Nucleic Acid-Mediated Delivery of Therapeutic Agents

Abstract

The present disclosure provides compositions comprising one or more therapeutic compounds complexed with nucleic acid fragments to form nanoparticles, and uses thereof.

Description

Cross - Reference to Related Applications

[0001] This application claims priority to Provisional Application Serial No. 62 / 897,254, filed on September 6, 2019, under 35 U.S.C.§119. The disclosure thereof is incorporated herein by reference.

Technical Field

[0002] The present disclosure provides nucleic acid - mediated delivery of therapeutic agents capable of associating or binding with DNA or RNA and uses thereof. Background

[0003] Therapeutic agents capable of associating or binding with DNA or RNA have great potential for treating cancer and other diseases. However, due to their inherent chemical structures, they are completely or partially insoluble, and their bioavailability is limited. Some of these therapeutic agents are soluble, but they lead to systemic toxicity and are often rapidly excreted from the body. Summary

[0004] Disclosed herein is a delivery platform for therapeutic agents that can associate or bind with DNA or RNA, is more effective than current formulations, and has further advantages in terms of manufacturing cost and ease of construction. In the test examples shown herein, a mixture of nucleic acid fragments in the range of 50 - 2,000 nucleotides was used as a bioactive nanocarrier for the intercalator doxorubicin (DOX). It was found that DOX can rapidly and easily complex with nucleic acid fragments. This DOX / nucleic acid formulation was better monodispersed than the nucleic acid fragments themselves and improved the therapeutic concentration range of DOX. As shown in the tests herein, it is clear that the delivery of therapeutic agents capable of associating or binding with DNA or RNA can generally be improved by the use of the delivery platform disclosed herein.

[0005] In certain embodiments, the present disclosure provides a composition comprising one or more therapeutic compounds that complex with nucleic acid fragments to form nanoparticles. In other embodiments or further embodiments of any of the foregoing embodiments, the one or more therapeutic compounds are small molecules that can associate or bind with DNA or RNA. In other embodiments or further embodiments of any of the foregoing embodiments, the nucleic acid fragment is complexed with the one or more therapeutic compounds at a weight ratio of 2:1 to 10:1. In other embodiments or further embodiments of any of the foregoing embodiments, the nucleic acid fragment is complexed with the one or more therapeutic compounds at a weight ratio of 4:1 to 7:1. In other embodiments or further embodiments of any of the foregoing embodiments, the nucleic acid fragment is complexed with the one or more therapeutic compounds at a weight ratio of about 6:1. In other embodiments or further embodiments of any of the foregoing embodiments, the size of the nanoparticles is from 20 nm to 200 nm. In other embodiments or further embodiments of any of the foregoing embodiments, the size of the nanoparticles is from 50 nm to 100 nm. In other embodiments or further embodiments of any of the foregoing embodiments, the one or more therapeutic compounds include anthracycline agents, anthraquinone agents, camptotheca compounds, podophyllum compounds, minor groove binders, bleomycin, and / or actinomycin D. In other embodiments or further embodiments of any of the foregoing embodiments, the one or more therapeutic compounds include aclarubicin, doxorubicin, daunorubicin, idarubicin, epirubicin, amrubicin, pirarubicin, valrubicin, and / or zorubicin. In other embodiments or further embodiments of any of the foregoing embodiments, the one or more therapeutic compounds include doxorubicin. In other embodiments or further embodiments of any of the foregoing embodiments, the one or more therapeutic compounds include mitoxantrone, topotecan, etoposide, teniposide, bleomycin, actinomycin D, and / or duocarmycin A. In other embodiments or further embodiments of any of the foregoing embodiments, one or more of the nucleic acid fragments include ligands that target the nanoparticles to specific cells, tissues, organs, or tumors.In other embodiments or any further embodiments of the foregoing embodiments, the nucleic acid fragment includes fragments of naturally occurring DNA, RNA, and / or DNA-RNA hybrids. In other embodiments or any further embodiments of the foregoing embodiments, the nucleic acid fragment includes chemically synthesized DNA, RNA, and / or DNA-RNA hybrids of different nucleotide lengths. In other embodiments or any further embodiments of the foregoing embodiments, the RNA is modified to replace the 2'-ribose hydroxyl group with an -O-alkyl group or a halide. In other embodiments or any further embodiments of the foregoing embodiments, the nucleic acid fragment is a DNA fragment. In other embodiments or any further embodiments of the foregoing embodiments, the DNA fragment is derived from salmon DNA. In other embodiments or any further embodiments of the foregoing embodiments, the length of the nucleic acid fragment is from 20 nt to 10,000 nt. In other embodiments or any further embodiments of the foregoing embodiments, the length of the nucleic acid fragment is from 50 nt to 2,000 nt. In other embodiments or any further embodiments of the foregoing embodiments, the composition includes nanoparticles of one or more therapeutic compounds complexed with a DNA fragment having a length of 50 nt to 2,000 nt. In other embodiments or any further embodiments of the foregoing embodiments, the one or more therapeutic compounds are selected from aclarubicin, doxorubicin, daunorubicin, idarubicin, epirubicin, amrubicin, pirarubicin, valrubicin, and / or zorubicin. In other embodiments or any further embodiments of the foregoing embodiments, the one or more therapeutic compounds is doxorubicin.

[0006] In certain embodiments, the disclosure also provides a pharmaceutical composition comprising the compositions disclosed herein and a pharmaceutically acceptable carrier, diluent, and / or excipient. In further embodiments, the pharmaceutical composition is formulated for parenteral administration.

[0007] In certain embodiments, the present disclosure further provides a method of treating a subject with cancer in need of treatment, the method comprising administering to the subject an effective amount of the pharmaceutical composition disclosed herein. In further embodiments, the cancer is selected from acute lymphoblastic leukemia, acute myeloblastic leukemia, osteosarcoma, breast cancer, endometrial cancer, gastric cancer, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, liver cancer, kidney cancer, multiple myeloma, neuroblastoma, ovarian cancer, small cell lung cancer, soft tissue sarcoma, thymoma, thyroid cancer, transitional cell bladder cancer, uterine sarcoma, Wilms tumor, and Waldenström macroglobulinemia.

[0008] In certain embodiments, the present disclosure provides a method of treating a human subject with cancer in need of treatment, the method comprising administering an effective amount of the composition disclosed herein. In further embodiments, the cancer is selected from acute lymphoblastic leukemia, acute myeloblastic leukemia, osteosarcoma, breast cancer, endometrial cancer, gastric cancer, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, liver cancer, kidney cancer, multiple myeloma, neuroblastoma, ovarian cancer, small cell lung cancer, soft tissue sarcoma, thymoma, thyroid cancer, transitional cell bladder cancer, uterine sarcoma, Wilms tumor, and Waldenström macroglobulinemia. In other embodiments or further embodiments of any of the foregoing embodiments, the method further comprises administering to the subject one or more anti-cancer agents selected from angiogenesis inhibitors, tyrosine kinase inhibitors, PARP inhibitors, alkylating agents, vinca alkaloids, anthracycline agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, aromatase inhibitors, mTor inhibitors, retinoids, and HDAC inhibitors. In other embodiments, or further embodiments of any of the foregoing embodiments, the method further comprises administering to the subject one or more anti-cancer agents selected from mitoxantrone, topotecan, etoposide, teniposide, bleomycin, actinomycin D, and / or duocarmycin A.

[0009] The accompanying drawings and the following description set forth in detail several embodiments of the present disclosure. Other features, objects, and advantages will be apparent from the description, drawings, and claims.

Brief Description of the Drawings

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[0035] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, "a vector" includes a plurality of such vectors, "the nucleic acid" includes one or more nucleic acids and equivalents thereof known to those of ordinary skill in the art, and the like.

[0036] Also, unless otherwise specified, "or" means "and / or". Similarly, "comprise", "comprising", "include", and "including" are interchangeable and not intended to be limiting.

[0037] It should further be understood that when the term "comprising" is used in the description of various embodiments, those of ordinary skill in the art will understand that in some specific examples, the embodiments can be described by substituting the expressions "consisting essentially of" or "consisting of".

[0038] Unless otherwise defined, 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. Many methods and reagents are similar or equivalent to those described herein, but exemplary methods and materials are disclosed herein.

[0039] All publications mentioned in this specification are hereby incorporated by reference in their entirety to describe and disclose the methodologies that may be used in connection with the description herein. Further, with respect to any terms presented in one or more publications that are similar or identical to terms explicitly defined in this disclosure, the definitions of the terms as explicitly set forth in this disclosure will be followed in all respects.

[0040] In the present disclosure, the term "cancer" is used to encompass cell proliferative disorders, neoplasms, precancerous cell disorders, and cancers, unless specifically described otherwise. Thus, "cancer" refers to any cell that undergoes abnormal cell proliferation leading to metastasis or tumor growth. Exemplary cancers include adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, anorectal cancer, anal canal cancer, appendiceal cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, cutaneous cancer (non-melanoma), biliary tract cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urothelial bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brainstem glioma, cerebellar astrocytoma, cerebellar astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer (including triple-negative breast cancer), bronchioloalveolar carcinoma / carcinoid, carcinogenic tumor, gastrointestinal and nervous system cancer, nervous system lymphoma, central nervous system cancer, primary central nervous system malignant lymphoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colorectal cancer, colon cancer, cutaneous T-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Sézary syndrome, endometrial cancer, esophageal cancer, germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoma tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor glioma, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypothalamic cancer, intraocular melanoma, eye cancer, pancreatic islet cell tumor (endocrine pancreas), Kaposi sarcoma, kidney cancer, renal cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin lymphoma, primary central nervous system malignant lymphoma, Waldenström macroglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma, mesothelioma, metastatic squamous neck cancer, oral cancer, tongue cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorder, chronic myelogenous leukemia, acute myelogenous leukemia, multiple myeloma, chronic myeloproliferative disorder, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, pancreatic cancer, pancreatic islet cell pancreatic cancer, paranasal and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma,Pineal blastoma / tentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, metastatic cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing sarcoma, soft tissue sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), papilloma, actinic keratosis and keratoacanthoma, Merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, extraosseous primitive neuroectodermal tumor, testicular cancer, laryngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, renal pelvis, ureter and other urothelial carcinomas, gestational trophoblastic tumor, urethral cancer, endometrial uterine cancer, uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer, and Wilms tumor are included, but not limited to these. In certain embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute myeloblastic leukemia, osteosarcoma, breast cancer, endometrial cancer, gastric cancer, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, liver cancer, kidney cancer, multiple myeloma, neuroblastoma, ovarian cancer, small cell lung cancer, soft tissue sarcoma, thymoma, thyroid cancer, transitional cell bladder cancer, uterine sarcoma, Wilms tumor, and Waldenström macroglobulinemia.,

[0041] As used herein, the term "disorder" reflects all physical abnormalities in a human or animal body, or parts that impair normal function, and is generally synonymous with the terms "disease", "syndrome", and "condition" (as medical conditions) in that it typically manifests by obvious signs and symptoms and is intended to be used interchangeably.,

[0042] As used herein, the term "uncontrolled-release excipient" refers to an excipient whose main function does not include modification of the duration or location of release of the active substance from the dosage form, compared to conventional immediate-release dosage forms.,

[0043] As used herein, the terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", "physiologically acceptable carrier", or "physiologically acceptable excipient" refer to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each component should be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the drug. It should also be suitable for use in contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications commensurate with a reasonable benefit / risk ratio. Examples of "pharmaceutically acceptable carriers" and "pharmaceutically acceptable excipients" can be found in Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 5th Edition; Edited by Rowe et al., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; Handbook of Pharmaceutical Additives, 3rd Edition; Edited by Ash, Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Edited by Gibson, CRC Press LLC: Boca Raton, Fla., 2004.

[0044] As used herein, the term "therapeutic agent capable of associating or binding with DNA or RNA" refers to a small molecule that can associate or bind with DNA or RNA and can be used to treat, typically, a disorder or disease such as cancer in a subject. Examples of "therapeutic agents capable of associating or binding with DNA or RNA" include anthracycline agents (e.g., aclarubicin, amrubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, pirarubicin, valrubicin, and zorubicin); anthracenedione agents (e.g., mitoxantrone and pixantrone); camptothecin genus compounds (e.g., belotecan, camptothecin, cositecan, exatecan, gimitecan, irinotecan, larototecan, rubitecan, silatecan, and topotecan); podophyllum genus compounds (e.g., etoposide and teniposide); bleomycin; actinomycin D; minor groove binders (e.g., duocarmycin A, adozelesin, bizelesin, and carzelesin); purine antagonists (e.g., cladribine, clofarabine, nelarabine, mercaptopurine, thioguanine, and pentostatin); pyrimidine antagonists (e.g., capecitabine, carmofur, doxifluridine, floxuridine, fluorouracil, tegafur, cytarabine, gemcitabine, azacitidine, and decitabine); folic acid antagonists (e.g., aminopterin, methotrexate, pemetrexed, and pralatrexate); and alkylating agents (e.g., cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, melphalan, prednimustine, bendamustine, chloromethine, uramustine, carmustine, fotemustine, lomustine, nimustine, ranimustine, streptozocin, mannosulfan, treosulfan, carbocian, thiotepa, triaziquone, triethylenemelamine, carboplatin, cisplatin, dicycloplatin, nedaplatin, oxaliplatin, satraplatin, temozolomide, dacarbazine, mitobronitol, pipobroman, and procarbazine), but are not limited thereto.

[0045] As used herein, the term "excipient with controlled release" refers to an excipient whose primary function is to modify the duration or location of release of an active substance from a dosage form, as compared to conventional immediate-release dosage forms.

[0046] The term "therapeutically acceptable" refers to a compound (or salt, prodrug, tautomer, zwitterionic form, etc.) that is suitable for use in contact with the tissues of a patient without undue toxicity, irritation, allergic response, or immunogenicity commensurate with a reasonable benefit / risk ratio and that is effective for its intended use.

[0047] As used herein, the terms "treat", "treating", and "treatment" refer to ameliorating a condition associated with a disease or disorder (e.g., cancer). This includes preventing or delaying the onset of symptoms of the disease or disorder and / or reducing the severity or frequency of symptoms of the disease or disorder.

[0048] As used herein, the term "subject" refers to an animal including, but not limited to, primates (e.g., humans, monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, guinea pigs, hamsters, ferrets, etc.), rabbits, pigs (e.g., pigs, mini-pigs), horses, dogs, and cats. The terms "subject" and "patient" are used interchangeably herein. For example, a mammalian subject can refer to a human patient.

[0049] Therapeutic agents capable of associating or binding with DNA or RNA have great potential for treating cancer and other diseases, but due to their inherent chemical structures, they are insoluble and their bioavailability can be insufficient. Some of these therapeutic agents are soluble, but they can lead to systemic toxicity and are often rapidly excreted from the body. Currently, as a solution to these problems, there is a method of delivering such compounds (e.g., anthracycline drugs) using nanocarriers, but this generally has drawbacks such as very low drug loading, immunogenicity, slow clearance of the carrier, resulting in low therapeutic efficacy, and a significant increase in cost. For example, doxorubicin (DOXIL), a polyethylene glycolylated (PEGylated) liposomal formulation of doxorubicin, has all of these limitations. This improves the safety profile of doxorubicin but is less effective than DOX. In fact, the long-term circulation of DOXIL in the bloodstream allows the immune system to express antibodies against the PEGylated moieties on the particles. Thus, the main drawback of the current solutions lies in the biocompatibility of the nanocarriers. Furthermore, the current solutions are not easy to assemble. In contrast, the compositions and methods of the present disclosure can be assembled in a simple manner and are more cost-effective. For example, for another DOX nanocarrier, HPMA-DOX (N-(2-hydroxypropyl)methylacrylamide polymer-doxorubicin), a shorter circulation time (20.1 hours) has been reported compared to the compositions described herein, but it is difficult to scale up to commercial levels and the assembly process is more complex. Others have synthetic nucleic acid systems for delivering chemotherapeutic agents (e.g., for DOX and cytosine deaminase delivery, nucleic acids are clicked), but such formulations are very costly and time-consuming and difficult to reach the commercial stage. Furthermore, PEGylation of such formulations will result in immunogenicity problems similar to those of other PEGylated delivery systems such as DOXIL reported previously.

[0050] As shown in the in vitro and in vivo tests presented herein, by using nucleic acid fragments as a delivery vehicle for DOX, safety and efficacy were improved in the treatment of induced solid tumors in mice. In particular, in the in vivo tests presented herein, treatment with DOX / DNA nanoparticles improved survival and delayed tumor growth compared to treatment with DOX alone. As demonstrated by in vitro cytotoxicity tests and in vivo blood circulation tests at 24, 48, and 72 hours, the aforementioned favorable results would be due to the longer circulation of DOX / DNA nanoparticles and the controlled release of DOX from DNA. By these two means, DOX / DNA nanoparticles exhibit a chemotherapeutic effect superior to treatment with DOX alone and superior to treatment with DOXIL. DOXIL has been shown to be effective in reducing systemic toxicity but has not resulted in an improvement in the therapeutic effect. Furthermore, PEGylation of DOXIL and repeated administration of the chemotherapeutic formulation have been shown to result in immunogenicity. Other delivery vehicles in the field of nanotherapeutics are, unfortunately, very complex in their formulation and production and thus pose difficulties for scale-up production. Since both the therapeutic agent and the nucleic acid are already manufactured on a commercial scale, the therapeutic agent / nucleic acid formulations, preparations, and compositions are easy for manufacturing and commercial scale-up production.

[0051] In certain embodiments, the present disclosure provides a composition, preparation or formulation comprising one or more therapeutic agents complexed with nucleic acids to form nanoparticles. Examples of therapeutic agents that can be complexed with nucleic acids to form nanoparticles include norepinephrine reuptake inhibitors (NRIs) such as atomoxetine; dopamine reuptake inhibitors (DARIs) such as methylphenidate; serotonin-norepinephrine reuptake inhibitors (SNRIs) such as milnacipran; sedatives such as diazepam; norepinephrine-dopamine reuptake inhibitors (NDRIs) such as bupropion; serotonin-norepinephrine-dopamine reuptake inhibitors (SNDRIs) such as venlafaxine; monoamine oxidase inhibitors such as selegiline; hypothalamic phospholipids; endothelin converting enzyme (ECE) such as phosphoramidon; thromboxane receptor antagonists such as ifetroban; potassium channel openers; thrombin inhibitors such as hirudin; hypothalamic phospholipids; growth factor inhibitors such as modifiers of PDGF activity; platelet activating factor (PAF) antagonists; low molecular weight heparins such as enoxaparin; factor VIIa inhibitors and factor Xa inhibitors; renin inhibitors; neutral endopeptidase (NEP) inhibitors; vasopeptidase inhibitors (dual NEP-ACE inhibitors) such as omapatrilat and gemopatrilat; HMG CoA reductase inhibitors such as pravastatin, lovastatin, atorvastatin, simvastatin, NK-104 (also known as itavastatin, nisvastatin, or nisvastatin) and ZD-4522 (also known as rosuvastatin, or atavasin or bisvastatin); squalene synthase inhibitors; fibrate drugs; bile acid sequestrants such as cholestyramine; niacin; antiatherosclerotic agents such as ACAT inhibitors; MTP inhibitors; calcium channel blockers such as amlodipine besylate; potassium channel activators; α-muscarinic drugs; β-muscarinic drugs such as carvedilol and metoprolol; antiarrhythmic agents;Diuretics such as chlorothiazide, hydrochlorothiazide, flumethiazide, hydroflumethiazide, bendroflumethiazide, methylchlorothiazide, trichloromethylthiazide, polythiazide, benzothiazide, ethacrynic acid, tricrinaphene, chlorthalidone, furosemide, muzolimine, bumetanide, triamterene, amiloride, and spironolactone; antidiabetic agents such as biguanides (e.g., metformin), glucosidase inhibitors (e.g., acarbose), insulin, meglitinides (e.g., repaglinide), sulfonylureas (e.g., glimepiride, glipizide, and glyburide), thiazolidinediones (e.g., troglitazone, rosiglitazone, and pioglitazone); mineralocorticoid receptor antagonists such as PPAR-γ agonists, spironolactone and eplerenone; growth hormone secretagogues; aP2 inhibitors; phosphodiesterase inhibitors such as PDE III inhibitors (e.g., cilostazol) and PDE V inhibitors (e.g., sildenafil, tadalafil, and vardenafil); protein tyrosine kinase inhibitors; antiproliferative agents such as methotrexate, FK506 (tacrolimus, prograf), mycophenolate mofetil; chemotherapeutic agents; immunosuppressive agents; anticancer agents and cytotoxic agents (e.g., alkylating agents such as nitrogen mustard, alkyl sulfonates, nitrosoureas, ethyleneimines, and triazenes); antimetabolites such as folic acid antagonists, purine analogs, and pyridone analogs; antibiotics such as anthracycline agents, bleomycin, mitomycin, dactinomycin, and plicamycin; enzymes such as L-asparaginase; farnesyl-protein transferase inhibitors; hormonal agents such as glucocorticoids (e.g., cortisone), estrogen / antiestrogen, androgen / antiandrogen, progestin, luteinizing hormone-releasing hormone antagonists, and octreotide acetate; microtubule disrupting agents such as ecteinascidin; microtubule stabilizing agents such as paclitaxel, docetaxel, and epothilones A-F; plant-derived products such as vinca alkaloids, epipodophyllotoxins, and taxanes; topoisomerase inhibitors; polyphenol compounds; polyketide compounds; prenyl-protein transferase inhibitors; and cyclosporine;Cytotoxic drugs such as azathioprine and cyclophosphamide; TNF-α inhibitors such as tenidap; anti-TNF antibodies or soluble TNF receptors such as etanercept, rapamycin, and leflunomide; cyclooxygenase-2 (COX-2) inhibitors such as celecoxib and rofecoxib; various agents such as hydroxyurea, procarbazine, mitotane, and hexamethylmelamine; gold compounds; platinum coordination complexes such as cisplatin, satraplatin, and carboplatin, but not limited to these. As demonstrated in the test examples presented herein, the DOX / nucleic acid nanoparticles disclosed herein can be used for effective treatment of cancer. However, it should be understood that all diseases or disorders treatable by therapeutic agents are encompassed by this disclosure.;

[0052] In certain embodiments, the present disclosure provides a composition, preparation or formulation of a therapeutic agent comprising a polyphenol complexed with a nucleic acid to form nanoparticles. Polyphenols are a class of organic compounds that are not only natural products, but also synthetic or semi-synthetic, characterized by having a number of phenolic structural units. The specific physical, chemical, and biological properties (metabolism, toxicity, treatment, etc.) of each member of this class are determined by the number and characteristics of these phenolic structures. Many polyphenols are micronutrients produced as secondary metabolites by edible plants. These compounds have poor bioavailability (only a fraction of the intake is absorbed and the excretion is rapid), exhibit complex pharmacokinetics and metabolism, but exhibit therapeutic properties. Substantial evidence (epidemiological studies, animal studies, and clinical trials in humans) indicates that polyphenols reduce the associated conditions of cardiovascular diseases including thrombosis (Navarro-Nunez et al., J Agric Food Chem. 2008;56:2970-2976) and atherosclerosis (Chiva-Blanch et al., Am J Clin Nutr. 2012;95:326-3434) and inflammation (Rieder et al., Br J Pharmacol. 2012;167:1244-1258), and exhibit anti-cancer effects (Gali et al., Cancer Res. 1991;51:2820-2825) and neuroprotective effects (Gatson et al., J Trauma Acute Care Surg. 2013;74:470-475). The activities of these compounds are achieved through various mechanisms including, namely, a very characteristic antioxidant action (Pignatelli et al., Atherosclerosis. 2006;188:77-83), inhibition of intracellular kinase activity (Wright et al., Regen Med.2012;7:295-307), binding to cell surface receptors (Jacobson et al., Adv Exp Med Biol. 2002 505:163-171), and complete disruption of the cell plasma membrane (Pawlikowska-Pawlega et al., Biochim Biophys Acta. 2007;1768:2195-2204). Research on the application of polyphenols is increasing particularly in the functional food, dietary supplement, and pharmaceutical industries.However, as a problem in human health, the effectiveness of polyphenols depends on maintaining the stability, bioactivity, and bioavailability of bioactive compounds. Also, some phenolic compounds have an unpleasant taste, which limits their pharmaceutical use. Encapsulation or complexation of the polyphenols and nucleic acids disclosed herein can be effectively useful in solving some of the drawbacks found in free polyphenol compounds. Since the compositions and methods disclosed herein relate to a platform-based polyphenol delivery system, it is expected that any type of polyphenol can be complexed or encapsulated by the nucleic acids disclosed herein. Examples of such polyphenol compounds include, but are not limited to, xanthohumol, epicatechin, epigallocatechin, EGCG, flavanols such as procyanidins, flavanones such as hesperidin and naringenin, flavones such as apigenin, chrysin, and luteolin, flavonols such as quercetin, kaempferol, myricetin, isorhamnetin, and galangin; isoflavonoids such as genistein and daidzein; phenolic acids such as ellagic acid, gallic acid, ferulic acid, and chlorogenic acids; lignans such as sesamin and secoisolariciresinol diglucoside; and stilbenes such as resveratrol, pterostilbene, and piceatannol. Accordingly, the present disclosure provides a platform technology that enables the delivery of safe, efficient, and controlled polyphenols in a subject for treating any number of diseases or disorders treatable with polyphenol compounds. For example, as demonstrated in numerous studies, Polyphenols limit the development of coronary heart disease (Renaud et al., Lancet. 1992; 339:1523-1526; Dubick et al., J Nutraceut Functional & Med Foods. 2001;3:67-93; Nardini et al., Platelets. 2007;18:224-243; and Vita et al., Am Clin Nutr. 2005;81:292-297), type II diabetes (Rizvi et al., Clin Exp Pharmacol Physiol. 2005;32:70-75; Matsui et al., J Agric Food Chem. 2002;50:7244-7248; Dembinska-Kiec et al., Br J Nutr 20. 2008; 99:109-117; and Chen et al., Eur J Pharmacol. 2007;568:269-277), obstructive pulmonary disease (Tabak et al., Am J Respir Crit Care Med. 2001;164:61-64; and Woods et al., Am J Clin Nutr. 2003;78:414-421), and neurodegenerative diseases (Ajami et al., Neuroscience & Biobehavioral Reviews 2007;73:39-47; and Mandel et al., Free Radical Biology and Medicine 2004;37(3):304-317). Further note that any number of polyphenolic compounds can be complexed with the nucleic acids disclosed herein to produce polyphenol / nucleic acid nanoparticles, so the preparations, compositions, or formulations disclosed herein are not limited to the delivery of one specific polyphenolic compound.

[0053] In addition, the polyketide compound can complex with the nucleic acids disclosed herein, or both the polyketide compound and the polyphenol compound can complex with the nucleic acids disclosed herein. Polyketides are a large group of secondary metabolites that contain substituted carbonyl and methylene groups (-CO-CH2-) or are derived from precursors containing such substituents. Many polyketides have antibacterial and immunosuppressive properties. Similar to polyphenol compounds, polyketide compounds can form π-π stacking interactions with the nucleic acid species disclosed herein to form polyketide / nucleic acid nanoparticles.

[0054] In certain embodiments, the present disclosure provides a composition, preparation, or formulation comprising one or more therapeutic agents capable of associating or binding to the disclosed DNA or RNA. These complex with the nucleic acid to form nanoparticles. Examples of therapeutic agents that can complex with nucleic acids to form nanoparticles include anthracycline agents (e.g., aclarubicin, amrubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, pirarubicin, valrubicin, and zorubicin); anthracenedione agents (e.g., mitoxantrone and pixantrone); camptotheca compounds (e.g., belotecan, camptothecin, cositecan, exatecan, gimitecan, irinotecan, larototecan, rubitecan, silatecan, and topotecan); podophyllum compounds (e.g., etoposide and teniposide); bleomycin; actinomycin D; minor groove binders (e.g., duocarmycin A, adozelesin, bizelesin, and carzelesin); purine antagonists (e.g., cladribine, clofarabine, nelarabine, mercaptopurine, thioguanine, and pentostatin); pyrimidine antagonists (e.g., capecitabine, carmofur, doxifluridine, floxuridine, fluorouracil, tegafur, cytarabine, gemcitabine, azacitidine, and decitabine); folic acid antagonists (e.g., aminopterin, methotrexate, pemetrexed, and pralatrexate); and alkylating agents (e.g., cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, melphalan, prednimustine, bendamustine, chloromethine, uramustine, carmustine, fotemustine, lomustine, nimustine, ranimustine, streptozocin, mannosulfan, treosulfan, carbocine, thiotepa, triaziquone, triethylenemelamine, carboplatin, cisplatin, dicroplatin, nedaplatin, oxaliplatin, satraplatin, temozolomide, dacarbazine, mitobronitol, pipobroman, and procarbazine), but are not limited thereto.In certain embodiments, one or more therapeutic agents capable of associating or binding with DNA or RNA are selected from anthracycline agents (e.g., aclarubicin, amrubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, pirarubicin, valrubicin, and zorubicin); anthracenedione agents (e.g., mitoxantrone and pixantrone); camptotheca compounds (e.g., belotecan, camptothecin, cositecan, exatecan, gimitecan, irinotecan, larototecan, rubitecan, silatecan, and topotecan); podophyllum compounds (e.g., etoposide and teniposide); bleomycin; actinomycin D; and minor groove binders (e.g., duocarmycin A, adozelesin, bizelesin, and carzelesin). In further embodiments, one or more therapeutic agents capable of associating or binding with DNA or RNA include aclavin, ambrabicin, daunorubicin, doxorubicin, epirubicin, izrubicin, pirarubicin, valrubicin, and / or zrubicin. In another embodiment, one or more therapeutic agents capable of associating or binding with DNA or RNA include mitoxantrone, topotecan, etoposide, teniposide, bleomycin, actinomycin D, and / or duocarmycin A.

[0055] The compositions and methods disclosed herein relate to platform-based therapeutic delivery systems, and thus any type of therapeutic compound that associates or binds with DNA or RNA is expected to be complexed or encapsulated by the nucleic acids disclosed herein. Examples of such therapeutic compounds include anthracycline agents (e.g., aclarubicin, amrubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, pirarubicin, valrubicin, and zorubicin); anthracenedione agents (e.g., mitoxantrone and pixantrone); camptotheca compounds (e.g., belotecan, camptothecin, cositecan, exatecan, gimitecan, irinotecan, larotecan, rubitecan, silatecan, and topotecan); podophyllum compounds (e.g., etoposide and teniposide); bleomycin; actinomycin D, but are not limited thereto. Accordingly, the present disclosure provides formulations, compositions, or platform technologies that can be used for the safe, efficient, and controlled delivery of therapeutic agents that can associate or bind with DNA or RNA in a subject for treating any number of diseases or disorders treatable by such therapeutic agents. As demonstrated in the test examples presented herein, the DOX / nucleic acid nanoparticles disclosed herein can be used for the effective treatment of cancer. However, it should be understood that any disease or disorder treatable by a therapeutic agent that can associate or bind with DNA or RNA is encompassed by the present disclosure. Further note that since any number of therapeutic agents that can associate or bind with DNA or RNA can be complexed with the nucleic acids disclosed herein to produce therapeutic agent / nucleic acid nanoparticles, the preparations, compositions, or formulations disclosed herein are not limited to the delivery of a single specific therapeutic agent that associates or binds with a particular DNA or RNA.

[0056] Regarding the nucleic acid component of the therapeutic agent / nucleic acid nanoparticles, any type and length of nucleic acid species can be used and complexed with a therapeutic agent that can be associated with or bind to DNA or RNA. That is, the nucleic acid species should be able to form π-π stacking interactions with a therapeutic agent that can be associated with or bind to DNA or RNA. Although DNA was used in the tests presented herein, DNA, RNA, DNA-RNA hybrids, or mixtures thereof can also be used to form the therapeutic agent / nucleic acid nanoparticles disclosed herein. Further, for the purposes of this disclosure, "nucleic acid" includes nucleic acid analogs. A nucleic acid is a chain of nucleotides consisting of three parts: namely, a phosphate backbone, a pentose sugar (ribose or deoxyribose), and one of four nucleobases. Nucleic acid analogs may have any of these modifications.

[0057] DNA (abbreviation for deoxyribonucleic acid) is an organic chemical substance having a complex molecular structure found in all prokaryotic and eukaryotic cells, as well as many viruses. DNA encodes genetic information for the transmission of genetic traits. Each strand of the DNA molecule consists of a long chain of monomer nucleotides. The nucleotides of DNA consist of a deoxyribose sugar molecule to which a phosphate group is attached and one of four nitrogenous bases (i.e., two purines (adenine and guanine) and two pyrimidines (cytosine and thymine)). The nucleotides are joined together by covalent bonds between the phosphate of one nucleotide and the sugar of the next, forming a phosphate-sugar backbone with the nitrogenous bases protruding. One strand is held to the other by hydrogen bonds between the bases, and the sequence of these bonds is specific, i.e., adenine binds only to thymine and cytosine binds only to guanine. The structure of the DNA molecule is very stable, so that it can act as a template for the replication of new DNA molecules and also as a template for the production (transcription) of related RNA (ribonucleic acid) molecules.

[0058] RNA (abbreviation for ribonucleic acid) is a high-molecular-weight complex compound that functions in protein synthesis in cells and alternates with DNA (deoxyribonucleic acid) as a carrier of the genetic code in some viruses. RNA consists of ribonucleotides (nitrogenous bases attached to ribose sugars) linked by phosphodiester bonds and forms chains of various lengths. The nitrogenous bases in RNA are adenine, guanine, cytosine, and uracil, which replaces thymine in DNA. The ribose sugar of RNA is a ring structure consisting of five carbons and one oxygen. The presence of a chemically reactive hydroxyl group (-OH group) attached to the second carbon group within the ribose sugar molecule makes RNA prone to hydrolysis. On the other hand, DNA does not have a reactive -OH group at the same position on the sugar moiety (deoxyribose). This chemical property of RNA, compared to DNA, is considered one of the reasons why DNA is the preferred carrier of genetic information in most organisms. In certain embodiments, the reactive -OH group of RNA may be substituted with a less reactive -O-alkyl group or halide group, thereby rendering the RNA resistant to the action of ribonucleases.

[0059] DNA-RNA hybrids are abundant in human cells. They are formed during transcription when nascent RNA is in proximity to its DNA template. The resulting RNA / DNA hybrids and displaced single-stranded (ss) DNA are called R-loops. RNA / DNA hybrids are structurally different from the corresponding double-stranded DNA and are more stable than it. RNA / DNA hybrids are found at replication origins, immunoglobulin class switch regions, and transcription complexes. RNA / DNA hybrids do not adopt the traditional B conformation of DNA or the A conformation of RNA but occur as a mixture or heteroduplex.

[0060] For the purposes of the present disclosure, nucleic acid fragments can result from enzymatic cleavage or physical disruption of naturally occurring nucleic acids, chemical synthesis of nucleic acids of various sizes, or some combination thereof. Nucleic acids of any species from prokaryotes, eukaryotes, eukaryotes, etc. can be used. In certain embodiments, the nucleic acid fragment is derived from salmon DNA. Further, the size and length of the nucleic acid fragment can be varied to suit the particular treatment being used. For example, the nucleic acid fragment can have a length of 20 nt, 30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 100 nt, 110 nt, 120 nt, 130 nt, 140 nt, 150 nt, 160 nt, 170 nt, 180 nt, 190 nt, 200 nt, 250 nt, 300 nt, 350 nt, 400 nt, 450 nt, 500 nt, 550 nt, 600 nt, 650 nt, 700 nt, 750 nt, 800 nt, 850 nt, 900 nt, 950 nt, 1,000 nt, 1,500 nt, 2,000 nt, 2,500 nt, 3,000 nt, 3,500 nt, 4,000 nt, 4,500 nt, 5,000 nt, 5,500 nt, 6,000 nt, 6,500 nt, 7,000 nt, 7,500 nt, 8,000 nt, 8,500 nt, 9,000 nt, 9,500 nt, 10,000 nt, or a length between any two of the foregoing lengths (e.g., 20 nt to 10,000 nt, 50 nt to 2,000 nt, etc.). The sequence of the nucleic acid can be random or selected to have a desired sequence. In the latter case, the sequence can be selected to target a transcription factor (TF), TLR, or other DNA or RNA binding protein, or be an aptamer. In such cases, a specific sequence or ligand for a tumor-specific antigen can be selected to target the therapeutic agent / nucleic acid nanoparticles to a specific tissue, organ, or tumor.Ligands for tumor-specific antigens are commercially available from various vendors and thus do not need to be newly produced (see, for example, Elabscience, Santa Cruz biotechnology, Biospacific, Novus Biologicals, etc.). In certain embodiments, the ligand conjugated to the therapeutic agent / nucleic acid nanoparticle binds to a tumor-specific antigen selected from alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, CA15-3, CA19-9, MUC-1, epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), ras or p53, CTAG1B, MAGEA1, the abnormal product of HER2 / neu. The ligand that binds to the tumor-specific antigen has a high affinity (K) to efficiently uptake into the target tumor cells. dIt must bind to the target antigen at a concentration of <10 nM) and have minimal immunogenicity. In a further embodiment, the ligand that binds to the tumor-specific antigen binds to the therapeutic agent / nucleic acid nanoparticles disclosed herein via the use of cleavable linkers (acid-labile linkers, protease-cleavable linkers, and disulfide linkers). Acid-labile linkers are designed to be stable at blood pH levels but become unstable and degrade when in the low pH environment of lysosomes. Protease-cleavable linkers are also designed to be stable in blood / plasma but rapidly release the free drug in the lysosomes within cancer cells upon cleavage by lysosomal enzymes. They utilize the high level of protease activity within lysosomes and contain peptide sequences that are recognized and cleaved by these proteases, such as the dipeptide Val-Cit bond that is rapidly hydrolyzed by cathepsin. A third type of linker that can be used to attach the ligand to the therapeutic agent / nucleic acid nanoparticles contains a disulfide bond. The linker utilizes the high level of intracellular reduced glutathione to release the free drug intracellularly. Reagents such as Traut's reagent (2-iminothiolane), MBS (3-maleimidobenzoic acid N-hydroxysuccinimide ester), and SATA (N-succinimidyl S-acetylthioacetate) can convert such primary amine groups to sulfhydryls and then form disulfide bonds with ligands containing cysteine residues. Other reagents, such as SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), and sulfo-SMCC, can be used as linkers to attach ligands to the nucleic acids of the therapeutic agent / nucleic acid nanoparticles. Examples of how such groups can be used to attach ligands to the therapeutic agent / nucleic acid nanoparticles can be found at www:labome.com / method / Antibody-Conjugation.html and the following references cited therein: Safdari et al., Monoclon Antib Immunodiagn Immunother.2013 32:409-12; Joosten V et al., Microb Cell Fact. 2003 2:1; Winter et al., Trends Pharmacol Sci. 1993 14:139-43; Arbabi et al., Front Immunol. 2017 8:1589; Brinkley et al., Bioconjug Chem. 1992 3:2-13; Vlasak et al., MAbs. 2011 3:253-63; Ducancel et al., MAbs. 2012 4:445-57; McCombs et al., AAPS J. 2015;17:339-51; Hondal R., Protein Pept Lett. 2005 12:757-64; Zimmerman et al., Bioconjug Chem. 2014 25:351-61; Traut et al., Biochemistry. 1973 12:3266-73; Knight P., Biochem J. 1979 179:191-7; Carlsson et al., Biochem J. 1978 173:723-37; Peeters J et al., J Immunol Methods. 1989 120:133-43; Hashida et al., J Appl Biochem. 1984 6:56-63; Avrameas et al., Immunochemistry. 1971 8:1175-9; Richards et al., J Mol Biol. 1968 37:231-3; Chandler et al., J Immunol Methods. 1982 53:187-94; Coulepis et al., J Clin Microbiol. 1985 22:119-24; White et al., J Clin Microbiol. 1989 27:2300-4; Liu et al., J Immunol Methods. 2000 234:P153-67; Tian et al., Bioconjug Chem. 2015 26:1144-55; Vira et al., Anal Biochem. 2010 402:146-50; Szabo et al., Biophys J. 2018 114:688-700; Hagan et al., Lanthanide-Anal Bioanal Chem. 2011 400:2847-64; Han et al., Nat Protoc.2018 13 2121-2148; Bottrill et al., Chem Soc Rev. 2006 35:557-71; Ye et al., J Clin Lab Anal. 2014 28:335-40; Fernandez Moreira et al., Analyst. 2010 135:42-52; Brouwers et al., J Nucl Med. 2004 45:327-37; Vera et al., Nucl Med Biol. 2012 39:3-13; Stein et al., J Nucl Med. 2001 42:967-74; Bratthauer G., Methods Mol Biol. 2010 588:257-70; Engle et al., Science. 2019 364:1156-1162; Sano et al., Science. 1992 258:120-2; Malou et al., Trends Microbiol. 2011 19:295-302; Cardoso et al., Curr Med Chem. 2012;19:3103-27; East et al., Methods Mol Biol. 2014 1199:67-83; Tan et al., Nanomaterials(Basel). 2015 5:1297-1316; Geng et al., Bioconjug Chem. 2016 27:2287-2300; Pecanha et al., J Immunol.1991 146:833-9; Pecanha et al., J Immunol.1993 150:2160-8; and Chen Y., Methods Mol Biol.2013 1045:267-73. These disclosures are incorporated herein by reference.

[0061] The examples of DOX / DNA nanoparticles disclosed in the tests shown in this specification are polydisperse depending on the nature of the nucleic acid used, but it is expected that monodisperse nanoparticles can be produced by selecting the nucleic acid. Such monodisperse nanoparticles may result in more improved therapeutic outcomes. Also, a cationic molecule (e.g., PTD domain) can be used to complex the nucleic acid constituting the nanoparticles disclosed herein (e.g., by minimizing degradation by nucleases), providing or improving the controlled release characteristics of the nanoparticles. Further, by utilizing the hygroscopicity of the nucleic acid, a hydrogel loaded with a therapeutic agent can be produced, and the nucleic acid can be conjugated to a protein (e.g., thymosin-α1) to provide a multimodal approach in the treatment of diseases or disorders by the nanoparticles disclosed herein. As a multimodal approach by stimulating the immune system to fight cancer while delivering an anti-cancer therapeutic compound, for example, a therapeutic agent / nucleic acid nanoparticle can be conjugated to an immune-enhancing protein such as thymosin-α1.

[0062] A therapeutic agent can be complexed with a nucleic acid at a certain weight ratio (wt / wt) to form nanoparticles. For example, the nucleic acid fragment can be complexed with one or more therapeutic compounds at a weight ratio of about 1:20, 1:15, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, or a range that includes any two of the aforementioned ratios or is between them (including their separate increments (e.g., 2:1 to 10:1, 4:1 to 7:1, 4.5:1 to 6.5:1, etc.)). In certain embodiments, the nucleic acid fragment is complexed with one or more therapeutic compounds at a weight ratio of about 6:1. Also, based on the concentration of the raw materials, reaction parameters (e.g., temperature, time, etc.), and the addition of agents (e.g., surfactants, salts, etc.), the size of the therapeutic agent / nucleic acid nanoparticles can also be controlled.In certain embodiments, the size of the therapeutic agent / nucleic acid nanoparticles is about 10 nm, 12 nm, 14 nm, 15 nm, 16 nm, 18 nm, 20 nm, 22 nm, 24 nm, 25 nm, 26 nm, 28 nm, 30 nm, 32 nm, 34 nm, 35 nm, 36 nm, 38 nm, 40 nm, 42 nm, 44 nm, 45 nm, 46 nm, 48 nm, 50 nm, 52 nm, 54 nm, 55 nm, 56 nm, 58 nm, 60 nm, 62 nm, 64 nm, 65 nm, 66 nm, 68 nm, 70 nm, 72 nm, 74 nm, 75 nm, 76 nm, 78 nm, 80 nm, 82 nm, 84 nm, 85 nm, 86 nm, 88 nm, 90 nm, 92 nm, 94 nm, 95 nm, 96 nm, 98 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, or a range including any two of the foregoing or therebetween (including their respective increments (e.g., 20 nm to 200 nm, 50 nm to 100 nm, etc.)). In certain embodiments, the size of the therapeutic agent / nucleic acid nanoparticles is about 70 nm. The nanoparticles can generally have any shape including spherical, oval, cubic, hexagonal, prismatic, rod-shaped, helical, triangular, star-shaped, or irregular shapes.

[0063] In one embodiment, the present disclosure provides a pharmaceutical composition comprising the therapeutic agent / nucleic acid nanoparticles disclosed herein. The pharmaceutical composition can be formulated using carriers, excipients, additives, or adjuvants into a form suitable for administration to a subject. Frequently used carriers or adjuvants include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk proteins, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycol, and solvents (e.g., sterile water, alcohol, glycerol, and polyhydric alcohols). Intravenous media include fluids and nutrient supplements. Preservatives include antibacterial agents, antioxidants, chelating agents, cryoprotectants, and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions containing salts, preservatives, buffers, etc., and non-toxic excipients. See, for example, Remington's Pharmaceutical Sciences, 15th Edition, Easton: Mack Publishing Co., 1405-1412, 1461-1487 (1975), and The National Formulary XIV, 14th Edition, Washington: American Pharmaceutical Association (1975). These contents are incorporated herein by reference. The pH and exact concentrations of the various components of the pharmaceutical composition are adjusted according to routine skill in the art. See Goodman and Gilman’s, The Pharmacological Basis for Therapeutics (7th Edition).

[0064] The pharmaceutical composition according to the present disclosure can be administered locally or systemically in a therapeutically effective amount. As used herein, "administering a therapeutically effective amount" is intended to include a method of imparting or applying the pharmaceutical composition of the present disclosure to a subject to effect the intended therapeutic function. The therapeutically effective amount varies depending on factors such as the degree of infection, age, gender, and body weight of the subject. The dosing schedule can be adjusted to provide an optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally decreased depending on the urgency of the therapeutic situation.

[0065] The pharmaceutical composition can be administered by convenient methods such as injection (e.g., subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the pharmaceutical composition may be coated with a material to protect it from enzymes, acids, and other natural state actions that can inactivate the pharmaceutical composition. The pharmaceutical composition can also be administered parenterally or intraperitoneally. Dispersants can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under normal storage and use conditions, these formulations may contain preservatives to prevent the growth of microorganisms.

[0066] A pharmaceutical composition suitable for injection comprises a sterile aqueous solution (water-soluble) or dispersion, and sterile powder, for immediate preparation of a sterile injectable solution or dispersion. The composition is typically sterile and fluid to an extent that allows for easy injection. Typically, the composition is stable under the conditions of manufacture and storage and is preserved against contamination with microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium including, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. For example, coating agents such as lecithin can be used to maintain the required particle size in the case of a dispersant, or surfactants can be used to maintain appropriate fluidity. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc., can prevent the action of microorganisms. In many cases, isotonic agents, such as sugars and polyhydric alcohols (such as mannitol, sorbitol or sodium chloride), are used in the composition. By including an agent in the composition that delays absorption (such as aluminum monostearate or gelatin), the absorption of the injectable composition can be delayed.

[0067] If necessary, a sterile injectable solution can be prepared by incorporating the required amount of the pharmaceutical composition into a suitable solvent containing one or a combination of the above-described ingredients and then filter-sterilizing. Generally, a dispersion can be prepared by incorporating the pharmaceutical composition into a sterile medium containing a basic dispersion medium as described above and other required ingredients.

[0068] The pharmaceutical composition can be administered orally, for example, using an inert diluent or an assimilable edible carrier. Also, the pharmaceutical composition and other ingredients can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or directly incorporated into the subject's food. For therapeutic treatment by oral administration, the pharmaceutical composition can be formulated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. Such compositions and formulations should contain at least 1% by weight of the active compound. Of course, the percentages of the composition and preparation may vary and, for convenience, can be about 5% to about 80% by weight per unit.

[0069] Tablets, troches, pills, capsules, etc. can also contain, for example, binders such as gum tragacanth, acacia, corn starch, gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch, potato starch, alginic acid, lubricants such as magnesium stearate, sweetening agents such as sucrose, lactose, saccharin, or flavoring agents such as peppermint, oil of licorice, cherry flavor. When the dosage unit form is a capsule, it can contain, in addition to the materials of the above type, a liquid carrier. Various other materials can be present as coating agents or the physical form of the dosage unit can be modified in other ways. For example, tablets, pills, or capsules can be coated with shellac, sugar, or both. Syrups or elixirs can contain sweetening agents such as sucrose, preservatives such as methyl and propyl parabens, dyes, and flavorings such as raspberry, orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts used. Also, the pharmaceutical composition can be formulated into sustained-release preparations and formulations.

[0070] Accordingly, "pharmaceutically acceptable carriers" are intended to include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc. The use of such media and reagents for pharmaceutical active substances is well known in the art. Their use in the said therapeutic compositions and methods of treatment is contemplated, except when any of the commonly used media and reagents are incompatible with the pharmaceutical composition. Supplementary active compounds can also be incorporated into the composition.

[0071] For ease of administration and uniformity of formulation, it is particularly advantageous to formulate parenteral compositions in unit dosage form. As used herein, "unit dosage form" refers to physically discrete units suitable as unit dosages for the subjects to be treated. Each unit containing a predetermined quantity of the pharmaceutical composition is calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carriers. The specification of the unit dosage forms of the present disclosure is related to the characteristics of the pharmaceutical composition and the particular therapeutic effect to be achieved.

[0072] For convenient and effective administration, the main pharmaceutical composition is formulated in acceptable unit dosages with an effective amount together with a suitable pharmaceutically acceptable carrier. In the case of compositions containing supplementary active ingredients, the dosage is determined with reference to the normal dosage and method of administration of the said ingredients.

[0073] In certain embodiments, for treating a cancer subject, the therapeutic agent / nucleic acid nanoparticles disclosed herein can be administered in combination with anti-cancer agents known in the art. The therapeutic agent / nucleic acid nanoparticles disclosed herein can be administered simultaneously or sequentially with an anti-cancer agent to treat a cancer subject. By using the therapeutic agent / nucleic acid nanoparticles of the present disclosure together with an anti-cancer agent, a multimodal therapy is provided that can treat cancer more effectively than the use of the anti-cancer agent alone or the therapeutic agent / nucleic acid nanoparticles alone. Examples of anti-cancer agents that can be used with the therapeutic agent / nucleic acid nanoparticles disclosed herein include alkylating agents (e.g., thiotepa and CYTOXAN® cyclophosphamide); alkyl sulfonates (e.g., busulfan, improsulfan, and piposulfan); aziridines (e.g., benzodopa, carboquone, meturedopa, and uredopa); ethyleneimines and methylamelamines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and thiotepa); acetogenins (e.g., bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; calicheamicin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); ellipticine; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (e.g., chlorambucil, chloronaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobenbiquine, phenesterine, prednimustine, trofosfamide, and uracil mustard); nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine); vinca alkaloids; epipodophyllotoxins; antibiotics (e.g., enediyne antibiotics (e.g., calicheamicin, particularly, calicheamicin gamma 1 and calicheamicin omega 1)); L-asparaginases;Anthracenedione-substituted urea; methylhydrazine derivative; dynemicin (including dynemicin A); bisphosphonates (e.g., clodronate); esperamicin; neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, actinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calvisine, calminomycin, cardinophilin, chromomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (e.g., mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, keramycin, rhodomycin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin); antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (e.g., denopterin, methotrexate, pteropterin, and trimethoprim); purine analogs (e.g., fludarabine, 6-mercaptopurine, thiampurine, and thioguanine); pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, and floxuridine); androgens (e.g., calusterone, drostanolone propionate, epitostanol, mepitiostane, and testolactone); adrenal antagonists (e.g., aminoglutethimide, mitotane, and trilostane); folic acid supplements (e.g., folinic acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diaziquone; eflornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;Mitansinoids (e.g., mitansine and ansamitocins); mitoguazone; mitoxantrone; mopidamol; nidamycin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2 2''-trichlorothieethylenamine; trichothecenes (especially, T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gasitocin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids (e.g., TAXOL (registered trademark) paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE (registered trademark) Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE (registered trademark) (docetaxel) (Rhone-Poulenc Rorer, Antony, France)); chlorambucil; GEMZAR (registered trademark) (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes (e.g., cisplatin, oxaliplatin, and carboplatin); vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE (registered trademark) vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids (e.g., retinoic acid); capecitabine; leucovorin (LV); irinotecan; adrenocortical suppressants; adrenocortical steroids; progestins; estrogens; androgens; gonadotropin-releasing hormone analogs;and includes, but is not limited to, any of the above pharmaceutically acceptable salts, acids, or analogs. Also included are antihormonal agents that act to regulate or inhibit hormonal action on tumors (e.g., antiestrogens and selective estrogen receptor modulators (SERMs), such as tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® toremifene); aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands (e.g., 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestane, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARTMIDEX® anastrozole); androgen antagonists (e.g., flutamide, nilutamide, bicalutamide, and leuprolide, goserelin); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation (e.g., PKC-alpha, Ralf, and H-Ras); ribozymes (e.g., VEGF-A expression inhibitors (e.g., ANGIOZYME® ribozyme) and HER2 expression inhibitors); vaccines (e.g., gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine); PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELLX® rmRH;An anti-cancer agent that includes an antibiotic (e.g., trastuzumab), and any pharmaceutically acceptable salt, acid, or analog thereof. In certain embodiments, the therapeutic agent / nucleic acid nanoparticles disclosed herein are used in combination with one or more anti-cancer agents selected from cyclophosphamide, tamoxifen, tegafur, paclitaxel, apatinib, cisplatin, docetaxel, 5-fluorouracil, capecitabine, carboplatin, vinorelbine, capecitabine, gemcitabine, ixabepilone, eribulin, ifosfamide, rituximab, vincristine, prednisone, bleomycin, and dacarbazine.;

[0074] Also described herein are kits and manufacturer's instructions for use in the therapeutic or biological uses described herein. Such kits can include a container that is partitioned to contain a carrier, package, or one or more containers such as vials and tubes. Each container contains one of the individual elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and tubes. The containers can be formed from various materials such as glass or plastic.

[0075] For example, the container can contain one or more of the therapeutic / nucleic acid nanoparticles described herein, optionally in a composition as disclosed herein or in combination with another reagent (e.g., mRNA and / or ssRNA). The container optionally has a sterile access port (e.g., the container can be a bag of solution for intravenous administration or a vial with a stopper pierceable by a hypodermic needle). Such kits optionally include an explanatory statement, or label, or instructions related to its use in the methods described herein.

[0076] The kit typically includes one or more other containers, each having one or more materials (e.g., optionally, reagents in concentrated form, and / or devices) that are desirable from a commercial and user perspective for the use of the compounds described herein. Examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes, carriers, packages, containers, vials, and / or tube labels listing the contents and / or instructions for use and / or an attached document with instructions for use. Typically, a set of instructions is also included.

[0077] The label may be on the container or associated with the container. The label may be on the container when the letters, numbers, or other features forming the label are attached, molded, or etched onto the container itself. For example, the label may be associated with the container when it is present in a receptacle or carrier that holds the container, as an attached document. The label can be used to indicate that the contents are used for a specific therapeutic use. The label can also indicate how to use the contents, such as the methods described herein. These other therapeutic agents can be used, for example, in the amounts indicated in the Physician's Desk Reference (PDR) or as determined by one of ordinary skill in the art.

[0078] The methods and compositions described herein can be further defined by the following aspects (Aspects 1 - 29): 1. A composition comprising one or more therapeutic compounds complexed with a nucleic acid fragment to form nanoparticles, wherein the one or more therapeutic compounds are small molecules capable of associating or binding with DNA or RNA. 2. The composition according to aspect 1, wherein the nucleic acid fragment is complexed with the one or more therapeutic compounds at a weight ratio of 2:1 to 10:1. 3. The composition according to aspect 1 or 2, wherein the nucleic acid fragment is complexed with the one or more therapeutic compounds at a weight ratio of 4:1 to 7:1. 4. The composition according to any one of the preceding aspects, wherein the nucleic acid fragment is complexed with the one or more therapeutic compounds at a weight ratio of about 6:1. 5. The composition according to any one of the above aspects, wherein the size of the nanoparticles is 20 nm to 200 nm. 6. The composition according to any one of the above aspects, wherein the size of the nanoparticles is 50 nm to 100 nm. 7. The composition according to any one of the above aspects, wherein the one or more therapeutic compounds include anthracycline agents, anthraquinone agents, camptotheca genus compounds, podophyllum genus compounds, minor groove binders, bleomycin, and / or actinomycin D. 8. The composition according to any one of the above aspects, wherein the one or more therapeutic compounds include aclarubicin, doxorubicin, daunorubicin, idarubicin, epirubicin, amrubicin, pirarubicin, valrubicin, and / or zorubicin. 9. The composition according to any one of the above aspects, wherein the one or more therapeutic compounds include doxorubicin. 10. The composition according to any one of the above aspects, wherein the one or more therapeutic compounds include mitoxantrone, topotecan, etoposide, teniposide, bleomycin, actinomycin D, and / or duocarmycin A. 11. The composition according to any one of the above aspects, wherein one or more of the nucleic acid fragments include a ligand that targets the nanoparticles to a specific cell, tissue, organ, or tumor. 12. The composition according to any one of the above aspects, wherein the nucleic acid fragment includes a fragment of naturally occurring DNA, RNA, and / or DNA-RNA hybrid. 13. The composition according to any one of the above aspects, wherein the nucleic acid fragment includes chemically synthesized DNA, RNA, and / or DNA-RNA hybrid of different nucleotide lengths. 14. The composition according to any one of the above aspects, wherein the RNA is modified such that the 2'-ribose hydroxyl group is replaced with an -O-alkyl group or a halide. 15. The composition according to any one of the above aspects, wherein the nucleic acid fragment is a DNA fragment. 16. The composition according to any one of the above aspects, wherein the DNA fragment is derived from salmon DNA. 17. The composition according to any one of the above aspects, wherein the length of the nucleic acid fragment is 20 nt to 10,000 nt. 18. The composition according to any one of the above aspects, wherein the length of the nucleic acid fragment is 50 nt to 2,000 nt. 19. The composition according to any one of the above aspects, wherein the composition comprises nanoparticles of one or more therapeutic compounds complexed with a DNA fragment having a length of 50 nt to 2,000 nt. 20. The composition according to any one of the above aspects, wherein the one or more therapeutic compounds are selected from aclarubicin, doxorubicin, daunorubicin, idarubicin, epirubicin, amrubicin, pirarubicin, valrubicin, and / or zorubicin. 21. The composition according to any one of the above aspects, wherein the one or more therapeutic compounds is doxorubicin. 22. A pharmaceutical composition comprising the composition according to any one of the above aspects and a pharmaceutically acceptable carrier, diluent, and / or excipient. 23. The pharmaceutical composition according to aspect 22, wherein the pharmaceutical composition is formulated for parenteral delivery. 24. A method of treating a subject with cancer in need of treatment, the method comprising administering to the subject an effective amount of the pharmaceutical composition according to aspect 22 or aspect 23. 25. The method according to aspect 24, wherein the cancer is selected from acute lymphoblastic leukemia, acute myeloblastic leukemia, osteosarcoma, breast cancer, endometrial cancer, gastric cancer, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, liver cancer, kidney cancer, multiple myeloma, neuroblastoma, ovarian cancer, small cell lung cancer, soft tissue sarcoma, thymoma, thyroid cancer, transitional cell bladder cancer, uterine sarcoma, Wilms tumor, and Waldenström macroglobulinemia. 26. A method of treating a human subject with cancer in need of treatment, the method comprising administering to the subject an effective amount of the composition according to any one of aspects 1 to 21. 27. The method according to aspect 26, wherein the cancer is selected from acute lymphoblastic leukemia, acute myeloblastic leukemia, osteosarcoma, breast cancer, endometrial cancer, gastric cancer, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, liver cancer, kidney cancer, multiple myeloma, neuroblastoma, ovarian cancer, small cell lung cancer, soft tissue sarcoma, thymoma, thyroid cancer, transitional cell bladder cancer, uterine sarcoma, Wilms tumor, and Waldenström macroglobulinemia. 28. The method according to aspect 26 or aspect 27, further comprising administering to the subject one or more anti-cancer agents selected from angiogenesis inhibitors, tyrosine kinase inhibitors, PARP inhibitors, alkylating agents, vinca alkaloids, anthracycline agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, aromatase inhibitors, mTor inhibitors, retinoids, and HDAC inhibitors. 29. The method according to any one of aspects 26 to 28, further comprising administering to the subject one or more anti-cancer agents selected from mitoxantrone, topotecan, etoposide, teniposide, bleomycin, actinomycin D, and / or duocarmycin A.

[0079] The following examples are intended to illustrate, but not to limit, the present disclosure. They are typical ones that can be used, but other procedures known to those skilled in the art may be substituted.

Examples

[0080] Materials Doxorubicin (DOX) and ethidium bromide were purchased from Thermo Fisher Scientific (Waltham, MA). Deoxyribonucleic acid (DNA) (50 - 2000 nucleotide fragments with an MW range of 16.88 kDa - 1350 kDa) was provided by Pharma Research Products (Seongam, Korea). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Millipore Sigma (Burlington, MA). The ULYSIS® Alexa Fluor® 488 Nucleic Acid Labeling Kit was purchased from Thermo Fisher scientific. The Label IT® Nucleic Acid Labeling Kit, Cy® 5 was purchased from Mirus Bio. EL4 cells (ATCC, Rockville, MD) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (MediaTech, Manassas, VA) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA) and 1% antibiotics (100 units / mL penicillin, 100 μg / ml streptomycin) (Gibco, Grand Island, NY). All materials were used as purchased.

[0081] DOX Quenching with DNA To optimize the encapsulation efficiency, quenching tests were performed. First, fluorescence spectra were measured between DNA:DOX ratios of 1 - 100, and then between DNA:DOX ratios of 1 - 10. Excitation was performed at 490 nm and emission was measured at 590 nm. Fluorescence was read using a xenon laser and a Synergy H1 Hybrid Multi-Mode Reader (Biotek Instruments, Winooski, VT).

[0082] Preparation of DOX / DNA Nanoparticles Briefly stated, DNA was added to DOX at a weight ratio of 6:1. Time was allotted for self-organization. Finally, phosphate-buffered saline (PBS) was added to the mixture, and an additional 30 minutes was allotted for ionic stabilization of the complex.

[0083] DNA was pipetted into an equal volume of DOX and mixed by pipetting up and down. After maintaining the reactants and reaction at 70 °C, they were allowed to stand for 15 minutes for self-organization. Next, an equal volume of 2X PBS was pipetted into the reaction and mixed by pipetting up and down. After standing for 30 minutes, the DOX / DNA nanoparticles were used in the next experiment. For volumes of 25 mL or less, a 96-well plate was used. For volumes greater than 25 mL, a round-bottom flask was used. Mixing was carried out using a magnetic stir bar rotating at 500 RPM with a multi-plate stirrer (IKA Works, Inc., Wilmington, NC). In this case, DNA was added dropwise to DOX using a glass pipette. To confirm the formation of nanoparticles, images of DOX / DNA were taken using a transmission electron microscope (TEM).

[0084] Transmission electron images of DOX / DNA and DNA DNA was pipetted into an equal volume of DOX and mixed by pipetting up and down. After maintaining the reactants and reaction at 70 °C, they were allowed to stand for 15 minutes for self-organization. Next, an equal volume of 2X PBS was pipetted into the reaction and mixed by pipetting up and down. Before using the DOX / DNA nanoparticles in the next experiment, they were allowed to stand for 30 minutes. For volumes of 25 mL or less, a 96-well plate was used. For volumes greater than 25 mL, a round-bottom flask was used. Mixing was carried out using a magnetic stir bar rotating at 500 RPM with a multi-plate stirrer (IKA Works, Inc., Wilmington, NC). In this case, DNA was added dropwise to DOX using a glass pipette. To confirm that nanoparticles were generated, images of DOX / DNA were taken using a transmission electron microscope (TEM).

[0085] A DOX / DNA solution (10 μL) was dropped onto a carbon-coated grid (Thermo Fisher Scientific) and dried overnight at room temperature. The morphology and size of the nanoparticles were observed at 200 kV using a JEOL 2800 transmission electron microscope (JEOL, Peabody, MA).

[0086] Degradation of DNA in 10% FBS / PBS The degradation of DNA in 10% FBS / PBS (for 48 h) was characterized using a 1% (w / v) agarose gel (a solution of 1X Tris-Acetate-EDTA (TAE) containing 1 μg / mL ethidium bromide). DNA was incubated with serum-containing PBS at 37 °C over time. At each time point, the samples were stored at -20 °C to stop enzymatic degradation by nucleases. The concentration of DNA was 100 μg / ml. The gel was run at 100 V for 40 min and the DNA was imaged on a UV transilluminator (Fotodyne, Heartland, WI).

[0087] Binding kinetics The binding kinetics of DOX and DNA were measured by observing the fluorescence of DOX / DNA based on the concentration of DOX. The fluorescence of DOX / DNA was measured as DOX. The DNA was kept constant at 400 μg / ml. Fluorescence was measured using a multimode reader. The binding kinetics of DOX and DOX / DNA were tested in PBS, serum-containing PBS, or FBS.

[0088] Cytotoxicity of EL4 EL4 cells (ATCC, Rockville, MD) were seeded in 96-well plates at 10 k cells / well and the cells were treated with DOX or its equivalent in the concentration range of 0.001 μg / mL to 10 μg / mL for 24, 48, or 72 h (n = 3), after which cell viability was analyzed by MTT assay. The cells were incubated under conditions of 37 °C, 5% CO2, and 100% humidity. Absorbance was read at 571 nm.

[0089] Endocytosis inhibition Flow cytometry or fluorescence analysis was used to measure the EL4 DOX uptake from inhibitor NaN3 (120 mM), PS2 (12 μg / mL), filipin III (5 μg / mL), CPZ (20 μM), EIPA (20 μg / mL), or DOX or DOX / DNA treatment after exposure to 4°C. Briefly, cells were stimulated with the inhibitor for 15 minutes and treated with DOX or DOX / DNA for 1 hour. Cells containing DOX or DOX / DNA were counted using yellow fluorescence on a flow cytometer. Also, cells containing the DOX or DOX / DNA were counted using fluorescence analysis. Fluorescence was measured using a Guava® easyCyte™ flow cytometer (Millipore Sigma, Burlington, MA) or a multimode reader (Biotek Instruments, Winooski, VT).

[0090] Confocal imaging First, EL4 cells were seeded at 200k cells / mL onto a 35 mm Ibidi μ-dish (Ibidi USA Inc., Fitchburg, WI), their cell nuclei were stained, and they were incubated for 15 minutes. These cells were centrifuged, washed with DPBS, and treated with DOX / DNA-Cy5 for 3 hours. Finally, these cells were centrifuged at 500×g, washed with DPBS, left in DMEM, and imaged using a Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems, Buffalo Grove, IL).

[0091] In vivo tests All animal experiments were conducted using IACUC-approved procedures. EL4 tumors were established in the right posterior flank of 6- to 12-week-old female C57BL / 6 / 027 mice (Charles River Laboratories, Wilmington, MA, USA) by subcutaneous injection of approximately 100 μL of a PBS solution of 1E6 EL4 cells. When the tumors were visible and measurable at 2 mm, they were administered to the mice via tail vein injection. Tumor size was measured using digital calipers, and the tumor volume was calculated using the formula: V = W 2 L / 2 (where V is the tumor volume, W is the tumor width, and L is the tumor length). These mice were appropriately supplied with food and water.

[0092] Pharmacokinetics Pharmacokinetic studies were performed in EL4-challenged C57BL / 6 mice (6-8 weeks old, female, n = 3) that were treated i.v. with either 20 mg / kg DOX or 20 mg / kg DOX-equivalent DOX / DNA. Blood samples were collected from the mice's reclining veins at each time point and centrifuged in tubes for serum collection. For the resulting supernatants, DOX fluorescence was analyzed in acidified alcohol using a multimode reader.

[0093] Hematotoxicity and Liver Enzyme Panel The mice used in this study were 6- to 12-week-old female C57BL / 6 / 027 mice (Charles River Laboratories, Wilmington, MA, USA). They were administered to these mice by tail vein injection. Twenty-four hours later, complete blood counts (CBCs) and liver enzyme levels were measured. Blood for CBC measurement was collected from the reclining vein, mixed with EDTA, and analyzed for white blood cells (WBC), red blood cells (RBC), hemoglobin (Hgb), platelets (Plt), and hematocrit (HCT) using a blood analyzer. For the liver enzyme panel, serum was isolated from the blood and sent to IDEXX Laboratories, Inc. (Westbrook, ME) for analysis of alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin.

[0094] Binding kinetics The binding dynamics of DOX and DNA were measured by observing the fluorescence of DOX / DNA based on the concentration of DOX. The fluorescence of DOX / DNA measured as [DOX] was enhanced. [DNA] was maintained at 400 μg / mL constantly. Fluorescence was measured using a multimode reader. The binding dynamics of DOX and DOX / DNA were tested in PBS, serum-containing PBS, or FBS.

[0095] In vivo distribution The accumulation of DOX in organs and tumors was measured at 1, 3, 6, and 12 hours after i.v. administration of DOX or DOX / DNA (equivalent to 20 mg / kg DOX) to EL4-challenged C57BL / 6 mice (female, 6 - 8 weeks old) (n = 5). Briefly, organs and tumors were collected, ground under cooling, homogenized in acidified alcohol, and then centrifuged. For the obtained supernatant, DOX fluorescence was analyzed using a multimode reader.

[0096] Acute toxicity The acute toxicity in C57BL / 6 mice (female, 6 - 8 weeks old) was observed at 24 hours after injection at a dose of 10 - 40 mg / kg DOX or equivalent (n = 7).

[0097] Tumor growth and survival rate After i.v. treatment with a certain dose range of DOX, DOX / DNA, or DOXIL, the tumor growth and survival rate of EL4-challenged mice (female mice, 6 - 12 weeks old) were regularly tracked for 30 days (n = 5). An initial tumor challenge composed of 1E6 EL4 cells was subcutaneously injected into the right posterior flank of the mice. When the tumor grew to 2 mm and became measurable, treatment was administered via the tail vein. Mice were euthanized when the tumor exceeded 15 mm, when tumor lesions appeared, or when the body weight decreased by less than 75%.

[0098] Tumor growth and survival rate in repeated administration From the day of the first i.v. treatment (day 0) with 20 mg / kg of DOX, DOX / DNA, or DOXIL, the tumor growth, body weight, and survival rate of EL4 challenge mice (6-week-old, female) were tracked for 22 days. Subsequently, on days 7 and 14, 20 mg / kg of DOX, DOX / DNA or DOXIL was administered (n = 5). An initial tumor challenge consisting of 1E6 EL4 cells was subcutaneously injected into the right posterior flank of the mice. When the tumor grew to 2 mm and became measurable, treatment was administered via the tail vein. Mice were euthanized when tumor lesions appeared, when the weight decreased by less than 75%, or when the tumor exceeded 15 mm.

[0099] Physical Characterization of the DOX / DNA Complex To evaluate the loading capacity and efficiency of DOX by DNA, a quenching test of DOX was performed using DNA, and the loading capacity and encapsulation efficiency of DOX / DNA nanoparticles were evaluated (see Figure 1). As a result of measuring the loading capacity and encapsulation efficiency of DOX / DNA, they were approximately 14% and approximately 88%, respectively. Using excitation at 490 nm, first, the fluorescence spectra between DNA:DOX ratios of 1 to 100 were measured (Figure 1, upper panel). Next, the fluorescence spectra between DNA:DOX ratios of 1 to 10 were measured (Figure 1, lower panel). As a result of measuring the optimal weight ratio from this, the DNA:DOX ratio was 6:1.

[0100] Size Characterization of the DOX / DNA Complex The size and morphology of DOX / DNA were evaluated using a transmission electron microscope. DOX / DNA nanoparticles were prepared as described above before being diluted to a concentration of 1 μg / mL (equivalent to DOX) in water (see Figure 2) or in PBS (see Figure 3). The solution was allowed to stand for an additional 30 minutes before being dropped onto a carbon grid for TEM imaging. The TEM of DOX / DNA showed that the size of the nanoparticles was approximately 70 nm. These characterization tests indicated that the particles could carry chemotherapeutic agents such as DOX, and in particular, the size characterization test demonstrated that these particles could reach cancer cells.

[0101] Possibility of long-term storage of DOXDNA nanoparticles Briefly, DOX / DNA was prepared in PBS, diluted with H20, lyophilized overnight, reconstituted with H2O, and subsequently imaged. Final [DNA] = 6 μg / mL, final [DOX] = 1 μg / mL. The stability of DOX / DNA was not significantly affected by the lyophilization process (see Figures 4 - 6).

[0102] Stability of DNA in water and PBS DNA was prepared in PBS, diluted with H20, and imaged. Final [DNA] = 6 μg / mL. DNA remained very stable in water or PBS (see Figures 7 - 8).

[0103] DNA degradation in 10% FBS / PBS To measure the stability of DNA against serum exposure, DNA (100 μg / mL) was incubated with PBS containing serum at 37 °C for 0 - 48 hours. At each time point, the samples were stored at -20 °C to stop enzymatic degradation from nucleases. DNA degraded over time in serum (see Figure 9). Nucleases in serum-containing medium are likely to contribute to the degradation of DNA. It can be speculated that the release of DOX is delayed by the time-dependent degradation of DNA in 10% FBS.

[0104] In vitro cytotoxicity of the DOX / DNA complex at 24 hours, 48 hours, and 72 hours A test was conducted to examine the in vitro cytotoxicity of DOX / DNA in EL4 cells at 24 hours (left curve), 48 hours (middle curve), and 72 hours (right curve). EL4 cells were treated three times at 24, 48, and 72 hours over a concentration range, and then cell viability was analyzed by MTT assay. Reported IC 50 values: For DOX / DNA, IC 50 = 1.143 μg / mL or 2.1 μM, for DOX, IC 50As seen at = 0.313 μg / mL or 0.576 μM (see Figure 10), DOX / DNA showed lower cytotoxicity than DOX against these cells by approximately 3.5-fold after 24 hours of incubation. Reported IC 50 values: At 48 hours, for DOX / DNA, IC 50 = 0.072 μg / mL, for DOX, IC 50 = 0.093 μg / mL, or at 72 hours, for DOX / DNA, IC 50 = 0.055 μg / mL, for DOX, IC 50 = 0.048 μg / mL. As seen, DOX / DNA showed lower cytotoxicity against these cells compared to DOX after 48 - and 72-hour incubations. These results, in parallel with the 24-hour cytotoxicity data, suggest that the release of DOX from DOX / DNA was delayed. Furthermore, these results indicate that the nanoparticles are less toxic compared to their free small molecule counterparts.

[0105] Pharmacokinetics of DOX / DNA in vivo The results of pharmacokinetic studies conducted in EL4-challenged C57BL / 6 mice treated i.v. with either 20 mg / kg DOX or 20 mg / kg of DOX equivalent of DOX / DNA are shown (see Figure 11). The mice were 6 - 8-week-old female mice. DOX / DNA had a longer blood circulation retention half-life (T 1 / 2 = 3 minutes, n = 3) in EL4-challenged C57BL / 6 mice compared to mice treated with DOX (T 1 / 2It was found to have (for about 75 minutes). DOX was absorbed into the tissue in about 15 minutes (as indicated by the steep initial gradient of the curve), and thereafter, a profile showing liver and kidney clearance was observed. However, DOX / DNA showed a profile of slow tissue absorption lasting for 1 hour. From the above results, it can be inferred that the circulation of DOX and the protection / shielding of DOX were enhanced by DNA. Thereafter, a profile showing liver and kidney clearance was observed. Therefore, the drug delivery system of the present disclosure may change the dissolution and absorption of doxorubicin, thereby realizing the sustained release of the active ingredient.

[0106] In vitro dissociation kinetics of DOX / DNA By observing the fluorescence of DOX / DNA based on the concentration of DOX, the binding kinetics between DOX and DNA were measured. The fluorescence of DOX / DNA measured as [DOX] was enhanced. [DNA] remained constant at 400 μg / ml. Fluorescence was measured using a multimode reader. The binding kinetics of DOX with DOX / DNA were tested in PBS, serum-containing PBS, or FBS. The dissociation of DOX from DOX / DNA increased with the increase in serum content and with the passage of time (see Figures 12 - 13). This data supports the data from the DNA degradation assay. The K values of DOX dissociation from DOX / DNA in PBS, 10% FBS, 25% FBS, 50% FBS, and FBS were calculated and were 76.8 nM, 152.7 nM, 317.7 nM, 565.1 nM, and 1329.7 nM, respectively. This experiment clearly shows that DOX is released from DNA by FBS. d The results showed that they were 76.8 nM, 152.7 nM, 317.7 nM, 565.1 nM, and 1329.7 nM, respectively. This experiment clearly shows that DOX is released from DNA by FBS.

[0107] DOX release test from DOX / DNA Cumulative DOX release from DOX / DNA was performed over 72 hours in 100% PBS, 10% FBS / PBS, 25% FBS / PBS, 50% FBS / PBS, or 100% FBS. When using FBS, it was found that DOX was released maximally from DOX / DNA (see Figure 14). This data, in parallel with the binding kinetics experiments, suggests that DOX is released from DOX / DNA over time depending on the amount of serum in the medium. At least according to this model, most of the DOX should be released from the nanoparticles over 72 hours.

[0108] Complete blood count and liver enzyme panel of DOXDNA and DOX Complete blood counts and liver enzyme panels were performed in C57BL / 6 mice treated with 20 mg / kg DOX, 20 mg / kg DOX equivalent of DOX / DNA, PBS, or 120 mg / kg DNA (n = 3). Female C57BL / 6 / 027 mice (Charles River Laboratories, Wilmington, MA, USA) aged 6 - 12 weeks were used in this study. These mice were administered via tail vein injection. Twenty-four hours post p.i., complete blood counts (CBC) and liver enzyme levels were measured. Blood was collected by retro-orbital venipuncture, mixed with EDTA, and white blood cells (WBC), red blood cells (RBC), hemoglobin (Hgb), platelets (Plt), and hematocrit (HCT) were analyzed using a blood analyzer. For the liver enzyme panel, serum was isolated from the blood and sent to IDEXX Laboratories, Inc. (Westbrook, ME) for analysis of alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin. DOX was shown to have a greater effect on circulating blood cells and liver enzymes compared to DOX / DNA and DOXIL. Based on these panels, DOX / DNA had a significantly different regulatory effect on blood components and liver enzymes compared to when DOX was used alone (see Figure 15).

[0109] In vivo distribution of DOXDNA and DOX The accumulation of DOX in organs and tumor tissues was characterized at 1, 3, 6, and 12 hours after i.v. administration of DOX, DOXIL (equivalent to 20 mg / kg DOX), or DOX / DNA (equivalent to 20 mg / kg DOX) to EL4-challenge C57BL / 6 mice (female, 6 - 8 weeks old). In the DOX / DNA group, the accumulation of DOX in the lung was lower (n = 5) (except for the 12-hour case of DOXIL (n = 3)) (see Figures 16 - 18). The maximum tumor accumulation of DOX was observed in mice treated with DOX / DNA. Therefore, DOX / DNA improves drug delivery to the tumor site. Also, less organ toxicity was observed with DOX / DNA, specifically in the lung and spleen. Here, it is also emphasized that the liver and kidneys removed high levels of DOX. Larger particles such as DOX / DNA allow uptake by macrophages and are removed from the lung. This results in a decrease in the lung toxicity of DOX when delivered as DOX / DNA.

[0110] Survival curves of acute toxicity in C57BL / 6 mice Acute toxicity was not observed at dose regimens of 20 mg / kg or less (n = 7). In mice treated with DOX, acute toxicity (cardiac arrest) was seen with administration of a 40 mg / kg dose (see Figure 19). Therefore, DOX / DNA is safer than DOX. DOX / DNA has a larger therapeutic concentration range than DOX. Regarding DOXIL, DOX / DNA has an easier assembly process and can be manufactured more efficiently compared to DOXIL.

[0111] Efficacy of DOX, DOX / DNA, and DOXIL treatments on tumor growth and survival in the EL4-cancer model To confirm the safety and efficacy of the drug delivery system, the tumor growth and survival rate of EL4 challenged mice were regularly monitored for 30 days after i.v. administration of a certain dose range of DOX or DOX / DNA to female mice aged 2 - 3 months. DOX / DNA delayed tumor growth and improved the survival rate (n = 5) in EL4 challenged C57BL / 6 mice, which was superior to that when treated with DOX alone (see Figure 20). At a dose of 20 mg / kg, long - term survival was shown and tumor growth was retarded when using the nano - carrier formulation. Interestingly, complete tumor regression was observed in mice treated with 40 mg / kg of DOX / DNA by day 28. Furthermore, 60% of these mice survived until the end of the experiment. When treated at high doses, DOX / DNA treatment reduced the body weight of EL4 challenged C57BL / 6 mice (n = 5, see Figure 21). These results not only demonstrated that DNA had a protective effect against systemic toxicity, but also effectively verified that it could increase the maximum tolerated dose of DOX. Also, treatment with DNA alone was the same as that with PBS, indicating the safety of the drug delivery vehicle in the solid tumor model of these mice.

[0112] Endocytosis inhibition and its impact on the uptake of DOX / DNA and DOX For DOX / DNA, the inhibitors CPZ ((20 mM), Filipin III (5 μM), EIPA (20 μM), and their various combinations, or EL4 cell uptake at 4 °C were evaluated (see Figure 22). Chlorpromazine (CPZ) is an inhibitor of the clathrin - dependent pathway. On the other hand, Filipin III is an inhibitor of the caveolin - dependent pathway. EIPA is an inhibitor of the macropinocytosis pathway. The concentrations selected for the assay were measured using a dose - response assay for each inhibitor. DOX / DNA was found to be taken up by cells via the clathrin - dependent pathway and the caveolin - dependent pathway. Also, membrane fusion was involved as shown by the inhibition of DOX / DNA uptake at 4 °C.

[0113] The uptake of DOX by EL4 cells was tested at 4 °C using the inhibitors NaN3 (120 mM), PS2 (12 μg / mL), filipin III (5 μg / mL), and EIPA (20 μM). Uptake was measured using flow cytometry. Briefly, cells were stimulated with the inhibitors for 15 minutes before treatment with DOX for 1 hour. Cells containing DOX were counted using a flow cytometer with yellow fluorescence. In contrast to DOX / DNA, these inhibition tests suggest that the uptake of DOX is mainly through membrane fusion (see Figure 23).

[0114] Confocal imaging of EL4 cells treated with DOX / DNA shows the localization of the treatment in EL4 cells. The CLSM images show that DOX / DNA was taken up by EL4 cells over time (see Figure 24). These CLSM images also suggest the internal movement of not only DOX but also nanoparticles.

[0115] Titration of DOX, DNA, and DOX / DNA with weak bases In water, DOX, DNA, or DOX / DNA with an initial volume of 1 mL was prepared. DOX was equivalent to 600 μg / mL. After lowering the pH to 2 or less with 1 M HCl, the pH was adjusted upward with a small amount of 0.1 M NaOH (100 μL or 20 μL). DOX, DNA, and DOX / DNA all showed similar titration curves (see Figure 25).

[0116] Although numerous embodiments are described herein, it will be understood that various modifications can be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. A composition comprising one or more therapeutic compounds forming nanoparticles, wherein the size of the nanoparticles is 20 nm to 200 nm and is complexed with nucleic acid fragments, and the one or more therapeutic compounds are anthracyclines or polyphenols capable of associating or binding with DNA, the nucleic acid fragments are complexed with the one or more therapeutic compounds at a weight ratio of 2:1 to 10:1, and the nucleic acid fragments are DNA fragments.

2. The composition according to claim 1, wherein the DNA fragment is complexed with the one or more therapeutic compounds at a weight ratio of 4:1 to 7:

1.

3. The composition according to claim 1, wherein the DNA fragment is complexed with the one or more therapeutic compounds at a weight ratio of about 6:

1.

4. The composition according to claim 1, wherein the size of the nanoparticles is 50 nm to 100 nm.

5. The composition according to claim 1, wherein the one or more therapeutic compounds are polyphenols comprising epigallocatechin gallate (EGCG) and / or resveratrol.

6. The composition according to claim 1, wherein the one or more therapeutic compounds are anthracyclines selected from aclarubicin, doxorubicin, daunorubicin, idarubicin, epirubicin, amrubicin, pirarubicin, valrubicin, and zorubicin.

7. The composition according to claim 6, wherein the one or more therapeutic compounds are doxorubicin.

8. The composition according to claim 1, wherein one or more of the DNA fragments comprise a ligand that targets the nanoparticles to a specific cell, tissue, organ, or tumor.

9. The composition according to claim 1, wherein the DNA fragment is a naturally occurring DNA fragment.

10. The composition according to claim 1, wherein the DNA fragment is chemically synthesized DNA of different nucleotide lengths.

11. The composition according to claim 9, wherein the naturally occurring DNA fragment is derived from salmon DNA.

12. The composition according to claim 1, wherein the length of the DNA fragment is 20 nt to 10,000 nt.

13. The composition according to claim 12, wherein the length of the DNA fragment is 50 nt to 2,000 nt.

14. The composition according to claim 1, comprising nanoparticles of one or more therapeutic compounds complexed with a DNA fragment having a length of 50 nt to 2,000 nt.

15. The composition according to claim 14, wherein the one or more therapeutic compounds are anthracyclines including aclarubicin, doxorubicin, daunorubicin, idarubicin, epirubicin, amrubicin, pirarubicin, valrubicin, and / or zorubicin.

16. The composition according to claim 15, wherein the one or more therapeutic compounds are doxorubicin.

17. A pharmaceutical composition comprising the composition according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier, diluent, and / or excipient.

18. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is formulated for parenteral delivery.

19. The pharmaceutical composition according to claim 17 for treating cancer.

20. The pharmaceutical composition according to claim 19, wherein the cancer is selected from acute lymphoblastic leukemia, acute myeloblastic leukemia, osteosarcoma, breast cancer, endometrial cancer, gastric cancer, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, liver cancer, kidney cancer, multiple myeloma, neuroblastoma, ovarian cancer, small cell lung cancer, soft tissue sarcoma, thymoma, thyroid cancer, transitional cell bladder cancer, uterine sarcoma, Wilms tumor, and Waldenström macroglobulinemia.

21. Use of the composition according to any one of claims 1 to 16 in the manufacture of a drug for treating cancer.

22. The use according to claim 21, wherein the cancer is selected from acute lymphoblastic leukemia, acute myeloblastic leukemia, osteosarcoma, breast cancer, endometrial cancer, gastric cancer, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, liver cancer, kidney cancer, multiple myeloma, neuroblastoma, ovarian cancer, small cell lung cancer, soft tissue sarcoma, thymoma, thyroid cancer, transitional cell bladder cancer, uterine sarcoma, Wilms tumor, and Waldenström macroglobulinemia.

23. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is administered in combination with one or more anticancer agents selected from angiogenesis inhibitors, tyrosine kinase inhibitors, PARP inhibitors, alkylating agents, vinca alkaloids, anthracycline agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, aromatase inhibitors, mTor inhibitors, retinoids, and HDAC inhibitors.

24. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is administered in combination with one or more anti-cancer agents selected from mitoxantrone, topotecan, etoposide, teniposide, bleomycin, actinomycin D, and / or duocarmycin A.

25. The composition according to claim 14, wherein the one or more therapeutic compounds are polyphenols comprising epigallocatechin gallate (EGCG) and / or resveratrol.

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