Alkylated nucleosides, as well as compositions thereof and methods for nucleic acid delivery

Abstract

The present invention provides novel compounds, their liposomes, microbubbles and / or nanodroplets, and emulsion compositions and formulations useful for delivery of various nucleic acids and genes (e.g., single-stranded RNA, DNA, si-RNA, and CRISPR constructs), as well as methods for their preparation and use, including methods of imaging and gene delivery using ultrasound activation.

Description

Technical Field

[0001] Priority Claim and Related Patent Applications This application claims the benefit of priority from U.S. Provisional Application Serial No. 62 / 958,328, filed on January 8, 2020, the entire contents of which are incorporated herein by reference for all purposes.

Background Art

[0002] Technical Field of the Invention The present invention relates to compounds and pharmaceutical compositions and methods for their preparation and diagnostic or therapeutic use. More specifically, the present invention relates to novel compounds useful for the delivery of various nucleic acids and genes (e.g., single-stranded RNA, DNA, si-RNA, and CRISPR constructs), their liposomes, microbubbles, and / or nanodroplets, and compositions and formulations of emulsions, and methods for their preparation and methods of use including imaging and gene delivery using ultrasound activation.

[0003] Background of the Invention Gene therapy is a new medical field focused on the use of nucleic acids to deliver them to a patient's cells for therapeutic purposes as drugs to treat or prevent disease. Gene therapy includes oligonucleotide-based drugs such as DNA, RNA, CRISPR, and combinations thereof. A particularly interesting area today is CRISPR (clustered, regularly spaced short palindromic repeats), e.g., CRISPR-Cas9 (CRISPR-related protein 9), where RNA binds to this Cas9 enzyme. In CRISPR-Cas9, modified RNA is used to recognize a DNA sequence, and the Cas9 enzyme cleaves the DNA at the target site. Double-stranded RNA can be used as siRNA, which can be used as catalytic RNA to prevent the expression of a target gene. Currently, several approved products based on antisense oligonucleotides (ASOs) are available. ASOs typically contain single-stranded RNA constructs that, depending on the target, can block or enhance gene expression and protein translation. All ASOs approved to date utilize topical administration.

[0004] Novel and improved delivery technologies that enable systemic and / or local delivery of various gene-based therapeutics, including ASOs, remain in continuous demand. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] This invention describes alkylated compounds comprising one or more nucleoside analogs (including both deoxyribonucleosides and ribonucleosides) that enable the generation of micelles, liposomes, nanoparticles, microspheres, emulsions, fluorocarbon emulsions, and microbubbles. The alkylated nucleosides disclosed herein incorporate genetic material ("payload") into a corresponding structure by binding to a corresponding complementary nucleoside on the payload. The alkylated nucleoside ("carrier") is preferably electrically neutral and stabilizes the genetic material, storing it until the carrier delivers the payload to the target cells. Furthermore, the invention optionally includes one or more targeted ligands to assist delivery to selected desired cells. Optionally, ultrasound or other energy sources are used to monitor the delivery of the genetic payload and "activate" the carrier at the target site to release the genetic payload. Activation refers to an energy-mediated interaction with the carrier that facilitates the release, delivery to the cells, and intracellular delivery of the genetic payload.

[0006] In one embodiment, the present invention generally relates to compounds comprising one or more nucleosides, or derivatives or analogs thereof, or pharmaceutically acceptable forms thereof, each covalently bonded to one or more alkyl groups having at least nine (e.g., at least twelve, at least eighteen) carbon atoms.

[0007] In a particular embodiment, one or more nucleosides, or derivatives or analogs thereof, are covalently bonded to one or more alkyl groups via linking groups containing a diphosphate moiety.

[0008] In certain embodiments, the one or more nucleosides, or their derivatives or analogs, comprise one or more parts selected from cytosine, adenine, guanine, uracil, and thymine. In certain embodiments, the one or more nucleosides, or their derivatives or analogs, comprise two or more parts selected from cytosine, adenine, guanine, uracil, and thymine. In certain embodiments, the one or more nucleosides, or their derivatives or analogs, comprise four parts selected from cytosine, adenine, guanine, uracil, and thymine.

[0009] In certain embodiments, the one or more nucleosides, or derivatives or analogs thereof, comprise one or more portions selected from cytidine, adenosine, 5-methyluridine, uridine, and guanosine.

[0010] In certain embodiments, the one or more nucleosides, or derivatives or analogs thereof, comprise two or more portions selected from cytidine, adenosine, 5-methyluridine, uridine, and guanosine.

[0011] In certain embodiments, the one or more nucleosides are charge-neutral nucleosides.

[0012] In certain embodiments, each of the one or more alkyl groups has about 12 to about 24 carbon atoms (e.g., 12 to 16, 16 to 18, 18 to 24). In certain embodiments, the compound has two alkyl groups, each having about 12 to about 24 carbon atoms.

[0013] In certain embodiments, the one or more nucleosides include deoxyribonucleic acid. In certain embodiments, the one or more nucleosides include ribonucleic acid.

[0014] In certain embodiments, the compound further comprises a targeted ligand.

[0015] In another embodiment, the present invention generally relates to a complex comprising a compound disclosed herein, wherein the compound non-covalently binds to a nucleic acid molecule to form a complex. In certain embodiments, the nucleic acid molecule includes a gene, RNA, or CRISPR sequence.

[0016] In yet another aspect, the present invention generally relates to compositions comprising a compound or nucleic acid disclosed herein conjugated thereto.

[0017] In yet another aspect, the present invention generally relates to micelles or liposomes comprising a complex conjugated with a compound or nucleic acid disclosed herein.

[0018] In yet another aspect, the present invention generally relates to microbubbles comprising a complex thereof conjugated with a compound or nucleic acid disclosed herein.

[0019] In yet another aspect, the present invention generally relates to nanodroplets comprising a complex thereof conjugated with a compound or nucleic acid disclosed herein.

[0020] In yet another aspect, the present invention generally relates to compositions comprising micelles or liposomes as disclosed herein.

[0021] In yet another aspect, the present invention generally relates to compositions comprising microbubbles as disclosed herein.

[0022] In yet another aspect, the present invention generally relates to compositions comprising nanodroplets as disclosed herein.

[0023] In certain embodiments, fluorocarbons are used to form microbubbles or nanodroplets. In certain embodiments, the fluorocarbons are selected from perfluoropropane, perfluorobutane, and perfluoropentane.

[0024] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising a conjugate of a compound or nucleic acid disclosed herein with its complex, or a micelle, liposome, microbubble or nanodroplet comprising such a compound or complex, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0025] In yet another aspect, the invention generally relates to a method for treating a disease or condition, comprising administering to a subject in need thereof a pharmaceutical composition comprising a conjugate of a compound or nucleic acid disclosed herein with its complex, or a micelle, liposome, microbubble or nanodroplet comprising such a compound or complex, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0026] In certain embodiments, the disease or condition is selected from eye diseases (uveitis, retinitis and retinal dystrophy), vascular and heart diseases, cancer (acute lymphoblastic leukemia, B-cell lymphoma, head and neck squamous cell carcinoma and a variety of tumor phenotypes), lung diseases, Alzheimer's disease, other neurodegenerative conditions, and lipoprotein lipase deficiency.

[0027] In yet another aspect, the invention generally relates to a method for delivering a nucleic acid to a target site, the method comprising administering to a subject a composition comprising a conjugate of a compound or nucleic acid disclosed herein with its complex, or a micelle, liposome, microbubble or nanodroplet comprising such a compound or complex, and a pharmaceutically acceptable excipient, carrier, or diluent. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The invention will be better understood from the following detailed description read in conjunction with the drawings in which like reference numerals are used to designate like elements.

[0029] [Figure 1]Figure 1 shows the chemical structure of dipalmitoylphosphatidylcytidine (phosphatidylcytidine).

[0030] [Figure 2] Figure 2 shows a diagram of a ribose nucleoside, which is useful for preparing alkylated nucleoside moieties.

[0031] [Figure 3A] Figure 3A shows the binding of fluorescent polyguanosine (Poly-G) to the lipid 16:0 CDP DP. A 96-well plate was coated with 16:0 CDP DP lipid and dried overnight. DNA sequences were added to the plate while increasing the concentration of Poly-G (fluorescence), and incubated for 1 hour to confirm whether the sequences were bound to the lipid. A control lipid (DPPC) was used to confirm whether the sequences were bound. After incubation for 1 hour, fluorescence intensity was measured using a plate reader at Ex=488 nm and Em=525 nm.

[0032] [Figure 3B] Figure 3B shows graphs of the binding of different concentrations of fluorescent poly-guanosine (Poly-G) to microbubbles containing phosphatidylcytidine.

[0033] [Figure 4A] Figure 4A shows the amount of fluorescent poly-G bound to microbubbles (MBs) containing the lipid 16:0 CDP DP (1,2-dipalmitoyl-sn-glycero-3-(cytidine diphosphate)(ammonium salt)). Microbubbles containing the CDP DP lipid were activated and then incubated for 1 hour in the same dilution from the previous assay. The MBs were washed three times to remove unbound fluorescent sequences. A portion of the MBs was plated into a 96-well plate (Figure 4A) and incubated at Ex=488 nm and Em=525 nm for 1 hour, after which the fluorescence intensity was measured using a plate reader. The remaining portion was observed under a fluorescence microscope (Figure 4B).

[0034] [Figure 4B] Figure 4B shows a micrograph of microbubbles containing phosphatidylcytidine bound to fluorescent poly-G.

[0035] [Figure 5] Figure 5 shows micrographs of cells in which microbubbles containing phosphatidylcytidine bound to poly-G are shown inside and on the target cells.

[0036] [Figure 6] Figure 6 shows the synthetic scheme for generating the dialkyl-diphosphate nucleoside moiety. The conjugation of the described nucleoside diphosphate to the free-OH group of the described diacylglycerol proceeds via dicyclohexylcarbodiimide (DCC) coupling in the presence of a tertiary amine base to produce the described dialkyl-diphosphate nucleotide product. Purification of the crude material is achieved by silica gel chromatography.

[0037] [Figure 7A]Figure 7A shows a synthetic scheme for generating a neutral dialkyl-nucleoside moiety. Conjugation of both the described diacylglycerol and the described nucleoside moiety to the propionic acid-PEG4-propionic acid linker proceeds under DCC-mediated coupling in the presence of a tertiary amine base. Due to the symmetry of the linker, either the diacylglycerol linker or the nucleoside linker product can be independently generated and isolated for subsequent conjugation to the appropriate moiety or for the potential one-pot synthesis of the dialkyl-nucleoside under appropriate stoichiometric control. Propionic acid-PEG4-propionic acid (50 mg) was dissolved in 1 mL of diethyl ether, and 55 μL of SOCl2 (5 equivalents) was added and the mixture was stirred for 40 minutes. Pyridine (1.5 equivalents) was added along with 84 mg of 1,2-dipalmitoylglycerol (1 equivalent) dissolved in 2 mL of ether. Once the fuming subsided, 50 mg of guanosine (1 equivalent) dissolved in 1 mL of DMSO was added, and the reaction mixture was stirred for 1 hour. The reaction mixture was diluted with 5 mL each of DMSO and ether, and then diluted with 5 mL of water to remove unreacted thionyl chloride. The aqueous layer was separated and washed with ethyl acetate, and both organic layers were washed together with water. The solvent was evaporated to obtain 58.7 mg of white powder (34% yield).

[0038] [Figure 7B] Figure 7B shows a mass spectrometry graph representing the nucleoside product obtained using the chemical scheme in Figure 7A.

[0039] [Figure 8]Figure 8 shows a synthetic scheme to prevent side reactions at the ribose moiety. Undesirable reactions can occur in both synthetic procedures for the nucleoside conjugation, involving the free hydroxyl group of ribose and the amine of the nucleic acid base. Protecting the carbohydrate moiety with 2,2-dimethoxypropane (acetone dimethyl acetal) yields the main 2,3-isopropylidene product with a free 5-OH group available for subsequent bonding steps. This protection strategy is likely to provide imine derivatives of the nucleic acid base amine and may also hinder lateral reactivity at these sites. Bonding of the protected moiety proceeds as described, followed by hydrolysis of the protecting group under weakly acidic aqueous conditions to obtain the product. [Modes for carrying out the invention]

[0040] The present invention is described in the following description in preferred embodiments with reference to the figures, where similar numbers represent the same or similar elements. Throughout this specification, any reference to “one embodiment,” “embodiment,” or similar language means that a particular feature, structure, or property described in relation to an embodiment is included in at least one embodiment of the present invention. Thus, occurrences of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may all, though not necessarily, refer to the same embodiment. The described features, structures, or properties of the present invention can be combined in any suitable way in one or more embodiments. In the following description, numerous specific details are listed to provide a full understanding of the embodiments of the present invention. However, those skilled in the art will recognize that the present invention may be carried out without one or more specific details, or using other methods, components, materials, etc. In other examples, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the present invention. In certain embodiments, the present applicant’s invention comprises one or more alkylated nucleoside analogs mixed with one or more gene constructs.

[0041] In one embodiment, alkylated nucleoside materials are formulated either by themselves or with one or more other alkylated moieties. Preferred alkylated moieties include fatty acids, cholesterol and its derivatives, phospholipids, and fluorinated surfactants. Generally, one or more alkylated nucleoside analogs are formulated with a gene construct to form a corresponding structure, generally ranging in size from nanoscale domains to microscale sizes, for example, in the range of about 5 nm to about 5 microns. In one embodiment, the fluorinated material is incorporated into a composition containing an alkylated nucleoside moiety with genetic material to form emulsions, nanodroplets (NDs), and microbubbles (MBs). In this specification, emulsion may refer to a liquid structure that generally remains unchanged after administration to a subject. ND may refer to a material that is liquid but can change to a gas or other state upon activation by changes in temperature or energy such as light, magnetic, electrical or ultrasonic energy. MB refers to a gas within the structure. Preferred gases include fluorocarbon gases.

[0042] In one embodiment, the nucleoside analog comprises a monoalkyl group, for example, a fatty acid bonded to the nucleoside. In another embodiment, they comprise a cholesterol nucleoside analog. In yet another embodiment, the present invention comprises a fluoroalkyl moiety attached to the nucleoside. In a preferred embodiment, the present invention comprises a dialkyl moiety bonded to the nucleoside head group.

[0043] The alkylated nucleoside is optionally formulated with one or more additional lipids. In certain embodiments, the applicant's phospholipid composition comprises one or more substantially charge-neutral phospholipids. In certain embodiments, the applicant's phospholipid composition comprises dipalmitoylphosphatidylcholine ("DPPC"). DPPC is a zwitterionic compound and is a substantially neutral phospholipid. In certain embodiments, the applicant's phospholipid composition comprises a second phospholipid comprising a polyhydroxyhead group and / or more than 350 daltons of head groups, where M is Na+ , K + Li + , and NH4 +Selected from the group consisting of the following. The phospholipid may contain an ammonium counterion and a polyethylene glycol ("PEG") head group bonded to the phosphoryl moiety. In certain embodiments, the applicant's composition contains a PEGylated lipid. In certain embodiments, the PEG group MW is about 1,000 to about 10,000 daltons. In certain embodiments, the PEG group MW is about 2,000 to about 5,000 daltons. In certain embodiments, the PEG group MW is about 5,000 daltons. In certain embodiments, the applicant's lipid composition comprises one or more of the following pegylated lipids: 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000](ammonium salt), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000](ammonium salt), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000](ammonium salt), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000](ammonium salt), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine n-N-[methoxy(polyethylene glycol)-3000](ammonium salt), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](ammonium salt), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](ammonium salt), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol))-5000](ammonium salt) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](ammonium salt), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](ammonium salt), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](ammonium salt), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) glycol)-2000](ammonium salt), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000](ammonium salt), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000](ammonium salt), 1,2-Dioleoyl-sn- Licelo-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000](ammonium salt), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](ammonium salt), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](ammonium salt), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](ammonium salt), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](ammonium salt). The phospholipid 5 shown above represents dipalmitoylphosphatidylethanolamine, or DPPE. PE, particularly DPPE, is a preferred lipid in the present invention, preferably in formulations with other lipids at concentrations between 5 and 20 mol%, most preferably 10 mol%. In certain embodiments, the applicant's invention includes one or more conical or hexagonal HII-forming lipids. Useful conical lipids in the present invention include monogalactosyldiacylglycerol (MGDG), monoglucosyldiacylglycerol (MGDG), diphosphatidylglycerol (DPG), also known as cardiolipin, phosphatidylserine (PS), phosphatidylethanolamine (PE), and diacylglycerol. Phosphatidic acid (PA) is also a conical lipid, but is undesirable because it is prone to hydrolysis and may cause biological effects. The most preferred conical phospholipid is phosphatidylethanolamine (PE). Potentially useful conical cationic lipids include, but are not limited to, 1,2-dioleoyl-3-trimethylammonium-propane(chloride), 1,2-dioleoyl-3-trimethylammonium-propane(methylsulfate), 1,2-dimyristoyl-3-trimethylammonium-propane(chloride), 1,2-dipalmitoyl-3-trimethylammonium-propane(chloride), 1,2-distearoyl-3-trimethylanimonium-propane(chloride), and 1,2-dioleoyl- It contains 3-dimethylammonium-propane, 1,2-dimyristoyl-3-dimethylammonium-propane, 1,2-dipalmitoyl-3-dimethylammonium-propane, 1,2-distearoyl-3-dimethylammonium-propane, dimethyldioctadecylammonium (sodium salt or bromide salt), 1,2-di-O-octadecenyl-3-trimethylanimoniumpropane (chloride salt), and O,O-di-O-octadecenyl-3-ta-trimethylammonioacetyl-diethanolamine. Potentially useful additional cationic lipids include N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butyl-carboxamide)ethyl]-3,4-di[oleyloxy]-benzamide, 1,2-di-O-octadecenyl-3-trimethyl-ammoniumpropane (chloride salt) (DOTMA), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (saturated, unsaturated, or mixed chains of various chain lengths with 12-18 carbon atoms, e.g., saturated with unsaturated), and dimethyl dioctadecenyl Silammonium (bromide salt), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-ammonium (saturated, unsaturated, or mixed chains of various lengths with 14 to 18 carbon atoms, e.g., saturated with unsaturated), 1,2-dipalmitoyl-3-trimethylammonium-propane (chloride salt) (saturated, unsaturated, or mixed chains of various lengths with 14 to 18 carbon atoms, e.g., saturated with unsaturated), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride, N 4-It contains cholesteryl-spermine HCl salt and 1,2-dioleyloxy-3-dimethylaminopropane.

[0044] Cationic lipids can be used to neutralize the charge of RNA or DNA constructs, which are generally polyanions. The alkylated nucleoside forms a base pair with a complementary nucleoside in the RNA or DNA construct, and this base pair is thought to result in a stronger attraction than the electrostatic interaction mediated by the cationic lipid. In this regard, when cationic lipids are used, they are not used to bind genetic material, but rather to modulate the charge of the resulting nanostructure.

[0045] Generally, when the alkylated nucleoside is used with lipids, the concentration of the alkylated nucleoside represents about 1 to about 95 mol% of the total lipids in the formulation. More preferably, the alkylated nucleoside is in the range of about 5 to 50 mol% of the total lipids, and even more preferably, the alkylated nucleoside represents about 10 mol% of the total lipids in the formulation. Those skilled in the art will recognize that the alkyl chain of the alkylated nucleoside portion can be saturated or unsaturated, and when dialkyl nucleosides are used, they may also be mixed systems, for example, containing both saturated and unsaturated alkyl chains. Similarly, lipids used in addition to the alkylated nucleoside in the formulation may be saturated or unsaturated, and when dialkylated lipids are used (e.g., phosphocholine), they may also be mixed systems, for example, containing both saturated and unsaturated alkyl chains. In one embodiment, the alkylated nucleoside analog is generally incorporated into the liposome in a molar ratio of about 5 mol% to about 50 mol%. As is known in the art, various lipids can be used in this embodiment.

[0046] In another embodiment, the nucleoside lipid is used in an emulsion and may constitute up to 100% of the lipids, but generally less than 90%, 80%, or preferably about 70-75% of the total lipids.

[0047] In other embodiments, the alkylated nucleoside analogs are used in microbubbles or nanodroplets, as shown in the examples.

[0048] The present invention generates a variety of different constructs useful for multiple different applications. The route of administration varies depending on the condition being treated. The materials of the present invention can be administered intravenously, pulmonaryly (e.g., by inhalation), orally, subcutaneously, transdermally, by inhalation, nasally, intraperitoneally, intravaginally, intrasacrally, and rectally.

[0049] Microbubbles and liposomes prepared using the aforementioned nucleoside lipids are useful for treating lung diseases. Because microbubbles are filled with gas, they have a very small effective hydrodynamic diameter and are well-suited for delivery to the lungs. The constructs can be administered to the lungs via inhalation. Nebulizers can be used for inhalation. Useful nebulizers include jet nebulizers that use compressed gas to produce aerosols (small particles of drug in the air), ultrasonic nebulizers that produce aerosols by high-frequency vibrations, and mesh nebulizers in which liquid passes through a very fine mesh to form an aerosol. In addition, the product can be administered to the lungs using an inhaler. Exemplary inhalers include hydrofluoroalkane inhalers or HFA inhalers (formerly called metered-dose inhalers or MDIs), dry powder inhalers (DPIs), and soft mist inhalers (SMIs). For use with dry powder inhalers, the formulation may be supplied as a dry powder.

[0050] One or more bifunctional PEGylated lipids can be used. Examples of bifunctional PEGylated lipids include, but are not limited to, DSPE-PEG(2000), succinyl 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000](ammonium salt), DSPE-PEG(2000), PDP1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000](ammonium salt), and DSPE-PEG(2000)maleimide 1,2-distearoyl-s n-Glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000](ammonium salt), DSPE-PEG(2000)biotin 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000](ammonium salt), DSPE-PEG(2000)cyanur 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethylene glycol)-2000](ammonium salt) DSPE-PEG(2000)amine 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000](ammonium salt), DPPE-PEG(5,000)-maleimide, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[dibenzocyclooctyl(polyethylene glycol)-2000](ammonium salt), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azide(polyethylene glycol) 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000](ammonium salt), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000](ammonium salt), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000](ammonium salt), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000](ammonium salt), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000](ammonium salt), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000](ammonium salt), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethylene glycol)] This includes -2000] (ammonium salt), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] (ammonium salt), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-5000] (ammonium salt), N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000 and N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)5000]}.

[0051] The bifunctional lipid can be used to attach antibodies, peptides, vitamins, glycopeptides, and other targeted ligands to the structure containing the alkylated nucleoside. One or more targeted ligands can be incorporated into the corresponding structure. The MW of the PEG chain can vary from about 1,000 to about 10,000 daltons. In certain embodiments, the MW of the PEG chain is about 2,000 to about 5,000 daltons. The lipid chains of the lipids used in this invention can vary in length from about 12 to about 24 carbon atoms. Most preferably, the chain length is about 16 to about 18 carbon atoms. The chains can be saturated or unsaturated, but are preferably saturated. Cholesterol and cholesterol derivatives can also be used in this invention, provided that they are neutral or, if charged, contain more than 350 MW of head groups juxtaposed with their charge to protect the charge from the biological environment.

[0052] When using bifunctional lipids to construct targeted ligands, also known as bioconjugates, these targeted moieties are generally incorporated into the structure at a concentration of about 0.25 to about 10 mol%, more preferably about 0.5 to about 5 mol%, and most preferably about 1 mol%, of the total lipids.

[0053] In various embodiments, the alkylated nucleoside is formulated as MB or ND. The core of the structure described above may contain a gas or a gaseous precursor. Typical gaseous and gaseous precursors include nitrogen, oxygen, sulfur hexafluoride, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, or mixtures thereof. For imaging and gene / ASO / siRNA / CRISPR delivery purposes, an ideal MB / gaseous precursor contains a core gas with low water solubility coupled with a boiling point below body temperature. This results in an MB / ND with a long circulation time, a long useful lifetime, and high echogenicity for ultrasound visualization and ultrasound activation to facilitate gene delivery. The applicant's gaseous precursors include, for example, fluorinated carbon, perfluorocarbon, sulfur hexafluoride, perfluoroether, and combinations thereof. As those skilled in the art will understand, certain fluorinated compounds such as sulfur hexafluoride, perfluorocarbon, or perfluoroether are used as gaseous precursors because they exist in a liquid state when the composition is first prepared. Whether the aforementioned fluorine compound is a liquid or not generally depends on its liquid-to-gas transition temperature or boiling point. For example, the liquid-to-gas transition temperature (boiling point) of perfluoropentane, a preferred perfluorocarbon, is 29.5°C. This means that while perfluoropentane is generally a liquid at room temperature (about 25°C), it can turn into a gas inside the human body, where the normal human body temperature is about 37°C, exceeding the transition temperature of perfluoropentane. Therefore, under normal circumstances, perfluoropentane is a gaseous precursor. As is known to those skilled in the art, the effective boiling point of a substance can be related to the pressure to which it is subjected. This relationship is illustrated by the law of ideal gases: PV=nRT, where P is pressure, V is volume, n is moles of substance, R is the gas constant, and T is temperature (°K). The law of ideal gases shows that as pressure increases, the effective boiling point also increases. Conversely, as pressure decreases, the effective boiling point decreases. The fluorocarbons used as gaseous precursors in the compositions of the present invention include partially or completely fluorinated carbons, preferably saturated, unsaturated, or cyclic perfluorocarbons.Preferred perfluorocarbons include, for example, perfluoromethane, perfluoroethane, perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, perfluorohexane, perfluorocyclohexane, and mixtures thereof. More preferably, the perfluorocarbon is perfluorohexane, perfluoropentane, perfluoropropane, or perfluorobutane.

[0054] Preferred ethers include partially or completely fluorinated ethers, preferably perfluorinated ethers having a boiling point of about 36°C to about 60°C. Fluorinated ethers are ethers in which one or more hydrogen atoms are replaced by fluorine atoms. Preferred perfluorinated ethers for use as gaseous precursors in the present invention include, for example, perfluorotetrahydropyran, perfluoromethyltetrahydrofuran, perfluorobutyl methyl ether (e.g., perfluoro-t-butyl methyl ether, perfluoroisobutyl methyl ether, perfluoro-n-butyl methyl ether, perfluoropropyl methyl ether, perfluoroisopropyl ethyl ether, perfluoro-n-propyl ethyl ether, perfluorocyclobutyl methyl ether, perfluorocyclopropyl ethyl ether, perfluoropropyl methyl ether (e.g., perfluoroisopropyl methyl ether, perfluoro-n-propyl methyl ether)), perfluorodiethyl ether, perfluorocyclopropyl methyl ether, perfluoroethyl ethyl ether, and perfluorodimethyl ether.

[0055] Other preferred perfluoroether analogs include four to six carbon atoms and optionally one halide ion, preferably Br. -This includes, for example, compounds having the structure Cn Fy Hx OBr (where n is an integer from 1 to about 40, y is an integer from 0 to about 13, and x is an integer from 0 to about 13) are useful as gaseous precursors. Other preferred fluorinated compounds for use as gaseous precursors in the present invention are sulfur hexafluoride and octafluoropropane.

[0056] A mixture of different types of compounds can be used, for example, a mixture of a fluorinated compound (e.g., perfluorocarbon or perfluoroether) and another type of gas such as nitrogen.

[0057] Generally, preferred gaseous precursors undergo a phase transition to gas at temperatures up to about 57°C, preferably from about 20°C to about 52°C, preferably from about 37°C to about 50°C, more preferably from about 38°C to about 48°C, even more preferably from about 38°C to about 46°C, even more preferably from about 38°C to about 44°C, and even more preferably from about 38°C to about 42°C. Most preferably, the gaseous precursor undergoes a phase transition at temperatures below about 40°C. As will be recognized by those skilled in the art, the optimal phase transition temperature of a gaseous precursor for use in a particular application depends on the material under consideration, e.g., the specific patient being treated, the tissue being targeted, the nature of the physiological stress condition causing the temperature rise (e.g., cancer, infection, or inflammation), the stabilizing material used, and / or the herbal agent being delivered.

[0058] Furthermore, those skilled in the art will recognize that the phase transition temperature of a compound can be affected by local conditions within the structure, such as local pressure (e.g., interstitial, interface, or other pressure within the region). For example, if the pressure within the structure is higher than the ambient pressure, this is expected to increase the phase transition temperature. The extent of such an effect can be estimated using predictions from standard gas laws, such as Charles's Law and Boyle's Law. As a rough estimate, for a compound with a liquid-to-gas phase transition temperature of about 30°C to about 50°C, the phase transition temperature is expected to increase by about 1°C for every 25 mmHg increase in pressure. For example, the liquid-to-gas phase transition temperature (boiling point) of perfluoropentane is 29.5°C at a standard pressure of about 760 mmHg, but the boiling point is about 30.5°C at an interstitial pressure of 795 mmHg.

[0059] In one embodiment of the present invention, the alkylated nucleoside is incorporated into a lipid blend and stirred with a fluorocarbon gas to form MB. The resulting MB is then subjected to low temperature and high pressure, and their gaseous core is liquefied into ND. In this application, preferred gases are perfluoropropane, perfluorobutane, and perfluoropentane. To form ND of perfluoropropane, for example, a microbubble suspension may be cooled to about -17°C and then pressurized to about 50 PSI (for example, by injecting nitrogen gas or air into the vial). The milky suspension of MB then becomes translucent and bluish as ND is formed. The temperature and pressure required to form ND from perfluorobutane MB are not as low and high, and in the case of perfluoropentane MB, even lower temperatures and pressures are not required. When administered intravenously, the ND remains liquefied (due to LaPlace pressure), but returns to MB upon ultrasound irradiation. The acoustic pressure required to activate MB to ND is lowest with perfluoropropane, intermediate with perfluorobutane, and highest with perfluoropentane ND. By mixing these gases at various concentrations, the composition can be adjusted to the specific acoustic pressure at which ND is converted to MB. When ultrasound is applied to biomedical imaging, power levels are limited to avoid biological effects. Alkylated nucleosides containing a perfluoropropane core carrying a genetic drug payload can be activated at safe acoustic pressures, e.g., a mechanical index of ultrasound less than approximately 1.0. The advantage of ND is its small diameter (e.g., nanometer size) compared to the micron size of MB. To deliver the genetic material payload, it is desirable for the material to pass through the intracellular space. Smaller particles are considered advantageous for cellular delivery. In this regard, it is desirable for targeted ligands to bind the particles to cellular targets. Targeted regimes that induce intracellular delivery are advantageous. As an example, E-selectin targeted particles are internalized by cells. By incorporating a second ligand, such as a vitamin, intracellular delivery of, for example, folic acid or transferrin can be achieved.When these particles migrate internally, they can enter endosomes and be hydrolyzed. However, sonication-induced activation releases the contents of these particles (e.g., gene payloads) from the endosomes into the intracellular environment. The genetic payloads can then enter the desired intracellular space, such as the nucleus in CRISPR or the ribosome in ASO.

[0060] In one embodiment of the present invention, the alkylated nucleoside (which may be formulated with one or more other lipids) may include high-boiling point fluorocarbon materials, such as perfluorodecalin, perfluorooctyl bromide, perfluorotripropylamine, and other such fluorocarbons known to those skilled in the art. Note that when therapeutic genetic material is attached to the alkylated nucleoside, these structures may shift positions to form a “raft.” In doing so, the alkylated nucleoside may be reoriented to form base pairs with RNA or DNA in the delivered construct. In this regard, the fluorocarbon may act as an interface that reduces surface tension, acts thermodynamically favorably, and allows the alkylated nucleoside to shift positions to provide the most effective base pairing.

[0061] In another embodiment, the alkylated nucleoside and associated genetic drugs are provided as dry powders using spray drying and / or freeze-drying. Various antifreezes and stabilizers well known in the art can be used therein, including but not limited to trehalose, formamide, dimethyl sulfoxide, and mixtures of formamide with DMSO, propylene glycol, glycerol, ethylene glycol, treitol, and 2-methyl-2,4-pentanediol. Those skilled in the art will recognize that the above antifreezes can be used individually, in combination, or in combination with other antifreezes known in the art. By use of solvents such as glycerol and propylene glycol, the present invention may be provided in a form that is substantially water-free and to be rehydrated with water or saline before use.

[0062] The applicant's invention is useful for delivering various RNA and DNA-based therapeutics. Antisense oligonucleotides are small pieces of DNA or RNA that bind to specific molecules of RNA. This typically blocks the RNA's ability to make a particular protein. Antisense oligonucleotides (ASOs) are sometimes so named because their base sequence is complementary to the messenger RNA of a gene called the "sense" sequence (thus, the sense segment "5'-AAGGUC-3'" of the messenger RNA is blocked by the antisense messenger RNA segment "3'-UUCCAG-5'"). Historically, unmodified phosphodiester RNA ASOs are degraded after IV administration before reaching their targets. The applicant's invention is thought to help stabilize ASOs so that they can reach their intended targets. However, other DNA and RNA derivatives, such as morpholino oligomers, e.g., DNA or RNA bases bound to a methylenemorpholine ring backbone linked via a phosphorodiamidate group, can also be used in the invention. The binding of nuclear small ribonucleoprotein complexes prevents binding at the intron boundaries of the premRNA strand, and through other mechanisms, by blocking or ribosome activity, and through other mechanisms, ASO (modified or unmodified) can interfere with premRNA. Peptide nucleic acids (the peptide nucleic acid backbone consists of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds) can also be used in the present invention. Locked nucleic acids, modified RNA nucleotides in which the ribose portion is modified with an additional bridge connecting the 2' oxygen and 4' carbon, can also be used in the present invention. In locked nucleic acids, the bridge locks the ribose to the 3'-end conformation. Locked nucleic acids can be mixed with DNA or RNA residues in oligonucleotides to enhance hybridization properties. RNA interference can also benefit from the applicant's inventions in which two types of small ribonucleic acid molecules (microRNA and small interfering RNA (siRNA)) are used for RNA interference. siRNA is generally double-stranded and includes a passenger strand and a guide strand.The passenger strand is degraded, and the guide strand is incorporated into the RNA-induced silencing complex. For base pairing in the applicant's invention, this allows the guide strand to be used without the passenger strand for RNA interference. Plasmids, circular constructs of double-stranded DNA, are generally in the size range of 1 to over 1,000 kilobase pairs and can be used in the applicant's invention. If necessary, the plasmid DNA can be heated or subjected to chemical means to partially degrade the two strands and optimize base pairing between the alkylated nucleoside and DNA of the applicant. Alternatively, CRISPR, for example CRISPR-Cas9, can be used in the present invention, in which a single guide RNA of the system recognizes its target sequence in the genome, and the Cas9 nuclease acts as scissors for cutting the double strand of DNA. The guide RNA can be designed to base pair with the alkylated nucleoside of the present invention and to release the guide RNA when the CRISPR-Cas9 complex enters the cell. Cationic lipids can be incorporated into formulations together with the applicant's alkylated nucleosides to improve the binding of double-stranded DNA, RNA, and CRISPR constructs. Similarly, cell-permeable peptides and nuclear localization motifs can be incorporated into the applicant's inventions.

[0063] As those skilled in the art will recognize, the applicant's invention can be used to treat a wide variety of diseases, using corresponding gene constructs to carry out treatment. Without limiting itself, the invention can be used to treat eye diseases (uveitis, retinitis, and retinal dystrophy), vascular and heart diseases, cancers (acute lymphoblastic leukemia, B-cell lymphoma, head and neck squamous cell carcinoma, and a wide variety of neoplastic conditions), lung diseases, Alzheimer's disease, and other neurodegenerative conditions and lipoprotein lipase deficiencies. The applicant's invention can also be used ex vivo, for example, to introduce one or more genes or other gene constructs into cells, such as CAR T cells, for administration to a patient to treat a disease. A possible example is using the applicant's invention to target CAR T cells to treat p53-positive cancer. The invention can be used as a preclinical discovery tool in in vivo and in vitro studies. The embodiments of the present invention are listed below. (1) A compound comprising one or more nucleosides, or derivatives or analogs thereof, or a pharmaceutically acceptable form thereof, each covalently bonded to one or more alkyl groups having at least nine carbon atoms. (2) The compound according to (1), wherein one or more nucleosides, or derivatives or analogs thereof, are covalently bonded to one or more alkyl groups via a linking group containing a diphosphate moiety. (3) The compound according to (2), wherein the one or more nucleosides, or derivatives or analogs thereof, comprises one or more moieties selected from cytosine, adenine, guanine, uracil, and thymine. (4) The compound according to (3), wherein the one or more nucleosides, or derivatives or analogs thereof, comprises two or more moieties selected from cytosine, adenine, guanine, uracil, and thymine. (5) The compound according to (2), wherein the one or more nucleosides, or derivatives or analogs thereof, comprises one or more moieties selected from cytidine, adenosine, 5-methyluridine, uridine, and guanosine. (6) The compound according to (5), wherein one or more nucleosides, or derivatives or analogs thereof, comprises two or more moieties selected from cytidine, adenosine, 5-methyluridine, uridine, and guanosine. (7) The compound according to any one of (1) to (6), wherein each of the one or more alkyl groups has about 12 to about 24 carbon atoms. (8) The compound according to (7), wherein the compound comprises two alkyl groups, each having about 12 to about 24 carbon atoms. (9) The compound described in (7), wherein the one or more nucleosides include deoxyribonucleic acid. (10) The compound according to (7), wherein the one or more nucleosides include ribonucleic acid. (11) The compound according to (7), wherein one or more nucleosides are charge-neutral. (12) The compound further comprises a targeted ligand, as described in (7). (13) The targeting ligand is a compound according to (12), selected from antibodies, peptides, vitamins, and glycopeptides. (14) The targeting ligand is an antibody, the compound described in (13). (15) A complex comprising the compound described in (7) which is non-covalently bound to a nucleic acid molecule to form a complex, wherein the nucleic acid molecule comprises a single-stranded RNA molecule, a DNA molecule, a siRNA molecule, a CRISPR construct, or an antisense oligonucleotide. (16) A composition comprising the compound described in (7). (17) (15) A micelle or liposome containing the complex described above. (18) (15) Microbubbles containing the composite described above. (19) (15) Microdroplets or nanodroplets containing the composite described above. (20) (17) A composition comprising micelles or liposomes as described in (18), microbubbles as described in (18), or microdroplets or nanodroplets as described in (19). (twenty one) The composition according to (20), wherein a fluorocarbon is used to form microbubbles, microdroplets, nanodroplets, micelles, or liposomes. (twenty two) The composition according to (21), wherein the fluorocarbon is selected from perfluoropropane, perfluorobutane, and perfluoropentane. (twenty three) A pharmaceutical composition comprising any one of the compounds described in (1) to (6) for the treatment of a disease or condition selected from eye diseases (uveitis, retinitis and retinal dystrophy), vascular and heart diseases, cancer (acute lymphoblastic leukemia, B-cell lymphoma, head and neck squamous cell carcinoma and a wide variety of neoplastic conditions), lung diseases, Alzheimer's disease and other neurodegenerative conditions, and lipoprotein lipase deficiency. (twenty four) Use for manufacturing any compound, disease or condition described in any one of items (1) to (6). (twenty five) The aforementioned diseases or conditions are selected from eye diseases (uveitis, retinitis and retinal dystrophy), vascular and cardiac diseases, cancers (acute lymphoblastic leukemia, B-cell lymphoma, head and neck squamous cell carcinoma and a wide variety of neoplastic conditions), lung diseases, Alzheimer's disease and other neurodegenerative conditions, and lipoprotein lipase deficiencies, as described in (24). (26) Use of any one of the compounds described in (1) to (6) for the purpose of manufacturing a pharmaceutical product for delivering nucleic acids to a target that requires it. (27) The use according to (26), wherein delivering nucleic acids to the subject includes applying energy selected from light, magnetic, electrical, and ultrasonic energy to a target site of the subject to activate the delivery of the nucleic acid molecules. (28) The energy to be applied is ultrasonic energy, as described in (27). (29) The aforementioned delivery is via pulmonary delivery, as described in (27). (30) The use described in (29) is by inhalation of the subject into the lungs. Further embodiments of the present invention are listed below. (A-1) A complex comprising a compound that non-covalently binds to a nucleic acid molecule to form a complex, wherein each compound comprises a nucleoside covalently bonded to one or more alkyl groups having at least nine carbon atoms, The nucleoside is selected from cytosine, adenine, guanine, uracil and thymine, or selected from cytidine, adenosine, 5-methyluridine, uridine and guanosine, and The nucleic acid molecule is a complex comprising a single-stranded RNA molecule, a DNA molecule, a siRNA molecule, a CRISPR construct, or an antisense oligonucleotide. (A-2) The complex according to (A-1), wherein the nucleoside is covalently bonded to one or more alkyl groups via a linking group containing a diphosphate moiety. (A-3) The composite according to (A-2), wherein each of the one or more alkyl groups has 12 to 24 carbon atoms. (A-4) The complex is the complex according to any one of (A-1 to 3), wherein the complex comprises two alkyl groups, each having 12 to 24 carbon atoms. (A-5) The nucleoside is a complex according to (A-4), comprising deoxyribonucleic acid. (A-6) The nucleoside is a complex according to (A-4), comprising ribonucleic acid. (A-7) The nucleoside is charge-neutral, as described in (A-4). (A-8) The complex according to (A-4), further comprising a targeted ligand selected from an antibody, peptide, vitamin, and glycopeptide. (A-9) The targeting ligand is an antibody, the complex described in (A-8). (A-10) A composition containing the complex described in (A-1). (A-11) Microbubbles containing the composite described in (A-1). (A-12) Microdroplets or nanodroplets containing the composite described in (A-1). (A-13) A composition comprising the microbubbles described in (A-11), or the microdroplets or nanodroplets described in (A-12). (A-14) The composition according to (A-13), wherein a fluorocarbon is used to form microbubbles, microdroplets, nanodroplets, micelles, or liposomes. (A-15) The composition according to (A-14), wherein the fluorocarbon is selected from perfluoropropane, perfluorobutane, and perfluoropentane. (A-16) A pharmaceutical composition comprising the complex described in any one of (A-1 to A-3) for treating a disease or condition selected from eye diseases (uveitis, retinitis and retinal dystrophy), vascular and heart diseases, cancer (acute lymphoblastic leukemia, B-cell lymphoma, head and neck squamous cell carcinoma and a wide variety of neoplastic conditions), lung diseases, Alzheimer's disease and other neurodegenerative conditions, and lipoprotein lipase deficiency. (A-17) Use of any one of the complexes described in paragraph (A-1 to A-3) for the manufacture of a pharmaceutical product for the treatment of a disease or condition, wherein the disease or condition is selected from eye diseases (uveitis, retinitis and retinal dystrophy), vascular and cardiac diseases, cancers (acute lymphoblastic leukemia, B-cell lymphoma, head and neck squamous cell carcinoma and a wide variety of neoplastic conditions), lung diseases, Alzheimer's disease and other neurodegenerative conditions, and lipoprotein lipase deficiency.

[0064] The following examples are intended to illustrate, and not limit, the implementation of the present invention. [Examples]

[0065] Example 1. Preparation of microbubbles (MBs) of perfluoropropane coated with phosphatidylcholine, called MVT-100. A lipid blend containing dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), and dipalmitoylphosphatidylethanolamine-polyethylene glycol-5,000 (DPPE-MEPG-5000) was prepared. The lipids, suspended in propylene glycol, were heated to 70±50°C until dissolved. The resulting lipid solution was then added to an aqueous solution containing sodium chloride, phosphate buffer, and glycerol, and thoroughly mixed by stirring. Each 1 ml of the resulting lipid blend contained 0.75 mg of total lipids (consisting of 0.400 mg of DPPC, 0.046 mg of DPPE, and 0.32 mg of MPEG-5000-DPPE). Furthermore, 1 ml of each of the lipid blends contained 103.5 mg of propylene glycol, 126.2 mg of glycerin, 2.34 mg of sodium phosphate monobasic monohydrate, 2.16 mg of sodium phosphate dibasic heptahydrate, and 4.87 mg of sodium chloride in water for injection. The pH was 6.2 to 6.8. The materials were prepared in sealed vials with headspace containing octafluoropropane (OFP) gas (>80%) along with balanced air.

[0066] Microbubbles generated by the MVT-100 formulation remain stable over time in terms of concentration and size distribution, even when suspended in normal physiological saline. This lipid blend, called MVT-100, was used as the base MB for ND preparation and ASO binding. Specifically, phosphatidylcytidine was added to alter the molar ratio of nucleoside phospholipids to facilitate ASO binding.

[0067] Example 2. Preparation of phosphatidylcytidine-containing MB and loading of ASO. Fluorescently labeled poly-G (Alexa Fluor 488) was obtained from Integrated DNA Technologies (IDT) and added to a lipid mixture (0.75 mg / ml, 73.8 mol% DPPC, 9 mol% DPPE, 6.3 mol% DPPE-PEG5000, and 10 mol% 16:0 CDP DG (cytidine diphosphate)). MB was prepared by stirring. 10 μl of MB was incubated with various dilutions of fluorescently labeled poly-G, and then incubated for 1 hour. After 1 hour of incubation, unbound fluorescent poly-G was removed by centrifugation in an Eppendorf tube (1500 rpm for 3 minutes). The clear liquid at the bottom was withdrawn with a syringe, and the milky MB layer on top was resuspended in fresh PBS. This was repeated three times.

[0068] Example 3. Conjugation of ASO to both MB containing phosphatidylcytidine and MB without phosphatidylcytidine. Aliquotes of MB bound to ASO were seeded in a black 96-well plate, and the fluorescence intensity was measured using a plate reader (Molecular Devices, SpetraMax M3) at Exλ (stroke lambda, hereafter the same) = 488 nm and Emλ = 525 nm. Aliquots of MB bound to ASO were seeded on a poly-d-lysine coated glass-bottom dish (Mat Tek, Ashland, Massachusetts) and allowed to adhere to the surface. Fluorescent MB was observed under a microscope using a Leica DMI6000 multifunction motorized inverted microscope.

[0069] Example 4. Preparation of nanodroplets containing phosphatidylcytidine and nanodroplets without phosphatidylcytidine. A perfluoropropane (PFP) microbiota (MB) based on MVT-100 containing a proprietary noncritical excipient was liquefied at low temperatures by incubation in an ethylene glycol bath at -17°C and 50 PSI for 5 minutes. The MB appeared as a whitish foam, but after the liquefaction process, the ND appeared as a pale bluish translucent emulsion. The resulting ND had an average particle size of 600 nm and a PFP concentration of 90%, compared to the MB of the parent MVT-100, which had an average particle size of 830 nm and a PFP concentration of 90% in headspace.

[0070] Example 5. Incubation of cells with phosphatidylcytidine MB Aliquots of the MB conjugated to the ASO were added to human epithelial colorectal adenocarcinoma cells (CaCO2). The cells were grown in a T25 flask using Eagle's Minimum Essential Medium (EMEM) formulated with ATCC supplemented with 20% fetal bovine serum and 1% penicillin-streptomycin. The cells were incubated at 37°C in a humid atmosphere with 5% carbon dioxide. After confluence, the cells were detached with trypsin and transferred to a glass-bottom dish coated with poly-d-lysine, and then incubated for a further 24 hours to ensure proper adhesion. MB was added and incubated with the cells for 2 hours. After 2 hours, the cells were washed to remove unbound fluorescent MB. The cells were observed under a microscope using a Leica DMI6000 multifunction motorized inverted microscope.

[0071] Predicted example 1. Preparation of MB containing four different nucleoside lipids. MB was prepared from the lipids described in Example 1, except that 10 mol% of the lipids were replaced with four different nucleoside lipids. Phosphatidyl-cytidine, adenine, thymine, and guanine were prepared according to the synthesis scheme shown in Figure 6. Each of the corresponding phosphatidyl nucleosides was purified by HPLC. Each phosphatidyl nucleoside portion was added to the formulation at a concentration of 2.5 mol% to achieve a total molar percentage of 10 mol% of the total phosphatidyl nucleoside aggregate. The resulting MB was shown to bind favorably to ASO. Serum stability assays showed that MB containing phosphatidyl nucleosides provided a significant improvement in stability compared to ASO without MB.

[0072] Predicted example 2. Preparation of ND containing four different nucleoside lipids. MB was prepared as in Predicted Example 1. The obtained MB was exposed to low temperature and high pressure as in Example 4. Next, the obtained ND was incubated with ASO.

[0073] Predicted example 3. Preparation of ND(tND) targeting E-selectin for ASO delivery. A bioconjugate of E-selectin-binding peptide is prepared from DSPE-PEG-maleimide and DK12-OH peptide to obtain the resulting bioconjugate. The bioconjugate is purified by HPLC and its structure is confirmed by mass spectrometry. To prepare tND, typically 1 mol% of the bioconjugate is mixed with 76 mol% DPPC, 7 mol% DPPE-MPEG(5000), 7 mol% DPPE, and 10 mol% nucleoside phosphatidylcholine lipid as described in Predicted Example 1. The lipids are dissolved in buffered ordinary physiological saline, propylene glycol, and glycerol in a 76 / 7 / 7 / 10 molar ratio diluent. The clear lipid mixture is placed in a sealed vial, and the vial is filled with octafluoropropane gas. For ND formulations, particle size and concentration characteristics are evaluated using Accusizer® 780 (Particle Sizing Systems, Port Ritchie, Florida) and NanoBrook 90 Plus (Brookhaven), respectively, to ensure the uniformity of the ND formulations. The concentration of perfluoropropane in various formulations is measured by Raman spectroscopy (DXR2 Smart Raman, ThermoScientific). ASOs related to E-selectin and ICAM-1, as phosphorothioate analogs, are incubated with ND, and unbound ASOs are removed by dialysis. The resulting ND targets E-selectin and is useful in alleviating inflammatory conditions such as uveitis, arthritis, and other related conditions.

[0074] Predicted example 4. Preparation of liposomes that bind to ASO. Liposomes are generated using the lipids described in Predicted Example 4. Fluorocarbon gases are not used in this application. After the lipids are rehydrated, liposomes are generated by freeze-thawing and subsequent extrusion of the liposomes. ASO is added to the liposomes, and unbound ASO is removed by dialysis. The resulting E-selectin-targeting liposomes are useful for delivering ASO to inflammatory conditions.

[0075] Example 5. Preparation of emulsions useful for binding with ASO. The neutral lipids shown in Figures 7A-7B are prepared with cytidinyl, adenine, thymine, and guanine head groups. These lipids are formulated without additional lipids to form micelles. ASO is added to the resulting micelles to form a complex.

[0076] Predicted example 6. Preparation of nucleoside lipids to eliminate ribose side reactions. The nucleoside lipids shown in Figure 8 are generated and purified by HPLC. The resulting nucleoside lipids are useful for ASO binding.

[0077] Predicted example 7. Imaging and treatment of patients with uveitis. ND is prepared as described above to contain 10 mol% nucleoside lipids having cytidinyl, adenine, thymine, and guanine head groups. ASO for E-selectin and ICAM-1 is added to the ND and bound to them. The ND contains a trace amount of fluorophore DiO. About 10 10 Patients with uveitis are intravenously injected with approximately 2.5 ml of a solution containing approximately 2 mg each of ND, E-selectin, and ICAM-1-targeted ASO. Fundus examination shows ND uptake in the inflamed retina. Ultrasonography using a 20 MHz transducer shows ND uptake into the inflamed area of ​​the eye. Next, a 1.0 MHz transducer is used at a power level of 720 milliwatts to cavitate ND / MB, causing ASO release from the endosomes of inflamed endothelial cells and macrophages. Follow-up imaging at 2 weeks using E-selectin-targeted MB shows much lower uptake, reflecting a reduction in inflammation.

[0078] Predicted example 8. Lung delivery and treatment of lung diseases Microbubbles and liposomes prepared using the aforementioned nucleoside lipids are useful for treating lung diseases.

[0079] A. The neutral nucleoside lipids are mixed with the lipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), and DPPE-PEG (5,000). The final lipid concentrations are approximately 10-20 mol% for the nucleoside lipids and 80-90 mol% for DPPC, DPPE, and DPPE-PEG. The proportion of non-nucleoside lipids is approximately 82 mol% for DPPC, 10 mol% for DPPE, and 8 mol% for DPPE-PEG. The lipids are suspended in a liquid containing approximately 80 w / vol% physiological saline with 10 v / vol% propylene glycol and 10 w / vol% glycerol. Approximately 2 mg of total lipids per 1 ml are placed in a 1.5 ml glass Wheaton vial with a perfluorobutane headspace, and the vial is sealed. The vial is shaken on an amalgameter device at a speed of approximately 4,500 RPM to generate microbubbles containing approximately 10-20 mol% of nucleoside lipids. The microbubbles are then mixed with approximately 1:1 wt / vol of lipids and w / vol of TGF-β mRNA-targeting antisense oligonucleotides, gently agitated, and administered to a patient with idiopathic pulmonary fibrosis via an ultrasonic nebulizer. The patient inhales the atomized microbubbles carrying the antisense oligonucleotides. Multiple treatments are administered, resulting in disease improvement over a period of several months.

[0080] B. The above is substantially repeated, except that the antisense oligonucleotide (ASO) is added to the aqueous suspension of lipids in the vial, and the stirring step is repeated so that when microbubbles are formed, the microbubbles bind to the ASO.

[0081] C. The above Example A is substantially repeated, except that the lipid is dissolved in propylene glycol, heated with ASO to 55°C, and filtered through a 0.2 micron filter. The vial is then filled with this material and sealed with perfluorobutane gas. The resulting product is essentially anhydrous. The vial is shaken at about 4,500 RPM for 45 seconds, and the microbubbles are rehydrated by injecting the vial with about 80% w / vol saline containing about 10% w / vol glycerol. The material is gently stirred in the vial, withdrawn with a syringe, and placed in an ultrasonic nebulizer for administration to the patient.

[0082] The applicant's disclosures described herein are described in preferred embodiments with reference to drawings where similar numbers represent the same or similar elements. Throughout this specification, references to “one embodiment,” “embodiment,” or similar language mean that certain features, structures, or characteristics described in relation to such embodiments are included in at least one embodiment of the present invention. Thus, occurrences of the phrases “in one embodiment,” “in embodiment,” and similar language throughout this specification may, but not necessarily, all refer to the same embodiment.

[0083] The features, structures, or properties described in the applicant's disclosure can be combined in any suitable manner in one or more embodiments. Numerous specific details are listed in this description to provide a full understanding of embodiments of the invention. However, those skilled in the art will recognize that the applicant's compositions and / or methods may be carried out without one or more specific details, or using other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the disclosure.

[0084] In this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0085] As used herein, unless otherwise specifically stated or evident from the context, the term “about” is understood to mean within the normal tolerance range in the art, e.g., within two standard deviations of the mean. “About” may be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise evident from the context, all numerical values ​​provided herein may be modified by the term “about.”

[0086] As used herein, unless otherwise specifically stated or evident from the context, the term “or” is understood to be inclusive.

[0087] When used to define compositions and methods, the term “contains” is intended to mean that the composition and method includes the enumerated elements but does not exclude other elements. When used to define compositions and methods, the term “essentially consists of” shall mean that the composition and method includes the enumerated elements but excludes other elements that are essentially important to the composition and method. For example, “essentially consists of” means the administration of a pharmacologically active agent as explicitly stated and excludes pharmacologically active agents not explicitly stated. The term “essentially consists of” does not exclude pharmacologically inactive or inert agents, such as pharmaceutically acceptable excipients, carriers, or diluents. When used to define compositions and methods, the term “consistes of” shall mean the exclusion of trace elements of other components and substantial method steps. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this disclosure, but preferred methods and materials are described herein. The methods described herein may be performed in any logically possible order, in addition to the specific order disclosed.

[0089] Reference This disclosure makes references and quotations to other documents, including patents, patent applications, patent publications, journals, books, articles, and web content. All such documents are incorporated herein by reference in their entirety for all purposes. Any document or part thereof that is said to be incorporated herein by reference but which contradicts existing definitions, references, or other disclosures expressly made herein is incorporated only to the extent that it does not create a conflict between the incorporated document and the disclosure herein. In the event of a conflict, the present disclosure shall prevail as the preferred disclosure to resolve the conflict.

[0090] Equal parts Representative examples are intended to aid in illustrating the present invention and are not intended to limit the scope of the invention, nor should they be construed as limiting the scope of the invention. In fact, various modifications of the invention and many further embodiments, in addition to those shown and described herein, will be apparent to those skilled in the art from the entirety of this document, including the examples and scientific and patent references contained herein. The aforementioned examples include important additional information, examples and guidance that can be adapted to the implementation of the invention in its various embodiments and equivalents.

Claims

1. A complex comprising a compound that non-covalently binds to a nucleic acid molecule to form a complex, wherein the compound comprises a nucleoside covalently bonded to two alkyl groups each having 12 to 24 carbon atoms, The nucleoside comprises two or more moieties selected from cytosine, adenine, guanine, uracil, and thymine, and The nucleic acid molecule is a complex containing an antisense oligonucleotide.

2. The composite according to claim 1, wherein the nucleoside is covalently bonded to one or more alkyl groups via a linking group containing a diphosphate moiety.

3. The composite according to claim 1, wherein the nucleoside is charge-neutral.

4. The complex according to claim 1, further comprising a targeted ligand selected from an antibody, a peptide, a vitamin, and a glycopeptide.

5. The complex according to claim 4, wherein the targeted ligand is an antibody.

6. A microbubble comprising the composite according to claim 1.

7. A microdroplet or nanodroplet comprising the composite according to claim 1.

8. The microbubble according to claim 6, wherein the microbubble is formed using fluorocarbon.

9. The microdroplets or nanodroplets according to claim 7, wherein the microdroplets or nanodroplets are formed using fluorocarbon.

10. The microbubble according to claim 8, wherein the fluorocarbon is selected from perfluoropropane, perfluorobutane, and perfluoropentane.

11. The microdroplets or nanodroplets according to claim 9, wherein the fluorocarbon is selected from perfluoropropane, perfluorobutane, and perfluoropentane.

12. A pharmaceutical composition comprising the complex according to claim 1 or 2 for treating a disease or condition selected from eye diseases (uveitis, retinitis and retinal dystrophy), vascular and heart diseases, cancer (acute lymphoblastic leukemia, B-cell lymphoma, head and neck squamous cell carcinoma and a wide variety of neoplastic conditions), lung diseases, Alzheimer's disease and other neurodegenerative conditions, and lipoprotein lipase deficiency.

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