and Liposome Technology, Gregoriadis, G., ed., Vol. I, pp. 29-31, 51-67 and 79-108 (CRC Press Inc., Boca Raton, Fla. 1984). The disclosures of each of the foregoing patents, publications and patent applications are incorporated by reference herein, in their entirety.

[0103] Filter pore sizes are selected for sizing as well as to remove any potential contaminants. The filter pore size may be between 10 nm and 1 pm, more preferably between 30 nm and 1 pm, and even more preferably between 100 nm and 1 pm. Two or more filters may be stacked in a series to maximize the effectiveness of filtration. Useful materials for formation of the filters include polymers such as poly sulfonate, polycarbonate, and polyvinylidene chloride. In addition, glass, ceramics, and metal filters may also be utilized. Additionally, wire, polymer, or ceramic meshes may also be utilized. Filtration mayeither be utilized. Filtration may be performed as part of the manufacturing process or during administration through an in-line filter.

[0104] In some embodiments, each of the nanobubbles can have a size that facilitates internalization of the nanobubbles by the cells. For example, each of the nanobubbles can have a size (diameter) of about 30 nm to about 600 nm or about 100 nm to about 500 nm e.g., about 300 nm), depending upon the particular lipids, edge activator, and membrane stiffener as well as the method used to form the nanobubble.

[0105] For storage prior to complexing with the negatively charged genetic material, the nanobubbles may be suspended in an aqueous solution, such as a saline solution (for example, a phosphate buffered saline solution), or simply water, and stored preferably at a temperature of between about 2°C and about 10°C, preferably at about 4°C. Preferably, the water is sterile. Most preferably, the nanobubbles are stored in an isotonic saline solution, although, if desired, the saline solution may be a hypotonic saline solution (e.g., about 0.3 to about 0.5% NaCl). The solution also may be buffered, if desired, to provide a pH range of about pH 5 to about pH 7.4. Suitable buffers for use in the storage media include, but are not limited to, acetate, citrate, phosphate and bicarbonate.

[0106] Bacteriostatic agents may also be included with the nanobubbles to prevent bacterial degradation on storage. Suitable bacteriostatic agents include but are not limited to benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, butylparaben, cetylpyridinium chloride, chlorobutanol, chlorocresol, methylparaben, phenol, potassium benzoate, potassium sorbate, sodium benzoate and sorbic acid. One or more antioxidants may further be included with the gaseous precursor- filled liposomes to prevent oxidation of the lipid. Suitable antioxidants include tocopherol, ascorbic acid and ascorbyl palmitate.

[0107] The negatively charged genetic material or nucleic acid can be complexed with the cationic nanobubbles by initially diluting the negatively charged genetic material in PBS. The solution containing the cationic nanobubbles can then be added dropwise to solution containing the negatively charged genetic material. The combined solutions can be continuously stirred or mixed and incubated for a duration of time effective to complex the negatively charged genetic material to the cationic nanobubbles.

[0108] The cationic nanobubbles complexed with the negatively charged genetic material or nucleic acids can be used for in vitro, ex vivo, or in vivo methods of delivery or transfection of the genetic material to a cell or tissue of interest. The cationic nanobubblescomplexed with negatively charged genetic material can be readily up taken or internalized by cells or tissue and advantageously only readily release the complexed negatively charged genetic material upon insonation with ultrasound energy after cellular uptake.

[0001] For example, in vitro or ex vivo cells can be transfected using the cationic nanobubbles complexed with the negatively charged genetic material or nucleic acids by adding to the cells or mixing with the cells the cationic nanobubbles complexed with the negatively charged genetic material and co-culturing or incubating the cells and the plurality of cationic nanobubbles until at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or more of the cells uptake or internalize the cationic nanobubbles. Following incubation, unbound nanobubbles or nanobubbles not internalized by the cells can be removed.

[0002] In some embodiments, acoustic pulses can be applied to the cells and nanobubbles during co-culturing or incubation to stimulate nanobubble endocytosis by the cells. A combination of acoustic vibrations and nanobubble oscillations can help drive small nanobubbles into cells without active targeting approaches.

[0003] In other embodiments, the nanobubbles mixed with the cells can include a targeting moieties that targets the nanobubbles to the cells and promotes uptake of the nanobubbles by the cells as well as cell membrane transport peptides, such as HIV TAT, that facilitate transfer of the nanobubbles across a cell membrane. Targeting of the nanobubbles to the cells in culture or facilitating transport of the nanobubbles across the cell membrane can have the advantage of providing a highly efficient strategy to drive nanobubble uptake into the cells. The nanobubbles can be labeled with antibodies to cell surface receptors or protein or cell membrane transport peptides. Nanobubbles can be functionalized with the targeting moiety or cell membrane transport peptide using a linker, such as standard EDC / NHS conjugation chemistry and purified by size exclusion chromatography.

[0109] Following uptake of the nanobubbles in the cells the nanobubbles can be insonated with ultrasound energy effective to release the negatively charged genetic material within the cells to, for example, genetically modify the cell.

[0110] For in vivo delivery of the cationic nanobubbles to cells or tissue of a subject in need thereof, the cationic nanobubbles complexed with the negatively charged genetic material can be formulated for administration (e.g., injection) to a subject diagnosed and administered to a subject via any known route, such as via an intravenous injection. By wayof example, a composition comprising a plurality of cationic nanobubbles complexed with the negatively charged genetic material can be intravenously administered to a subject. It will be appreciated that other routes of injection may be used including, for example, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal routes.

[0111] The cationic nanobubbles complexed with the negatively charged genetic material administered to the subject can circulate in the subject and if targeted using a targeting moiety bind to and / or complex with the targeted cells by binding and / or complexing of the targeting moiety with the cell surface molecule of the targeted cell. Typically, cell targeted nanobubbles can bind to and / or complex with the targeted cells within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 1 hour or less.

[0112] The location(s) where the nanobubble composition is administered to the subject may be determined based on the subject’s individual need, for example the location of neoplastic cells (e.g., the position of a tumor, the size of a tumor, and the location of a tumor on or near a particular organ).

[0113] Once the cell targeted nanobubbles are bound to and / or complexed with the target cell, the size and / or diameter of the cationic nanobubbles complexed with the negatively charged genetic material allows the nanobubbles to be internalized by or enter the targeted cell by, for example, endocytosis and / or phagocytosis. The nanobubbles can accumulate within the cell or targeted cell and remain within the cells for an extended period, for example, at least one hour, two hours, three hours, or more.

[0114] Following internalization of the nanobubbles complexed with the negatively charged genetic material into the cell or target cell, the internalized nanobubbles can be insonated with ultrasound energy of a given frequency, acoustic pressure, and time effective to promote oscillation, vibration, and / or inertial cavitation of the internalized nanobubbles resulting release of the complexed negatively charged genetic material from the cationic nanobubble within the cell.

[0115] Insonation of the internalized cationic nanobubbles can be achieved by using a non-invasive, minimally invasive, and / or external ultrasound source that produces ultrasoundenergy effective to promote inertial cavitation. The intensity and frequency of the applied ultrasound signal, as well as the duty cycle and pattern for activating the ultrasound source are controllable and configured to suit a given application. Monitoring nanobubble dynamics and correlating treatment parameters presents a strategy for gaining further insights on the mechanism of action as well as intra-treatment monitoring for improving clinical outcomes.

[0116] The ultrasound source can provide specific acoustic sequences that can drive nanobubble oscillation without disrupting and / or adversely affecting normal cells and tissues. These sequences can be applied from a non-focused transducer, which is distinct from typical focused ultrasound transducers used for drug delivery and ultrasound therapy, such as histotripsy.

[0117] In some embodiments, the ultrasound energy is provided by a non-focused ultrasound transducer, a diagnostic imaging ultrasound transducer, a focused ultrasound transducer, or high-intensity focused ultrasound (HIFU)transducer.

[0118] In some embodiments, the ultrasound energy can be provided from a nonfocused transducer, which is distinct from typical focused ultrasound transducers used for drug delivery and ultrasound therapy, such as high intensity focused ultrasound (HIFU) or histotripsy.

[0119] In some embodiments, the insonation can be at a duty cycle of about 1% to about 50%, about 1% to about 40%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, or about 5% to about 15%, an ultrasound frequency of about 0.2 kHz to about 50 MHz, 0.1 MHz to about 40 MHz, 1 MHz to about 30 MHz, 1 MHz to about 20 MHz, 1 MHz to about 15 MHz, or about 1 MHz to about 10 MHz, an intensity of about 0. 1 W / cm2to about 10 W / cm2, about 0.1 W / cm2to about 9 W / cm2, about 0.1 W / cm2to about 8 W / cm2, about 0.1 W / cm2to about 7 W / cm2, about 0.1 W / cm2to about 6 W / cm2, about 0.1 W / cm2to about 5 W / cm2, about 0.1 W / cm2to about 4 W / cm2, or about 1 W / cm2to about 4 W / cm2, a pressure amplitude of about 1 kPa to about 10 MPa, about 50 kPa to about 5 MPa, about 50 kPa to about 5 MPa, about 50 kPa to about 1 MPa, about 100 kPa to about 750 KPa, or about 150 kPa to about 750 KPa, and a time of 5 sec to about 30 minutes, about 5 sec to about 25 minutes, about 5 sec to about 20 minutes, about 5 sec to about 15 minutes, about 5 sec to about 10 minutes, about 5 sec to about 5 minutes, or about 5 sec to about 1 minute.

[0120] In other embodiments, to induce maximum inertial cavitation, the overall pulse length may also be longer (10-30 cycles) than a typical imaging pulse (3-6 cycles).

[0121] In some embodiments, a system that includes the ultrasound source may also be equipped with both the ultrasound source (transmitter) as well as a passive cavitation detection and monitoring acoustic sensor (receiver). The acoustic sensor may be integrated into the transmitting ultrasound source as a transducer element in an array of a plurality of elements, or the acoustic sensor may be implemented as a stand-alone sensor, such as a hydrophone which is suitably placed with respect to the ultrasound source and target region. Instead of simply detecting reflected ultrasound signals at the source frequency, this system can rely on detection of inertial oscillation or cavitation signals arising from the nanobubbles that selectively accumulate the targeted cells.

[0122] Although the use of nanobubbles in medicine is in an early development stage, it is possible that in the future, the applications of nanobubbles in medicine will be as far reaching if not more than that of microbubbles whose applications span across the areas of malignant, infectious, cardiovascular and autoimmune diseases.

[0123] The following example is for the purpose of illustration only and is not intended to limit the scope of the claims, which are appended hereto.Examples

[0124] In these Examples, we present data on a cationic bubble formulation called DPTAP and compare its performance to the other formulations. We performed in-vitro optimizations to study: 1) the effect of lipid shell composition; and 2) the impact of cargo loading on nanobubble physical (size and surface charge) and acoustic properties and stability. We determined the optimal cargo loading capacity and optimized the transfection parameters in PC3-pip cells, including NB concentration and sonication settings. Additionally, we examined the in-vivo stability of the formulation in mice.

[0125] The results provided below are significant because they demonstrate a platform technology capable of delivering mRNA, DNA, and plasmids. We have shown that loading cargos influences the ultrasound activity of the nanobubbles, enhancing both their signal and stability. The preparation and genetic material loading protocol for these cationic nanobubbles is straightforward, facilitating future translational applications. Our delivery platform offers distinct benefits: (a) enhanced genetic material loading capacity, (b)visualization of tumor kinetics and increased extravasation, and (c) noninvasive, on-demand deployment to improve transfection in cancer cells. Importantly, this approach is not limited to cancer and could be applied to many other diseases.

[0126] We started formulating cationic nanobubbles by replacing DPPA used in our anionic nanobubble formulations with cationic lipid DOTAP, which is commonly used for gene delivery reagent and in microbubble formulation. We found though replacement of DPPA with DOTAP caused the cationic nanobubbles coalesce making them unsuitable for gene therapy applications. To mimic our anionic nanobubble, DPTAP was used since it is more similar in tail structure to the DPPA lipid that DOTAP replaced in the anionic formulation.Materials a. C22 (DBPC) - (l,2-dibehenoyl-sn-glycero-3-phosphocholine) b. DPPA - (1,2 Dipalmitoyl-sn-Glycero-3-Phosphate) c. DOTAP - (l,2-dioleoyl-3-trimethylammonium-propane) d. DPTAP - (1 ,2-Dipalmitoyl-3-trimethylammonium propane) e. DPPE - (l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine) f. DSPE-mPEG 2000 - (l,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)) g. PBS - phosphate buffered saline h. Propylene glycol i. Glycerol

[0127] Fig. 2 illustrates a schematic of a lipid solution production protocol for use in forming cationic nanobubbles (NBs). The schematic shows cationic NBs are made following our standard NB preparation / activation protocols described in Nature Protocol (Manuscript IDNP=PI230563). Briefly, lipids and propylene glycol are added with the amount shown in Table 1 and heated in 82°C water bath until dissolved (~ 1 hour). A PBS / glycerol solution is heated in water bath to 82°C. The heated PBS / glycerol solution is added to dissolved lipids, ImL at a time, keeping in water bath. The bath is sonicated for 10 min and the base lipid solution is stored at 4°C.Table 1 - Detail of cationic NB formulations per 1 mL of solution

[0137] Fig. 3 illustrates a schematic of nanobubble activation and pDNA loading protocol. The schematic of Fig. 3, shows for gas exchange, remove headspace air from the 3 mL serum vial and flush headspace air with C4F10 gas. The DPTAP lipid solution is activated through mechanical agitation with a Vial Mix shaker for 45 seconds. Following activation, cationic NBs (CNBs) are isolated from the solution by centrifugation at 50 ref for 5 minutes. The vial containing the DPTAP lipid solution is inverted for this step. After centrifugation, only 250 pL of the nanobubbles is gradually removed from the vial with a 21- gauge needle.

[0138] For the pDNA loading, we dilute the plasmid first in a larger volume, then gradually add NBs to promote homogenous coating. pDNA is diluted in PBS in a 3 mL glass vial and placed on ice DPTAP nanobubble is added dropwise using pipette, to the diluted pDNA. The vial is capped with a septa stopper and incubated for 15 minutes while continuously stirring on ice.

[0139] Figs. 4(A-B) illustrate graphs comparing the (A) size and (B) surface charge (zeta potential) of unloaded anionic nanobubbles and cationic nanobubbles prepared using DOTAP lipid solution and DPTAP solution. The size, and surface charge (zeta potential), of the unloaded CNB formulations were examined using DLS at 25°C. Here, the anionic NBs are referred to as SOP. Rhodamine was incorporated into the CNB formulation for further in vitro cell uptake and transfection study.

[0140] Fig. 5 illustrates an image showing concentrations of nanobubbles prepared using DOTAP lipid solution and DPTAP solution. The nanobubble concentration of DPTAP is significantly increased compared to DOTAP.

[0141] Fig. 6 illustrates a graph comparing concentration of buoyant and non-buoyant nanobubbles prepared using DOTAP lipid solution and DPTAP solution. The buoyant particle (bubble) concentration of unloaded CNB was determined using Resonance measurement. As shown, the DPTAP concentration was increased about 8-fold compared to DOTAP.

[0142] Figs. 7(A-B) illustrates plots comparing ultrasound stabilities of nanobubbles prepared using DOTAP lipid solution and DPTAP solution. Fig. 8 illustrates ultrasound contrast images of nanobubbles prepared using DOTAP lipid solution and DPTAP solution of Figs. 6(A-B). The stability of the unloaded CNB formulations was examined by continuousexposure to ultrasound (18 MHz, 4% power, 1 fps) for 7 min using Vevo2100. The freshly prepared sample (0 min) was compared to a sample kept on ice for 120 min.

[0143] Fig. 9 illustrates images showing co-localization of a plasmid vector loaded on nanobubble prepared using a DPTAP solution. pMax GFP plasmid (pDNA) was labeled with Cy-5 and the co-localization of pDNA on DPTAP was then evaluated by fluorescent microscope. Cy-5 pMax GFP plasmid can be seen on the DPTAP bubble (ring). This proves that the plasmid is incorporated on the shell of the bubble.

[0144] Fig. 10 illustrates images showing loading capacity of pmaxGFP plasmid and cmvGFP nano-plasmid of nanobubbles prepared using a DPTAP solution by electrophoresis. Loading capacity and change in zetapotential of the 1) pmaxGFP plasmid (3486 bp); and 2) cmvGFP nano-plasmid (2473 bp) on DPTAP NBs was examined. Free DNA is seen at 5 and 10 g / mL. A faint streak is also seen at 2.5 pg. The wells visibly enhance starting at 1.25 pg upwards, indicative of NBs complexed with pDNA. This is the same result as the CMV plasmid.

[0145] Fig. 11 illustrates zeta potential of pmaxGFP plasmid and cmvGFP nanoplasmid of nanobubbles prepared using a DPTAP solution. Based on the gel, free DNA begins to be seen at 2.5pg / mL, corresponding to a zeta potential of -16.9mV. However, zeta potential can still drop further with more pDNA to -33mV at lOpg / mL.

[0146] Figs. 12(A-B) illustrate a plot and graph showing the effect of pDNA loading on concentration and ultrasound contrast stability of nanobubbles prepared using a DPTAP solution. pDNA concentration does not seem to change NB stability much on ultrasound; all concentrations appear like the 0 pg.

[0147] Figs. 13(A-B) illustrate an image and graph showing PC3-pip cell uptake of nanobubbles prepared using a DPTAP solution. Fig. 14 illustrates a graph showing PC3-pip cell uptake of nanobubbles prepared using a using anionic nanobubbles. Protocol: Seed 10k PC3-pip overnight add NBs at 5k, 10k, or 20k NBs / cell — > at each timepoint: wash 2x with PBS — > plate reader for fluorescence. For DPTAP, there seems to be a maximum uptake of rhodamine by the cells. This maximum is reached in the 20k NB / cell condition by Ihr and the 10k NB / cell condition at approximately 3hrs.

[0148] Figs. 15(A-D) illustrate plots, images, and graphs showing ultrasound contrast of nanobubbles prepared using a DPTAP solution in PC3-pip cells. Ultrasound result: DPTAP inside in cells are stable and shows increased ultrasound contrast compared to controlcell only. Incubating cells for 1 hr has fewer ultrasound contras. Contrast signal increases with increased NB: cell ratio. Flow cytometry result: Incubating the cells for Ihr increases the rhodamine uptake compared to 15min. Increasing the NB:cell ratio also increases the rhodamine MFI.

[0149] Fig. 16 illustrates images showing transfection efficiency of nanobubbles prepared using a DPTAP solution loaded with pmaxGFP in PC3-pip cells without and with administration of therapeutic ultrasound. Protocol: Seed 100k cells 24hrs — > incubate in PBS-loaded DPTAP NBs (300ug / pL pmaxGFP) 15min — > flood well with media, seal with Tegaderm — > apply therapeutic ultrasound (1MHz, 50%DC, 2.2 W / cm2, 1 min) — > image 24hrs later. Therapeutic ultrasound and pDNA-loaded DPTAP NB increase transfection efficiency in PC3-pip cells. Optimizing sonication parameters is necessary to maximize in vitro transfection.

[0150] Figs. 17(A-B) illustrates a plot and images showing stability of nanobubbles prepared using a DPTAP solution loaded with cmvGFP nano-plasmid in mouse liver.

[0151] Fig. 18 illustrates images showing transfection efficiency of nanobubbles prepared using a DPTAP solution loaded with pmaxGFP in prostate cancer cells without and with administration of therapeutic ultrasound. No transfection is visible with CNBs only when they carry plasmid into the cells. With combination of ultrasound (TUS) and CNBs carrying the plasmid, expression of GFP is seen in prostate cancer cells.

[0152] Fig. 19 illustrates images showing transfection efficiency of nanobubbles prepared using a DPTAP solution loaded with pmaxGFP in livers of mice administered the nanobubbles after therapeutic ultrasound. Significant transfection is visible with CNBs +TUS in the liver of mice. Treatment done daily for 5 days. Saline control shows minimal background.

[0153] From the above description of the invention, those skilled in the art will perceive improvements, changes, and modifications. Such improvements, changes, and modifications within the skill of the art are intended to be covered by the appended claims. All references, publications, and patents cited in the present application are herein incorporated by reference in their entirety.

Claims

Having described the invention, we claim:

1. A composition comprising: a plurality of nanobubbles, each nanobubble having a lipid membrane that defines a gas containing internal void, the membrane including a plurality of cationic lipids having a cationic charge effective to complex at least one negatively charged genetic material, an edge-activator incorporated between lipids of the membrane and that enhances the flexibility of the membranes; and a membrane stiffener incorporated on an outer surface of the membrane and that enhances the membrane's resistance to tearing.

2. The composition of claim 1, further including at least one negatively charged genetic material loaded on and / or complexed with the nanobubbles.

3. The composition of claim 2, wherein the negatively charged genetic material includes at least one of DNA, pDNA, mRNA, microRNA, siRNA, nanoplasmids, plasmids, or other negatively charged genetic material.

4. The method any of claims 1 to 3, wherein the plurality of cationic lipids include cationic lipids having saturated C16-C22 hydrophobic tails and optionally cationic lipids having unsaturated C16-C22 hydrophobic tails.

5. The method of any of claims 1 to 4, wherein the plurality of cationic lipids include l,2-distearoyl-3-trimethylammonium-propane (DSTAP), l,2-Dipalmitoyl-3- trimethylammonium propane (DPTAP), l,2-distearoyl-3-dimethylammonium-propane (DSDAP), N-[l-(2,3-dilauroyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DLTAP), N-[l-(2,3-dimyristoyloxy)propyl]-N,N,N-tri methylammonium methylsulfate (DMTAP), N-[l -(2- palmitoyloxy-3-lauroyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (LPTAP), or a combination thereof.

6. The composition of any of claim 1 to 5, wherein the lipid membrane further includes a plurality of anionic, neutral and / or ionizable lipids, preferably phospholipids, more preferably, each including a hydrophilic head containing a phosphate group and two C16-C22 hydrophobic tails, preferably, the C16-C22 hydrophobic tails are saturated.

7. The composition of claim 6, wherein the phospholipids are selected from are selected from dibehenoylglycerophosphocoline (DBPC), dipalmitoylphosphatidic acid (DPP A), dipalmitoylphosphatidylethanolamine (DPPE), PEG functionalized distearoylphosphatidylethanolamine (DSPE-PEG), or combinations thereof, preferably, a combination of DBPC, DPPE, and DSPE-PEG.

8. The composition of any of claims 1 to 7, wherein lipid membrane includes a mixture of dibehenoylglycerophosphocoline (DBPC), dipalmitoylphosphatidylethanolamine (DPPE), PEG functionalized distearoylphosphatidylethanolamine (DSPE-PEG), and 1,2- Dipalmitoyl-3-trimethylammonium propane (DPTAP).

9. The composition of claim 8, wherein the ratio of DBPC, DPPE, DSPE-PEG, and DPTAP is about 6:2: 1:0.25-110. The composition of any of claims 1 to 9, wherein the stabilized nanobubbles have a lipid concentration of at least about 5 mg / ml, preferably, about 5 mg / ml to about 30 mg / ml.

11. The composition of any of claims 1 to 10, wherein the edge activator consists of propylene glycol and the membrane stiffener consists of glycerol.

12. The composition of any of claims 1 to 11, wherein the gas includes at least one perfluorocarbon gas, preferably C Fs or C4F10.

13. The composition of any of claims 1 to 12, wherein the lipid membrane optionally includes at least one of a surfactant, alcohol, or cholesterol, that change the shape, modulus, and / or interfacial tension of the bubble membrane.

14. The composition of any of claims 1 to 13, and wherein the nanobubbles have an average diameter of about 30 nm to about 600 nm, preferably, about 50 nm to about 500 nm, more preferably about 300 nm.

15. The composition of any of claims 1 to 14, further comprising at least one targeting moiety that is linked to the membrane of each nanobubble.

16. The composition of claim 14, the targeting moiety being selected from the group consisting of polypeptides, polynucleotides, small molecules, elemental compounds, antibodies, and antibody fragments.

17. The composition of any of claims 1 to 16, wherein the plurality of gas-filled nanobubbles further including a polymer coating at least a portion of an outer surface of the lipid membrane.

18. The composition of claim 17, wherein the polymer coating inhibits degradation of the at least one negatively charged genetic material loaded on and / or within the nanobubbles.

19. The composition of claim 17 or 18, wherein the polymer coating includes a layer of polydopamine coated on at least the portion of the outer surface.

20. The composition of any of claims 1 to 19, having a filament, vermiform, or worm-like shape.

21. A method of delivering a genetic material to a cell of interest, the method comprising: providing a plurality of nanobubbles of any of claims 1 to 20 loaded with at least one negatively charged genetic material; administering the plurality of nanobubbles loaded with the negatively charged genetic material to the cell; andoptionally insonating the nanobubbles internalized by the cell with ultrasound energy effective to release the negatively charged genetic material.

22. The method of claim 21, wherein the insonation is at a duty cycle of about 1% to about 100%, an ultrasound frequency of about 0.2 kHz to about 50 MHz, an intensity of about 0.1 W / cm2to about 5 W / cm2, a pressure amplitude of about 50 kPa to about 10 MPa, and a time of about 5 sec to about 30 minutes.

23. The method of claim 20 or 21 , wherein the ultrasound energy is provided by a non-focused, diagnostic imaging, focused, or high-intensity focused (HIFU) ultrasound transducer.

Images