MAKING AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, co-pending U.S. provisional application entitled "LIPID-SURFACTANT NANOPARTICLES FOR DRUG DELIVERY AND METHODS OF MAKING AND USES THEREOF" having serial no. 62/631 ,582, filed February 16, 2018, the contents of which are incorporated by reference in their entirety.

TECHNICAL FIELD

[0002] The present disclosure generally relates to drug delivery, and in particular to nanoparticle drug delivery.

BACKGROUND

[0003] Lipid-based nanoparticles (LNPs) have been extensively utilized in the delivery of nucleic acids (using oligonucleotides (ON) as an example) and lipophilic drugs. For example, LNPs have been used in the delivery of plasmid DNA and messenger RNA for CAR-T gene therapy or cancer vaccine delivery, and for the delivery of siRNA, miRNA, and sgRNA for gene silencing or gene editing. However, there are a number of challenges associated with LNP formulation. It is difficult to make sub-100-nm LNPs at high ON concentration by self-assembly. LNP synthesis typically requires 40% ethanol during assembly, which needs to be removed afterwards. This complicates the manufacturing of LNPs. These limitations have impeded the clinical translation of LNP formulations of ON therapeutics.

[0004] There remains a need for improved nanoparticles for delivering nucleic acids and lipophilic drugs that overcome the aforementioned deficiencies.

SUMMARY

[0005] A variety of lipid-surfactant nanoparticles are provided that overcome one or more of the aforementioned deficiencies. In some aspects, the lipid-surfactant nanoparticles include (i) an aqueous core region; (ii) an outer lipid bilayer encapsulating the inner core region, wherein the lipid bilayer comprises an outer hydrophilic surface at an outer surface of the lipid-surfactant nanoparticle and an inner hydrophilic surface at the inner core region; and (iii) a nonionic surfactant having an HLB value of about 10 or higher and a CMC value of about 0.1 mM or less, wherein the lipid-surfactant nanoparticle has one or both of a nucleic acid in the aqueous core region and a lipophilic active agent in the outer lipid bilayer; and wherein the nonionic surfactant is associated with one or both of the outer hydrophilic surface and the inner hydrophilic surface. In some aspects, the nonionic surfactant is present in an amount from about 10 mole percent to about 50 mole percent based upon the total moles of lipid and surfactant in the lipid-surfactant nanoparticle.

[0006] The lipid-surfactant nanoparticles can be made with controllable size distributions. In some aspects, the lipid-surfactant nanoparticles have a diameter of about 30 nm to about 200 nm.

[0007] The lipid-surfactant nanoparticles include one or more lipids, for example a cationic lipid, a neutral lipid, a PEGylated lipid, cholesterol, a targeted conjugate thereof, or a combination thereof. In some aspects, the lipid-surfactant nanoparticles include a mixture of two, three, four, or more lipids in the outer lipid bilayer.

[0008] Suitable cationic lipids can include those selected from the group consisting of

N-[1-(2,3-dioleoyloxy)propyl]-N,N _l N-trimethyl ammonium salts (TAP lipids); dimethyldioctadecyl ammonium bromide (DDAB); 1 ,2-diacyloxy-3-trimethylammonium propane;

N-[1-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine (DODAP); 1 ,2-diacyloxy-3-dimethylammonium propane; N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);

1.2- dialkyloxy-3-dimethylammonium propane; dioctadecylamidoglycylspermine (DOGS);

3 -[N-(N',N'-dimethylamino-ethane)carbamoyl]cholesterol (DC-Choi);

2.3- dioleoyloxy-N-(2-(sperminecart30xamido)-ethyl)-N,N-dimethyl- 1-propanaminium

trifluoro-acetate (DOSPA); β-alanyl cholesterol; cetyl trimethyl ammonium bromide (CTAB); diCi ₄ -amidine; N-ferf-butyl-N'-tetradecyl-3-tetradecylamino-propionamidine;

N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG);

ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride;

1 ,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER);

N,N,N ^, ,N'-tetramethyl-N'-bis(2-hydroxylethyl)-2,3-dioleoylox y-1 ,4-butanediammonium iodide; 1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imid azolinium chloride derivatives;

2,3-dialkyloxypropyl quaternary ammonium compound derivatives containing a hydroxyalkyl moiety on the quaternary amine; and a combination thereof. [0009] Suitable cationic lipids can include those selected from the group consisting of a 1 ,2- dioleoyl-3-trimethylammonium-propane salt (DOTAP); a 1 ,2-dimyristoyl-3-trimethylammonium- propane salt (DMTAP); a 1 ,2-dipalmitoyl-3-trimethylammonium-propane salt (DPTAP); a 1,2- distearoyl-3-trimethylammonium-propane salt (DSTAP); dimethyldioctadecyl ammonium bromide (DDAB); 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1 ,2-Dioleyloxy-3- dimethylaminopropane (DODMA); a 1,2-Dioleyloxy-3-trimethylammonium propane salt (DOTMA);1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); and a combination thereof.

[0010] Suitable neutral lipids can include those selected from the group consisting of sterols, phospholipids, lysolipids, lysophospholipids, sphingolipids, and a combination thereof.

[0011] Suitable neutral lipids can include those selected from the group consisting of 1 ,2- dioleylphosphoethanolamine (DOPE), 1 ,2-dihexadecylphosphoethanolamine (DHPE), 1 ,2- distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), 1 ,2- dioleoylphosphatidylcholine (DOPC); 1 ,2-dipalmitoyl phosphatidylcholine (DPPC), 1 ,2- dimyristoylphosphatidylcholine (DMPC), dibehenoylphosphatidylcho- line (DBPC), ditricosanoylphosphatidylcholine (DTPC), and dilignoceroylphatidylcholine (DLPC).

[0012] In some aspects, the outer lipid bilayer includes a targeted conjugate of a lipid and a targeting moiety selected from the group consisting of peptides and polypeptides, antibody mimetics, nucleic acids, glycoproteins, small molecules, carbohydrate, and lipids. In some aspects, the targeted conjugate is a T-X-L conjugate, where T is a targeting moiety, X is a linker, and L is a lipid. For example. T can be a protein, peptide, or fragment thereof that binds to a cell- surface receptor. A suitable linker can include a C3-C12 polyethylene glycol linker. In some aspects, L is a lipid selected from the group consisting of 1 ,2-dioleylphosphoethanolamine (DOPE), 1 ,2-dihexadecylphosphoethanolamine (DHPE), 1 ,2-distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), 1 ,2- dioleoylphosphatidylcholine (DOPC); 1 ,2-dipalmitoyl phosphatidylcholine (DPPC), 1 ,2-dimyristoylphosphatidylcholine (DMPC), dibehenoylphosphatidylcho- line (DBPC), ditricosanoylphosphatidylcholine (DTPC), and dilignoceroylphatidylcholine (DLPC).

[0013] In some aspects, the targeting moiety is a T7 phage-display peptide comprising a sequence SEQ ID NO:1 (HAIYPRH). The targeting moiety can include any targeting moiety that selectively targets a cell surface marker. Suitable cell surface markers can include a breast cancer marker, a colon cancer marker, a rectal cancer marker, a lung cancer marker, a pancreatic cancer marker, an ovarian cancer marker, a bone cancer marker, a renal cancer marker, a liver cancer marker, a neurological cancer marker, a gastric cancer marker, a testicular cancer marker, a head and neck cancer marker, an esophageal cancer marker, or a cervical cancer marker.

[0014] The outer lipid bilayercan include a PEGylated lipid. Suitable PEGylated lipids can include those selected from the group consisting of, distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dimyristoyl phosphatidylethanolamine-poly ethylene glycol (DMPE-PEG), DPPE-PEG, DMG-PEG, DMA-PEG, DPA-PEG, DMG-PEG, dipalmitoylglycerosuccinate polyethylene glycol (DPGS-PEG), stearyl-polyethylene glycol, and cholesteryl-potyethylene glycol.

[0015] The lipid-surfactant nanoparticle includes a nonionic surfactant. Suitable nonionic surfactants can include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401 , stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. In some aspects, the nonionic surfactant is a fatty acid ester surfactant selected from the group consisting of sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, and polyoxyethylene stearates. In some aspects, the nonionic surfactant is a polyoxyethylene.

[0016] The lipid-surfactant nanoparticle can be used to deliver a variety of nucleic acids and/or lipophilic active agents with high loading efficiency. In some aspects, the nucleic acid and/or lipophilic active agent, individually or combined, have a loading efficiency in the nanoparticle of about 80% or more by weight. The nucleic acid can include those selected from the group consisting of a plasmid, a messenger RNA, an sgRNA (for CRISPR gene editing), an immuno stimulatory oligonucleotide, an siRNA, an antisense oligonucleotide, a microRNA, an antagomir, an aptamer, and a ribozyme. In some aspects, the nucleic acid is the antisense oligonucleotide G3139 (Genasense, oblimersen), an 18-mer antisense oligonucleotide targeting bcl-2. The lipophilic active agents can include those selected from the group consisting of lipophilic vitamins and derivatives thereof, antibiotics, antimicrobials, anti-inflammatory agents, hormones, adrenocortical steroids, non-steroidal anti-inflammatory agents, cancer therapeutics, and drugs acting on the CNS (Central Nervous System).

[0017] The mole% of the surfactant is an important parameter. At below 10 mole%, the surfactant is not very effective in promoting ON loading into LSNs or preventing LSN from aggregation at high ON concentration. At over 50 mole%, the LSNs become destabilized and unable to retain ONs. The higher the mole% of the surfactant (in the range of 10-50 mole%), the greater the colloidal stability of the LSNs at high ON concentration. In this range, the LSNs exhibit low cytotoxicity and hemolytic activity, therefore, can be safely administered as a therapeutic agent, either through injection or to the airway through instillation or nebulization into an aerosol.

[0018] The LSNs have excellent colloidal stability on the shelf. In vivo, the LSNs are able to gradually release the Tween 80 surfactants and generate highly active nanoparticles in vivo that can efficiently extravasate and facilitate efficient delivery at the cellular level. An example of LSN formulation is DOTAP/DODMA/DOPE/Tween80 (5:40:15:40 m/m).

[0019] DOTAP/DODMA/DOPE/Tween80 (5:40:15:40 m/m) is an excellent formulation of LSN for ON delivery based on ON loading and particle size characteristics

[0020] Addition of triethylammonium acetate (TEAA) introduces trimethylamine (TEA) counterion enhanced lipid-interaction of nucleic acids and facilitates LSN synthesis. TEA-containing buffer, such as TEAA, can also be used to improve classical LNP synthesis, leading to smaller particle size and greater ON loading efficiency. The acetate component of TEAA can serve as a buffering agent for pH between 4 and 5, which is useful when making LSNs containing tertiary amine cationic lipids, which have an ionizable headgroup.

[0021] ONs include antisense oligos, siRNA, miRNA mimics, antimiRs, CpG ODNs, aptamers, etc.

[0022] Although ON is used as an example, the LSNs can be used for other nucleic acids such as plasmid DNA, messenger RNA, IncRNA, pre-miR, dsRNA, sgRNA, etc.

[0023] LSNs can be synthesized in an ethanol-free protocol at high ON concentration with enhanced colloidal stability. LSNs are easily adapted for largescale production for clinical trials

[0024] Pharmaceutical formulations including the nanoparticles and a pharmaceutically acceptable carrier are also provided. Pharmaceutically acceptable carriers include diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.

[0025] Method of treating or preventing a disease or disorder in a subject or patient in need thereof are also provided. The methods can include administering a therapeutically effective amount of the nanoparticles in a pharmaceutical formulation to the subject. The administration can include one or more of parenteral administration, topical administration, enteral administration, and pulmonary administration. In some aspects, the subject has a cancer. . [0026] Other systems, methods, features, and advantages of lipid-surfactant nanoparticles and formulations thereof will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Further aspects of the present disclosure will be readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

[0028] FIG. 1 is a diagram of an exemplary lipid-surfactant nanoparticle loaded with nucleic acid.

[0029] FIG. 2 is a picture of an agarose gel-electrophoresis for oligonucleotide (ON) loaded DOTAP/DODMA/DOPE/Tween80 nanoparticles with a ratio of (DOTAP+DODMA+DOPE)/ON = 10 in 50 mM TEAA pH 4.5 to promote ON-lipid interaction. The mole percent of Tween 80 in the nanoparticles is (Lane 1) 0 mole%, (Lane 2) 5 mole%, (Lane 3) 10 mole%, (Lane 4) 20 mole%, (Lane 5) 50 mole%, (Lane 6) 60 mole%, and (Lane 7) 70 mole%. Lane 8 is the formulation with 70 mole% Tween 80 but without ON loading.

[0030] FIG. 3 is a picture of an agarose gel-electrophoresis for oligonucleotide (ON) loaded DOTAP/DODMA/DOPE/Tween80 nanoparticles with a weight ratio of DOTAP : DO DM A: DO P E of 15:25:40 in 50 mM TEAA pH 4.5 to promote ON-lipid interaction. Lane 1 is of the free ON. The mole percent of Tween 80 in the nanoparticles is (Lane 2) 10 mole%, (Lane 3) 20 mole%, (Lane 4) 35 mole%, (Lane 5) 50 mole%, (Lane 6) 60 mole%, (Lane 7) 70 mole %, and (Lane 8) 80 mole%.

[0031] FIG. 4 is a graph of the percent oligonucleotide loading (w/w) and the particle size for oligonucleotide (ON) loaded DOTAP/DODMA/DOPE/Tween80 (10:20:30:x m/m) where x is the mole% of Tween 80. The Y-axis shows the scale for both particle size (nm) and ON loading percentage. The x-axis is the mole percentage of tween 80. The data showed that the size and ODN loading into the nanoparticles can be controlled as a function of the mole% of Tween 80.

[0032] FIG. 5 is a picture of an agarose gel-electrophoresis for GFP mRNA loaded DOTAP/DOPE/Tween 80 nanoparticles from Example 3 (Lanes 1-2: free mRNA; Lanes 3-4 mRNA LSNs). DETAILED DESCRIPTION

[0033] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The skilled artisan will recognize many variants and adaptations of the embodiments described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0034] All publications and patents cited in this specification are cited to disclose and describe the methods and/or materials in connection with which the publications are cited. All such publications and patents are herein incorporated by references as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and/or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definition in the publications and patents cited that is not also expressly repeated in the instant specification should not be treated as such and should not be read as defining any terms appearing in the accompanying claims. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0035] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Functions or constructions well-known in the art may not be described in detail for brevity and/or clarity. Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of nanotechnology, organic chemistry, material science and engineering and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

[0036] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of "about 0.1 % to about 5% ^* should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase "x to y" includes the range from 'x' to 'y' as well as the range greater than 'x' and less than 'y'. The range can also be expressed as an upper limit, e.g. 'about x, y, z, or less' and should be interpreted to include the specific ranges of 'about x', 'about y', and 'about z' as well as the ranges of 'less than x', less than y', and 'less than z'. Likewise, the phrase 'about x, y, z, or greater ¹ should be interpreted to include the specific ranges of 'about x', 'about y', and 'about z' as well as the ranges of 'greater than x', greater than y', and 'greater than z'. In some embodiments, the term "about" can include traditional rounding according to significant figures of the numerical value. In addition, the phrase "about 'x' to 'y'", where 'x' and 'y' are numerical values, includes "about 'x' to about y.

Definitions

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0038] The articles "a" and "an," as used herein, mean one or more when applied to any feature in embodiments of the present invention described in the specification and claims. The use of "a" and "an" does not limit the meaning to a single feature unless such a limit is specifically stated. The article "the" preceding singular or plural nouns or noun phrases denotes a particular specified feature or particular specified features and may have a singular or plural connotation depending upon the context in which it is used.

[0039] The terms "subject" or "patient", as used herein, refer to any organism to which the particles may be administered, e.g., for experimental, therapeutic, diagnostic, and/or prophylactic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non- human primates, and humans) and/or plants. [0040] The terms "treating" or "preventing", as used herein, can include preventing a disease, disorder or condition from occurring in an animal which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0041] The term "therapeutic effect" is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human.

[0042] The term "modulation" is art-recognized and refers to up regulation (i.e., activation or stimulation), down regulation (i.e., inhibition or suppression) of a response, or the two in combination or apart.

[0043] "Parenteral administration", as used herein, means administration by any method other than through the digestive tract or non-invasive topical or regional routes. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraperitoneally, intrapleural^, intratracheally, intramuscularly, subcutaneously, subjunctivally, by injection, and by infusion.

[0044] "Topical administration", as used herein, means the non-invasive administration to the skin, orifices, or mucosa. Topical administrations can be administered locally, i.e., they are capable of providing a local effect in the region of application without systemic exposure. Topical formulations can provide systemic effect via adsorption into the blood stream of the individual. Topical administration can include, but is not limited to, cutaneous and transdermal administration, buccal administration, intranasal administration, intravaginal administration, intravesical administration, ophthalmic administration, and rectal administration.

[0045] "Enteral administration", as used herein, means administration via absorption through the gastrointestinal tract. Enteral administration can include oral and sublingual administration, gastric administration, or rectal administration. [0046] "Pulmonary administration", as used herein, means administration into the lungs by inhalation or endotracheal administration. As used herein, the term "inhalation" refers to intake of air to the alveoli. The intake of air can occur through the mouth or nose.

[0047] The terms "sufficient" and "effective", as used interchangeably herein, refer to an amount (e.g., mass, volume, dosage, concentration, and/or time period) needed to achieve one or more desired result(s). A "therapeutically effective amount" is at least the minimum concentration required to effect a measurable improvement or prevention of any symptom or a particular condition or disorder, to effect a measurable enhancement of life expectancy, or to generally improve patient quality of life. The therapeutically effective amount is thus dependent upon the specific biologically active molecule and the specific condition or disorder to be treated. Therapeutically effective amounts of many active agents, such as antibodies, are well known in the art. The therapeutically effective amounts of anionic proteins, protein analogues, or nucleic acids hereinafter discovered or for treating specific disorders with known proteins, protein analogues, or nucleic acids to treat additional disorders may be determined by standard techniques which are well within the craft of a skilled artisan, such as a physician.

[0048] The terms "bioactive agent" and "active agent", as used interchangeably herein, include, without limitation, physiologically or pharmacologically active substances that act locally or systemically in the body. A bioactive agent is a substance used for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), diagnosis (e.g., diagnostic agent), cure or mitigation of disease or illness, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.

[0049] The term "prodrug" refers to an agent, including a nucleic acid or proteins that is converted into a biologically active form in vitro and/or in vivo. Prodrugs can be useful because, in some situations, they may be easier to administer than the parent compound. For example, a prodrug may be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have improved solubility in pharmaceutical compositions compared to the parent drug. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Harper, N.J. (1962) Drug Latentiation in Jucker, ed. Progress in Drug Research, 4:221-294; Morozowich et al. (1977) Application of Physical Organic Principles to Prodrug Design in E. B. Roche ed. Design of Biopharmaceutical Properties through Prodrugs and Analogs, APhA; Acad. Pharm. Sci.; E. B. Roche, ed. (1977) Bioreversible Carriers in Drug in Drug Design, Theory and Application, APhA; H. Bundgaard, ed. (1985) Design of Prodrugs, Elsevier; Wang et al. (1999) Prodrug approaches to the improved delivery of peptide drug, Curr. Pharm. Design. 5(4):265-287; Pauletti et al. (1997) Improvement in peptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al. (1998). The Use of Esters as Prodrugs for Oral Delivery of β-Lactam antibiotics, Pharm. Biotech. 11 :345-365; Gaignault et al. (1996) Designing Prodrugs and Bioprecursors I. Carrier Prodrugs, Pract. Med. Chem. 671-696; M. Asghamejad (2000). Improving Oral Drug Transport Via Prodrugs, in G. L. Amidon, P. I. Lee and E. M. Topp, Eds., Transport Processes in Pharmaceutical Systems, Marcell Dekker, p. 185-218; Balant et al. (1990) Prodrugs for the improvement of drug absorption via different routes of administration, Eur. J. Drug Metab. Pharmacokinet., 15(2): 143-53; Balimane and Sinko (1999). Involvement of multiple transporters in the oral absorption of nucleoside analogues, Adv. Drug Delivery Rev., 39(1 -3): 183-209; Browne (1997). Fosphenytoin (Cerebyx), Clin. Neuropharmacol. 20(1): 1-12; Bundgaard (1979). Bioreversible derivatization of drugs-principle and applicability to improve the therapeutic effects of drugs, Arch. Pharm. Chemi. 86(1): 1-39; H. Bundgaard, ed. (1985) Design of Prodrugs, New York: Elsevier; Fleisher et al. (1996) Improved oral drug delivery: solubility limitations overcome by the use of prodrugs, Adv. Drug Delivery Rev. 19(2): 115-130; Fleisher et al. (1985) Design of prodrugs for improved gastrointestinal absorption by intestinal enzyme targeting, Methods Enzymol. 112: 360-81 ; Farquhar D, et al. (1983) Biologically Reversible Phosphate-Protective Groups, J. Pharm. Sci., 72(3): 324-325; Han, H.K. et al. (2000) Targeted prodrug design to optimize drug delivery, AAPS PharmSci., 2(1): E6; Sadzuka Y. (2000) Effective prodrug liposome and conversion to active metabolite, Curr. Drug Metab., 1 (1):31-48; D.M. Lambert (2000) Rationale and applications of lipids as prodrug carriers, Eur. J. Pharm. Sci., 11 Suppl. 2:S15-27; Wang, W. et al. (1999) Prodrug approaches to the improved delivery of peptide drugs. Curr. Pharm. Des., 5(4):265-87.

[0050] The term "biocompatible", as used herein, refers to a material that along with any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause any significant adverse effects to the recipient. Generally speaking, biocompatible materials are materials which do not elicit a significant inflammatory or immune response when administered to a patient.

[0051] The term "biodegradable" as used herein, generally refers to a material that will degrade or erode under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the subject. The degradation time is a function of composition and morphology. Degradation times can be from hours to weeks.

[0052] The term "pharmaceutically acceptable", as used herein, refers to compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio, in accordance with the guidelines of agencies such as the U.S. Food and Drug Administration. A "pharmaceutically acceptable carrier", as used herein, refers to all components of a pharmaceutical formulation that facilitate the delivery of the composition in vivo. Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.

[0053] The term "molecular weight", as used herein, generally refers to the mass or average mass of a material. If a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in various ways including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (M _w ) as opposed to the number-average molecular weight (M _n ). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.

[0054] The term "small molecule", as used herein, generally refers to an organic molecule that is less than 2000 g/mol in molecular weight, less than 1500 g/mol, less than 1000 g/mol, less than 800 g/mol, or less than 500 g/mol. Small molecules are non-polymeric and/or non-oligomeric.

[0055] The term "hydrophilic", as used herein, refers to substances that have strongly polar groups that readily interact with water.

[0056] The term "hydrophobic", as used herein, refers to substances that lack an affinity for water; tending to repel and not absorb water as well as not dissolve in or mix with water.

[0057] The term "lipophilic", as used herein, refers to compounds having an affinity for lipids.

[0058] The term "amphiphilic", as used herein, refers to a molecule combining hydrophilic and lipophilic (hydrophobic) properties. "Amphiphilic material" as used herein refers to a material containing a hydrophobic or more hydrophobic oligomer or polymer (e.g., biodegradable oligomer or polymer) and a hydrophilic or more hydrophilic oligomer or polymer.

[0059] The term "targeting moiety", as used herein, refers to a moiety that binds to or localizes to a specific locale. The moiety may be, for example, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule. The locale may be a tissue, a particular cell type, or a subcellular compartment. In some embodiments, a targeting moiety can specifically bind to a selected molecule.

[0060] The term "mean particle size", as used herein, generally refers to the statistical mean particle size (diameter) of the particles in the composition. The diameter of an essentially spherical particle may be referred to as the physical or hydrodynamic diameter. The diameter of a non- spherical particle may refer preferentially to the hydrodynamic diameter. As used herein, the diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art, such as dynamic light scattering. Two populations can be said to have a "substantially equivalent mean particle size" when the statistical mean particle size of the first population of nanoparticles is within 20% of the statistical mean particle size of the second population of nanoparticles; more preferably within 15%, most preferably within 10%.

[0061] The terms "monodisperse" and "homogeneous size distribution", as used interchangeably herein, describe a population of particles, microparticles, or nanoparticles all having the same or nearly the same size. As used herein, a monodisperse distribution refers to particle distributions in which 90% of the distribution lies within 5% of the mean particle size.

[0062] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably to refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. These terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base (e.g., pseudouridine), sugar (e.g., 2'-0-methyl, 2'-F, LNA), and/or phosphate moieties (e.g., phosphorothioate or methylphosphonate backbones). In general and unless otherwise specified, an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T or U. The term "nucleic acid" is a term of art that refers to a string of at least two base- sugar-phosphate monomeric units. Nucleotides are the monomeric units of nucleic acid polymers. The term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the form of a messenger RNA, antisense oligo, plasmid DNA, parts of a plasmid DNA or genetic material derived from a virus. Antisense is a polynucleotide that interferes with the function of DNA and/or RNA. The term nucleic acids refers to a string of at least two base-sugar-phosphate combinations. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, but contain the same bases. The term also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids.

[0063] A "functional fragment" of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains at least one function as the full-length protein, polypeptide or nucleic acid. A functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well-known in the art. Similarly, methods for determining protein function are well-known. For example, the DNA binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, e.g., genetic or biochemical. See, for example, Fields et al. (1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and PCT WO 98/44350.

[0064] The term "pharmaceutically acceptable counter ion" refers to a pharmaceutically acceptable anion or cation. In various embodiments, the pharmaceutically acceptable counter ion is a pharmaceutically acceptable ion. For example, the pharmaceutically acceptable counter ion is selected from citrate, malate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1 ,1'-methylene-bis-(2- hydroxy-3-naphthoate)). In some embodiments, the pharmaceutically acceptable counter ion is selected from chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, citrate, malate, acetate, oxalate, acetate, and lactate. In particular embodiments, the pharmaceutically acceptable counter ion is selected from chloride, bromide, iodide, nitrate, sulfate, bisulfate, and phosphate. [0065] The term 'pharmaceutically acceptable salt(s) ^* refers to salts of acidic or basic groups that may be present in compounds used in the present compositions. Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfate, citrate, matate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1 ,1'-methylene-bis-(2- hydroxy-3-naphthoate)) salts. Compounds included in the present compositions that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds included in the present compositions, that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.

[0066] If the compounds described herein are obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that may be used to prepare non-toxic pharmaceutically acceptable addition salts.

[0067] A pharmaceutically acceptable salt can be derived from an acid selected from 1-hydroxy-

2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4- acetamidobenzoic acid, 4-aminosalicylicacid, acetic acid, adipic acid, ascorbic acid, asparticacid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid

(decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1 ,2-disulfonic acid, ethanesulfonic acid, formic acid, fu marie acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, iseth ionic, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic, naphthalene- 1 ,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic, phosphoric acid, proprionic acid, pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, toluenesulfonic acid, trifluoroacetic, and undecylenic acid.

[0068] The term "bioavailable" is art-recognized and refers to a form of the subject invention that allows for it, or a portion of the amount administered, to be absorbed by, incorporated to, or otherwise physiologically available to a subject or patient to whom it is administered.

[0069] "Parenteral administration", as used herein, means administration by any method other than through the digestive tract or non-invasive topical or regional routes. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraperitoneally, intrapleurally, intratracheally, intramuscularly, subcutaneously, subjunctivally, by injection, and by infusion.

[0070] "Topical administration", as used herein, means the non-invasive administration to the skin, orifices, or mucosa. Topical administrations can be administered locally, i.e. they are capable of providing a local effect in the region of application without systemic exposure. Topical formulations can provide systemic effect via adsorption into the blood stream of the individual. Topical administration can include, but is not limited to, cutaneous and transdermal administration, buccal administration, intranasal administration, intravaginal administration, intravesical administration, ophthalmic administration, and rectal administration.

[0071] "Enteral administration", as used herein, means administration via absorption through the gastrointestinal tract. Enteral administration can include oral and sublingual administration, gastric administration, or rectal administration.

[0072] "Pulmonary administration", as used herein, means administration into the lungs by inhalation or endotracheal administration. As used herein, the term "inhalation" refers to intake of air to the alveoli. The intake of air can occur through the mouth or nose.

Lipid-Surfactant Nanoparticles for Oligonucleotide Delivery

[0073] Lipid-surfactant nanoparticles are provided that overcome many of the aforementioned deficiencies in existing lipid particle delivery vehicles. The lipid-surfactant nanoparticles described herein can, in many aspects, be made in higher concentrations that in previous methods. Additionally, the lipid-surfactant nanoparticles described herein can, in many aspects, be made without the use of ethanol or other volatile alcohol solvents and/or can be made with average diameters less than 200 nm. In many aspects, the lipid-surfactant nanoparticles are stable and can be loaded with one or both of nucleic acids and lipophilic active agents for delivery to a subject in need thereof.

[0074] In various aspects, the lipid-surfactant nanoparticles have an inner core region and an outer lipid bilayer encapsulation the inner core region. The lipid bilayer has an outer hydrophilic surface at an outer surface of the lipid-surfactant nanoparticles and an inner hydrophilic surface at the inner core region. A nonionic surfactant can be associated with one or both of the outer hydrophilic surface and the inner hydrophilic surface. The nonionic surfactant can be present in an effective amount such that the outer lipid bilayer is sufficiently permeable to achieve the high loading levels of the nucleic acid while not completely disrupting the lipid bilayer to prevent nanoparticle formation and nucleic acid loading. Lipophilic active agents can partition into the outer lipid bilayer for delivering various lipophilic active agent. In some aspects, the lipid-surfactant nanoparticles can include both a nucleic acid in the aqueous core and a lipophilic active agent in the outer lipid bilayer.

[0075] The lipid-surfactant nanoparticles described herein can be loaded with a variety of nucleic acids. The nucleic acid can be a therapeutic, prophylactic, or diagnostic agent. In some aspects, the nucleic acid has a loading efficiency in the nanoparticle of about 70%, about 80%, about 85%, or more by weight. The nucleic acid can include those selected from the group consisting of a plasmid, a messenger RNA, an sgRNA, an immuno stimulatory oligonucleotide, an siRNA, an antisense oligonucleotide, a microRNA, an antagomir, an aptamer, and a ribozyme. In some aspects, the nucleic acid is the antisense oligonucleotide G3139 (Genasense, oblimersen), an 18-mer antisense oligonucleotide targeting bcl-2.

[0076] The lipid-surfactant nanoparticles can be loaded with a variety of lipophilic drugs or active agents. The lipophilic active agents can include those selected from the group consisting of lipophilic vitamins and derivatives thereof, antibiotics, antimicrobials, anti-inflammatory agents, hormones, adrenocortical steroids, non-steroidal anti-inflammatory agents, cancer therapeutics, and drugs acting on the CNS (Central Nervous System). Lipophilic vitamins and vitamin derivatives can include Vitamin A, Vitamin E, and derivatives such as retinoic acid. Lipophilic antibiotics can include avermectin, ivermectin, spiramycin, ceftiofur, and derivatives thereof. Examples of lipophilic antimicrobials can include as amoxicillin, erythromycin, oxytetracycline, lincomycin, and derivatives thereof. Examples of lipophilic anti-inflammatory agents can include dexamethasone, phenylbutazone, and derivatives thereof. Examples of lipophilic hormones can include thyroxine and derivatives thereof. Examples of lipophilic adrenocortical steroids can include examethasone palmitate, triamcinolone acetonide acetate and prednisone halide pine. Examples of lipophilic non-steroidal anti-inflammatory agents can include indomethacin, aspirin, and examples thereof. Examples of lipophilic cancer therapeutics can include daunorubicin, actinomycin, and derivatives thereof. Examples of lipophilic drugs acting on the CNS (Central Nervous System) can include benzodiazepines, barbiturates, and derivatives thereof.

[0077] The lipid-surfactant nanoparticle includes a nonionic surfactant. Suitable nonionic surfactants can include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401 , stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. In some aspects, the nonionic surfactant is a fatty acid ester surfactant selected from the group consisting of sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, and polyoxyethylene stearates. In some aspects, the nonionic surfactant is a polyoxyethylene.

[0078] The mole% of the surfactant is an important parameter. At below 10 mole%, the surfactant is not very effective in promoting ON loading into LSNs or preventing LSN from aggregation at high ON concentration. At over 50 mole%, the LSNs become destabilized and unable to retain ONs. The higher the mole% of the surfactant (in the range of 10-50 mole%), the greater the colloidal stability of the LSNs at high ON concentration. In this range, the LSNs exhibit low cytotoxicity and hemolytic activity, therefore, can be safely administered as a therapeutic agent, either through injection or to the airway through instillation or nebulization into an aerosol. In some aspects, the nonionic surfactant is present in an amount from about 10 mole percent to about 50 mole percent, about 20 mole percent to about 50 mole percent, or about 30 more percent to about 50 mole percent based upon the total moles of lipid and surfactant in the lipid-surfactant nanoparticle.

[0079] The lipid-surfactant nanoparticles can be made with controllable size distributions. In some aspects, the lipid-surfactant nanoparticles have a diameter of about 30 nm to about 200 nm. The size of the particles can be adjusted for the intended application. The particles can be nanoparticles or microparticles, although nanoparticles are preferred. The particle can have a diameter of about 10 nm to about 10 microns, about 10 nm to about 1 micron, about 10 nm to about 500 nm, about 20 nm to about 500 nm, or about 25 nm to about 250 nm. In preferred embodiments the particle is a nanoparticle having a diameter from about 25 nm to about 250 nm. In various embodiments, a particle may be a nanoparticle, i.e., the particle has a characteristic dimension of less than about 1 micrometer, where the characteristic dimension of a particle is the diameter of a perfect sphere having the same volume as the particle. The plurality of particles can be characterized by an average diameter (e.g., the average diameter for the plurality of particles). In some embodiments, the diameter of the particles may have a Gaussian-type distribution. In some embodiments, the plurality of particles have an average diameter of less than about 300 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 50 nm, less than about 30 nm, less than about 10 nm, less than about 3 nm, or less than about 1 nm. In some embodiments, the particles have an average diameter of at least about 5 nm, at least about 10 nm, at least about 30 nm, at least about 50 nm, at least about 100 nm, at least about 150 nm, or greater. In certain embodiments, the plurality of the particles have an average diameter of about 10 nm, about 25 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 500 nm, or the like. In some embodiments, the plurality of particles have an average diameter between about 10 nm and about 500 nm, between about 50 nm and about 400 nm, between about 100 nm and about 300 nm, between about 150 nm and about 250 nm, between about 175 nm and about 225 nm, or the like. In some embodiments, the plurality of particles have an average diameter between about 10 nm and about 500 nm, between about 20 nm and about 400 nm, between about 30 nm and about 300 nm, between about 40 nm and about 200 nm, between about 50 nm and about 175 nm, between about 60 nm and about 150 nm, between about 70 nm and about 130 nm, or the like. For example, the average diameter can be between about 70 nm and 130 nm. In some embodiments, the plurality of particles have an average diameter between about 20 nm and about 220 nm, between about 30 nm and about 200 nm, between about 40 nm and about 180 nm, between about 50 nm and about 170 nm, between about 60 nm and about 150 nm, or between about 70 nm and about 130 nm. In one embodiment, the particles have a size of 40 to 120 nm with a zeta potential close to 0 mV at low to zero ionic strengths (1 to 10 mM), with zeta potential values between + 5 to - 5 mV, and a zero/neutral or a small surface charge.

[0080] The lipid-surfactant nanoparticles typically have an aqueous center. The aqueous center can contain water or a mixture of water and alcohol. Suitable alcohols include, but are not limited to, methanol, ethanol, propanol, (such as isopropanol), butanol (such as n-butanol, isobutanol, sec-butanol, terf-butanol, pentanol (such as amyl alcohol, isobutyl carbinol), hexanol (such as 1- hexanol, 2-hexanol, 3-hexanol), heptanol (such as 1-heptanol, 2-heptanol, 3-heptanol and 4- heptanol) or octanol (such as 1-octanol) or a combination thereof. One particular advantage is that, at least in some aspects, the lipid-surfactant nanoparticles can be made without ethanol.

[0081] The lipid-surfactant nanoparticles include one or more lipids, for example a cationic lipid, a neutral lipid, a PEGylated lipid, cholesterol, a targeted conjugate thereof, or a combination thereof. In some aspects, the lipid-surfactant nanoparticles include a mixture of two, three, four, or more lipids in the outer lipid bilayer.

[0082] Suitable cationic lipids can include those selected from the group consisting of

N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts (DOTMA); dimethyldioctadecyl ammonium bromide (DDAB); 1 ,2-diacyloxy-3-trimethylammonium propane;

N-[1-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine (DODAP); 1 ,2-diacyloxy-3-dimethylammonium propane; N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);

1.2- dialkyloxy-3-dimethylammonium propane; dioctadecylamidoglycylspermine (DOGS);

3 -[N-(N',N'-dimethylamino-ethane)carbamoyl]cholesterol (DC-Choi);

2.3- dioleoyloxy-N-(2-(sperminecart)oxamido)-ethyl)-N,N-dimethyl- 1-propanaminium

trifluoro-acetate (DOSPA); β-alanyl cholesterol; cetyl trimethyl ammonium bromide (CTAB); diCi4-amidine; N-ferf-butyl-N'-tetradecyl-3-tetradecylamino-propionamidine;

N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG);

ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride;

1 ,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER);

N,N,N',N ^, -tetramethyl-N'-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1 ,4-butanediammonium iodide; 1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imid azolinium chloride derivatives;

2,3-dialkyloxypropyl quaternary ammonium compound derivatives containing a hydroxyalkyl moiety on the quaternary amine; and a combination thereof.

[0083] Suitable cationic lipids can include those selected from the group consisting of a 1 ,2- dioleoyl-3-trimethylammonium-propane salt (DOTAP); a 1 ,2-dimyristoyl-3-trimethylammonium- propane salt (DMTAP); a 1 ,2-dipalmitoyl-3-trimethylammonium-propane salt (DPTAP); a 1 ,2- distearoyl-3-trimethylammonium-propane salt (DSTAP); dimethyldioctadecyl ammonium bromide (DDAB); 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1 ,2-Dioleyloxy-3- dimethylaminopropane (DODMA); a 1 ,2-Dioleyloxy-3-trimethylammonium propane salt (DOTMA); 1 ,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); and a combination thereof. [0084] Suitable neutral lipids can include those selected from the group consisting of sterols, phospholipids, lysolipids, lysophospholipids, sphingolipids, and a combination thereof.

[0085] Suitable neutral lipids can include those selected from the group consisting of 1 ,2- dioleylphosphoethanolamine (DOPE), 1 ,2-dihexadecylphosphoethanolamine (DHPE), 1 ,2- distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), 1 ,2- dioleoylphosphatidylcholine (DOPC); 1 ,2-dipalmitoyl phosphatidylcholine (DPPC), 1 ,2- dimyristoylphosphatidylcholine (DMPC), dibehenoylphosphatidylcho- line (DBPC), ditricosanoylphosphatidylcholine (DTPC), and dilignoceroylphatidylcholine (DLPC).

[0086] In some aspects, the outer lipid bilayer includes a targeted conjugate of a lipid and a targeting moiety selected from the group consisting of peptides and polypeptides, antibody mimetics, nucleic acids, glycoproteins, small molecules, carbohydrate, and lipids. In some aspects, the targeted conjugate is a T-X-L conjugate, where T is a targeting moiety, X is a linker, and L is a lipid. For example. T can be a protein, peptide, or fragment thereof that binds to a cell- surface receptor. A suitable linker can include a C3-C12 polyethylene glycol linker. In some aspects, L is a lipid selected from the group consisting of 1 ,2-dioleylphosphoethanolamine (DOPE), 1 ,2-dihexadecylphosphoethanolamine (DHPE), 1 ,2-distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), 1 ,2- dioleoylphosphatidylcholine (DOPC); 1 ,2-dipalmitoyl phosphatidylcholine (DPPC), 1 ,2-dimyristoylphosphatidylcholine (DMPC), dibehenoylphosphatidylcho- line (DBPC), ditricosanoylphosphatidylcholine (DTPC), and dilignoceroylphatidylcholine (DLPC).

[0087] In some aspects, the targeting moiety is a T7 phage-display peptide comprising a sequence SEQ ID NO:1 (HAIYPRH). The targeting moiety can include any targeting moiety that selectively targets a cell surface marker. Suitable cell surface markers can include a breast cancer marker, a colon cancer marker, a rectal cancer marker, a lung cancer marker, a pancreatic cancer marker, a ovarian cancer marker, a bone cancer marker, a renal cancer marker, a liver cancer marker, a neurological cancer marker, a gastric cancer marker, a testicular cancer marker, a head and neck cancer marker, an esophageal cancer marker, or a cervical cancer marker.

[0088] The outer lipid bilayer can include a PEGylated lipid. Suitable PEGylated lipids can include those selected from the group consisting of, distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dimyristoyl phosphatidylethanolamine-poly ethylene glycol (DMPE-PEG), dipalmitoylglycerosuccinate polyethylene glycol (DPGS-PEG), stearyl-polyethylene glycol, and cholesteryl-polyethylene glycol. Methods of Making the Lipid-Surfactant Nanoparticles

[0089] In various aspects, a method of making the lipid-surfactant nanoparticle includes providing a nucleic acid, one or more lipids, and a suitable nonionic surfactant for forming a particle and recovering particles. The methods can include combining the nucleic acid in an aqueous phase and the lipids and nonionic surfactants in an organic phase, combining the aqueous phase and the organic phase to form a second phase; emulsifying the second phase to form an emulsion phase; and recovering particles. In various embodiments, the emulsion phase is further homogenized.

[0090] Emulsifying the second phase to form an emulsion phase may be performed in one or two emulsification steps. For example, a primary emulsion may be prepared, and then emulsified to form a fine emulsion. The primary emulsion can be formed, for example, using simple mixing, a high pressure homogenizer, probe sonicator, stir bar, or a rotor stator homogenizer. The primary emulsion may be formed into a fine emulsion through the use of e.g. a probe sonicator or a high pressure homogenizer, e.g. by pass(es) through a homogenizer. For example, when a high pressure homogenizer is used, the pressure used may be about 4000 to about 8000 psi, or about 4000 to about 5000 psi.

[0091] Either solvent evaporation or dilution may be needed to complete the extraction of the solvent and solidify the particles. For better control over the kinetics of extraction and a more scalable process, a solvent dilution via aqueous quench may be used. For example, the emulsion can be diluted into cold water to a concentration sufficient to dissolve all of the organic solvent to form a quenched phase. Quenching may be performed at least partially at a temperature of about 5 °C or less. For example, water used in the quenching may be at a temperature that is less that room temperature (e.g. about 0 to about 10 °C, or about 0 to about 5 °C).

[0092] In various embodiments, the particles are recovered by filtration. For example, ultrafiltration membranes can be used. Exemplary filtration may be performed using a tangential flow filtration system. For example, by using a membrane with a pore size suitable to retain nanoparticles while allowing solutes, micelles, and organic solvent to pass, nanoparticles can be selectively separated. Exemplary membranes with molecular weight cut-offs of about 300-500 kDa (-5-25 nm) may be used.

[0093] In various embodiments, the particles are freeze-dried or lyophilized, in some instances, to extend their shelf life. In some embodiments, the composition also includes a lyoprotectant. In certain embodiments, a lyoprotectant is selected from a sugar, a polyalcohol, or a derivative thereof. In particular embodiments, a lyoprotectant is selected from a monosaccharide, a disaccharide, or a mixture thereof. For example, a lyoprotectant can be sucrose, lactulose, trehalose, lactose, glucose, maltose, mannitol, cellobiose, or a mixture thereof.

.Emulsion methods

[0094] In some embodiments, a nanoparticle is prepared using an emulsion solvent evaporation method. For example, the lipids and surfactants are dissolved in a water immiscible organic solvent and mixed with a solution of the nucleic acid or a combination of drug solutions. In some embodiments a solution of a therapeutic, prophylactic, or diagnostic agent to be encapsulated is mixed with the lipid/surfactant solution. The water immiscible organic solvent, can be, but is not limited to, one or more of the following: chloroform, dichloromethane, and acyl acetate. An aqueous solution is added into the resulting polymer solution to yield emulsion solution by emulsification. The emulsification technique can be, but not limited to, probe sonication or homogenization through a homogenizer.

Ultrasonication/hiah speed homoaenization methods

[0095] Lipid particles can be prepared by ultrasonication/high speed homogenization. The combination of both ultrasonication and high speed homogenization is particularly useful for the production of smaller lipid particles. The particles are formed in the size range from 10 nm to 200 nm, preferably 50 nm to 100 nm, by this process.

Solvent evaporation methods

[0096] Lipid particles can be prepared by solvent evaporation approaches. The lipophilic material is dissolved in a water-immiscible organic solvent (e.g. cyclohexane) that is emulsified in an aqueous phase. Upon evaporation of the solvent, nanoparticles dispersion is formed by precipitation of the lipid in the aqueous medium. Parameters such as temperature, pressure, choices of solvents can be used to control particle size and distribution. Solvent evaporation rate can be adjusted through increased/reduced pressure or increased/reduced temperature.

Solvent emulsification-diffusion methods

[0097] Lipid particles can be prepared by solvent emulsification-diffusion methods. The lipid is first dissolved in an organic phase, such as ethanol and acetone. An acidic aqueous phase is used to adjust the zeta potential to induce lipid coacervation. The continuous flow mode allows the continuous diffusion of water and alcohol, reducing lipid solubility, which causes thermodynamic instability and generates lipid particles. Pharmaceutical formulations

[0098] The formulations described herein contain an effective amount of nanoparticles in a pharmaceutical carrier appropriate for administration to an individual in need thereof The formulations can be administered parenterally (e.g., by injection or infusion), topically (e.g., to the eye), or via pulmonary administration.

Parenteral Formulations

[0099] The nanoparticles can be formulated for parenteral delivery, such as injection or infusion, in the form of a solution or suspension. The formulation can be administered via any route, such as, the blood stream or directly to the organ or tissue to be treated.

[0100] Parenteral formulations can be prepared as aqueous compositions using techniques is known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsions.

[0101] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, com oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

[0102] Solutions and dispersions of the nanoparticles can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, and combination thereof.

[0103] Suitable surfactants may be anionic, cation ic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethytthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-B-alanine, sodium N-lauryl-B-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0104] The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s) or nanoparticles.

[0105] The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0106] Water soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

[0107] Sterile injectable solutions can be prepared by incorporating the nanoparticles in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized nanoparticles into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the nanoparticle plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.

[0108] Pharmaceutical formulations for parenteral administration are preferably in the form of a sterile aqueous solution or suspension of lipid-surfactant nanoparticles. Acceptable solvents include, for example, water, Ringer's solution, phosphate buffered saline (PBS), and isotonic sodium chloride solution. The formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1 ,3-butanediol.

[0109] In some instances, the formulation is distributed or packaged in a liquid form. Alternatively, formulations for parenteral administration can be packed as a solid, obtained, for example by lyophilization of a suitable liquid formulation. The solid can be reconstituted with an appropriate carrier or diluent prior to administration.

[0110] Solutions, suspensions, or emulsions for parenteral administration may be buffered with an effective amount of buffer necessary to maintain a pH suitable for ocular administration. Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers.

[0111] Solutions, suspensions, or emulsions for parenteral administration may also contain one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art and some examples include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes.

[0112] Solutions, suspensions, or emulsions for parenteral administration may also contain one or more preservatives to prevent bacterial contamination of the ophthalmic preparations. Suitable preservatives are known in the art, and include polyhexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwise known as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixtures thereof.

[0113] Solutions, suspensions, or emulsions for parenteral administration may also contain one or more excipients known art, such as dispersing agents, wetting agents, and suspending agents.

Topical Formulations

[0114] The nanoparticles can be formulated for topical administration. Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, liquids, and transdermal patches. The formulation may be formulated for transmucosal, transepithelial, transendothelial, or transdermal administration. The compositions contain one or more chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, and combination thereof.

[0115] In some embodiments, the nanoparticles can be administered as a liquid formulation, such as a solution or suspension, a semi-solid formulation, such as a lotion or ointment, or a solid formulation. In some embodiments, the nanoparticles are formulated as liquids, including solutions and suspensions, such as eye drops or as a semi-solid formulation, such as ointment or lotion for topical application to the skin, to the mucosa, such as the eye or vaginally or rectally.

[0116] The formulation may contain one or more excipients, such as emollients, surfactants, emulsifiers, penetration enhancers, and the like.

[0117] "Emollients" are an externally applied agent that softens or soothes skin and are generally known in the art and listed in compendia, such as the "Handbook of Pharmaceutical Excipients', 4 ^th Ed., Pharmaceutical Press, 2003. These include, without limitation, almond oil, castor oil, ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycol palmitostearate, glycerin, glycerin monostearate, glyceryl monooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium- chain triglycerides, mineral oil and lanolin alcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol and combinations thereof. In one embodiment, the emollients are ethylhexylstearate and ethylhexyl palmitate.

[0118] "Surfactants" are surface-active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading and wetting properties of a product. Suitable non- ionic surfactants include emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof. In one embodiment, the non-ionic surfactant is stearyl alcohol.

[0119] "Emulsifiers" are surface active substances which promote the suspension of one liquid in another and promote the formation of a stable mixture, or emulsion, of oil and water. Common emulsifiers are: metallic soaps, certain animal and vegetable oils, and various polar compounds.

Suitable emulsifiers include acacia, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, self-emulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gum and combinations thereof. In one embodiment, the emulsifier is glycerol stearate.

[0120] Suitable classes of penetration enhancers are known in the art and include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithins, cholate salts, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses, and diimides), macrocyclics, such as macrocylic lactones, ketones, and anhydrides and cyclic ureas, surfactants, N-methyl pyrrolidones and derivatives thereof, DMSO and related compounds, ionic compounds, azone and related compounds, and solvents, such as alcohols, ketones, amides, polyols (e.g., glycols). Examples of these classes are known in the art.

[0121] An "oil" is a composition containing at least 95% wt of a lipophilic substance. Examples of lipophilic substances include but are not limited to naturally occurring and synthetic oils, fats, fatty acids, lecithins, triglycerides and combinations thereof.

[0122] An "emulsion" is a composition containing a mixture of non-miscible components homogenously blended together. In particular embodiments, the non-miscible components include a lipophilic component and an aqueous component. An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase, it is known as a water-in-oil emulsion. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non- ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile nonaqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers. [0123] An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase, it is known as a water-in-oil emulsion. The oil phase may consist at least in part of a propellant, such as an HFA propellant. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers.

[0124] A "lotion" is a low- to medium-viscosity liquid formulation. A lotion can contain finely powdered substances that are in soluble in the dispersion medium through the use of suspending agents and dispersing agents. Alternatively, lotions can have as the dispersed phase liquid substances that are immiscible with the vehicle and are usually dispersed by means of emulsifying agents or other suitable stabilizers. In one embodiment, the lotion is in the form of an emulsion having a viscosity of between 100 and 1000 centistokes. The fluidity of lotions permits rapid and uniform application over a wide surface area. Lotions are typically intended to dry on the skin leaving a thin coat of their medicinal components on the skin's surface.

[0125] A "cream" is a viscous liquid or semi-solid emulsion of either the "oil-in-water" or "water- in-oil type". Creams may contain emulsifying agents and/or other stabilizing agents. In one embodiment, the formulation is in the form of a cream having a viscosity of greater than 1000 centistokes, typically in the range of 20,000-50,000 centistokes. Creams are often time preferred over ointments as they are generally easier to spread and easier to remove.

[0126] The difference between a cream and a lotion is the viscosity, which is dependent on the amount/use of various oils and the percentage of water used to prepare the formulations. Creams are typically thicker than lotions, may have various uses and often one uses more varied oils/butters, depending upon the desired effect upon the skin. In a cream formulation, the water- base percentage is about 60-75 % and the oil-base is about 20-30 % of the total, with the other percentages being the emulsifier agent, preservatives and additives for a total of 100 %. [0127] An "ointment" is a semisolid preparation containing an ointment base and optionally one or more active agents. Examples of suitable ointment bases include hydrocarbon bases (e.g., petrolatum, white petrolatum, yellow ointment, and mineral oil); absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream); water-removable bases (e.g., hydrophilic ointment), and water-soluble bases (e.g., polyethylene glycol ointments). Pastes typically differ from ointments in that they contain a larger percentage of solids. Pastes are typically more absorptive and less greasy that ointments prepared with the same components.

[0128] A "gel" is a semisolid system containing dispersions of the nanoparticles in a liquid vehicle that is rendered semisolid by the action of a thickening agent or polymeric material dissolved or suspended in the liquid vehicle. The liquid may include a lipophilic component, an aqueous component or both. Some emulsions may be gels or otherwise include a gel component. Some gels, however, are not emulsions because they do not contain a homogenized blend of immiscible components. Suitable gelling agents include, but are not limited to, modified celluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose; Carbopol homopolymers and copolymers; and combinations thereof. Suitable solvents in the liquid vehicle include, but are not limited to, diglycol monoethyl ether; alklene glycols, such as propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl alcohol and ethanol. The solvents are typically selected for their ability to dissolve the drug. Other additives, which improve the skin feel and/or emolliency of the formulation, may also be incorporated. Examples of such additives include, but are not limited, isopropyl myristate, ethyl acetate, C ₁₂ -C ₁₅ alkyl benzoates, mineral oil, squalane, cyclomethicone, capric/caprylic triglycerides, and combinations thereof.

[0129] Foams consist of an emulsion in combination with a gaseous propellent. The gaseous propellant consists primarily of hydrofluoroalkanes (HFAs). Suitable propellents include HFAs such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1 ,1 ,1 , 2,3,3, 3-heptafluoropropane (HFA 227), but mixtures and admixtures of these and other HFAs that are currently approved or may become approved for medical use are suitable. The propellents preferably are not hydrocarbon propellant gases which can produce flammable or explosive vapors during spraying. Furthermore, the compositions preferably contain no volatile alcohols, which can produce flammable or explosive vapors during use.

[0130] Buffers are used to control pH of a composition. Preferably, the buffers buffer the composition from a pH of about 4 to a pH of about 7.5, more preferably from a pH of about 4 to a pH of about 7, and most preferably from a pH of about 5 to a pH of about 7. In a preferred embodiment, the buffer is triethanolamine.

[0131] Preservatives can be used to prevent the growth of fungi and microorganisms. Suitable antifungal and antimicrobial agents include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal.

[0132] In certain embodiments, it may be desirable to provide continuous delivery of one or more nanoparticles to a patient in need thereof. For topical applications, repeated application can be done or a patch can be used to provide continuous administration of the noscapine analogs over an extended period of time.

Enteral Formulations

[0133] The nanoparticles can be prepared in enteral formulations, such as for oral administration. Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.

[0134] Formulations are prepared using pharmaceutically acceptable carriers. As generally used herein "carrier" includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof. Polymers used in the dosage form include hydrophobic or hydrophilic polymers and pH dependent or independent polymers. Preferred hydrophobic and hydrophilic polymers include, but are not limited to, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxy methylcellulose, polyethylene glycol, ethylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate, and ion exchange resins.

[0135] Carrier also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants.

[0136] Formulations can be prepared using one or more pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof. [0137] Delayed release dosage formulations can be prepared as described in standard references such as "Pharmaceutical dosage form tablets", eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), "Remington - The science and practice of pharmacy", 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems", 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules. These references provide information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

[0138] The nanoparticles may be coated, for example to delay release once the particles have passed through the acidic environment of the stomach. Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0139] Coatings may be formed with a different ratio of water soluble polymer, water insoluble polymers and/or pH dependent polymers, with or without water insoluble/water soluble non polymeric excipient, to produce the desired release profile. The coating is either performed on dosage form (matrix or simple) which includes, but not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, "ingredient as is" formulated as, but not limited to, suspension form or as a sprinkle dosage form.

[0140] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0141] Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.

[0142] Optional pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Diluents, also referred to as "fillers," are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.

[0143] Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid and polyvinylpyrrolidone.

[0144] Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.

[0145] Disintegrate are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross- linked PVP (Polyplasdone® XL from GAF Chemical Corp).

[0146] Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA).

[0147] Diluents, also referred to as "fillers," are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar. The usual diluents include inert powdered substances such as starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders. A lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.

[0148] The preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials, method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies.

Pulmonary Formulations

[0149] The nanoparticle can be prepared for pulmonary administration. The nanoparticles may be alone or in combination with additional active agents, pharmaceutically acceptable carriers, pharmaceutically acceptable excipients, or combinations thereof. The pharmaceutical carrier and excipient are composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.

[0150] Formulations for pulmonary administration can be administered using methods known in the art. Suitable methods can include, but are not limited to, dry powder inhalers, pressurized metered-dose inhalers, nebulizers and aerosolizers, electrodynamic aerosol generators, and endotracheal aerosol generators.

[0151] In the case of pulmonary administration, formulations can be divided into dry powder formulations and liquid formulations. Both dry powder and liquid formulations can be used to form aerosol formulations. Useful formulations, and methods of manufacture, are described by Caryalho, et al., J Aerosol Med Pulm Drug Deliv. 2011 Apr;24(2):61-80. Epub 2011 Mar 16, for delivery of chemotherapeutic drugs to the lungs.

Dry Powder Formulations

[0152] Dry powder formulations are finely divided solid formulations containing one or more nanoparticles which are suitable for pulmonary administration. Dry powder formulations include one or more nanoparticles. Such dry powder formulations can be administered via pulmonary inhalation to a patient without the benefit of any carrier, other than air or a suitable propellant. Preferably, however, the dry powder formulations contain one or more nanoparticles in combination with a pharmaceutically acceptable carrier.

[0153] The pharmaceutical carrier may include a bulking agent, such as carbohydrates (including monosaccharides, polysaccharides, and cyclodextrins), polypeptides, amino acids, and combinations thereof. Suitable bulking agents include fructose, galactose, glucose, lactitol, lactose, maltitol, maltose, mannitol, melezitose, myoinositol, 35ryptococc, raffinose, stachyose, sucrose, trehalose, xylitol, hydrates thereof, and combinations thereof.

[0154] The pharmaceutical carrier may include a lipid or surfactant. Natural surfactants such as dipalmitovlphosphatidvlcholine (DPPC) are the most preferred. This is commercially available for treatment of respiratory distress syndrome in premature infants. Synthetic and animal derived pulmonary surfactants include: Synthetic Pulmonary Surfactants such as Exosurf (a mixture of DPPC with hexadecanol and tyloxapol added as spreading agents); Pumactant (Artificial Lung Expanding Compound or ALEC); a mixture of DPPC and PG; KL-4 (composed of DPPC, palmitoyl-oleoyl phosphatidylglycerol, and palmitic acid, combined with a 21 amino acid synthetic peptide that mimics the structural characteristics of SP-B); and Venticute (DPPC, PG, palmitic acid and recombinant SP-C) or animal derived surfactants such as Alveofact (extracted from cow lung lavage fluid); Curosurf (extracted from material derived from minced pig lung; Infasurf (extracted from calf lung lavage fluid); and Survanta (extracted from minced cow lung with additional DPPC, palmitic acid and tripalmitin). Exosurf, Curosurf, Infasurf, and Survanta are the surfactants currently FDA approved for use in the U.S.

[0155] The pharmaceutical carrier may also include one or more stabilizing agents or dispersing agents. The pharmaceutical carrier may also include one or more pH adjusters or buffers. Suitable buffers include organic salts prepared from organic acids and bases, such as sodium citrate or sodium ascorbate. The pharmaceutical carrier may also include one or more salts, such as sodium chloride or potassium chloride. [0156] Dry powder formulations are typically prepared by blending the one or more nanoparticles with a pharmaceutical carrier. Optionally, additional active agents may be incorporated into the mixture as discussed above. The mixture is then formed into particles suitable for pulmonary administration using techniques known in the art, such as lyophilization, spray drying, agglomeration, spray coating, extrusion processes, hot melt particle formation, phase separation particle formation (spontaneous emulsion particle formation, solvent evaporation particle formation, and solvent removal particle formation), coacervation, low temperature casting, grinding, milling (e.g., air-attrition milling (jet milling), ball milling), high pressure homogenization, and/or supercritical fluid crystallization.

[0157] An appropriate method of particle formation can be selected based on the desired particle size, particle size distribution, and particle morphology. In some cases, the method of particle formation is selected so as to produce a population of particles with the desired particle size, particle size distribution for pulmonary administration. Alternatively, the method of particle formation can produce a population of particles from which a population of particles with the desired particle size, particle size distribution for pulmonary administration is isolated, for example by sieving.

Liquid Formulations

[0158] Liquid formulations contain one or more nanoparticles, possibly with one or more additional active agents, dissolved or suspended in a liquid pharmaceutical carrier.

[0159] Suitable liquid carriers include, but are not limited to distilled water, de-ionized water, pure or ultrapure water, saline, and other physiologically acceptable aqueous solutions containing salts and/or buffers, such as phosphate buffered saline (PBS), Ringer's solution, and isotonic sodium chloride, or any other aqueous solution acceptable for administration to an animal or human.

[0160] Preferably, liquid formulations are isotonic relative to physiological fluids and of approximately the same pH, ranging e.g., from about pH 4.0 to about pH 7.4, more preferably from about pH 6.0 to pH 7.0. The liquid pharmaceutical carrier can include one or more physiologically compatible buffers, such as a phosphate buffers. One skilled in the art can readily determine a suitable saline content and pH for an aqueous solution for pulmonary administration.

[0161] Liquid formulations may include one or more suspending agents, such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, gum tragacanth, or lecithin. Liquid formulations may also include one or more preservatives, such as ethyl or /7-propyl p- hydroxybenzoate.

[0162] In some cases the liquid formulation may contain one or more solvents that are low toxicity organic (i.e. nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydofuran, ethyl ether, and propanol. These solvents can be selected based on their ability to readily aerosolize the formulation. Any such solvent included in the liquid formulation should not detrimentally react with the one or more active agents present in the liquid formulation. The solvent should be sufficiently volatile to enable formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as an alcohol, glycol, polyglycol, or fatty acid, can also be included in the liquid formulation as desired to increase the volatility and/or alter the aerosolizing behavior of the solution or suspension.

[0163] Liquid formulations may also contain minor amounts of polymers, surfactants, or other excipients well known to those of the art. In the context of pulmonary formulations, "minor amounts" means no excipients are present that might adversely affect uptake of the one or more active agents in the lungs.

Aerosol Formulations

[0164] The dry powder and liquid formulations described above can be used to form aerosol formulations for pulmonary administration. Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art. The term aerosol as used herein refers to any preparation of a fine mist of solid or liquid particles suspended in a gas. In some cases, the gas may be a propellent; however, this is not required. Aerosols may be produced using a number of standard techniques, including as ultrasonication or high pressure treatment.

[0165] Preferably, a dry powder or liquid formulation as described above is formulated into aerosol formulations using one or more propellents. Suitable propellents include air, hydrocarbons, such as pentane, isopentane, butane, 37ryptococ, propane and ethane, carbon dioxide, chlorofluorocarbons, fluorocarbons, and combinations thereof. Suitable fluorocarbons include 1-6 hydrogen containing fluorocarbons, such as CHF2CHF2, CF3CH2F, CH2F2CH3, and CF3CHFCF3 as well as fluorinated ethers such as CF3-O-CF3, CF ₂ H-0-CHF ₂ , and CF3-CF2-O- CF2-CH3. Suitable fluorocarbons also include perfluorocarbons, such as 1-4 carbon perfiuorocarbons including CF3CF3, CF3CF2CF3, and CF3CF2CF2CF3. Suitable hydrofluoroalkanes (HFA) propellents, include, but are not limited to, 1 ,1 ,1 ,2,3,3,-heptafluoro-n- propane (HFA 227), 1,1 ,1 ,2-tetrafluoroethane (HFA 134) 1 ,1 ,1 ,2, 25 3,3,3-heptafluoropropane (Propellent 227), or any mixture of these propellents.

[0166] Preferably, the one or more propellents have sufficient vapor pressure to render them effective as propellents. Preferably, the one or more propellents are selected so that the density of the mixture is matched to the density of the particles in the aerosol formulation in order to minimize settling or creaming of the particles in the aerosol formulation.

[0167] The propellant is preferably present in an amount sufficient to propel a plurality of the selected doses of the aerosol formulation from an aerosol canister.

EXAMPLES

[0168] Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.

Methods of Manufacturing of ODN-LSNs

[0169] The oligonucleotide loaded lipid-surfactant nanoparticles can be prepared via a variety of methods. A variety of oligonucleotide (ODN) loaded nanoparticles were investigated using a combination of cationic lipids (DOTAP and DOPE)

Method 1

Formulation 1

[0170] Dissolve ODN and sucrose in water, sterile filter (solution A)

[0171] Dissolve DOTAP/DOPE/Tween-80 (4:4:2) in tert-butyl alcohol (TBA), sterile filter (solution B)

[0172] Combine A and B at 1 :12 (DNA/lipid) into sterile vial, lyophilize into dry cake (Vial A: ODN/Lipid Complex)

[0173] Prepare 40% ethanol, sterile filter, fill vial (Vial B: Solvent) Formulation 2

[0174] Lipid composition DODMA/DOPE/Tween-80 (5:3:2); 40% ethanol+10mM citrate pH 4.0

Formulation 3

[0175] ODN in water with 40 mM TEAA pH 4.5 (Solution A)

[0176] DOTAP/DODMA/DOPE/PEG-DMG (20:30:30:20) (Solution B)

[0177] Lipid to DNA ratio of 16:1

[0178] Mix A and B at 3:2, adjust pH to 5, dilute 2x with saline, diafiltration to remove ethanol

[0179] At the clinic:

[0180] Add Solvent (Vial B) into ODN/Lipid Complex (Vial A), shake well to reconstitute the drug at room temperature

[0181] Dilute the reconstituted drug into 3-1 Ox volume of saline for injection. Use within 8 hrs

Method 2

[0182] Dissolve ODN in TE buffer (pH 8) (Solution A) sterile filtered

[0183] Dissolve lipid mixture in ethanol (Solution B) (with acetic acid for pH adjustment to 4 if tertiary amine lipid is used) sterile filtered

[0184] At point of use, combine equal volumes of A and B at 2:3 v/v

[0185] Dilute with saline for administration

Method 3

[0186] Dissolve ODN (1 mg/mL) in water, sterile filtration (Solution A) at 45 degree

[0187] Dissolve lipid mixture (DOTAP/DODMA/DOPE/Tween-80 (20:30:30:20)) in 80% ethanol (Solution B) with 50 mM TEAA pH 4, sterile filtration at 45 degree

[0188] Combine A and B at 1 :1 v/v and L/D = 10 using a T-mixer

[0189] Dilute with 3x volume of saline

[0190] Remove ethanol by diafiltration against saline and concentrate to 2.2 mg/mL

[0191] Add 10% trehalose, adjust concentration to 2 mg/mL, Sterile filter and sterile fill [0192] Lyophilization

[0193] At point of use reconstitute in saline

[0194] In early development, the drug can be prepared using the composition of the reconstituted drug in 40% ethanol and stored at -80 degree and bypass the manufacturing process described above. At small scale, the ODN/lipid mixture can be dried in a speedvac under vacuum. The drug is warmed to room temperature and then diluted into saline. Adding TEA can facilitate formulation.

Method 4

[0195] Dissolve ODN (4 mg/mL) in 20% sucrose, sterile filtration (Solution A)

[0196] Dissolve lipid mixture DOTAP/DODMA/DOPE/Tween-80 (5:40:25:30) in ethanol at 200 mg/mL and then dilute 1 :5 in with 50 mM ΤΕΞΑΑ pH 5, sonicate with a probe sonicator for 1 min to reduce particle size to < 80 nm, sterile filtration (40 mg/mL lipid concentration) (Solution B)

[0197] Combine A and B at 1:1 v/v rapidly and fill vial (2 mg/mL ON and 10% ethanol in final product)

[0198] At point of use dilute in saline

[0199] This is a micelle-like ethanol low preparation with optional lyophilization. If lyophilized, replace ethanol with tert-butanol. This is due to realization that with Tween-80 and mild heating, 40% ethanol is unnecessary for LNP production.

Alternate protocol:

[0200] Dissolve ODN (2 mg/mL) in 20% sucrose, sterile filtration (Solution A)

[0201] Dissolve lipid mixture DOTAP/DODMA/DOPE/Tween-80 (5:40:25:30) in ethanol at 100 mg/mL and then dilute 1 :5 in with 50 mM TEAA pH 5, sonicate with a probe sonicator for 1 min to reduce particle size to < 80 nm, sterile filtration (20 mg/mL lipid concentration) (Solution B)

[0202] Combine A and B at 1:1 v/v rapidly and fill vial (1 mg/mL ON and 10% ethanol in final product)

[0203] At point of use dilute in saline

Example 1 : On Loaded DOTAP/DODMA/DOPE/Tween80 (15:25:40:20 m/m)

[0204] DOTAP/DODMA/DOPE/Tween80 (15:25:40:20 m/m) LSN formulations were prepared via Method 4 described above in (DOTAP+DODMA+DOPE)/ON = 10; 50 mM TEAA pH 4.5 to promote ON-lipid interaction. [0205] When the mole% of Tween 80 was below 10 mole %, LSNs assembly was inefficient, resulting in reduced ON loading (FIG. 2: Lanes 1&2, corresponding to 0% and 5%) and LSNs that are prone to aggregate. This is because the LSNs were not sufficiently permeable to enable penetration of ON into the core of the nanoparticles. When the mole% of Tween 80 was between 10 mole % and 50 mole %, LSNs were permeabilized, enabling ON penetration and assembly of stable LSNs via electrostatic interaction with no detectable free ON at lipid-to-ON weight ratio of 10 (FIG. 2: Lanes 3, 4, and 5, corresponding to 10%, 20% and 50%). LSN particle size is tunable and decreases with increase in mole% of Tween 80. The formulation with 40% Tween 80 had an average particle size = 60 nm

[0206] When the mole% of Tween 80 is over 50%, the LSNs became micelle-like and did not retain ONs in the core, resulting in mostly free ON in the preparation (FIG. 2: Lanes 6 & 7, corresponding to 60% and 70%)

Example 2: ON Loaded POTAP/DOPMA/POPE/Tween80 (15:25:40:20 m/rm

[0207] An ON loaded LSN formulation of DOTAP/DODMA/DOPE/Tween80 (15:25:40:20 m/m) were prepared via Method 4 described above.

[0208] FIG. 3 an agarose gel-electrophoresis for Tween80 mole % ranging from 10 mole % to about 80 mole % and compared to free ON (Lane 1). In summary, in this formulation, Tween 80 of 10-50 mole% resulted in stable nanoparticles with full ON entrapment t by gel electrophoresis. Tween 80 of 40% seems to be optimal, resulting in small particle size, with the composition DOTAP/DODMA/DOPE/Tween80 (10:20:30:40 m/m)

[0209] FIG. 4 is a graph of the percent oligonucleotide loading (w/w) and the particle size for oligonucleotide (ON) loaded DOTAP/DODMA/DOPE/Tween80 (10:20:30:40 m/m) nanoparticles as a function of the mole% of Tween 80.

Example 3: GFP mRNA Loaded DOTAP/DODMA/DOPE/Tween80 (67/23/10 m/m)

[0210] Non-capped GFP mRNA was incorporated into the LSN formulation at an mRNA concentration of 0.25 mg/mL. Lipid composition used was DOTAP/DOPE/Tween 80 (67/23/10 mol/mol) and mRNA-to-lipids (w/w)=1 :15. The mean particle size of the LSNs was 112.5 nm (PDI 0.16) with a positive zeta potential of 30 mV. The vehicle is 10% sucrose (isotonic). The encapsulation efficiency is 100% based on agarose gel electrophoresis. The mRNA-LSN were stable at 4°C for at least for 72 hrs and can be stored frozen at -20 °C. [0211] Gel electrophoresis demonstrated that no free mRNA bands were visible (FIG. 5 lanes 3- 4) indicating absence of free mRNA in the formulation.

PROPHETIC EXAMPLES

Evaluation of LSN loaded with RX-0201. an antisense oliao against akt-1

[0212] RX-0201 is a 20-mer phosphorothioate oligodeoxyribonucleotide (ODN) against akt-1 , with sequence of 5' gclgcatgatctccttggcg 3'. AKT-1 activation is found in a large percentage of human tumors. The oligonucleotide will be custom synthesized by IDT. LSNs will be synthesized with the formulation of DOTAP/DODMA/DOPC/Tween 80 (5:40:35:20 mole/mole). The lipids and surfactants will be co-dissolved in ethanol or tert-butanol at 200 mg/mL and diluted in 40 nM triethylamine acetate (TEAA) buffer at pH 4.5 to generate lipid-surfactant vesicles (LSVs). The mean particle size will be reduced to < 80 nm based on dynamic light scattering measurement on a NICOMP 370 Particle Size Analyzer by probe sonication for 1 min. The LSNs will then be synthesized by combining the LSVs with ODN diluted in 20% sucrose with heating to 60 °C to produce LSNs. The LSNs will be analyzed by dynamic light scattering for particle size. Zeta potential of the LSNs will be measured as well. The LSNs can be synthesized at a series of lipid- to-ODN ratios (lipid to-ODN weight ratio of 6 to 20 will be studied). LSNs at weight ratio of 8-10:1 are likely slightly negatively charged based on agarose gel-electrophoresis and optimal for in vivo delivery of ODNs. The ratio will vary depending on the sequence and composition of the oligo (e.g., AG vs TC content, nucleotide modifications), which affects charge density per unit weight of the oligo. We will vary the composition of the LSNs as well by varying the ratio of DOTAP and DODMA or using DOTAP or DODMA only, or substitute with other cation ic lipids such as DOTMA and DODAP. We expect to obtain LSNs with mean diameter of ~ 100 nm, e.g. about 90 nm to about 120 nm. The LSNs can be optionally lyophilized by a 2-stage program with primary drying followed by secondary drying. The dried cake can be reconstituted in sterile water for injection prior to administration.

[0213] We will evaluate the colloidal stability of the LSNs by incubating them at 4 °C or 37°C and monitoring changes in mean particle size and polydispersity index over time.

[0214] We will investigate akt-1 down regulation by the LSNs loaded with RX-0201 in a series of cancer cell lines, including A549 lung cancer cells and HepG2 hepatocellular carcinoma cells as well as several other cell lines. Akt-1 expression will be assessed by qRT-PCR after isolation of mRNA from the cells treated with the LSNs. Western blot will be used to analyze akt-1 protein expression. We also plan to investigate the pharmacokinetic properties of the LSNs by measuring ODN plasma concentration following i.v. injection, generating a plasma concentration versus time curve and calculating PK parameters of the LSN-ODN vs free ODN. We will study the synergistic effect between the LSN-ODN and either chemotherapeutic agent paclitaxel (for lung cancer) or tyrosine kinase inhibitor sorafenib (for HCC). We expect to see chemosensitization effect with the LSN-ODN. We will then investigate the tumor inhibitory activity of the LSN-ODN in xenograft tumor models in athymic mice. Mice with established tumor nodules will be given i.v. or i.p. injections of LSN-ODN alone or in combination with paclitaxel or sorafenib. Tumor size will monitored in the course of treatment and animal survival recorded. We expect the LSN-ODN to more effectively inhibit tumor growth than free ODN and prolong the survival of tumor-bearing mice. Meanwhile, a dose escalation toxicity study will be carried out to investigate the maximum tolerated dose of the LSN-ODN. Cytokines will be measured by ELISA to monitor cytokine induction as a result of LSN-ODN treatment.

Synthesis and evaluation of T7-coniuaated LSNs for delivery of G3139

Background

[0215] Bcl-2 is an anti-apoptotic protein. Bcl-2 inhibitor Venetoclax (ABT-199) has been approved in 2016 for chronic lymphocytic leukemia (CLL). G3139 (Genasense, oblimersen) is an 18-mer antisense oligo targeting bcl-2. Phase 3 trials of G3139 in melanoma and in CLL have shown clinical activities, resulting in NDA submissions to the FDA. However, the FDA determined the activities to be insufficient to warrant approval. We have since developed LNP formulations of G3139 and shown promising antitumor activities in murine xenograft models, likely due to improved delivery of G3139 to the tumor cells. Further improvement in delivery efficiency using the LSN formulation can result in additional efficacy gains, therefore, elevating G3139's activity in future clinical trials. T7 (HAIYPRH) is a phage-display peptide shown to target the transferrin receptor (TfR, CD71), which is highly expressed in tumor cells including lymphoma cells. T7- coated liposomes have been shown to selectively target tumor cells through the TfR and to cross the BBB through TfR-mediated transcytosis.

Significance

[0216] Targeting Bcl-2 in CLL has been validated through the approval of Venetoclax. Additional indications include non-Hodgkin's lymphoma and multiple myeloma. G3139 targets mRNA, which is upstream of Bcl-2 protein expression, is more specific than venetoclax, a small molecule BH3 mimetic. Tf-LSN has been shown to greatly improve delivery efficiency of oligos in tumor cell lines.

Tf-LSN is a platform for delivery that can be used for delivery of other classes of oligonucleotide therapeutics. Improvement of delivery efficiency for G3139 is likely to result in clinical utility of the drug in a variety of human malignancies.

[0217] In solid tumors, co-inhibition of bcl-2 related bcl-xl and mcl-1 may be highly critical since inhibiting a single target can trigger compensatory upregulation of other factors that offset its activity.

[0218] Dual targeting using binary combinations of oligos is likely to be much more effective than single-target oligos. The T7-LSN nanocarrier with our proprietary composition is one of the most active nanoparticles available and is an enabling platform technology. It is relatively easy to take the T7-LSNs into the clinic since the API and excipients are all available in cGMP quality from commercial suppliers and LSN production can be easily scaled up for commercial production.

Research Plan

[0219] T7-PEG-DSPE will be synthesized as a targeting ligand using Cys-T7 (CHAIYPRH) coupled to maleimide-PEG-DSPE and purified. T7-LSN-G3139 formulations will be synthesized using a series of compositions with varying amounts of T7-PEG-DSPE. An example of this composition is DOTAP/DODMA/DOPC/Tween 80/T7-PEG-DSPE (5:40:34:20:1 mole/mole). The formulations will be characterized for particle size, zeta potential, oligonucleotide encapsulation, and colloidal stability. Fluorescence-labeled T7-LSN-G3139 will be assessed for cellular uptake by confocal microscopy and by flow cytometry in A549, Raji and Ramos cells. Biological activity of T7-LSN-G3139 will be assessed by qRT-PCR analysis of bcl-2 expression. Effect of deferoxamine-induced TfR expression will also be studied as a potential mechanism to increase TfR-mediated cellular uptake. Non-targeted LSN-G3139 and free G3139 will be used as controls. T7-LSNs with the best targeting ratio versus control LSN will be selected for further evaluation in vivo. Cytotoxicity and apoptosis induction of T7-LSNs will also be assessed.

[0220] The T7-LSN composition will be optimized and validated. Product specifications will be developed including composition, particle size, zeta potential, oligo load, etc. Analytical methods will be developed and validated for the APIs and excipients. T7-LSNs will be subjected to stability testing. Its production method will be validated at pre-pilot scale, which must be compatible to scaling up to commercial scale under cGMP. These developmental efforts will support rapid clinical translation.

[0221] Sensitization of 3 tumor cell lines (A549, KB, and HepG2) to paclitaxel will be used to assess efficacy. The most effective combination will be further investigated in vivo. [0222] Plasma pharmacokinetics and cytokine induction by the T7-LSNs will be investigated in ICR mice. ELISA will be used to measure cytokines at 6 h post injection. Biodistribution of fluorescence-labeled T7-LSNs will be studied in mice carrying xenograft tumor. The effect of treatment on tumor growth will be investigated at dosing levels of 2 mg/kg, 5 mg/kg and 10 mg/kg in ASO dose given every 4 ^th day for 4 treatments total. In vivo bcl-2 down regulation in tumors and tumor growth inhibition will be measured along with animal survival.

Additional studies

[0223] The use of LSNs, with or without T7-PEG-DSPE targeting, will be explored for therapeutic delivery of microRNA mimics and antimiRs. T7 can be substituted with any other targeting ligand. The miRNAs we are currently interested are:

[0224] miR-29b (for Bronchopulmonary dysplasia (BPD)). We have custom designed miR-29b mimic as a pair of oligos custom synthesized by IDT. These will be incorporated in to the LSNs as described above and instilled into the lung of murine models of BPD. This formulation can be nebulized to form an aerosol for inhalation. Alternatively, the lyophilized formulation can be converted into dry powder aerosol for pulmonary administration in future clinical settings. We expect that the LSN-miR-29b will exert a beneficial therapeutic effect by preventing fibrosis and lesson BPD symptoms. The BPD models and the relevant experimental procedures have been published by the Rogers group using AAV viral vectors for miR-29b delivery. We expect the LSNs to be as effective and more practical for clinical applications.

[0225] miR-29b is also a tumor suppressor miR so we will also evaluated the LSNs for antitumor activities in lung cancer cell lines and animal models when possible. We will also look into miR- 34a mimic as an alternative. Meanwhile, LSNs carrying antimiR-21 , antimiR-155, antimiR-17-92, including antimiRs based on short seed-targeting LNA oligos, will also be evaluated since these miRs are known to be oncomiRs. The LSNs will be studied in relevant animal models for tumor inhibition and survival prolongation and biomarker modulation, as has been described previously.

[0226] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations, and are set forth only for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above- described embodiments of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure. [0227] The present disclosure will be better understood upon viewing the following aspects which should not be confused with the claims.

[0228] Aspect 1. A lipid-surfactant nanoparticle comprising: (i) an aqueous core region; (ii) an outer lipid bilayer encapsulating the inner core region, wherein the lipid bilayer comprises an outer hydrophilic surface at an outer surface of the lipid-surfactant nanoparticle and an inner hydrophilic surface at the inner core region; and (iii) a nonionic surfactant having a an HLB value of about 10 or higher and a CMC value of about 0.1 mM or less, wherein the nonionic surfactant is associated with one or both of the outer hydrophilic surface and the inner hydrophilic surface; wherein the lipid-surfactant nanoparticle comprises one or both of a nucleic acid in the aqueous core region and a lipophilic active agent in the outer lipid bilayer; and wherein the nonionic surfactant is present in an amount from about 10 mole percent to about 50 mole percent based upon the total moles of lipid and surfactant in the lipid-surfactant nanoparticle.

[0229] Aspect 2. The lipid-surfactant nanoparticle according to any one of aspects 1-21, wherein the lipid-surfactant nanoparticle has a diameter of about 30 nm to about 200 nm.

[0230] Aspect 3. The lipid-surfactant nanoparticle according to any one of aspects 1-21, wherein the outer lipid bilayer comprises a mixture of two or more lipids selected from the group consisting of a cationic lipid, a neutral lipid, a PEGylated lipid, cholesterol, a targeted conjugate thereof, and a combination thereof.

[0231] Aspect 4. The lipid-surfactant nanoparticle according to any one of aspects 1-21, wherein the outer lipid bilayer comprises a cationic lipid selected from the group consisting of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts (DOTMA); dimethyldioctadecyl ammonium bromide (DDAB); 1 ,2-diacyloxy-3-trimethylammonium propane; N-[1-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine (DODAP); 1 ,2-diacyloxy-3-dimethylammonium propane; N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);

1.2- dialkyloxy-3-dimethylammonium propane; dioctadecylamidoglycylspermine (DOGS); 3 -[N-(N',N ^, -dimethylamino-ethane)carbamoyl]cholesterol (DC-Choi);

2.3- dioleoyloxy-N-(2-(sperminecart)oxamido)-ethyl)-N,N-dimethyl- 1-propanaminium

trifluoro-acetate (DOSPA); B-alanyl cholesterol; cetyl trimethyl ammonium bromide (CTAB); diCi ₄ -amidine; N-ferf-butyl-N'-tetradecyl-3-tetradecylamino-propionamidine;

N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG); ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride; 1 ,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER); N,N,N\N'-tetramethyl-N'-bis(2-hydroxylethyl)-2,3-dioleoyloxy -1 ,4-butanediammonium iodide; 1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imid azolinium chloride derivatives; 2,3-dialkyloxypropyl quaternary ammonium compound derivatives containing a hydroxyalkyl moiety on the quaternary amine; and a combination thereof.

[0232] Aspect 5. The lipid-surfactant nanoparticle according to any one of aspects 1-21, wherein the outer lipid bilayer comprises a cationic lipid selected from the group consisting of a 1,2-dioleoyl-3-trimethylammonium-propane salt (DOTAP); a 1 ,2-dimyristoyl-3- trimethylammonium-propane salt (DMTAP); a 1 ,2-dipalmitoyl-3-trimethylammonium-propane salt (DPTAP); a 1 ,2- distearoyl-3-trimethylammonium-propane salt (DSTAP); dimethyldioctadecyl ammonium bromide (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2- Dioleyloxy-3-dimethylaminopropane (DODMA); a 1 ,2-Dioleyloxy-3-trimethylammonium propane salt (DOTMA);1 ,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); and a combination thereof.

[0233] Aspect 6 The lipid-surfactant nanoparticle according to any one of aspects 1-21 , wherein the outer lipid bilayer comprises a neutral lipid selected from the group consisting of sterols, phospholipids, lysolipids, lysophospholipids, sphingolipids, and a combination thereof.

[0234] Aspect 7. The lipid-surfactant nanoparticle according to any one of aspects 1-21, wherein the outer lipid bilayer comprises a neutral lipid selected from the group consisting of 1 ,2- dioleylphosphoethanolamine (DOPE), 1 ,2-dihexadecylphosphoethanolamine (DHPE), 1 ,2- distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), 1 ,2- dioleoylphosphatidylcholine (DOPC); 1 ,2-dipalmitoyl phosphatidylcholine (DPPC), 1 ,2- dimyristoylphosphatidylcholine (DMPC), dibehenoylphosphatidylcho- line (DBPC), ditricosanoylphosphatidylcholine (DTPC), and dilignoceroylphatidylcholine (DLPC).

[0235] Aspect 8. The lipid-surfactant nanoparticle according to any one of aspects 1-21, wherein the outer lipid bilayer comprises a targeted conjugate of a lipid and a targeting moiety selected from the group consisting of peptides and polypeptides, antibody mimetics, nucleic acids, glycoproteins, small molecules, carbohydrate, and lipids.

[0236] Aspect 9. The lipid-surfactant nanoparticle according to any one of aspects 1-21, wherein the outer lipid bilayer comprises a targeted conjugate, and wherein the targeted conjugate comprises a T-X-L conjugate, where T is a protein, peptide, or fragment thereof that binds to a cell-surface receptor, where X is a C3-C12 polyethylene glycol linker, and where L is a lipid selected from the group consisting of 1 ,2-dioleylphosphoethanolamine (DOPE), 1 ,2- dihexadecylphosphoethanolamine (DHPE), 1 ,2-distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), 1 ,2- dioleoylphosphatidylcholine (DOPC); 1 ,2- dipalmitoyl phosphatidylcholine (DPPC), 1 ,2-dimyristoylphosphatidylcholine (DMPC), dibehenoylphosphatidylcho- line (DBPC), ditricosanoylphosphatidylcholine (DTPC), and dilignoceroylphatidylcholine (DLPC).

[0237] Aspect 10. The lipid-surfactant nanoparticle according to any one of aspects 1-21 , wherein the targeting moiety is a T7 phage-display peptide comprising a sequence SEQ ID NO:1 (HAIYPRH).

[0238] Aspect 11. The lipid-surfactant nanoparticle according to any one of aspects 1-21 , wherein the targeting moiety selectively targets a cell surface marker, wherein the cell surface marker is a breast cancer marker, a colon cancer marker, a rectal cancer marker, a lung cancer marker, a pancreatic cancer marker, a ovarian cancer marker, a bone cancer marker, a renal cancer marker, a liver cancer marker, a neurological cancer marker, a gastric cancer marker, a testicular cancer marker, a head and neck cancer marker, an esophageal cancer marker, or a cervical cancer marker.

[0239] Aspect 12. The lipid-surfactant nanoparticle according to any one of aspects 1-21 , wherein the outer lipid bilayer comprises a PEGylated lipid selected from the group consisting of, distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dimyristoyl phosphatidylethanolamine-poly ethylene glycol (DMPE-PEG), DPPE-PEG, DMA-PEG, DPA- PEG, DPG-PEG, DMG-PEG, dipalmitoylglycerosuccinate polyethylene glycol (DPGS-PEG), stearyl-polyethylene glycol, and cholesteryl-polyethylene glycol.

[0240] Aspect 13. The lipid-surfactant nanoparticle according to any one of aspects 1-21 , wherein the outer lipid bilayer comprises a mixture of two, three, four, or more lipids.

[0241] Aspect 14. The lipid-surfactant nanoparticle according to any one of aspects 1-21 , wherein the nonionic surfactant comprises ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401 , stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. [0242] Aspect 15. The lipid-surfactant nanoparticle according to any one of aspects 1-21 , wherein the nonionic surfactant comprises a fatty acid ester surfactant selected from the group consisting of sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, and polyoxyethylene stearates.

[0243] Aspect 16. The lipid-surfactant nanoparticle according to any one of aspects 1-21 , wherein the nonionic surfactant comprises a polyoxyethylene.

[0244] Aspect 17. The lipid-surfactant nanoparticle according to any one of aspects 1-21 , wherein a loading efficiency of the nucleic acid and/or the lipophilic drug in the nanoparticle of about 80% or more by weight.

[0245] Aspect 18. The lipid-surfactant nanoparticle according to any one of aspects 1-21 , wherein lipid-surfactant nanoparticle comprises the nucleic acid in the aqueous core region, and the nucleic acid is selected from the group consisting of a plasmid, a messenger RNA, an sgRNA, an immuno stimulatory oligonucleotide, an siRNA, an antisense oligonucleotide, a microRNA, an antagomir, an aptamer, and a ribozyme.

[0246] Aspect 19. The lipid-surfactant nanoparticle according to any one of aspects 1-21 , wherein the nucleic acid is the antisense oligonucleotide G3139 (Genasense, oblimersen), an 18- mer antisense oligonucleotide targeting bcl-2.

[0247] Aspect 20. The lipid-surfactant nanoparticle according to any one of aspects 1-21 , wherein the lipid-surfactant nanoparticle comprises the lipophilic active agent in the outer lipid bilayer; and wherein the lipophilic active agent is selected from the group consisting of lipophilic vitamins and derivatives thereof, antibiotics, antimicrobials, anti-inflammatory agents, hormones, adrenocortical steroids, non-steroidal anti-inflammatory agents, cancer therapeutics, and drugs acting on the CNS (Central Nervous System).

[0248] Aspect 21. The lipid-surfactant nanoparticle according to any one of aspects 1-21 , wherein the lipophilic active agent is retinoic acid or a derivative thereof.

[0249] Aspect 22. A pharmaceutical formulation comprising a plurality of nanoparticles according to any one of aspects 1-21 , and a pharmaceutically acceptable carrier.

[0250] Aspect 23. The pharmaceutical formulation according to aspect 22, wherein the pharmaceutically acceptable carrier is selected from the group consisting of diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof. [0251] Aspect 24. A method of treating or preventing a disease or disorder in a patient in need thereof, the method comprising administering a therapeutically affective amount of the nanoparticles of any one of aspects 1-21 or the formulations of aspects 22-23 to the subject.

[0252] Aspect 25. The method of aspect 24, wherein the administration comprises one or more of parenteral administration, topical administration, enteral administration, and pulmonary administration.