Pharmaceutical composition comprising sialic acid for treating diseases related to the lung

Abstract

The present invention provides a pharmaceutical composition capable of delivering therapeutic agents such as nucleic acids to the lung via systemic administration to treat lung-related diseases. Said composition comprises a non-covalent complex formed by co-precipitating a sialic acid (SA) entity, a complexing agent, and a nucleic acid cargo.

Description

[0001] PHARMACEUTICAL COMPOSITION COMPRISING SIALIC ACID FOR

[0002] TREATING DISEASES RELATED TO THE LUNG

[0003] RELATED APPLICATION

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 653,177, filed on May 29, 2024. The entire teachings of the above application are incorporated herein by reference.

[0005] BACKGROUND OF THE INVENTION

[0006] Many therapeutic agents need to be delivered to certain targets in the human body. Such a target can be an organ, a certain type of tissue, or a specific receptor on a type of cell. In some cases, it is desirable to deliver a therapeutic agent to a specific tissue or organ where the disease of interest is located.

[0007] Recent advancements in in vivo delivery of nucleic acid therapeutics, including messenger RNA (mRNA), small interfering RNA (siRNA), and antisense oligonucleotide (ASO), have brought hope to treat and prevent a variety of infectious diseases, genetic diseases, cancer, and other types of diseases. Lipid nanoparticle (LNP)- based mRNA vaccines have demonstrated an excellent safety profile and efficacy against the SARS-CoV-2 virus during the COVID-19 pandemic. New therapeutic modalities, including cancer vaccines, protein replacement therapy, in vivo cell therapy, and genome editing, are emerging and are in various development stages. Many of these new therapeutic modalities rely on viral delivery. However, viral delivery suffers from several shortcomings. For example, viral gene delivery systems exhibit limitations such as immunogenicity, mutagenesis, carcinogenesis, and low loading capacity. Therefore, more and more drug developers are exploring LNP systems for the delivery of their therapeutic cargos.

[0008] Delivery of nucleic acid cargos to the lung using LNP technology presents significant opportunities to treat lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia (PCD), a-1 antitrypsin deficiency (AAT), pulmonary fibrosis, cystic fibrosis (CF), sarcoidosis, and lung cancer. In addition, delivery of vaccines to the deep lung may provide enhanced protection against respiratory infections compared with intramuscular or intranasal vaccines.

[0009] There are various methods to deliver therapeutic agents to the lungs, but systemic administration of nucleic acids offers several advantages. These include bypassing airway barriers, precise dosing, targeting specific cell types, avoiding local irritation, and being suitable for unstable patients. However, using nanoparticle technologies to deliver drugs to the lung via systemic administration has been challenging because nanoparticles, especially conventional LNPs, predominantly accumulate in the liver. Even for most of those “optimized” systems, only a portion of the LNP delivered accumulates in the lung.

[0010] Cheng, Q., et al., in Nat. Nanotechnol. 2020, 15(4): 313-320 introduced a strategy called Selective Organ Targeting (SORT). This approach involves systematically engineering various classes of LNPs to enhance the delivery of RNAs to extrahepatic tissues, such as the lungs. Their strategy includes incorporating an additional SORT molecule into the standard LNP formulation, which modifies the organ tropism of the delivery system. For instance, by adding a cationic lipid to the conventional LNP setup, they created positively charged nanoparticles, which showed a strong preference for targeting the lungs. Their findings revealed that positively charged LNPs are primarily lung-tropic, while negatively charged LNPs tend to accumulate in the spleen, and neutral LNPs are directed toward the liver. (Cheng, Q., Wei, T., Farbiak, L. et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat. Nanotechnol. 15, 313-320 (2020). https: / / doi.org / 10.1038 / s41565-020-0669-6).

[0011] However, Omo-Lamai, S. et al., in Adv Mater. 2024 Feb 23 found that the lung-tropic LNPs employing cationic lipids induce massive thrombosis. They demonstrated that thrombosis was induced in the lungs and other organs and greatly exacerbated by pre-existing inflammation. This clotting was induced by a variety of formulations with cationic lipids, including LNPs and non-LNP nanoparticles. (Omo- Lamai S, et al., Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions. Adv Mater, doi: 10.1002 / adma.202312026)

[0012] Therefore, there is an unmet need to develop a novel lipid nanoparticle system that can deliver nucleic acid cargo to the lung via systemic administration without causing thrombosis and inflammation.

[0013] SUMMARY OF THE INVENTION

[0014] The present invention is based on our surprising discovery in our animal studies that negatively charged nanoparticles (NPs) can target the lung. This discovery is against conventional wisdom, as it has been generally established within the scientific community that positively charged NPs tend to accumulate in the lung, negatively charged NPs tend to accumulate in the spleen, and neutrally charged NPs tend to accumulate in the liver.

[0015] Accordingly, the present invention provides a pharmaceutical composition capable of delivering therapeutic agents such as nucleic acids to the lung via systemic administration to treat lung-related diseases. Said composition comprises a non-covalent complex formed by co-precipitating a sialic acid (SA)-containing entity, a complexing agent, and optionally, an active pharmaceutical agent (or API) such as a nucleic acid cargo.

[0016] Said SA entity can be selected from the group consisting of sialic acid, poly(sialic acid), sialic acid derivatives, mimetics, and other entities that contain a sialic acid moiety in their chemical structure, herein collectively referred to as “SA entity” or “SA-containing entity”. Preferably, the SA-containing agent can be a poly sialic acid.

[0017] The complexing agent can be a small molecule, a lipid, or a polymer bearing a net positive charge under physiological conditions. Ionizable lipids or polymers that are neutral under ambient conditions but form positively charged entities at certain pHs may also be used to form the nanoparticles of the invention.

[0018] Said nucleic acid cargo is a therapeutic agent selected from the group containing DNA, mRNA, siRNA, anti-sense oligonucleotide (ASO), the components suitable for geneediting systems, including but not limited to CRISPR-Cas9, prime editing, base-editing, TALENs, ZFNs, etc.

[0019] The pharmaceutical composition can be formed by forming a non-covalent complex among the SA-containing entity, a complexing counterion, and the nucleic acid cargo, wherein said non-covalent complex may be a soluble entity or an insoluble particle. It is preferred that the non-covalent complex is an insoluble particle (e.g., a nanoparticle) that forms a stable suspension in an aqueous solution.

[0020] Preferably, the nanoparticle of the pharmaceutical composition of the present invention has a particle size from 1 nm to about 1 pm, preferably from about 10 nm to about 1 pm, and more preferably from about 20 nm to about 500 nm, and most preferably from about 50 nm to about 300 nm. For example, the nanoparticles may have an average size between about 50 and 300 nm, such as about 50, 75, 100, 125, 150, 175, 200, 225, 250, 300 nm.

[0021] Preferably, the nanoparticle of the pharmaceutical composition of the present invention has a negative charge, with a zeta potential of from 0 to -100 mV, -2 to -80 mV, -5 to -60 mV, -10 to -40 mV, or -20 to -35 mV. The polysialic acid is preferably water-soluble. The polysialic acid can comprise a plurality of sialic acid residues selected from the group consisting of Neu5Ac, Neu5Gc, and Kdn, or a combination thereof. The polysialic acid can have a molecular weight of from 500 to 50,000,000, from 1,000 to 5,000,000, and from 2,000 to 500,000 Da.

[0022] In some embodiments, the polysialic acid is a polysialic acid comprising only sialic acid repeating units. This type of polymer is often referred to as a “homopolymer.” One example of such homopolymer of polysialic acid is colominic acid, commercially available, for example, from Carbosynth, Oakbrook Terrace, IL, USA. Colominic acid, also referred to as polysialic acid, is a linear small polysaccharide containing a-2,8-linked sialic acid (neuraminic acid) with (n = 8 to >100) residues. The sialic acid unit in the SA-containing agent or polysialic acid may also have an alpha-2,3 linkage, an alpha-2,6 linkage, an alpha- 2,8 linkage, or combinations thereof.

[0023] In some embodiments, the polysialic acid is a “copolymer” comprising sialic acid repeating units and the repeating units of at least one different chemical entity. Non-limiting examples of such copolymers include PLGA-PSia, PEG-PSia, PLGA-PEG-PSia, etc. Here, PLGA is poly(lactide-co-glycolide), PEG is polyethylene glycol, and PSia is polysialic acid.

[0024] In some embodiments, the polysialic acid is an oligomer of sialic acid, such as a dimer, a trimer, a tetramer, a pentamer or a hexamer available as N-acetylneuraminic acid oligomers or their sodium salts, available from Nacalai USA, Inc., San Diego, CA, United States.

[0025] In some embodiments, polysialic acid is a pharmaceutically acceptable polymer with a sialic acid moiety at the terminal of its chemical structure. For example, PEG-Sia, or PLGA-PEG-Sia, where Sia represents the sialic acid moiety.

[0026] Preferably, the polysialic acid can be a homopolymer or colominic acid.

[0027] Preferably, a complexing agent can be added to the SA-agent to co-precipitate and form said nanoparticle formulation. The complexing agent may be neutral, anionic, or cationic. A cationic complexing agent is preferred in the present invention. A cationic complexing agent may be a nitrogen, sulfur, or phosphorus-containing molecule, such as a small molecule compound, a lipid, a polymer, or a dendrimer. The molecule is preferably amphiphilic, having one or more cationic moieties and one or more hydrophobic moieties. Cationic moieties can be nitrogen, sulfur, or phosphorus-containing groups. Preferred cationic moieties include primary or secondary amines, ammoniums, or phosphoniums. The hydrophobic moiety can be an organic or inorganic group. Preferred hydrophobic groups include substituted or unsubstituted, saturated or unsaturated higher alkyls, acyls or esters (C3-C20 or more). The hydrophobic groups can be linear, branched or cyclized (such as aryl groups or cholesterol and analogs thereof).

[0028] The small-molecule complexing agent used in the present invention is typically a nitrogen, sulfur, or phosphorus-containing compound or salt thereof. Non-limiting examples of small molecule complexing agents include ethyl lauroyl arginate HC1 (LAE), tripropyl amine, tributyl amine, triphenyl amine, hexadecylamine, hexylamine, Didodecyldimethylammonium bromide, Dodecyltrimethylammonium bromide (DTAB), Cetrimonium bromide (CTAB), benzathine, dimethyldioctadecylammonium bromide, benethamine, hydrabamine, stearalkonium chloride, DC-Cholesterol-HCl, cetylpyridinium chloride, l,2-distearoyl-3-dimethylammonium-propane, DODMA, lipofectin, etc.

[0029] The cationic lipid is typically pharmaceutically acceptable and can be natural or synthetic. An example of a cationic lipid is an ammonium lipid, or lipid characterized by a positively charged nitrogen moiety. For example, the cationic lipid can be substituted by a tertiary ammonium group, such as a trialkyl ammonium, preferably a trimethyl ammonium. The cationic lipid can be further substituted by one or more substituted or unsubstituted long- chain alkyls or alkenyls, such as a G4-C20 alkyl or alkenyl. Examples of commonly used lipids include multivalent cationic lipids, DOTMA, ethyl PC’s, DDAB, pH-sensitive lipids, di oleoyl-3 -trimethylammonium propane (DOTAP), DC-cholesterol, GL67, and DODMA.

[0030] The complexing agent can also be a cationic polymer or an ionizable polymer.

[0031] A cationic or ionizable polymer can be of natural or synthetic origin. Natural cationic or ionizable polymer can be a protein, such as gelatin, or a polysaccharide, such as cationic chitosan, cellulose, and dextran. Examples of synthetic cationic polymers include poly(2- N,N’ -dimethylaminoethylmethacrylate), polylysine, polyethyleneimine, poly(amidoamine)s, poly(amino ester)s, poly(P-amino ester)s (PBAE), and poly(amino acid)s.

[0032] The nanoparticles preferably encapsulate an active pharmaceutical agent (or API), such as an anti-cancer agent or immunotherapeutic agent, within the particles. Alternatively or additionally, the API is covalently or ionically attached to the surface of the nanoparticles. For example, the API can be covalently attached to the particle surface via a hydrolysable bond that facilitates in vivo release.

[0033] Preferably, the API encapsulated in the nanoparticles of the present invention is a nucleic acid selected from the group containing DNA, mRNA, circular RNA, micro RNA, siRNA, ASO, gene-editing materials such as CasX (X can be 9 or any other number), gRNA, etc.

[0034] The invention further provides methods for delivering an active pharmaceutical ingredient to the lung of a subject in need of it. The invention comprises administering the composition to the subject.

[0035] The invention further relates to methods for treating a disease or disorder, such as an autoimmune or inflammatory disease of the lung or lung cancer, in a subject in need thereof, comprising administering to the subject the composition of the invention.

[0036] The invention also provides a method for targeting active pharmaceutical ingredients (APIs) to the lungs for the treatment, prevention, or diagnosis of various pulmonary conditions, lung-related diseases or disorders. Such pulmonary conditions, or lung-related disease or disorders encompass, without limitation, any disorder affecting the respiratory system, including the airways, lung parenchyma, pulmonary vasculature, pleura, or respiratory muscles. These conditions may be acute or chronic, inflammatory, infectious (e.g., bacterial pneumonias including Streptococcus pneumoniae and Pseudomonas aeruginosa, viral infections such as influenza, RSV, and SARS-CoV-2, fungal infections, or mycobacterial infections including tuberculosis), genetic (e.g., cystic fibrosis, alpha- 1 antitrypsin deficiency), neoplastic (e.g., non-small cell lung cancer, small cell lung cancer, mesothelioma, or metastatic lung cancers), autoimmune (e.g., sarcoidosis, lupus pneumonitis, rheumatoid lung disease), or degenerative in nature. Further non-limiting examples include obstructive airway diseases such as asthma, chronic obstructive pulmonary disease (COPD) encompassing chronic bronchitis and emphysema, and bronchiolitis; restrictive lung diseases including idiopathic pulmonary fibrosis (IPF), hypersensitivity pneumonitis, asbestosis, and silicosis; pulmonary vascular diseases such as pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH); acute lung injury and acute respiratory distress syndrome (ARDS); allergic reactions such as allergic bronchopulmonary aspergillosis (ABPA); and conditions amenable to regenerative medicine applications, gene therapy, or localized vaccination strategies.

[0037] The invention also relates to methods for the preparation of nanoparticles. The method can comprise the steps of:

[0038] Dissolving a polysialic acid and, a water-soluble active pharmaceutical ingredient in an aqueous solvent to form an aqueous solution; dissolving a cationic lipid or a cationic polymer and, optionally, a water-insoluble active pharmaceutical ingredient in a water- miscible organic solvent to form an organic solution; combining the aqueous solution and organic solution, thereby precipitating nanoparticles; and collecting the nanoparticles.

[0039] In a preferred embodiment, the particles are prepared by first mixing an aqueous solution of a nucleic acid and an organic solution of a cationic lipid to form an intermediate lipoplex nanoparticle, followed by the addition of an aqueous solution of a polysialic acid to form the final nanoparticle. Preferably, said intermediate lipoplex nanoparticle bears a positive charge with a preferred zeta potential from 0 to + 50 mV, preferably from +5 to +30 mV.

[0040] DESCRIPTION OF THE FIGURES

[0041] The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

[0042] Figure 1 shows the luciferase expression in lung, spleen, and liver 6 hours after a single tail-vein injection of Sigfinity NPs loaded with a luciferase-mRNA (Example 1) into a BALB / C female mouse.

[0043] Figure 2 shows the tdTomato expression in lung, spleen, and liver 6 days after a single tail-vein injection of Sigfinity NPs loaded with a Cre-recombinase payload (Example 2) into an Ail 4 mouse.

[0044] Figure 3 A: In-life IVIS images of BALB / C female mice 6 hours after a single tailvein injection of Sigfinity NPs loaded with a luciferase-mRNA (Example 5).

[0045] Figure 3B: Ex-vivo IVIS images showing the luciferase expression in lung, spleen, and liver 6 hours after a single tail-vein injection of Sigfinity NPs loaded with a luciferase- mRNA (Example 5).

[0046] Figure 4A: Ex-vivo IVIS images showing the tdTomato expression in lung, spleen, and liver 72 hours after a single tail-vein injection of Sigfinity NPs loaded with a Cre-mRNA (Example 6).

[0047] Figure 4B: Flow cytometry results showing the tdTomato expression in various cells in the lung tissue. DETAILED DESCRIPTION OF THE INVENTION

[0048] Overview

[0049] The present invention is based on our surprising discovery in our animal studies that negatively charged nanoparticles can target the lung. This discovery is against conventional wisdom, as it has been generally established within the scientific community that positively charged NPs tend to accumulate in the lung, negatively charged NPs tend to accumulate in the spleen, and neutrally charged NPs tend to accumulate in the liver.

[0050] Positively charged LNPs that maintain a positive zeta potential at physiological pH, exhibit a pronounced tropism for the pulmonary vasculature following intravenous administration. However, despite their efficacy in lung targeting, a significant safety concern associated with positively charged LNPs is their inherent ability to induce thrombosis. Omo- Lamai et al. (2024) elucidated that the mechanism involves the LNP's direct interaction with fibrinogen, a soluble glycoprotein with a net negative charge at physiological pH. This electrostatic binding drives a critical conformational change in the fibrinogen molecule, altering its three-dimensional structure. This altered fibrinogen subsequently triggers the coagulation cascade, promoting both platelet activation and the generation of thrombin, ultimately culminating in the formation of pathological blood clots. This pro-thrombotic effect is further exacerbated by pre-existing inflammation, highlighting a major barrier to the clinical translation of such lung-tropic LNP formulations.

[0051] To ameliorate this LNP-induced thrombosis, the researchers propose the coadministration of pharmacological anticoagulants, which directly inhibit various stages of the coagulation cascade, thereby preventing clot formation. While effective in suppressing thrombosis, this approach introduces significant systemic risks, primarily an increased propensity for bleeding, ranging from minor bruising to severe and life-threatening hemorrhages. Anticoagulants also carry potential for complex drug interactions and often necessitate stringent patient monitoring due to their narrow therapeutic windows. The overarching goal in developing novel lung-targeting delivery vehicles is therefore to achieve an optimal balance between precise tissue accumulation and a robust safety profile, addressing these critical thrombotic concerns through advanced drug delivery design.

[0052] The present invention provides lung-tropic nanoparticles that contain non-conjugated sialic acid residues, compositions, and methods of use thereof, as well as non-conjugation methods to produce nanoparticles containing sialic acid moieties. The non-conjugation methods described herein avoid the side reactions and side-products that have been observed when using conjugation methods to incorporate sialic acid residues on the nanoparticle surfaces.

[0053] The invention described herein provides pharmaceutical formulations comprising nanoparticles containing sialic acid residues (with or without agent / drug / API load), as well as processes capable of producing such pharmaceutical formulations comprising nanoparticles for treating pulmonary diseases.

[0054] The invention includes methods for preparing nanoparticles containing sialic acid residues, the methods comprising coprecipitation or coacervation of a polycation, such as a cationic lipid or polymer, and the polysialic acid. Using the invention methods, polysialic acid is integrated into the nanoparticles that are produced.

[0055] Preferably, the nanoparticles encapsulate an active pharmaceutical agent (or API) within the nanoparticles, such as an anti-cancer or immunotherapeutic agent. Preferably, said API is a nucleic acid.

[0056] With the invention generally described above, specific aspects are described further in the sections below.

[0057] Definitions

[0058] As used herein, “pharmaceutically acceptable” includes those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for medical or veterinary use when in contact with the tissues of human beings and animals at the concentration, dosage or amount present in the product, without causing excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Preferably, a pharmaceutically acceptable material (e.g., polymer, excipient, surfactant, solvent, or microparticles / nanoparticles produced therefrom) is suitable or approved for human medical use.

[0059] As used herein, “nanoparticles” are preferably roughly round, sphere, or sphere-like in shape, and are generally within the size range of, e.g., between about 1-1,000 nm, between about 10-1,000 nm, or between about 20-500 nm, or between about 50-300 nm, as measured by dynamic light scattering, for example. The subject nanoparticles may also include particles that are less likely to clump in vivo.

[0060] Particle size and size distribution can be measured by a dynamic light scattering instrument, e.g., a Malvern Zetasizer. The particle size is typically reported as a mass mean diameter. Alternative techniques include, for example, sedimentation field flow fractionation, photon correlation spectroscopy, light scattering, dynamic light scattering, light diffraction, and disk centrifugation. The term “nanoparticle” is not intended to convey any specific shape limitation. Such particles include, but are not limited to, those having a generally polyhedral or spherical geometry. Preferred particles are characterized by a spherical geometry typically produced by emulsion-based encapsulation processes. It is understood that the terms “microparticle” and “nanoparticle” are used interchangeably herein, unless accompanied by a specific description of size. For example, the term “microparticles” is intended to also embrace “nanoparticles” as if stated as “microparticles and / or nanoparticles” unless the context demands otherwise.

[0061] The term “particle” encompasses both nanoparticle and microparticles.

[0062] As used herein “a” or “an” means one or more unless otherwise specified.

[0063] As used herein, “about” generally means up to ±10% of the particular term being modified.

[0064] A “polysialic acid” is a polymer comprising sialic acid monomers. Polysialic acids are described in more detail below.

[0065] The terms “sialic acid residue” and “sialic acid moiety” as well as their plural referents, and the like, are used interchangeably herein.

[0066] As used herein, the term “subject” is used to mean an animal, preferably a mammal, including a human or non-human. The terms “patient” and “subject” may be used herein interchangeably.

[0067] “Treatment” or “therapy” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing own or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease. As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) includes to clinical intervention to alter the natural course of a disease in the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, combinations of the invention are used to delay development of a disease or to slow the progression of a disease. Polysialic acid

[0068] Polysialic acid (PSia) includes homopolymers of sialic acid. Naturally occurring PSia was first found in E. coli and is one of the ingredients of bacterial capsular materials, such as Neisseria miningitidis B, Salmonella toucra 048 and Citrobacter freundii 05. PSia can be in a conformation of a-2,8 (A in the figure below) or a-2,9 linkages (B in the figure below) or a mixture of a-2,8 and a-2,9. PSia constituted of a-2,8 bond is non-immunogenic and biodegradable and can reduce the immunogenicity of protein polypeptides. PSia possess the properties of escaping phagocytes and prolonging circulation time in vivo.

[0069] Therefore, nanoparticles having a sialic acid moiety may also facilitate RES escape and render the nanoparticles prolonged circulation in the bloodstream. In addition, since sialic acid also binds several receptors on tumor cells, sialic acid-coated nanoparticles can be leveraged to target tumor site via the high-avidity binding of sialic acid to lectins.

[0070] As described above, a polysialic acid is a polymer comprising a chain of sialic acid monomers. In certain aspects, the polymer is a homopolymer (e.g., all the sialic acid monomer units are the same). In other aspects, the polymer is a heteropolymer (e.g., the polysialic acid comprises at least two different sialic acid monomer units). In yet other aspects, the polymer comprises at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 75, at least 100, at least 200, or at least 300 sialic acid monomers. The sialic acid monomer can be any derivative of a neuraminic acid. Sialic acid monomers include, for example, N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), or deaminated neuraminic acid (Kdn; 3-deoxy-D-glycero-D- galactononulosonic acid. The sialic acid monomer is exemplified by Formula (I):

[0071] In Neu5Ac, R is -NH-C(O)-CH3. In Neu5Gc, R is -NH-C(O)-CH2-OH. In Kdn, R is OH. Other examples of sialic acid monomers are N-sialic acid, O-sialic acid, 9-O-acetyl-8-O- methyl-N-acetylneuraminic acid (Neu5,9Ac28Me), and 7,8,9-tri-O-acetyl-N- glycolylneuraminic acid (Neu5Gc7,8,9Ac3), Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac 3; Neu5,7,9Ac 3; Neu5,8,9Ac 3; Neu5,7,8,9Ac 4; Neu5Ac9Lt; Neu4,5Ac 29Lt; Neu5Ac8Me; Neu5,9Ac28Me; Neu5Ac8S; Neu5Ac9P; Neu2en5Ac; Neu2en5,9Ac 2; Neu2en5Ac9Lt; Neu2,7an5Ac; Neu5Gc; Neu4Ac5Gc; Neu7Ac5Gc; Neu8Ac5Gc; Neu9Ac5Gc; Neu7,9Ac 25Gc; Neu8,9Ac 25Gc; Neu7,8,9Ac 35Gc; Neu5Gc9Lt; Neu5Gc8Me; Neu9Ac5Gc8Me; Neu7,9Ac 25Gc8Me; Neu5Gc8S; Neu5GcAc; Neu5GcMe; Neu2en5Gc; Neu2en9Ac5Gc; Neu2en5Gc9Lt; Neu2en5Gc8Me; Neu2,7an5Gc; Neu2,7an5Gc8Me; and Knd9Ac.

[0072] As described above, sialic acid monomers can be joined by a-2,8-, a-2,9, or a-2,8 / a- 2-9-ketosidic linkages, for example. a-2,4-ketosidic linkages and a-2,5-ketosidic linkages have also been described (Janas et al. (2011), Biochimica et Biophysica Acta 1808: 2923- 2932). The sialic acid monomers can be joined in any bonding arrangement. In certain embodiments, the polysialic acid comprises monomers that are 2~>8 linked, 2~>9 linked, or a combination thereof. In yet other aspects, the monomers are all 2- 8 linked or all 2- 9 linked. In further aspects, the polysialic acid comprises Neu5Ac monomers that are 2~>8 linked, 2~>9 linked, or a combination thereof. In yet other aspects, the polysialic acid comprises Neu5Gc monomers that are 2- 8 linked, 2- 9 linked, or a combination thereof. In further embodiments, the polysialic acid comprises Kdn monomers that are 2~>8 linked, 2~>9 linked, or a combination thereof. The polysialic acid can be a homopolymer comprising monomers selected from Neu5Ac, Neu5Gc, and Kdn, or the polysialic acid can be a heteropolymer comprising 2 or 3 monomers selected from Neu5Ac, Neu5Gc, and Kdn. In certain specific embodiments, the homopolymer comprises Neu5Ac monomers. The homopolymer can be a poly(Neu5Ac)n, a poly(Neu5Gc)n, or a poly(Kdn)npolymer, wherein n is an integer greater than 10, greater than 15, or greater than 20; and optionally, wherein the monomers are 2- 8 linked, 2- 9 linked, or a combination thereof. In yet other specific embodiments, the heteropolymer comprising Neu5Ac and Neu5Gc monomers.

[0073] The polysialic acid can be a branched or unbranched polymer. An “unbranched” polymer is straight-chain polysialic acid polymer comprising a linear sequence of monomers. A “branched” polymer is a polysialic acid polymer that comprises a main chain with one more substituent side chains or branched. An example of a branched polymer is one that comprises a sialic acid unit bonded to three or more different sialic acid units, thereby creating a branch point within the polysialic acid.

[0074] The polysialic acid can, for example, have a molecular weight of at least 500 Da, at least IkDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 75 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc.

[0075] Preferably, said polysialic acid has a molecular weight of from 50 to 50,000,000, from 100 to 5,000,000, or from 500 to 500,000 Da.

[0076] In some embodiments, the polysialic acid is a polysialic acid comprising only sialic acid repeating units. This type of polymer is often referred as a “homopolymer.” One example of such homopolymer of polysialic acid is colominic acid, commercially available from Carbosynth, Oakbrook Terrace, IL, USA. Colominic acid, also referred as polysialic acid, is a linear small polysaccharide containing a-2,8-linked sialic acid (neuraminic acid) with (n = 8 to >100) residues.

[0077] In some embodiments, the polysialic acid is a “copolymer” comprising sialic acid repeating units and the repeating units of at least one different chemical entity. Non-limiting examples of such copolymers include PLGA-PSia, PEG-PSia, PLGA-PEG-PSia, etc. Here, PLGA is poly(lactide-co-glycolide), PEG is polyethylene glycol and PSia is polysialic acid.

[0078] In some embodiments, said polysialic acid is an oligomer of sialic acid, such as a dimer, a trimer, a tetramer, a pentamer and a hexamer available as N-acetylneuraminic acid oligomers or their sodium salts, available from Nacalai USA, Inc., San Diego, CA, United States.

[0079] In some embodiments, said polysialic acid is a pharmaceutically acceptable polymer having a sialic acid moiety at the terminal of its chemical structure. For example, PEG-Sia, or PLGA-PEG-Sia, where Sia represents the sialic acid moiety. The sialic acid can also be a water-soluble salt and water-soluble derivative of sialic acid. For example, the sialic acid salt can be the sodium salt, the potassium salt, the magnesium salt, the calcium salt, or the zinc salt. As described above, the polysialic acid can comprising a combination of more than one type of sialic acid.

[0080] In one set of embodiments, one or more of sialic acid monomers within the polysialic acid is modified. For example, one or more sialic acid units can be modified by attachment to polyethylene glycol, or an alkyl group. In other embodiments, the polysialic acid is not modified.

[0081] The polysialic acid can also comprise other monomers or units in addition to sialic acid monomer. In certain examples, the polysialic acid is a conjugate of a polymer of sialic acid monomer units and another polymer, for example, a synthetic polymer, including for example, polyethylene glycol (PEG) (e.g., a polysialic acid-PEG copolymer). An example of such a conjugate has been described, for example, in Zhang et al. (2018), Drug Delivery and Translational Research 8, 602-616. The PEG can have the formula: H-(O-CH2-CH2)n-OH, where n is an integer representing the PEG polymerization degree. For example, n is at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500. In some cases, n is no more than 1000, no more than 500, no more than 200, no more than 100, no more than 50, no more than 30, or no more than 10.

[0082] The polysialic acids in a particle can be the same or can be different.

[0083] In addition, the polysialic acid can be substituted covalently or ionically along the length of the chain or at the termini of the chain. For example, one or more monomer units can be substituted by a targeting moiety, such as a cell ligand (or fragment), peptide, or carbohydrate. The substitution or conjugation step of the targeting moiety can occur before the nanoparticle is formed or after.

[0084] In specific embodiments, the polysialic acid can be a ganglioside comprising multiple sialic acid units. A water-soluble ganglioside is preferred for formulating the nanoparticles of the present invention. Examples of water-soluble gangliosides include ganglioside GT1, GT2, GT3, and GQlb.

[0085] Cationic lipid

[0086] Thus, a lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino) lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge. In some embodiments, the lipid can be selected from, for example, Dioleoyl-3- trimethylammonium propane (DOTAP), l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), 3 -(didodecylamino)-N 1 ,N 1 ,4-tridodecyl- 1 -piperazineethanamine (KL 10), N 1 -[2-(didodecylamino)ethyl]-N 1 ,N4,N4-tridodecyl- 1 ,4-piperazinediethanami- ne (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy- N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]- dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)- [1,3] -di oxolane (DLin-KC2-DMA), l,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- -octadeca-9,12- dien-l-yl oxy]propan-l -amine (Octyl-CLinDMA), (2R)-2-({8-[(3.beta.)-cholest-5-en-3- yloxy]octyl})oxy)-N,N-dimethyl-3-[(9- Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA (2R)), and (2S)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N- dimethyl-3-[(9Z- ,12Z)-octadeca-9,12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA (2S)).

[0087] Polyethylene Glycol (PEG) Lipids can also be used. The term "PEG lipid" refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3 -amines. Such lipids are also referred to as PEGylated lipids. In some embodiments, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0088] Lipids that can be used in the present invention as the complexing agent may be cationic lipids. Cationic lipids are amphiphilic molecules that have a cationic head group and a hydrophobic tail group connected by either stable or degradable linkages. Guanidine, imidazole, pyridinium, piperizine, and amino acid (e.g., lysine, arginine, ornithine, and tryptophan) are common head groups used in lipid modification. Lipids that can be used as complexing agent in the present invention include but not limit to monovalent aliphatic lipids with single amine functionality in their head group, e.g., N[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA),), N-(2-hydroxyethyl)- N,N-dimethyl-2,3- bis(tetradecyloxy- 1 -propanaminiumbromide) (DMRIE), Dimethyldioctadecylammonium (DDAB), ethyl PC’s, pH sensitive lipids, multivalent aliphatic lipids with several amine functionalities in head group, e.g., spermine groups, e.g., dioctadecylamidoglycylspermine (DOGS), N4-Cholesteryl-Spermine HC1 Salt (GL67) or cationic cholesterol derivatives, e.g., 3b-[N-(N0, NO-dimethylaminoethane) carbamoyl]cholesterol (DC-Chol), bis-guanidium-tren- cholesterol (BGTC), and neutral helper lipids such as l,2-dioleyl-sn-glycerol-3- phosphoethanolamine (DOPE) or cholesterol, which were added to complex of DNA and RNA and cationic lipids to improve transfection efficiency.

[0089] An ionizable lipid is a class of lipid molecules that are neutral and non-ionic at physiological pH, but will be protonated to become positively charged at lower pHs. Ionizable lipids can also form a complex with the SA-containing entity while promoting endosome escape and reducing toxicity. Examples of commercially available ionizable lipids include DLin-KC2-DMA, DLin-MC3-DMA, DLin-DMA, DODMA, SM-102, ALC-0315, and DODAP.

[0090] In order to increase the stability, functionality and other performance properties of the complex nanoparticles, other chemical entities commonly used in lipid nanoparticle (LNP) formulations such as structural lipids, PEGylated lipids, cholesterol, phospholipids, etc. may be added to the nanoparticle formulations of the present invention.

[0091] Cationic polymer

[0092] Polymeric complexing agents can be cationic polymers comprising one or more cationic monomers and include polylysine, cell-penetrating peptides (such as polyarginine), polyethyleneimine, chitosan, and poly(amino ester).

[0093] Polylysine is a cationic homopolypeptide and can be an a-polylysine or s-polylysine. Polylysine contains a positively charged amino group at neutral pH. a-Polylysine is a synthetic polymer and can be in the form of poly-L-lysine (PLL) and poly-D-lysine (PDL), respectively. s-Polylysine (s-poly-L-lysine, EPL) is typically produced as a homopolypeptide of approximately 25-30 L-lysine residues. Polylysine used in the present invention can be a copolymer of lysine and other chemical entity. Polylysine can also be modified to possess specific properties. For example, modified polylysine may be more hydrophobic by, for example, alkylating or acylating an amine group on one or more lysines.

[0094] Cell-penetrating peptides (CPPs) have the ability to translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or an organelle. Some cell-penetrating peptides such as polyarginine are cationic and are suitable as complexing agents for nucleic acids.

[0095] Polyethyleneimine (PEI) is a polymer with a repeating unit composed of an amine group and two carbon aliphatic (CH2CH2) spacer. There are linear and branched PEIs. The linear structure facilitates crystallization of the polymer and, as a result, linear PEIs can be crystalline and solid at room temperatures. Branched PEIs can be liquid at room temperatures. Linear PEIs contain primarily secondary amines, in contrast to branched PEIs which contain primary, secondary and tertiary amino groups.

[0096] Chitosan is a linear polysaccharide composed of randomly distributed P-(l — >4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine. The amino group in chitosan has a pKa value of ~6.5, which leads to significant protonation in neutral solution and a positive charge. Thus, chitosan can be used to form complexes with nucleic acid via ionic interaction.

[0097] Preferred poly(amino ester)s are biodegradable and biocompatible polymers. One example of poly(amino ester) is poly [a-(4-aminobutyl)-l-gly colic acid],

[0098] Poly(P-amino ester)s (PBAEs) are a class of polymers obtained from di-acrylates and functional amines including a primary and a secondary amine, preferably formed via Michael addition reaction. PBAEs are pH-sensitive, biodegradable and biocompatible. The pH buffering capability of PBAEs, which results from the presence of tertiary amines in the PBAE structure, facilitates endosomal escape and hence intracellular delivery of therapeutics.

[0099] Poly(amidoamine), or PAMAM, is a class of dendrimer which is made of repetitively branched subunits of amide and amine functionality.

[0100] The cationic complexing agents can also be modified to render other desired properties. For example, the cationic complexing agents, such as PBAE, can be PEGylated, thereby extending the in vivo circulation time. The cationic complexing agents, particularly polymers, can be optimized for molecule weight, degradation profile, in vivo half-life, pH responsiveness, and other properties that may be desired for specific applications.

[0101] Active Agent

[0102] The particles described can further comprise an active agent. The composition can comprise an API, and the API can be covalently or ionically attached to the surface of the nanoparticles via covalent bonds, such as a bond formed between an amide group of a protein and a carboxyl group on the surface of the nanoparticle. The API can also be encapsulated within the nanoparticles. The amount of the API can be about 0.01 to about 50% (w / w) of the nanoparticle, or about 0.05 to about 25%, about 0.1 to about 10%, about 0.2 to about 5%, about 0.5 to about 3%, about 1 to about 5%, or about 2 to about 5% (w / w) of the nanoparticle.

[0103] In certain aspects, the active agent is advantageously a drug (also referred to herein as an active pharmaceutical ingredient, or API). However, active agents that are non-therapeutic can also be included as part of the particles according to the methods. For example, agents useful in diagnostics, agriculture, cosmetics, personal products, home products, industrial chemicals, dyes, fluorescing agents or coloring agents and the like can be included. Preferred active ingredients include small molecules and macromolecules. For example, biomolecules, such as peptides, peptidomimetics, oligonucleotides, nucleic acid molecules and mimics thereof, such as DNA, RNA, PNA, siRNA, microRNA, antisense, proteins, antibodies and antigen binding fragments thereof, enzymes, hormones, growth factors, antigens, neoantigens, saccharides, oligosaccharides, polysaccharides, gene-editing materials such as CasX (X can be 9 or any other number), gRNA, and a combination thereof. The composition can be free from other active pharmaceutical ingredients or API, such as attached peptide or antigenic moieties. It is understood that an API can be substituted with non-therapeutic compounds, such as diagnostic, agricultural, or chemical agents. Therefore, in each instance where the term API is used, it shall be understood that the term “active agent,” including diagnostic, agricultural or chemical agents can be used in lieu thereof. The term “API” and “drug” are used interchangeably herein.

[0104] The API can be water-soluble or have relatively poor water-solubility. For example, a poorly water-soluble API may be dissolved in the same solvent used to dissolve the cationic lipid or polymer or the SA agent.

[0105] An API or active agent can include a wide variety of different compounds, including chemical compounds and mixtures of chemical compounds, e.g., small organic or inorganic molecules; saccharins; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; antibodies and antigen binding fragments thereof; nucleic acids; nucleic acid analogs and derivatives; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof. Preferably, the therapeutic agent is a small molecule.

[0106] As used herein, the term "small molecule" can refer to compounds that are "natural product-like," however, the term "small molecule" is not limited to "natural product-like" compounds. Rather, a small molecule is typically characterized in that it contains several carbon-carbon bonds and has a molecular weight of less than 5000 Daltons (5 kDa), preferably less than 3 kDa, still more preferably less than 2 kDa, and most preferably less than 1 kDa. In some cases it is preferred that a small molecule have a molecular weight equal to or less than 700 Daltons.

[0107] As used herein a “peptide” is an oligopeptide, for example, a sequence of 2 to 25 amino acids. The term "peptide", unless otherwise specified, includes in its scope a peptide that contains an already known analog of a naturally-occurring amino acid having a function as well as the naturally-occurring amino acid. A "protein" comprises one or more peptide (polypeptide) chains and can comprise more amino acids than a peptide. The terms “peptide,” “polypeptide,” and “protein,” may be used interchangeably herein.

[0108] Exemplary therapeutic agents include, but are not limited to, those approved by the FDA, subject to a new drug application with the FDA, in clinical trials or in preclinical research.

[0109] APIs include the herein disclosed categories and specific examples. It is not intended that the category be limited by the specific examples. Those of ordinary skill in the art will recognize also numerous other compounds that fall within the categories and that are useful according to the present disclosure. Examples include a radiosensitizer, a steroid, a xanthine, a beta-2-agonist bronchodilator, an anti-inflammatory agent, an analgesic agent, a calcium antagonist, an angiotensin-converting enzyme inhibitors, a beta-blocker, a centrally active alpha-agonist, an alpha- 1 -antagonist, an anticholinergic / antispasmodic agent, a vasopressin analogue, an anti arrhythmic agent, an anti-parkinsonian agent, an anti- angina / antihypertensive agent, an anticoagulant agent, an antiplatelet agent, a sedative, an anxiolytic agent, a peptidic agent, a biopolymeric agent, an antineoplastic agent, a laxative, an antidiarrheal agent, an antimicrobial agent, an antifungal agent, a vaccine, a protein, or a nucleic acid. In a further aspect, the pharmaceutically active agent can be coumarin, albumin, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives; xanthines such as theophylline and doxophylline; beta-2-agonist bronchodilators such as salbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol; anti-inflammatory agents, including anti-asthmatic anti-inflammatory agents, anti-arthritis anti-inflammatory agents, and non-steroidal anti-inflammatory agents, examples of which include but are not limited to sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesulide, naproxen, acetaminophen, ibuprofen, ketoprofen and piroxicam; analgesic agents such as salicylates; calcium channel blockers such as nifedipine, amlodipine, and nicardipine; angiotensin-converting enzyme inhibitors such as captopril, benazepril hydrochloride, fosinopril sodium, trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride, and moexipril hydrochloride; betablockers (i.e., beta adrenergic blocking agents) such as sotalol hydrochloride, timolol maleate, esmolol hydrochloride, carteolol, propanolol hydrochloride, betaxolol hydrochloride, penbutolol sulfate, metoprolol tartrate, metoprolol succinate, acebutolol hydrochloride, atenolol, pindolol, and bisoprolol fumarate; centrally active alpha-2-agonists such as clonidine; alpha- 1 -antagonists such as doxazosin and prazosin; anticholinergic / antispasmodic agents such as dicyclomine hydrochloride, scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate, and oxybutynin; vasopressin analogues such as vasopressin and desmopressin; anti arrhythmic agents such as quinidine, lidocaine, tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamil hydrochloride, propafenone hydrochloride, flecainide acetate, procainamide hydrochloride, moricizine hydrochloride, and disopyramide phosphate; antiparkinsonian agents, such as dopamine, L-Dopa / Carbidopa, selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine, and bromocryptine; anti-angina agents and antihypertensive agents such as isosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol and verapamil; anticoagulant and antiplatelet agents such as Coumadin, warfarin, acetylsalicylic acid, and ticlopidine; sedatives such as benzodiazapines and barbiturates; ansiolytic agents such as lorazepam, bromazepam, and diazepam; peptidic and biopolymeric agents such as calcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporin, insulin, somatostatin, protirelin, interferon, desmopressin, somatotropin, thymopentin, pidotimod, erythropoietin, interleukins, melatonin, granulocyte / macrophage-CSF, and heparin; antineoplastic agents such as etoposide, etoposide phosphate, cyclophosphamide, methotrexate, 5 -fluorouracil, vincristine, doxorubicin, cisplatin, hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase, altretamine, mitotane, and procarbazine hydrochloride; laxatives such as senna concentrate, casanthranol, bisacodyl, and sodium picosulphate; antidiarrheal agents such as difenoxine hydrochloride, loperamide hydrochloride, furazolidone, diphenoxylate hydrochloride, and microorganisms; vaccines such as bacterial and viral vaccines; antimicrobial agents such as penicillins, cephalosporins, and macrolides, antifungal agents such as imidazolic and triazolic derivatives; and nucleic acids such as DNA sequences encoding for biological proteins, and antisense oligonucleotides.

[0110] Examples of suitable APIs include infliximab, etanercept, bevacizumab, ranibizumab, adalimumab, certolizumab pegol, golimumab, Interleukin 1 (IL-1) blockers such as anakinra, T cell costimulation blockers such as abatacept, Interleukin 6 (IL-6) blockers such as tocilizumab; Interleukin 13 (IL- 13) blockers such as lebrikizumab; Interferon alpha (IFN) blockers such as Rontalizumab; Beta 7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as Anti-Ml prime; Secreted homotrimeric LTa3 and membrane bound heterotrimer LTal / .beta.2 blockers such as Anti-lymphotoxin alpha (LTa) or anti-VEGF agents and the like. Drugs or API include proteins or peptides, including but not limited, monoclonal antibodies (e.g., humanized, human, and / or mouse / human chimeric), polyclonal antibodies, and antibody-drug conjugates. Exemplary peptide / protein therapeutics include insulin, etanercept, pegfilgrastim, salmon calcitonin, cyclosporine, octreotide, liraglutide, bivalirudin, desmopressin, Cl esterase inhibitor (RUCONSET®), human glucocerebrosidase (ELELYSO®), humanized anti-CD20 monoclonal antibody (GAVYZA®), VEGFR Fc- fusion (EYLEA®), glucagon-like peptide-1 receptor agonist Fc-fusion (TRULICITY®), VEGFR Fc-fusion (ZALTRAP), Recombinant factor IX Fc fusion (ALPROLIX), Recombinant factor VIII Fc-fusion (ELOCTATE), GLP-1 receptor agonist-albumin fusion (TANZEUM®), Recombinant factor IX albumin fusion (IDELIVION®), PEGylated IFNb-la (PLEGRIDY®), Recombinant factor VIII PEGylated (ADYNOVATE®), humanized anti- HER2 / neu conjugated to emtansine (KADCYLA®), belimumab, ipilimumab, belatacept, brentuximab vedotin, aflibercept, asparaginase erwinia chrsanthemi, glucarpidase, taliglucerase alfa, pertuzumab, ziv-afilbercept, tbo-filgrastm, ocriplasmin, raxibacumab, ado- trastuzmab emtansine, golimumab, tocilizumab, Obinutuzumab, elosulfase alfa, metreleptin, albiglutide, ramucirumab, siltuxiumab, vedolizumab, peginterferon beta- la, pembrolizumab, dulaglutide, bintumomab, nivolumab, secukinumab, parathyroid hormone, filgrastim-sndz, dinutuximab, alirocumab, evolocumab, idaracizumab, asfotase-alfa, mepolizumab, dratumumab, necitumumab, elotuzumab, sebelipase alfa, obiltoxaximab, ixekizumab, reslizumab, infliximab-dyyb, atezolizumab, daclizumab, etancerpt-szzs, coagulation factor IX recombinant human, antihemophilic factor (recombinant), coagulation factor XIII A-subunit (recombinant), coagulation factor IX (recombinant), Fc fusion protein, antihemophilic factor (recombinant), Fc fusion protein, Cl esterase inhibitor recombinant, antihemophilic factor porcine, B-domain truncated recombinant, coagulation factor IX (recombinant), antihemophilic factor (recombinant), antihemophilic factor (recombinant) PEGylated, von Willebrand factor (recombinant), coagulation factor IX recombinant human, and antihemophilic factor (recombinant).

[0111] The present invention is applicable to the administration of anti-cancer agents. For example, the agent can be a DNA demethylating agents 5-azacytidine (azacitidine) or 5-aza- 2'-deoxycytidine (decitabine), (Cytarabine or ara-C); pseudoiso-cytidine (psi ICR); 5-fluoro- 2'-deoxycytidine (FCdR); 2'-deoxy-2',2'-difluorocytidine (Gemcitabine); 5-aza-2'-deoxy-2',2'- difluorocytidine; 5-aza-2'-deoxy-2'-fluorocytidine; Zebularine; 2',3'-dideoxy-5-fluoro-3'- thiacytidine (Emtriva); 2'-cyclocytidine (Ancitabine); Fazarabine or ara-AC; 6-azacytidine (6-aza-CR); 5,6-dihydro-5-azacytidine (dH-aza-CR); N.sup.4-pentyloxy-carbonyl-5'-deoxy- 5-fluorocytidine (Capecitabine); N4-octadecyl-cytarabine; or elaidic acid cytarabine. The cytidine analog can also be structurally related to cytidine or deoxy cytidine and functionally mimics and / or antagonizes the action of cytidine or deoxycytidine. The agents can also include 5 -fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL- 101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatinum, COX-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, DM1, DM3, DM4, doxorubicin, 2- pyrrolinodoxorubicine (2-PDox), a pro-drug form of 2-PDox (pro-2 -PDox), cyanomorpholino doxorubicin, doxorubicin glucuronide, endostatin, epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP 16), etoposide glucuronide, etoposide phosphate, exemestane, fmgolimod, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib, ganetespib, GDC-0834, GS- 1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, monomethylauristatin F (MMAF), monomethylauristatin D (MMAD), monomethylauristatin E (MMAE), navelbine, neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, SN-38, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide, transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD 1839 or a pharmaceutically acceptable salt thereof.

[0112] The anticancer agents include, but are not limited to, an inhibitor, agonist, antagonist, ligand, modulator, stimulator, blocker, activator or suppressor of a gene, ligand, receptor, protein, factor such as an adenosine receptor (such as A2B, A2a, A3), Abelson murine leukemia viral oncogene homolog 1 gene (ABL, such as ABL1), Acetyl-CoA carboxylase (such as ACC 1 / 2), adrenocorticotropic hormone receptor (ACTH), activated CDC kinase (ACK, such as ACK1), Adenosine deaminase, Adenylate cyclase, ADP ribosyl cyclase- 1, Aerolysin, Angiotensinogen (AGT) gene, murine thymoma viral oncogene homolog 1 (AKT) protein kinase (such as AKT1, AKT2, AKT3), AKT1 gene, Alkaline phosphatase, Alpha 1 adrenoceptor, Alpha 2 adrenoceptor, Alpha-ketoglutarate dehydrogenase (KGDH), Aminopeptidase N, Arginine deiminase, Beta adrenoceptor, Anaplastic lymphoma kinase receptor, anaplastic lymphoma kinase (ALK, such as ALK1), Alk-5 protein kinase, AMP activated protein kinase, Androgen receptor, Angiopoietin (such as ligand- 1, ligand-2), apolipoprotein A-I (APOA1) gene, apoptosis signal-regulating kinase (ASK, such as ASK1), Apoptosis inducing factor, apoptosis protein (such as 1, 2), Arginase (I), asparaginase, Asteroid homolog 1 (ASTE1) gene, ataxia telangiectasia and Rad 3 related (ATR) serine / threonine protein kinase, Axl tyrosine kinase receptor, Aromatase, Aurora protein kinase (such as 1, 2), Basigin, BCR (breakpoint cluster region) protein and gene, B-cell lymphoma 2 (BCL2) gene, Bcl2 protein, Bcl2 binding component 3, BCL2L11 gene, Baculoviral IAP repeat containing 5 (BIRCS) gene, B-Raf proto-oncogene (BRAF), Brc-Abl tyrosine kinase, Beta-catenin, B-lymphocyte antigen CD 19, B-lymphocyte antigen CD20, B- lymphocyte stimulator ligand, B-lymphocyte cell adhesion molecule, Bone morphogenetic protein- 10 ligand, Bone morphogenetic protein-9 ligand modulator, Brachyury protein, Bradykinin receptor, Bruton's tyrosine kinase (BTK), Bromodomain and external domain (BET) bromodomain containing protein (such as BRD2, BRD3, BRD4), Calmodulin, calmodulin-dependent protein kinase (CaMK, such as CAMKII), Cancer testis antigen 2, Cancer testis antigen NY-ESO-1, Cannabinoid receptor (such as CB1, CB2), Carbonic anhydrase, caspase 8 apoptosis-related cysteine peptidase CASP8-FADD-like regulator, Caspase (such as caspase-3, caspase-7, Caspase-9), Caspase recruitment domain protein-15, Cathepsin G, chemokine (C-C motif) receptor (such as CCR2, CCR4, CCR5), CCR5 gene, Chemokine CC21 ligand, cluster of differentiation (CD) such as CD4, CD27, CD29, CD30, CD33, CD37, CD40, CD40 ligand receptor, CD40 ligand, CD40LG gene, CD44, CD45, CD47, CD49b, CD51, CD52, CD55, CD58, CD66e, CD70 gene, CD74, CD79, CD79b, CD79B gene, CD80, CD95, CD99, CD117, CD122, CDwl23, CD134, CDwl37, CD158a, CD158bl, CD158b2, CD223, CD276 antigen; Chorionic gonadotropin, Cyclin Gl, Cyclin DI, cyclin-dependent kinases (CDK, such as CDK1, CDK1B, CDK2-9), casein kinase (CK, such as CM, CMI), c-Kit (tyrosine-protein kinase Kit or CD117), c-Met (hepatocyte growth factor receptor (HGFR)), CDK-activating kinase (CAK), Checkpoint kinase (such as CHK1, CHK2), Cholecystokinin CCK2 receptor, Claudin (such as 6, 18), Clusterin, Complement C3, COP9 signalosome subunit 5, CSF-1 (colony-stimulating factor 1 receptor), CSF2 gene, clusterin (CLU) gene, Connective tissue growth factor, cyclooxygenase (such as 1, 2), cancer / testis antigen IB (CTAG1) gene, CTLA-4 (cytotoxic T-lymphocyte protein 4) receptor, CYP2B1 gene, Cysteine palmitoyltransferase porcupine, cytokine signalling- 1, cytokine signalling-3, Cytochrome P450 11B2, Cytochrome P450 reductase, cytochrome P450 3A4, cytochrome P450 17A1, Cytochrome P450 17, Cytochrome P450 2D6, (provided the anticancer or cytrochrome modifying agents are something other than cobicistat), Cytoplasmic isocitrate dehydrogenase, Cytosine deaminase, cytosine DNA methyltransferase, cytotoxic T-lymphocyte protein-4, chemokine (C— X— C motif) receptor (such as CXCR4, CXCR1 and CXCR2), Delta-like protein ligand (such as 3, 4), Deoxyribonuclease, Dickkopf- 1 ligand, Dihydropyrimidine dehydrogenase, DNA binding protein (such as HU-beta), DNA dependent protein kinase, DNA gyrase, DNA methyltransferase, DNA polymerase (such as alpha), DNA primase, discoidin domain receptor (DDR, such as DDR1), DDR2 gene, dihydrofolate reductase (DHFR), Dipeptidyl peptidase IV, L-dopachrome tautomerase, dUTP pyrophosphatase, echinoderm microtubule like protein 4, epidermal growth factor receptor (EGFR) gene, EGFR tyrosine kinase receptor, Eukaryotic translation initiation factor 5 A (EIFSA) gene, Elastase, Elongation factor 1 alpha 2, Elongation factor 2, Endoglin, Endonuclease, Endoplasmin, Endosialin, Endostatin, endothelin (such as ET-A, ET-B), Enhancer of zeste homolog 2 (EZH2), epidermal growth factor, epidermal growth factor receptors (EGFR), Epithelial cell adhesion molecule (EpCAM), Ephrin (EPH) tyrosine kinase (such as Epha3, Ephb4), Ephrin B2 ligand, Epigen, Erb-b2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2) tyrosine kinase receptor, Erb-b3 tyrosine kinase receptor, Erb-b4 tyrosine kinase receptor, Extracellular signal-regulated kinases (ERK), E- selectin, Estradiol 17 beta dehydrogenase, Estrogen receptor (such as alpha, beta), Estrogen related receptor, Exportin 1, Extracellular signal related kinase (such as 1, 2), Factor (such as Xa, Vila), Fas ligand, Fatty acid synthase, Ferritin, focal adhesion kinase (FAK, such as FAK2), fibroblast growth factor (FGF, such as FGF1, FGF2, FGF4), FGF-2 ligand, FGF-5 ligand, Fibronectin, Fms-related tyrosine kinase 3 (Flt3), famesoid x receptor (FXR), Folate, Folate transporter 1, Folate receptor (such as alpha), folate hydrolase prostate-specific membrane antigen 1 (FOLH1), paired basic amino acid cleaving enzyme (FURIN), FYN tyrosine kinase, Galactosyltransferase, Galectin-3, glucocorticoid-induced TNFR-related protein GITR receptor, Glucocorticoid, Beta-glucuronidase, Glutamate carboxypeptidase II, glutaminase, Glutathione S-transferase P, Glypican 3 (GPC3), glycogen synthase kinase (GSK, such as 3-beta), Granulocyte-colony stimulating factor (GCSF) ligand, Granulocyte macrophage colony stimulating factor (GM-CSF) receptor, gonadotropin-releasing hormone (GNRH), growth factor receptor-bound protein 2 (GRB2), molecular chaperone groEL2 gene, Grp78 (78 kDa glucose-regulated protein) calcium binding protein, Imprinted Maternally Expressed Transcript (Hl 9) gene, Heat stable enterotoxin receptor, Heparanase, Hepatocyte growth factor, Heat shock protein gene, Heat shock protein (such as 27, 70, 90 alpha, beta), Hedgehog protein, HERV-H LTR associating protein 2, Hexose kinase, tyrosine-protein kinase HCK, Histamine H2 receptor, histone deacetylase (HD AC, such as 1, 2, 3, 6, 10, 11), Histone Hl, Histone H3, Histone methyltransferase (DOT IL), Human leukocyte antigen (HLA), HLA class I antigen (A-2 alpha), HLA class II antigen, Homeobox protein NANOG, mitogen-activated protein kinase kinase 1 (MAP4K1, HPK1), HSPB1 gene, Human papillomavirus (such as E6, E7) protein, Hyaluronidase, Hyaluronic acid, Hypoxia inducible factor- 1 alpha, Intercellular adhesion molecule 1 (ICAM-1), immunoglobulin (such as G, Gl, G2, K, M), indoleamine 2, 3 -dioxygenase (IDO, such as IDO1), indoleamine pyrrole 2,3 -dioxygenase 1 inhibitor, LKappa-B kinase (IKK, such as IKK.beta.. epsilon.), Immunoglobulin Fc receptor, Immunoglobulin gamma Fc receptor (such as I, III, IIIA), Interleukin 1 ligand, interleukin 2 ligand, Interleukin-2, IL-2 gene, IL-1 alpha, IL-1 beta, IL- 2, IL-2 receptor alpha subunit, IL-3 receptor, IL-4, IL-6, IL-7, IL-8, IL-12, IL-15, IL-12 gene, IL- 17, Interleukin 13 receptor alpha 2, Interleukin-29 ligand, interleukin- 1 receptor- associated kinase 4 (IRAK4), Insulin-like growth factor (such as 1, 2), insulin receptor, Integrin alpha- V / beta-3, Integrin alpha-V / beta-5, Integrin alpha-V / beta-6, Integrin alpha- 5 / beta-l, Integrin alpha-4 / beta-l, integrin alpha-4 / beta-7, Interferon inducible protein absent in melanoma 2 (AIM2), interferon (such as alpha, alpha 2, beta, gamma), interferon type I receptor, isocitrate dehydrogenase (such as IDH1, IDH2), Janus kinase (JAK, such as JAK1, JAK2), Jun N terminal kinase, Kinase insert domain receptor (KDR), Killer cell Ig like receptor, Kisspeptin (KISS-1) receptor, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) tyrosine kinase, KIT gene, Kinesin-like protein KIF11, kallikrein- related peptidase 3 (KLK3) gene, Kirsten rat sarcoma viral oncogene homolog (KRAS) gene, lactoferrin, lymphocyte activation gene 3 protein (LAG-3), lysosomal-associated membrane protein family (LAMP) gene, Lanosterol- 14 demethylase, LDL receptor related protein- 1, Leukotriene A4 hydrolase, Listeriolysin, L-Selectin, Luteinizing hormone receptor, Lyase, Lymphocyte antigen 75, lysine demethylases (such as KDM1, KDM2, KDM4, KDM5, KDM6, A / B / C / D), Lymphocyte function antigen-3 receptor, lymphocyte-specific protein tyrosine kinase (LCK), Lymphotactin, Lyn (Lck / Yes novel) tyrosine kinase, Lysophosphatidate-1 receptor, lysyl oxidase protein (LOX), lysyl oxidase-like protein (LOXL, such as LOXL2), Lysyl oxidase homolog 2, Macrophage migration inhibitory fact, melanoma antigen family A3 (MAGEA3) gene, MAGECI gene, MAGEC2 gene, Major vault protein, myristoylated alanine-rich protein kinase C substrate (MARCKS) protein, Melan-A (MART-1) melanoma antigen, Mas-related G-protein coupled receptor, matrix metalloprotease (MMP, such as MMP2, MMP9), myeloid cell leukemia 1 (MCL1) gene, Mcl-1 differentiation protein, macrophage colony-stimulating factor (MCSF) ligand, Melanoma associated antigen (such as 1, 2, 3, 6), melanocyte stimulating hormone ligand, Melanocyte protein Pmel 17, Membrane copper amine oxidase, Mesothelin, Metabotropic glutamate receptor 1, mitogen-activated protein kinase (MEK, such as MEK1, MEK2), Hepatocyte growth factor receptor (MET) gene, MET tyrosine kinase, methionine aminopeptidase-2, mitogen-activate protein kinase (MAPK), Mdm2 p53-binding protein, Mdm4 protein, Metalloreductase STEAP1 (six transmembrane epithelial antigen of the prostate 1), Metastin, Methyltransferase, Mitochondrial 3 ketoacyl CoA thiolase, MAPK- activated protein kinase (such as MK2), mTOR (mechanistic target of rapamycin (serine / threonine kinase), mTOR complex (such as 1, 2), mucin (such as 1, 5 A, 16), mut T homolog (MTH, such as MTH1), Myc proto-oncogene protein, NAD ADP ribosyltransferase, natriuretic peptide receptor C, Neural cell adhesion molecule 1, Neurokinin receptor, Neuropilin 2, Nitric oxide synthase, Nuclear Factor (NF) kappa B, NF kappa B activating protein, Neurokinin 1 (NK1) receptor, NK cell receptor, NK3 receptor, NKG2 A B activating NK receptor, NIMA-related kinase 9 (NEK9), Noradrenaline transporter, Notch (such as Notch-2 receptor, Notch-3 receptor), nucleophosmin-anaplastic lymphoma kinase (NPM- ALK), 2,5-oligoadenylate synthetase, Nuclear erythroid 2-related factor 2, Nucleolin, Nucleophosmin, O-methylguanine DNA methyltransferase, Ornithine decarboxylase, Orotate phosphoribosyltransferase, orphan nuclear hormone receptor NR4 Al, Opioid receptor (such as delta), Osteocalcin, Osteoclast differentiation factor, Osteopontin, OX-40 (tumor necrosis factor receptor superfamily member 4 TNFRSF4, or CD 134) receptor, 2 oxoglutarate dehydrogenase, purinergic receptor P2X ligand gated ion channel 7 (P2X7), Parathyroid hormone ligand, p53 tumor suppressor protein, P3 protein, Programmed cell death 1 (PD-1), Proto-oncogene serine / threonine-protein kinase (PIM, such as PIM-1, PIM-2, PIM-3), Poly ADP ribose polymerase (PARP, such as PARP1, 2 and 3), p38 kinase, p38 MAP kinase, platelet-derived growth factor (PDGF, such as alpha, beta), P-Gly coprotein (such as 1), Platelet-derived growth factor (PDGF, such as alpha, beta), PKN3 gene, P-Selectin, phosphatidylinositol 3 -kinase (PI3K), phosphoinositide-3 kinase (PI3K such as alpha, delta, gamma), phosphorylase kinase (PK), placenta growth factor, Pleiotropic drug resistance transporter, Pl exin Bl, Polo-like kinase 1, peroxisome proliferator-activated receptors (PPAR, such as alpha, delta, gamma), Preferentially expressed antigen in melanoma (PRAME) gene, Probable transcription factor PML, Programmed cell death ligand 1 inhibitor (PD-L1), Progesterone receptor, prostate specific antigen, Prostatic acid phosphatase, Prostanoid receptor (EP4), proteasome, Protein famesyltransferase, protein kinase (PK, such as A, B, C), Protein E7, protein tyrosine kinase, Protein tyrosine phosphatase beta, polo-like kinase (PLK), PLK1 gene, Prenyl-binding protein (PrPB), protoporphyrinogen oxidase, Prosaposin (PSAP) gene, phosphatase and tensin homolog (PTEN), Purine nucleoside phosphorylase, Pyruvate kinase (PYK), Pyruvate dehydrogenase (PDH), Pyruvate dehydrogenase kinase, Raf protein kinase (such as 1, B), RAFI gene, Ras GTPase, Ras gene, 5 -Alpha-reductase, RET gene, Ret tyrosine kinase receptor, retinoblastoma associated protein, retinoic acid receptor (such as gamma), Retinoid X receptor, Rheb (Ras homolog enriched in brain) GTPase, Rho (Ras homolog) associated protein kinase 2, ribonuclease, Ribonucleotide reductase (such as M2 subunit), Ribosomal protein S6 kinase, RNA polymerase (such as I, II), Ron (Recepteur d'Origine Nantais) tyrosine kinase, ROS1 (ROS proto-oncogene 1, receptor tyrosine kinase) gene, Rosl tyrosine kinase, Runt-related transcription factor 3, 5100 calcium binding protein A9, Sarco endoplasmic calcium ATPase, Gamma-secretase, Secreted frizzled related protein-2, Semaphorin-4D, SL cytokine ligand, Serine protease, Signaling lymphocytic activation molecule (SLAM) family member 7, spleen tyrosine kinase (SYK), Src tyrosine kinase, tumor progression locus 2 (TPL2), serine / threonine kinase (STK), signal transduction and transcription (STAT, such as STAT-1, STAT-3, STAT-5), Second mitochondria-derived activator of caspases (SMAC) protein, smoothened (SMO) receptor, Sodium phosphate cotransporter 2B, Sodium iodide cotransporter, Somatostatin receptor (such as 1, 2, 3, 4, 5), Sonic hedgehog protein, Specific protein 1 (Spl) transcription factor, Sphingomyelin synthase, Sphingosine- 1 -phosphate receptor-1, Sphingosine kinase (such as 1, 2), SRC gene, STAT3 gene, six-transmembrane epithelial antigen of the prostate (STEAP) gene, Steroid sulfatase, stimulator of interferon genes protein, Stimulator of interferon genes (STING) receptor, Stromal cell-derived factor 1 ligand, SUMO (small ubiquitin-like modifier), Superoxide dismutase, Survivin protein, Synapsin 3, Syndecan-1, Synuclein alpha, serine / threonine-protein kinase (TBK, such as TBK1), TATA box-binding protein-associated factor RNA polymerase I subunit B (TAF1B) gene, T-cell surface glycoprotein CD8, T-cell CD3 glycoprotein zeta chain, T-cell differentiation antigen CD6, T cell surface glycoprotein CD28, Tec protein tyrosine kinase, Tek tyrosine kinase receptor, telomerase, Tenascin, Telomerase reverse transcriptase (TERT) gene, Transforming growth factor (TGF, such as beta) kinase, TGF beta 2 ligand, T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), Tissue factor, Tumor necrosis factor (TNF, such as alpha, beta), TNF related apoptosis inducing ligand, TNFR1 associated death domain protein, TNFSF9 gene, TNFSF11 gene, trophoblast glycoprotein (TPBG) gene, Transferrin, Tropomyosin receptor kinase (Trk) receptor (such as TrkA, TrkB, TrkC), Trophoblast glycoprotein, Thymidylate synthase, Tyrosine kinase with immunoglobulin-like and EGF-like domains (TIE) receptor, Toll-like receptor (TLR such as 1-13), topoisomerase (such as I, II, III), Tumor protein 53 (TP53) gene, Transcription factor, Transferase, Transforming growth factor TGF-.beta. receptor kinase, Transglutaminase, Translocation associated protein, Transmembrane glycoprotein NMB, Tumor necrosis factor 13C receptor, Thymidine kinase, Thymidine phosphorylase, Thymidylate synthase, Thymosin (such as alpha 1), Thyroid hormone receptor, Trop-2 calcium signal transducer, Thyroid stimulating hormone receptor, Tryptophan 5-hydroxylase, Tyrosinase, tyrosine kinase (TK), Tyrosine kinase receptor, Tyrosine protein kinase ABL1 inhibitor, tank-binding kinase (TBK), Thrombopoietin receptor, TNF -related apoptosis-inducing ligand (TRAIL) receptor, Tubulin, Tumor suppressor candidate 2 (TUSC2) gene, Tyrosine hydroxylase, Ubiquitin-conjugating enzyme E2I (UBE2I, UBC9), Ubiquitin, Ubiquitin carboxyl hydrolase isozyme L5, Ubiquitin thioesterase- 14, Urease, Urokinase plasminogen activator, Uteroglobin, Vanilloid VR1, Vascular cell adhesion protein 1, vascular endothelial growth factor receptor (VEGFR), V- domain Ig suppressor of T-cell activation (VISTA), VEGF-1 receptor, VEGF-2 receptor, VEGF-3 receptor, VEGF-A, VEGF-B, Vimentin, Vitamin D3 receptor, Proto-oncogene tyrosine-protein kinase Yes, Wee-1 protein kinase, Wilms' tumor protein, Wilms' tumor antigen 1, X-linked inhibitor of apoptosis protein, Zinc finger protein transcription factor or any combination thereof.

[0113] The anticancer agent includes agents defined by their mechanism of action or class, including: anti-metabolites / anti-cancer agents such as pyrimidine analogs floxuridine, capecitabine, cytarabine, CPX-351 (liposomal cytarabine, daunorubicin), TAS-118; purine analogs, folate antagonists (such as pralatr exate), and related inhibitors; antiproliferative / antimitotic agents including natural products such as vinca alkaloid (vinblastine, vincristine) and microtubule such as taxane (paclitaxel, docetaxel), vinblastin, nocodazole, epothilones, vinorelbine) (NAVELBINE), and epipodophyllotoxins (etoposide, teniposide); DNA damaging agents such as actinomycin, amsacrine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide) (CYTOXAN), dactinomycin, daunorubicin, doxorubicin, epirubicin, iphosphamide, melphalan, merchlorethamine, mitomycin C, mitoxantrone, nitrosourea, procarbazine, taxol, Taxotere, teniposide, etoposide, and tri ethylenethiophosphoramide; DNA-hypom ethylating agent such as guadecitabine (SGI- 110) antibiotics such as dactinomycin, daunorubicin, doxorubicin, idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin), and; enzymes such as L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine; antiplatelet agents; a DNAi oligonucleotide targeting Bcl-2 such as PNT2258; agents that activate or reactivate latent human immunodeficiency virus (HIV) such as panobinostat or romidepsin asparaginase stimulators, such as crisantaspase (ERWINASE ®) and GRASPA (ERY-001, ERY-ASP); pan-Trk, ROS1 and ALK inhibitors such as entrectinib anaplastic lymphoma kinase (ALK) inhibitors such as alectinib antiproliferative / antimitotic alkylating agents such as nitrogen mustards cyclophosphamide and analogs (melphalan, chlorambucil, hexamethylmelamine, and thiotepa), alkyl nitrosoureas (carmustine) and analogs, streptozocin, and triazenes (dacarbazine); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, oxiloplatinim, and carboplatin), procarbazine, hydroxyurea, mitotane, and aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, and nilutamide), and aromatase inhibitors (letrozole and anastrozole); anticoagulants such as heparin, synthetic heparin salts, and other inhibitors of thrombin; fibrinolytic agents such as tissue plasminogen activator, streptokinase, urokinase, aspirin, dipyridamole, ticlopidine, and clopidogrel; antimigratory agents; antisecretory agents (breveldin); immunosuppressives tacrolimus, sirolimus, azathioprine, and mycophenolate; compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor inhibitors, and fibroblast growth factor inhibitors such as FPA14; angiotensin receptor blockers, nitric oxide donors; antisense oligonucleotides, such as AEG35156; DNA interference oligonucleotides, such as PNT2258, AZD-9150 antibodies such as trastuzumab and rituximab; anti-HER3 antibodies, such as LJM716 anti-HER2 antibodies such as margetuximab; anti-HLA-DR antibodies such as IMMU-114; anti-IL-3 antibodies, such as JNJ-56022473; anti-OX40 antibodies such as MEDI6469 anti-EphA3 antibodies, such as KB-004; an anti-CD20 antibody such as obinutuzumab; an antiprogrammed cell death protein 1 (anti-PD-1) antibody such as nivolumab (OPDIVO, BMS- 936558, MDX-1106), pembrolizumab (KEYTRUDA, MK-3477, SCH-900475, lambrolizumab, CAS Reg. No. 1374853-91-4), pidilizumab, and anti-programmed deathligand 1 (anti-PD-Ll) antibodies such as BMS-936559, atezolizumab (MPDL3280A), durvalumab (MEDI4736), avelumab (MSB0010718C), and MDX1105-01, CXCR4 antagonists such as BL-8040; CXCR2 antagonist such as AZD-5069; GM-CSF antibodies such as lenzilumab. Selective estrogen receptor downregulator (SERD) such as fulvestrant (Faslodex); a transforming growth factor-beta (TGF-beta) kinase antagonist such as galunisertib; a bispecific antibody such as MM-141 (IGF-l / ErbB3), MM-111 (Erb2ZErb3), JNJ-64052781 (CD19 / CD3). Mutant selective EGFR inhibitors, such as PF-06747775, EGF816, ASP8273, ACEA-0010, BI-1482694. Alpha-ketoglutarate dehydrogenase (KGDH) inhibitors such as CPI-613, XPO1 inhibitors such as selinexor (KPT-330). Isocitrate dehydrogenase 2 (IDH2) inhibitors such as enasidenib (AG-221), and IDH1 inhibitors such as AG-120, and AG-881 (IDH1 and IDH2). Agents that target the interleukin-3 receptor (IL- 3R) such as SL-401. Arginine deiminase stimulators, such as pegargiminase (ADI-PEG-20) antibody-drug conjugates, such as MLN0264 (anti-GCC, guanylyl cyclase C), T-DM1 (trastuzumab emtansine, Kadcycla), milatuzumab-doxorubicin (hCD74-DOX), brentuximab vedotin, DCDT2980S, polatuzumab vedotin, SGN-CD70A, SGN-CD19A, inotuzumab ozogamicin, lorvotuzumab mertansine, SAR3419, isactuzumab govitecan, anti-claudin-18.2 antibodies such as IMAB362 .beta.-catenin inhibitors, such as CWP-291 a CD73 antagonist such as MEDI-9447; c-PIM inhibitors, such as PIM447, a BRAF inhibitor such as dabrafenib, vemurafenib, a sphingosine kinase-2 (SK2) inhibitor such as Yeliva. (ABC294640) cell cycle inhibitors such as selumetinib (MEK1 / 2), sapacitabine, AKT inhibitors such as MK-2206, ipatasertib, afuresertib, anti-CTLA-4 (cytotoxic T-lymphocyte protein-4) inhibitor such as tremelimumab, c-MET inhibitors, such as AMG-337, savolitinib, tivantinib (ARQ-197), capmatinib, tepotinib inhibitors of CSF1R / KIT and FLT3 such as PLX3397, a kinase inhibitor such as vandetanib; E selectin antagonists such as GMI-1271, differentiation inducers such as tretinoin; epidermal growth factor receptor (EGFR) inhibitors such as osimertinib (AZD-9291) topoisomerase inhibitors (doxorubicin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mitoxantrone, pixantrone, sobuzoxane, topotecan, and irinotecan, MM-398 (liposomal irinotecan), vosaroxin and corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signal transduction kinase inhibitors; dysfunction inducers; nucleoside analogs such as DFP-10917 Axl inhibitors such as BGB-324; BET inhibitors such as INCB-054329, PARP inhibitors such as olaparib, rucaparib, veliparib, Proteasome inhibitors such as ixazomib, carfilzomib (Kyprolis); Glutaminase inhibitors such as CB-839; vaccines such as peptide vaccine TG-01 (RAS), bacterial vector vaccines such as CRS- 207 / GVAX, autologous Gp96 vaccine, dendritic cells vaccines, Oncoquest-L vaccine, DPX- Survivac, ProstAtak, DCVAC, ADXS31-142, demcizumab (anti-DLL4, Delta-like ligand 4, Notch pathway), napabucasin (BBI-608) smoothened (SMO) receptor inhibitors, such as 0D0MZ0®. (sonidegib, formerly LDE-225), LEQ506, vismodegib (GDC-0449), BMS- 833923, glasdegib (PF-04449913), LY2940680, and itraconazole; interferon alpha ligand modulators, such as interferon alfa-2b, interferon alpha-2a biosimilar (Biogenomics), ropeginterferon alfa-2b (AOP-2014, P-1101, PEG IFN alpha-2b), Multiferon (Alfanative, Viragen), interferon alpha lb, Roferon-A (Canferon, Ro-25-3036), interferon alfa-2a follow- on biologic (Biosidus) (Inmutag, Inter 2A), interferon alfa-2b follow-on biologic (Biosidus- Bioferon, Citopheron, Ganapar) (Beijing Kawin Technology -Kaferon) (AXXO-interferon alfa-2b), Alfaferone, pegylated interferon alpha- lb, peginterferon alfa-2b follow-on biologic (Amega), recombinant human interferon alpha-lb, recombinant human interferon alpha-2a, recombinant human interferon alpha-2b, veltuzumab-IFN alpha 2b conjugate, Dynavax (SD- 101), and interferon alfa-nl (Humoferon, SM-10500, Sumiferon); interferon gamma ligand modulators, such as interferon gamma (OH-6000, Ogamma 100); IL-6 receptor modulators, such as tocilizumab, siltuximab, AS-101 (CB-06-02, IVX-Q-101); Telomerase modulators, such as tertomotide (GV-1001, HR-2802, Riavax) and imetelstat (GRN-163, JNJ-63935937) DNA methyltransferases inhibitors, such as temozolomide (CCRG-81045), decitabine, guadecitabine (S-l 10, SGI- 110), KRX-0402, and azacitidine; DNA gyrase inhibitors, such as pixantrone and sobuzoxane; Bcl-2 family protein inhibitor ABT-263, venetoclax (ABT-199), ABT-737, and AT-101; Notch inhibitors such as LY3039478, tarextumab (anti-Notch2 / 3), BMS-906024 anti-myostatin inhibitors such as landogrozumab, hyaluronidase stimulators such as PEGPH-20, Wnt pathway inhibitors such as SM-04755, PRI-724, gamma-secretase inhibitors such as PF-03084014, IDO inhibitors such as indoximod, Grb-2 (growth factor receptor bound protein-2) inhibitor BP 1001 (liposomal Grb-2), TRAIL pathway-inducing compounds, such as ONC201, Focal adhesion kinase inhibitors such as VS-4718, defactinib, hedgehog inhibitors such as saridegib, sonidegib (LDE225), glasdegib and vismodegib, Aurora kinase inhibitors such as alisertib (MLN-8237), modulators of HSPB1 activity (heat shock protein 27, HSP27), such as brivudine, apatorsen, ATR inhibitor such as AZD6738, and VX-970, mTOR inhibitors, such as sapanisertib, Hsp90 inhibitors such as AUY922. Murine double minute (mdm2) oncogene inhibitors such as DS-3032b CD137 agonist such as urelumab, Anti -KIR monoclonal antibodies such as lirilumab (IPH-2102). Antigen CD 19 inhibitors such as MOR208, MEDI-551, AFM-11, CD44 binders such as A6, CYP17 inhibitors, such as VT-464, ASN-001, ODM-204. RXR agonists such as IRX4204, TLRs (Toll-like receptors) agonists such as IMO-8400 A hedgehog / smoothened (hh / Smo) antagonist such as taladegib. Immunomodulators such as complement C3 modulators, such as Imprime PGG. Intratumural immune-oncology agents such as G100 (TLR4 agonist) IL-15 agonists such as ALT-803 EZH2 (enhancer of zeste homolog 2) inhibitors such as tazemetostat. Oncolytic viruses, such as pelareorep, and talimogene laherparepvec). DOT1L (histone methyltransferase) inhibitors such as pinometostat (EPZ-5676), toxins such as Cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, diphtheria toxin, and caspase activators; and chromatin. DNA plasmid such as BC-819. PLK inhibitors of PLK 1, 2, and 3, such as volasertib (PLK1). Apoptosis Signal-Regulating Kinase (ASK) Inhibitors: ASK inhibitors include ASK1 inhibitors. Examples of ASK1 inhibitors include, but are not limited to, those described in WO 2011 / 008709 (Gilead Sciences) and WO 2013 / 112741 (Gilead Sciences). Bruton's Tyrosine Kinase (BTK) Inhibitors: Examples of BTK inhibitors include, but are not limited to, (S)-6-amino-9-(l-(but-2-ynoyl)pyrrolidin-3- yl)-7-(4-phenoxyphenyl)-7H-pur- in-8(9H)-one, acalabrutinib (ACP-196), BGB-3111, HM71224, ibrutinib, M-2951, ONO-4059, PRN-1008, spebrutinib (CC-292), TAK-020. Cyclin-dependent Kinase (CDK) Inhibitors: CDK inhibitors include inhibitors of CDK 1, 2, 3, 4, 6 and 9, such as abemaciclib, alvocidib (HMR-1275, flavopiridol), AT-7519, FLX-925, LEE001, palbociclib, ribociclib, rigosertib, selinexor, UCN-01, and TG-02. Discoidin Domain Receptor (DDR) Inhibitors: DDR inhibitors include inhibitors of DDR1 and / or DDR2. Examples of DDR inhibitors include, but are not limited to, those disclosed in WO 2014 / 047624 (Gilead Sciences), US 2009-0142345 (Takeda Pharmaceutical), US 2011- 0287011 (Oncomed Pharmaceuticals), WO 2013 / 027802 (Chugai Pharmaceutical), and WO 2013 / 034933 (Imperial Innovations). Histone Deacetylase (HDAC) Inhibitors: Examples of HDAC inhibitors include, but are not limited to, abexinostat, ACY-241, AR-42, BEBT-908, belinostat, CKD-581, CS-055 (HB 1-8000), CUDC-907, entinostat, givinostat, mocetinostat, panobinostat, pracinostat, quisinostat (JNJ-26481585), resminostat, ricolinostat, SHP-141, valproic acid (VAL-001), vorinostat. Janus Kinase (JAK) Inhibitors: JAK inhibitors inhibit JAK1, JAK2, and / or JAK3. Examples of JAK inhibitors include, but are not limited to, AT9283, AZD1480, baricitinib, BMS-911543, fedratinib, filgotinib (GLPG0634), gandotinib (LY2784544), INCB039110, lestaurtinib, momelotinib (CYT0387), NS-018, pacritinib (SB 1518), peficitinib (ASP015K), ruxolitinib, tofacitinib (formerly tasocitinib), and XL019. Lysyl Oxidase-Like Protein (LOXL) Inhibitors: LOXL inhibitors include inhibitors of LOXL1, LOXL2, LOXL3, LOXL4, and / or LOXL5. Examples of LOXL inhibitors include, but are not limited to, the antibodies described in WO 2009 / 017833 (Arresto Biosciences). Examples of LOXL2 inhibitors include, but are not limited to, the antibodies described in WO 2009 / 017833 (Arresto Biosciences), WO 2009 / 035791 (Arresto Biosciences), and WO 2011 / 097513 (Gilead Biologies). Matrix Metalloprotease (MMP) Inhibitors: MMP inhibitors include inhibitors of MMP 1 through 10. Examples of MMP9 inhibitors include, but are not limited to, marimastat (BB-2516), cipemastat (Ro 32-3555) and those described in WO 2012 / 027721 (Gilead Biologies). Mitogen-activated Protein Kinase (MEK) Inhibitors: MEK inhibitors include antroquinonol, binimetinib, cobimetinib (GDC-0973, XL-518), MT- 144, selumetinib (AZD6244), sorafenib, trametinib (GSK1120212), uprosertib+trametinib. Phosphatidylinositol 3-kinase (PI3K) Inhibitors: PI3K inhibitors include inhibitors of PI3K. gamma., PI3K. delta., PI3.beta., PI3K. alpha., and / or pan-PI3K. Examples ofPI3K inhibitors include, but are not limited to, ACP-319, AEZA-129, AMG-319, AS252424, BAY 10824391, BEZ235, buparlisib (BKM120), BYL719 (alpelisib), CH5132799, copanlisib (BAY 80-6946), duvelisib, GDC-0941, GDC-0980, GSK2636771, GSK2269557, idelalisib (ZYDELIG®), IPI-145, IPI-443, KAR4141, LY294002, Ly-3023414, MLN1117, 0XY111A, PA799, PX-866, RG7604, rigosertib, RP5090, taselisib, TG100115, TGR-1202, TGX221, WX-037, X-339, X-414, XL147 (SAR245408), XL499, XL756, wortmannin, ZSTK474, and the compounds described in WO 2005 / 113556 (ICOS), WO 2013 / 052699 (Gilead Calistoga), WO 2013 / 116562 (Gilead Calistoga), WO 2014 / 100765 (Gilead Calistoga), WO 2014 / 100767 (Gilead Calistoga), and WO 2014 / 201409 (Gilead Sciences). Spleen Tyrosine Kinase (SYK) Inhibitors: Examples of SYK inhibitors include, but are not limited to, 6-(lH-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[l,2-alpyrazin-8-amine, BAY-61-3606, cerdulatinib (PRT-062607), entospletinib, fostamatinib (R788), HMPL-523, NVP-QAB 205 AA, R112, R343, tamatinib (R406), and those described in U.S. Pat. No. 8,450,321 (Gilead Conn.), and those described in U.S. 2015 / 0175616. Tyrosine-kinase Inhibitors (TKIs): TKIs may target epidermal growth factor receptors (EGFRs) and receptors for fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF). Examples of TKIs include, but are not limited to, afatinib, bosutinib, brigatinib, cabozantinib, crenolanib, dacomitinib, dasatinib, dovitinib, E-6201, erlotinib, gefitinib, gilteritinib (ASP-2215), HM61713, icotinib, imatinib, KX2-391 (Src), lapatinib, lestaurtinib, midostaurin, nintedanib, osimertinib (AZD-9291), ponatinib, poziotinib, quizartinib, radotinib, rociletinib, sunitinib, and TH-4000. Further anticancer agents include: alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimemylolomelamine; acetogenins, especially bullatacin and bullatacinone; a camptothecin, including synthetic analog topotecan; bryostatin, callystatin; 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 CBI-TMI; eleutherobin; 5-azacytidine; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cyclophosphamide, glufosfamide, evofosfamide, bendamustine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, foremustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin phill), dynemicin including dynemicin A, bisphosphonates such as clodronate, an esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromomophores, aclacinomycins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as demopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid replinishers such as frolinic acid; radiotherapeutic agents such as Radium-223; trichothecenes, especially T-2 toxin, verracurin A, roridin A, and anguidine; taxoids such as paclitaxel) (TAXOL), abraxane, docetaxel) (TAXOTERE), cabazitaxel, BIND-014; platinum analogs such as cisplatin and carboplatin, NC-6004 nanoplatin; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformthine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; leucovorin; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; fluoropyrimidine; folinic acid; podophyllinic acid; 2- ethylhydrazide; procarbazine; polysaccharide-K (PSK); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; trabectedin, triaziquone; 2,2',2"-tricUorotriemylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiopeta; chlorambucil; gemcitabine) (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitroxantrone; vancristine; vinorelbine) (NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids such as retinoic acid; capecitabine; FOLFIRI (fluorouracil, leucovorin, and irinotecan); and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0114] Also included in the definition of anticancer agents are anti-hormonal agents such as anti-estrogens and selective estrogen receptor modulators (SERMs), inhibitors of the enzyme aromatase, anti-androgens, and pharmaceutically acceptable salts, acids or derivatives of any of the above that act to regulate or inhibit hormone action on tumors. Examples of anti- estrogens and SERMs include, for example, tamoxifen (including NOLVADEX), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene) (FARESTON). Inhibitors of the enzyme aromatase regulate estrogen production in the adrenal glands. Examples include 4(5)-imidazoles, aminoglutethimide, megestrol acetate) (MEGACE), exemestane, formestane, fadrozole, vorozole) (RIVISOR), letrozole) (FEMARA), and anastrozole) (ARIMIDEX). Examples of anti-androgens include apalutamide, abiraterone, enzalutamide, flutamide, galeterone, nilutamide, bicalutamide, leuprolide, goserelin, ODM-201, APC-100, ODM-204. Examples of progesterone receptor antagonist include onapristone.

[0115] Anti-angiogenic agents include, but are not limited to, retinoid acid and derivatives thereof, 2-methoxyestradiol, ANGIOSTATIN, ENDOSTATIN, regorafenib, necuparanib, suramin, squalamine, tissue inhibitor of metalloproteinase- 1, tissue inhibitor of metalloproteinase-2, plasminogen activator inhibitor- 1, plasminogen activator inbibitor-2, cartilage-derived inhibitor, paclitaxel (nab-paclitaxel), platelet factor 4, protamine sulphate (clupeine), sulphated chitin derivatives (prepared from queen crab shells), sulphated polysaccharide peptidoglycan complex (sp-pg), staurosporine, modulators of matrix metabolism including proline analogs such as l-azetidine-2-carboxylic acid (LAC A), cishydroxyproline, d,I-3,4-dehydroproline, thiaproline, . alpha., .alpha.'-dipyridyl, betaaminopropionitrile fumarate, 4-propyl-5-(4-pyridinyl)-2(3h)-oxazolone, methotrexate, mitoxantrone, heparin, interferons, 2 macroglobulin-serum, chicken inhibitor of metalloproteinase-3 (ChIMP-3), chymostatin, beta-cyclodextrin tetradecasulfate, eponemycin, fumagillin, gold sodium thiomalate, d-penicillamine, beta- 1 -anticollagenase- serum, alpha-2-antiplasmin, bisantrene, lobenzarit disodium, n-2-carboxyphenyl-4- chloroanthronilic acid disodium or "CCA", thalidomide, angiostatic steroid, carboxy aminoimidazole, metalloproteinase inhibitors such as BB-94, inhibitors of SI 00 A9 such as tasquinimod. Other anti-angiogenesis agents include antibodies, preferably monoclonal antibodies against these angiogenic growth factors: beta-FGF, alpha-FGF, FGF-5, VEGF isoforms, VEGF-C, HGF / SF, and Ang-l / Ang-2.

[0116] Anti-fibrotic agents include, but are not limited to, the compounds such as beta- aminoproprionitrile (BAPN), as well as the compounds disclosed in U.S. Pat. No. 4,965,288 relating to inhibitors of lysyl oxidase and their use in the treatment of diseases and conditions associated with the abnormal deposition of collagen and U.S. Pat. No. 4,997,854 relating to compounds which inhibit LOX for the treatment of various pathological fibrotic states, which are herein incorporated by reference. Further exemplary inhibitors are described in U.S. Pat. No. 4,943,593 relating to compounds such as 2-isobutyl-3-fluoro-, chloro-, or bromoallylamine, U.S. Pat. No. 5,021,456, U.S. Pat. No. 5,059,714, U.S. Pat. No. 5,120,764, U.S. Pat. No. 5,182,297, U.S. Pat. No. 5,252,608 relating to 2-(l-naphthyloxymemyl)-3- fluoroallylamine, and US 2004-0248871, which are herein incorporated by reference.

[0117] Exemplary anti-fibrotic agents also include the primary amines reacting with the carbonyl group of the active site of the lysyl oxidases, and more particularly those which produce, after binding with the carbonyl, a product stabilized by resonance, such as the following primary amines: emylenemamine, hydrazine, phenylhydrazine, and their derivatives; semicarbazide and urea derivatives; aminonitriles such as BAPN or 2- nitroethylamine; unsaturated or saturated haloamines such as 2-bromo-ethylamine, 2- chloroethylamine, 2-trifluoroethylamine, 3 -bromopropylamine, and p-halobenzylamines; and selenohomocysteine lactone. Other anti-fibrotic agents are copper chelating agents penetrating or not penetrating the cells. Exemplary compounds include indirect inhibitors which block the aldehyde derivatives originating from the oxidative deamination of the lysyl and hydroxylysyl residues by the lysyl oxidases. Examples include the thiolamines, particularly D-penicillamine, and its analogs such as 2-amino-5-mercapto-5-methylhexanoic acid, D-2-amino-3-methyl-3-((2-acetamidoethy)dithio)butanoic acid, p-2-amino-3-methyl-3- ((2-aminoethy)dithio)butanoic acid, sodium-4-((p- 1 -dimethyl-2-amino-2- carboxyethyl)dithio)butane sulphurate, 2-acetamidoethyl-2-acetamidoethanethiol sulphanate, and sodium-4-mercaptobutanesulphinate trihydrate.

[0118] The API can be an immunotherapeutic agent. Immunotherapeutic agents include, and are not limited to, therapeutic antibodies suitable for treating patients. Some examples of therapeutic antibodies include simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, dinutuximab, ecromeximab, elotuzumab, emibetuzumab, ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab (YERVOY, MDX-010, BMS-734016, and MDX-101), iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, moxetumomab, pasudotox, narnatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, obinutuzumab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, ramucirumab (CYRAMZA®) rilotumumab, rituximab, robatumumab, samalizumab, satumomab, sibrotuzumab, siltuximab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, ABP- 980, tucotuzumab, ubilituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49, OBI-833 and 3F8. Rituximab can be used for treating indolent B-cell cancers, including marginal-zone lymphoma, WM, CLL and small lymphocytic lymphoma. A combination of Rituximab and chemotherapy agents is especially effective.

[0119] The exemplified therapeutic antibodies may be further labeled or combined with a radioisotope particle such as indium-i l l, yttrium-90 (90Y-clivatuzumab), or iodine-131.

[0120] The composition comprises, in place of an API or in addition thereto, a targeting moiety, such as a peptide or protein ligand or domain, covalently attached to the surface of the nanoparticles, which targeting moiety specifically or preferentially binds to a target site (such as a cell surface receptor or binding partner for the targeting moiety), such that the nanoparticle bearing such a targeting moiety will be specifically or preferentially directed to the target site in vivo. The targeting moiety bearing nanoparticle may further comprise an API that is encapsulated or embedded within the nanoparticle that can be released or otherwise effective at the target site. In fact, sialic acid can itself be a targeting moiety.

[0121] By having targeting moieties, target specific nanoparticles are able to efficiently bind to or otherwise associate with a biological entity, for example, a membrane component or cell surface receptor. Targeting of a therapeutic agent (e.g., to a particular tissue or cell type, to a specific diseased tissue but not to normal tissue, etc.) is desirable for the treatment of tissue specific diseases such as cancer (e.g., prostate cancer). For example, in contrast to systemic delivery of a cytotoxic anti-cancer agent, targeted delivery could prevent the agent from killing healthy cells. Additionally, targeted delivery would allow for the administration of a lower dose of the agent, which could reduce the undesirable side effects commonly associated with traditional chemotherapy. As discussed above, the target specificity of the nanoparticles of the invention will be maximized by optimizing the ligand density on the nanoparticle. Targeting moieties can be covalently bound to the surface of the nanoparticle. For example, targeting moieties can be covalently bound to the complexing agent (lipid or polymer).

[0122] For example, a targeting moiety can be a moiety able to bind to or otherwise associate with a biological entity, for example, a membrane component, a cell surface receptor, prostate specific membrane antigen, or the like. The term "bind" or "binding," as used herein, refers to the interaction between a corresponding pair of molecules or portions thereof that exhibit mutual affinity or binding capacity, typically due to specific or non-specific binding or interaction, including, but not limited to, biochemical, physiological, and / or chemical interactions. "Biological binding" defines a type of interaction that occurs between pairs of molecules including proteins, nucleic acids, glycoproteins, carbohydrates, hormones, or the like. The term "binding partner" refers to a molecule that can undergo binding with a particular molecule. "Specific binding" refers to molecules, such as polynucleotides, that are able to bind to or recognize a binding partner (or a limited number of binding partners) to a substantially higher degree than to other, similar biological entities. In one set of embodiments, the targeting moiety has an affinity (as measured via a disassociation constant) of less than about 1 micromolar, at least about 10 micromolar, or at least about 100 micromolar.

[0123] In preferred embodiments, the targeting moiety of the invention is a small molecule. In certain embodiments, the term "small molecule" refers to organic compounds, whether naturally occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Small molecules typically have multiple carbon-carbon bonds. In certain embodiments, small molecules are less than about 2000 g / mol in size. In some embodiments, small molecules are less than about 1500 g / mol or less than about 1000 g / mol. In some embodiments, small molecules are less than about 800 g / mol or less than about 500 g / mol.

[0124] In particularly preferred embodiments, the small molecule targeting moiety targets prostate cancer tumors, and, in particular, the small molecule targeting moiety is a PSMA peptidase inhibitor. These moieties are also referred to herein as "low-molecular weight PSMA ligands." When compared with expression in normal tissues, expression of prostate specific membrane antigen (PSMA) is at least 10-fold overexpressed in malignant prostate relative to normal tissue, and the level of PSMA expression is further up-regulated as the disease progresses into metastatic phases (Silver et al. 1997, Clin. Cancer Res., 3:81), as described in US Patent Publication 2014 / 0235706.

[0125] In some embodiments, small molecule targeting moieties that may be used to target cells associated with prostate cancer tumors include PSMA peptidase inhibitors such as 2- PMPA, GPI5232, VA-033, phenylalkylphosphonamidates (Jackson et al., 2001, Curr. Med. Chem., 8:949; Bennett et al, 1998, J. Am. Chem. Soc., 120: 12139; Jackson et al., 2001, J. Med. Chem., 44:4170; Tsulcarnoto et al, 2002, Bioorg. Med. Chem. Lett., 12:2189; Tang et al., 2003, Biochem. Biophys. Res. Commun., 307:8; Oliver et al., 2003, Bioorg. Med. Chem., 11 :4455; and Maung et al., 2004, Bioorg. Med. Chem., 12:4969), and / or analogs and derivatives thereof. In some embodiments, small molecule targeting moieties that may be used to target cells associated with prostate cancer tumors include thiol and indole thiol derivatives, such as 2-MPPA and 3-(2-mercaptoethyl)-lH-indole-2-carboxylic acid derivatives (Majer et al., 2003, J. Med. Chem., 46:1989; and U.S. Patent Publication 2005 / 0080128). In some embodiments, small molecule targeting moieties that may be used to target cells associated with prostate cancer tumors include hydroxamate derivatives (Stoermer et al., 2003, Bioorg. Med. Chem. Lett., 13:2097). In some embodiments, small molecule targeting moieties that may be used to target cells associated with prostate cancer tumors include PBDA- and urea-based inhibitors, such as Z J 43, Z J 11, ZJ 17, ZJ 38 (Nan et al. 2000, J. Med. Chem., 43:772; and Kozikowski et al., 2004, J. Med. Chem., 47: 1729), and / or and analogs and derivatives thereof. In some embodiments, small molecule targeting moieties that may be used to target cells associated with prostate cancer tumors include putrescine, spermine, and spermidine, androgen receptor targeting agents (ARTAs), such as those described in U.S. Pat. Nos. 7,026,500; 7,022,870; 6,998,500; 6,995,284; 6,838,484; 6,569,896; 6,492,554; and in U.S. Patent Publications 2006 / 0287547; 2006 / 0276540; 2006 / 0258628; 2006 / 0241180; 2006 / 0183931; 2006 / 0035966; 2006 / 0009529; 2006 / 0004042; 2005 / 0033074; 2004 / 0260108; 2004 / 0260092; 2004 / 0167103; 2004 / 0147550; 2004 / 0147489; 2004 / 0087810; 2004 / 0067979; 2004 / 0052727; 2004 / 0029913; 2004 / 0014975; 2003 / 0232792; 2003 / 0232013; 2003 / 0225040; 2003 / 0162761; 2004 / 0087810; 2003 / 0022868; 2002 / 0173495; 2002 / 0099096; 2002 / 0099036. A related aspect of the invention provides a pharmaceutical composition comprising the subject composition, and a pharmaceutically accepted carrier or excipient. Pharmaceutical compositions are described below in more detail in a separate section.

[0126] In a most preferred embodiment, the API is a nucleic acid selected from DNA, mRNA, siRNA, ASO, microRNA, circular RNA, gene-editing agents such as CRISPR-Cas9 system, prime editors, base-editors, TALENs, ZFNs, etc.

[0127] Production of the Particles

[0128] The particles can be manufactured from the coprecipitation of the polysialic acid (i.e., PSA) and a cationic lipid or polymer. In general, the polysialic acid can be dissolved in an aqueous medium or water. The cationic lipid or polymer can be dissolved in a preferably water-miscible organic solvent, such as ethanol. Any API can be dissolved in the aqueous medium or organic solvent. The two solutions are then combined. Preferably, the mixtures are combined slowly (e.g., dropwise) or with mixing (e.g., homogenization) to incur nanoprecipitation. For example, a small amount of organic solution can be added to the aqueous phase with mixing.

[0129] The particles may also be manufactured using an automated, microfluidic device such as NanoAssemblr of Cytiva and Automated Nanoparticle System of Dolomite Microfluidics.

[0130] In a preferred embodiment, the particles are prepared by first mixing an aqueous solution of a nucleic acid and an organic solution of a cationic lipid to form a lipoplex nanoparticle, followed by the addition of an aqueous solution of a polysialic acid. Preferably, said lipoplex nanoparticle bears a positive charge with a preferred zeta potential from 0 to + 50 mV, more preferably from +5 to +30 mV.

[0131] For example, the cationic complexing agent can be dissolved in a solvent or aqueous buffer or solution. In the case an organic solvent is used, the solvent is preferably a water- miscible solvent. Examples of water-miscible solvents include acetone, methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF).

[0132] The SA-containing agent can be dissolved in a solvent or aqueous buffer or solution. In the case an organic solvent is used, the solvent is preferably a water-miscible solvent. Examples of water-miscible solvents include acetone, methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF).

[0133] The relative amount of the SA-containing agent to that of the cationic complexing agent used in the pharmaceutical composition is from 10: 1 to 1 : 100, more preferably from 1 : 1 to 1 : 10. As used herein, “small (amount)” refers to a relatively small amount / volume of one solution as compared to the volume of the other solution with the cationic lipid, such that an emulsion is initially formed. Typically, the volume ratio between the small amount of the organic solution and the aqueous phase, is at least about 1 : n, wherein n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100.

[0134] Using the methods of preparation described herein, the polysialic acid is tightly integrated into the produced nanoparticles.

[0135] As used herein, miscibility is defined to be the property of liquids to mix in all proportions, forming a homogeneous solution. Substances / liquids are said to be immiscible or not miscible, if in some proportion, they do not form a solution.

[0136] Exemplary solvents miscible with water include ethanol, acetone, tetrahydrofuran (THF), acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF).

[0137] Solvent is then removed and / or particles collected, for example, by evaporation, solvent exchange, tangential flow filtration (TFF), centrifugation or filtration, followed by concentration to a desired particle or API concentration, or dehydration, e.g., lyophilization.

[0138] The aqueous solution can optionally comprise a surfactant comprising organic or inorganic pharmaceutical excipients; various polymers; oligomers; natural products; nonionic, cationic, zwitterionic, or ionic surfactants; and mixtures thereof. The surfactant may comprise polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), a polysorbate (Tween series) surfactant, a PEO-PPO-PEO (polyethylene oxide-polypropylene oxide-polyethylene oxide) triblock copolymer (Pluronic series or Poloxamer series) surfactant, or a t-octylphenyl- poly ethylene glycol (Triton X-100) surfactant or a salt, derivative, copolymer, or mixture thereof.

[0139] Combinations of more than one surfactant can be used in the invention. Useful surfactants or surface stabilizers which can be employed in the invention may include, but are not limited to, known organic and inorganic pharmaceutical excipients. Such excipients include various polymers, low molecular weight oligomers, natural products, and surfactants. Surfactants or surface stabilizers include nonionic, cationic, zwitterionic, and ionic surfactants.

[0140] Representative examples of other useful surfactants or surface stabilizers include hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, sodium lauryl sulfate, sodium dioctylsulfosuccinate, gelatin, casein, lecithin (phosphatides), dextran, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glycerol monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers (e.g., macrogol ethers such as cetomacrogol 1000), polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters (e.g., the commercially available TWEENS® such as e.g., TWEEN 20® and TWEEN 80® (ICI Specialty Chemicals)); polyethylene glycols (e.g., CARBOWAXS 3550® and 934® (Union Carbide)), polyoxyethylene stearates, colloidal silicon dioxide, phosphates, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), 4-(l, 1,3,3- tetramethylbutyl)-phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol, superione, and triton), poloxamers (e.g., PLURONICS F68® and Fl 08®, which are block copolymers of ethylene oxide and propylene oxide); pol oxamines (e.g., TETRONIC 908®, also known as POLOXAMINE 908®, which is a tetrafunctional block copolymer derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Wyandotte Corporation, Parsippany, N.J.)); TETRONIC 1508® (T- 1508) (BASF Wyandotte Corporation), TRITONS X-200®, which is an alkyl aryl poly ether sulfonate (Rohm and Haas); CRODESTAS F-l 10®, which is a mixture of sucrose stearate and sucrose distearate (Croda Inc.); p-isononylphenoxypoly-(glycidol), also known as OLIN- 1OG® or SURFACTANT 10-G® (Olin Chemicals, Stamford, Conn.); Crodestas SL- 40(Croda, Inc.); and SA9OHCO, which is C18H37CH2(CON(CH3)- CH2(CHOH)4(CH2OH)2 (Eastman Kodak Co.); decanoyl-N-methylglucamide; n-decyl P-D- glucopyranoside; n-decyl P-D-maltopyranoside; n-dodecyl P-D-glucopyranoside; n-dodecyl P-D-maltoside; heptanoyl-N-methylglucamide; n-heptyl-p-D-glucopyranoside; n-heptyl P-D- thioglucoside; n-hexyl P-D-glucopyranoside; nonanoyl-N-methylglucamide; n-noyl P-D- glucopyranoside; octanoyl-N-methylglucamide; n-octyl-P-D-glucopyranoside; octyl P-D- thioglucopyranoside; PEG-derivatized phospholipid, PEG-derivatized cholesterol, PEG- derivatized cholesterol derivative, PEG-derivatized vitamin A, PEG-derivatized vitamin E, lysozyme, random copolymers of vinyl pyrrolidone and vinyl acetate, and the like.

[0141] Examples of useful cationic surfactants or surface stabilizers include, but are not limited to, polymers, biopolymers, polysaccharides, cellulosics, alginates, phospholipids, and nonpolymeric compounds, such as zwitterionic stabilizers, poly-n-methylpyridinium, anthryul pyridinium chloride, cationic phospholipids, chitosan, polylysine, polyvinylimidazole, polybrene, polymethylmethacrylate trimethylammoniumbromide bromide (PMMTMABr), hexyldesyltrimethylammonium bromide (HDMAB), polyvinylpyrrolidone-2-dimethylaminoethyl methacrylate dimethyl sulfate, 1,2 Dipalmitoyl- sn-Glycero-3-Phosphoethanolamine-N-[Amino(Polyethylene Glycol)2000] (sodium salt) (also known as DPPE-PEG(2000)-Amine Na) (Avanti Polar Lipids, Alabaster, Al), Poly(2- methacryloxyethyl trimethylammonium bromide) (Polysciences, Inc., Warrington, Pa.) (also known as S1001), poloxamines such as TETRONIC 908®, also known as POLOXAMINE 908®, which is a tetrafunctional block copolymer derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Wyandotte Corporation, Parsippany, N. J.), lysozyme, long-chain polymers such as alginic acid, carrageenan (FMC Corp.), and POLYOX (Dow, Midland, Mich.).

[0142] Other useful cationic stabilizers include, but are not limited to, cationic lipids, sulfonium, phosphonium, and quaternary ammonium compounds, such as stearyltrimethylammonium chloride, benzyl-di(2-chloroethyl)ethylammonium bromide, coconut trimethyl ammonium chloride or bromide, coconut methyl dihydroxyethyl ammonium chloride or bromide, decyl triethyl ammonium chloride, decyl dimethyl hydroxyethyl ammonium chloride or bromide, Cl 2- 15 dimethyl hydroxyethyl ammonium chloride or bromide, coconut dimethyl hydroxyethyl ammonium chloride or bromide, myristyl trimethyl ammonium methyl sulphate, lauryl dimethyl benzyl ammonium chloride or bromide, lauryl dimethyl (ethenoxy) ammonium chloride or bromide, N-alkyl (C12- 18)dimethylbenzyl ammonium chloride, N-alkyl (C14-18)dimethyl-benzyl ammonium chloride, N-tetradecylidmethylbenzyl ammonium chloride monohydrate, dimethyl didecyl ammonium chloride, N-alkyl and (Cl 2- 14) dimethyl 1-napthylmethyl ammonium chloride, trimethylammonium halide, alkyl-trimethylammonium salts and dialkyl-dimethylammonium salts, lauryl trimethyl ammonium chloride, ethoxylated alkyamidoalkyldialkylammonium salt and / or an ethoxylated trialkyl ammonium salt, dialkylbenzene dialkylammonium chloride, N- didecyldimethyl ammonium chloride, N-tetradecyldimethylbenzyl ammonium, chloride monohydrate, N-alkyl(C12-14) dimethyl 1 -naphthylmethyl ammonium chloride and dodecyldimethylbenzyl ammonium chloride, dialkyl benzenealkyl ammonium chloride, lauryl trimethyl ammonium chloride, alkylbenzyl methyl ammonium chloride, alkyl benzyl dimethyl ammonium bromide, C12, C15, C17 trimethyl ammonium bromides, dodecylbenzyl triethyl ammonium chloride, poly-diallyldimethylammonium chloride (DADMAC), dimethyl ammonium chlorides, alkyldimethylammonium halogenides, tricetyl methyl ammonium chloride, decyltrimethylammonium bromide, dodecyltriethylammonium bromide, tetradecyltrimethylammonium bromide, methyl trioctylammonium chloride (ALIQUAT 336TM), POLYQUAT 10TM, tetrabutylammonium bromide, benzyl trimethylammonium bromide, choline esters (such as choline esters of fatty acids), benzalkonium chloride, stearalkonium chloride compounds (such as stearyltrimonium chloride and Di- stearyldimonium chloride), cetyl pyridinium bromide or chloride, halide salts of quaternized polyoxyethylalkylamines, MIRAPOL™ and ALKAQUAT™ (Alkaril Chemical Company), alkyl pyridinium salts; amines, such as alkylamines, dialkylamines, alkanolamines, polyethylenepolyamines, N,N-dialkylaminoalkyl acrylates, and vinyl pyridine, amine salts, such as lauryl amine acetate, stearyl amine acetate, alkylpyridinium salt, and alkylimidazolium salt, and amine oxides; imide azolinium salts; protonated quaternary acrylamides; methylated quaternary polymers, such as poly [diallyl dimethylammonium chloride] and poly-[N-methyl vinyl pyridinium chloride]; and cationic guar.

[0143] Such exemplary cationic surfactants or surface stabilizers and other useful cationic surfactants or surface stabilizers are described in J. Cross and E. Singer, Cationic Surfactants: Analytical and Biological Evaluation (Marcel Dekker, 1994); P. and D. Rubingh (Editor), Cationic Surfactants: Physical Chemistry (Marcel Dekker, 1991); and J. Richmond, Cationic Surfactants: Organic Chemistry, (Marcel Dekker, 1990), each of which is incorporated by reference herein in its entirety.

[0144] Nonpolymeric cationic surfactants or surface stabilizers are any nonpolymeric compound, such as benzalkonium chloride, a carbonium compound, a phosphonium compound, an oxonium compound, a halonium compound, a cationic organometallic compound, a quaternary phosphorous compound, a pyridinium compound, an anilinium compound, an ammonium compound, a hydroxylammonium compound, a primary ammonium compound, a secondary ammonium compound, a tertiary ammonium compound, and quaternary ammonium compounds of the formula NR1R2R3R4(+). For compounds of the formula NR1R2R3R4(+): (i) none of R1-R4 are CH3; (ii) one of R1-R4 is CH3; (iii) three of R1-R4 are CH3; (iv) all of R1-R4 are CH3; (v) two of R1-R4 are CH3, one of R1-R4 is C6H5CH2, and one of R1-R4 is an alkyl chain of seven carbon atoms or less; (vi) two of Rl- R4 are CH3, one of R1-R4 is C6H5CH2, and one of R1-R4 is an alkyl chain of nineteen carbon atoms or more; (vii) two of R1-R4 are CH3 and one of R1-R4 is the group C6H5 (CH2)n, where n>l; (viii) two of R1-R4 are CH3, one of R1-R4 is C6H5CH2, and one of Rl- R4 comprises at least one heteroatom; (ix) two of R1-R4 are CH3, one of R1-R4 is C6H5CH2, and one of R1-R4 comprises at least one halogen; (x) two of R1-R4 are CH3, one of R1-R4 is C6H5CH2, and one of R1-R4 comprises at least one cyclic fragment; (xi) two of R1-R4 are CH3 and one of R1-R4 is a phenyl ring; or (xii) two of R1-R4 are CH3 and two of R1-R4 are purely aliphatic fragments. Such compounds include, but are not limited to, behenalkonium chloride, benzethonium chloride, cetylpyridinium chloride, behentrimonium chloride, lauralkonium chloride, cetalkonium chloride, cetrimonium bromide, cetrimonium chloride, cethylamine hydrofluoride, chlorallylmethenamine chloride (Quaternium-15), distearyldimonium chloride (Quaternium-5), dodecyl dimethyl ethylbenzyl ammonium chloride(Quatemium-14), Quaternium-22, Quaternium-26, Quatemium-18 hectorite, dimethylaminoethylchloride hydrochloride, cysteine hydrochloride, di ethanolammonium POE (10) oletyl ether phosphate, diethanolammonium POE (3)oleyl ether phosphate, tallow alkonium chloride, dimethyl dioctadecylammoniumbentonite, stearalkonium chloride, domiphen bromide, denatonium benzoate, myristalkonium chloride, laurtrimonium chloride, ethylenediamine dihydrochloride, guanidine hydrochloride, pyridoxine HC1, iofetamine hydrochloride, meglumine hydrochloride, methylbenzethonium chloride, myrtrimonium bromide, oleyltrimonium chloride, polyquatemium-1, procainehydrochloride, cocobetaine, stearalkonium bentonite, stearalkoniumhectonite, stearyl trihydroxyethyl propylenediamine dihydrofluoride, tallowtrimonium chloride, and hexadecyltrimethyl ammonium bromide.

[0145] Most of these surfactants or surface stabilizers are known pharmaceutical excipients and are described in detail in the Handbook of Pharmaceutical Excipients, published jointly by the American Pharmaceutical Association and The Pharmaceutical Society of Great Britain (The Pharmaceutical Press, 2000), specifically incorporated by reference.

[0146] The surfactants or surface stabilizers are commercially available and / or can be prepared by techniques known in the art.

[0147] Due to its hydrophilic nature, poly sialic acid tends to reside on the surface of the nanoparticles formed, making the nanoparticles hydrophilic and negatively charged.

[0148] The present invention is based on the inventors’ surprising discovery that the nanoparticles, despite being negatively charged, can have an exclusive lung tropism. This discovery is against conventional wisdom, as it has been generally established within the scientific community that positively charged NPs tend to accumulate in the lung, negatively charged NPs tend to accumulate in the spleen, and neutrally charged NPs tend to accumulate in the liver.

[0149] Without being bound by any scientific theory, the inventors hypothesize that the sialic acid moieties on the surface of the nanoparticles of the present invention are capable of binding to the Siglec receptors on certain lung cells, and as a result, target these cells via receptor-mediated endocytosis. This hypothesis can at least partially explain the strong lung tropism that the nanoparticles exhibited. Particle Sizes

[0150] The Z-average size of the subject nanoparticles is from 1 nm to about 1 pm, preferably from about 10 nm to about 1 pm, and more preferably from about 20 nm to about 500 nm, and most preferably from about 50 nm to about 300 nm. For example, the nanoparticles may have an average size between about 50 and 300 nm, such as about 50, 75, 100, 125, 150, 175, 200, 225, 250, 300 nm.

[0151] As used herein, particle size can be determined by any conventional particle size measuring techniques well known to those skilled in the art. Such techniques include, for example, sedimentation, field flow fractionation, photon correlation spectroscopy, light scattering, dynamic light scattering, light diffraction, and disk centrifugation, with dynamic light scattering highly preferred.

[0152] Zeta Potential

[0153] Zeta potential is a physical property that is exhibited by any particle in suspension. It measures the charge of a particle suspension. A positive zeta potential indicates that the particles bear a positive charge. A negative zeta potential indicates that the particles bear a negative charge. A zeta potential value of zero or close to zero means that the particles are neutrally charged. Zeta potential can be used to optimize the formulations of suspensions, emulsions and protein solutions, predict interactions with surfaces, and optimize the formation of films and coatings. Knowledge of the zeta potential can reduce the time needed to produce trial formulations. It can also be used as an aid in predicting long-term stability as a particle suspension having a large absolute number of zeta potential (positive or negative) has high colloidal stability.

[0154] Perferably, the nanoparticle of the pharmaceutical composition of the present invention has a negative charge, with a zeta potential of from 0 to -100 mV, -2 to -80 mV, -5 to -60 mV, -10 to -40 mV, or -20 to -35 mV.

[0155] Exemplary Uses

[0156] The particles and compositions thereof have numerous applications including in therapeutic methods. There are 14 known functional Siglecs in humans, and they are Siglec-1 (sialoadhesin), Siglec-2 (CD22), Siglec-3 (CD33), Siglec-4 (MAG), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-14, Siglec-15, and Siglec-16. The cytoplasmic domain of most of these Siglecs has immune receptor tyrosine-based inhibitory motifs (ITIMs) and signals negatively via recruitment of tyrosine phosphatases such as SHP- 1 and SHP-2. A few Siglecs, such as Siglec-14, Siglec-15, and Siglec-16, associate with the tyrosine-based activation motif (IT AM) adaptor DAP12 via a positively charged amino acid in their transmembrane region. These Siglecs are expressed in various immune cells and have different functionalities. In general, binding Siglecs having ITIMs deactivates certain immune responses, and binding Siglecs having IT AMs activates immune actions.

[0157] Therefore, one application of the present invention is to use the SA-containing nanoparticles to bind Siglecs that have ITAMs to activate immune responses to pathogens such as infection and tumor, thus treating infectious disease and cancer.

[0158] Another application of the present invention is to use the SA-containing nanoparticles to bind Siglecs that have ITIMs to deactivate specific immune responses and can thus treat inflammatory and autoimmune diseases.

[0159] Specifically, a drug can be incorporated into the subject nanoparticle and be delivered to the lungs to treat lung diseases. For example, an mRNA can be incorporated into the subject nanoparticles and be intravenously delivered to the lung with enhanced tissue targeting and cell transfection.

[0160] Preferably, the nanoparticles or the composition comprising the nanoparticles can be used in a method of treating a lung disease or condition in a subject in need thereof, or a method of reducing the duration or severity of the disease or condition in the subject in need thereof, wherein the disease or condition is treatable with the particles (and optionally with a specific API), comprising administering a composition or a pharmaceutical composition comprising the particles to the subject, thereby treating the disease or condition. Where the particles comprise an API, the particles can be used in a method of administering or delivering the API to a subject in need thereof and / or for a method of treating a subject suffering from a disease or condition that can be treated with the API. For example, when the API is an anti-inflammatory agent, the particles can be administered to a subject with an inflammatory condition.

[0161] It is to be noted that the nanoparticle of the present invention itself is antiinflammatory without the presence of an anti-inflammatory API. The SA residue on the nanoparticle surface binds the Siglec receptor on immune cells to suppress the cell-mediated inflammation. The SA residue on the nanoparticle surface can also bind the complement factor H (CFH) to shut down the complement cascade. The bindings created a self-associated molecular pattern (SAMP) effect to help mitigate inflammation and maintain homeostasis.

[0162] In additional aspects, the particles comprise an immunotherapeutic agent and can be used in immunotherapy. The nanoparticles described herein can be used to treat an inflammatory condition.

[0163] Examples of such diseases and conditions include, but are not limited to, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, celiac disease, hyper-IgM immunodeficiency, arteriosclerosis, atherosclerosis, coronary artery disease, sepsis, myocarditis, encephalitis, transplant rejection, hepatitis, thyroiditis (e.g. Hashimoto's thyroiditis, Graves disease), osteoporosis, polymyositis, dermatomyositis, Type I diabetes, Type II diabetes, gout, dermatitis, alopecia areata, systemic lupus erythematosus, Sjogren’s syndrome, lichen sclerosis, scleroderma, ulcerative colitis, diabetic retinopathy, pelvic inflammatory disease, periodontal disease, arthritis, juvenile chronic arthritis (e.g. chronic iridocyclitis), psoriasis, osteoporosis, nephropathy in diabetes mellitus, asthma, pelvic inflammatory disease, chronic inflammatory liver disease, chronic inflammatory lung disease, lung fibrosis, liver fibrosis, chronic inflammatory lung disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, peritonitis, cardiovascular disease, reperfusion injury, ischemia injury, stroke, burns, and other acute and chronic inflammatory diseases of the Central Nervous System (CNS; e.g. multiple sclerosis), gastrointestinal system, the skin and associated structures, the immune system, the hepato-biliary system, or any site in the body where pathology can occur with an inflammatory component. Inflammatory diseases also include diseases involving the gastrointestinal tract and associated tissues (such as ileus, appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, coeliac disease, hepatitis, Crohn's disease, enteritis, and Whipple's disease); systemic or local inflammatory diseases and conditions (such as asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, and sarcoidosis); diseases involving the urogenital system and associated tissues (such as septic abortion, epididymitis, vaginitis, prostatitis, and urethritis); diseases involving the respiratory system and associated tissues (such as bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis, adult respiratory distress syndrome, pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis, pharyngitis, pleurisy, and sinusitis); diseases arising from infection by various viruses (such as influenza, respiratory syncytial virus, HIV, hepatitis B virus, hepatitis C virus and herpes), bacteria (such as disseminated bacteremia, Dengue fever), fungi (such as candidiasis) and protozoal and multicellular parasites (such as malaria, filariasis, amebiasis, and hydatid cysts); dermatological diseases and conditions of the skin (such as burns, dermatitis, dermatomyositis, sunburn, urticaria warts, and wheals); diseases involving the cardiovascular system and associated tissues (such as stenosis, restenosis, vasulitis, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, congestive heart failure, myocarditis, autoimmune myocarditis, myocardial ischemia, periarteritis nodosa, and rheumatic fever); diseases involving the central or peripheral nervous system and associated tissues (such as Alzheimer's disease, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, and uveitis); diseases of the bones, joints, muscles and connective tissues (such as the various arthritides and arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, and synovitis); other autoimmune and inflammatory disorders (such as myasthenia gravis, thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcets's syndrome, allograft rejection, graft-versus-host disease, Type I diabetes, ankylosing spondylitis, Berger's disease, and Retier's syndrome); as well as various cancers, tumors and proliferative disorders (such as Hodgkins disease); and, in any case the inflammatory or immune host response to any primary disease.

[0164] Diseases that can be treated or prevented also include allergic disorders or conditions, including allergic disease, allergy, eczema, asthma, allergic rhinitis or skin hypersensitivity.

[0165] The disease to be treated or prevented can also be a viral infection, including, for example, a coronavirus infection, a hepatitis virus infection, a West Nile virus infection, a flavivirus, an influenza infection, a rhinovirus infection, a papillomavirus infection, a paramyxovirus infection, or a parainfluenza virus infection. Preferably, the viral infection infects the central nervous system of said subject. Preferably, the viral infection causes viral encephalitis or viral meningitis. In yet other aspects, the disease to be treated is a bacterial infection. Exemplary bacterial infections are staphylococcus infections, streptococcus infections, mycobacterial infections, bacillus infections, Salmonella infections, Vibrio infections, spirochete infections, and Neisseria infections. Preferred are bacteria that infect the central nervous system of the subject. Most preferred are bacteria that cause encephalitis or meningitis.

[0166] A preferred condition for use in the claimed invention is treating cancers. The cancer to be treated can include Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), indolent non-Hodgkin's lymphoma (iNHL), refractory iNHL, multiple myeloma (MM), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), B- cell ALL, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), myelodysplastic syndrome (MDS), myeloproliferative disease (MPD), mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldestrom's macroglobulinemia (WM), T-cell lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), or marginal zone lymphoma (MZL). In one embodiment, the cancer is minimal residual disease (MRD). In additional embodiment, the cancer is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), indolent non-Hodgkin's lymphoma (iNHL), and refractory iNHL. In certain embodiment, the cancer is indolent non-Hodgkin's lymphoma (iNHL). In some embodiment, the cancer is refractory iNHL. In one embodiment, the cancer is chronic lymphocytic leukemia (CLL). In other embodiment, the cancer is diffuse large B-cell lymphoma (DLBCL).

[0167] In certain embodiments, the cancer is lung cancer, including, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung.

[0168] Any of the methods of treatment provided may be used to treat cancer at various stages. By way of example, the cancer stage includes but is not limited to early, advanced, locally advanced, remission, refractory, reoccurred after remission, and progressive.

[0169] Preferably, the nanoparticle of the invention can be used in combination with a second therapeutic that is effective for treating any one of the treatable conditions.

[0170] Preferably, the subject is a human patient. Preferably, the subject is a non-human mammal, such as a non-human primate, a livestock animal (horse, mule, cattle, bull, cow, sheep, goat, pig, camel, etc.), a rodent (rabbit, hamster, mouse, rat, etc.), or a pet (cat, dog).

[0171] Preferably, the method includes administering the subject composition or pharmaceutical composition comprising the subject nanoparticles by any suitable means or routes, such as orally, nasally, intravenously, intramuscularly, ocularly, transdermally, subcutaneously, intratumorally, intravesicularly, intra-articularly, intracranially, and intraperitoneally. Intravenous route is the most preferred method for administrating the nanoparticles of the present invention.

[0172] The effectiveness of the nanoparticles described herein against the treatable diseases and conditions can be tested using a number of efficacy tests, including suitable animal models.

[0173] Pharmaceutical Composition

[0174] One aspect of the present invention provides pharmaceutical compositions which comprise the subject nanoparticles, and optionally comprise a pharmaceutically acceptable carrier or excipient. Preferably, these compositions optionally further comprise one or more additional therapeutic agents. Alternatively, the subject particles of the current invention may be administered to a patient in need thereof in combination with the administration of one or more other therapeutic agents. For example, additional therapeutic agents for conjoint administration or inclusion in a pharmaceutical composition with a compound of this invention may be an approved anti-inflammatory agent, an immunotherapeutic agent, or a chemotherapeutic agent, or it may be any one of a number of agents undergoing approval in the Food and Drug Administration. It will also be appreciated that certain of the subject particles of the present invention can exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable derivative thereof.

[0175] Preferably, the pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, which, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington ’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

[0176] Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0177] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable colloids, emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0178] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0179] Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The particles can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose and starch. Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the modified particles only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

[0180] It will also be appreciated that the nanoparticles and pharmaceutical compositions of the present invention can be formulated and employed in combination therapies, that is, the compounds and pharmaceutical compositions can be formulated with or administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account the compatibility of the desired therapeutics and / or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with another antiinflammatory agent), or they may achieve different effects (e.g., control of any adverse effects).

[0181] Preferably, the pharmaceutical compositions containing the particles of the present invention further comprise one or more additional therapeutically active ingredients (e.g., anti-inflammatory and / or palliative). For purposes of the invention, the term “Palliative” refers to treatment that is focused on the relief of symptoms of a disease and / or side effects of a therapeutic regimen, but is not curative. For example, palliative treatment encompasses painkillers, anti-nausea medications and anti-sickness drugs.

[0182] The following examples are given to illustrate the present invention. It should be understood, however, that the invention is not to be limited to the specific conditions or details described in these examples. Sigfinity™ Nanoparticles

[0183] The nanoparticles described herein are referred to as “Sigfinity™” nanoparticles.

[0184] EXAMPLES

[0185] Example 1: Preparation of Sigfinity™ nanoparticles with Luciferase-mRNA

[0186] 13.5 mg of the cationic lipid DOTAP was dissolved in 1.35 mL of ethanol to create a stock lipid solution with a 10 mg / mL concentration. 7.4 mg of colominic acid (purchased from Carbosynth, Oakbrook Terrace, IL, USA) was dissolved in 0.74 mL of Ultrapure distilled water to create a stock PSA solution with a 10 mg / mL concentration.

[0187] 100 pL of a 1 mg / mL Firefly Luciferase (Luc) mRNA solution was introduced to a 50-mL beaker containing 20 mL Ultrapure RNAse / DNase-free water. The beaker was equipped with a magnetic stir bar and was placed on a magnetic stirrer.

[0188] A total of 172.9 pL of the DOTAP stock solution was mixed with 4.5 mL of ethanol. The mixture was then introduced into a 5-mL syringe with a 21-G needle, which had been positioned above the 50-mL beaker using a clamp.

[0189] While the mRNA solution in the 50-mL beaker was magnetically stirred at 300 rpm, the plunger of the syringe was removed, and the DOTAP / ethanol solution was transferred to the syringe to allow the DOTAP solution to be added dropwise to the mRNA solution. After the addition was complete, stirring was continued for 5 minutes, followed by a repetition of the addition process with a new syringe filled with 89.1 pL of the PSA solution and 4.6 mL of Ultrapure RNAse / DNase-free water. After the second addition was completed, stirring was continued for 30 minutes.

[0190] The nanoparticle suspension was transferred into a 15-mL Slide-A-Lyzer™ dialysis cassette with 20K MWCO, dialyzed against Ultrapure RNAse / DNase-free distilled water for 6 hours, concentrated with a tangential flow filtration device. The resulting nanoparticles had a Z-average particle size of 219.0 nm, a zeta potential of -31.4 mV, and an RNA encapsulation efficiency (EE) measured by Ribogreen Assay of 76.0%. Encapsulated mRNA concentration in the final particle suspension was 17.9 pg / ml.

[0191] The final sample was collected and stored at -80° C in 15% w / v sucrose solution.

[0192] Example 2: Preparation of Sigfinity nanoparticles with a Cre-recombinase-mRNA

[0193] 22.4 mg of the cationic lipid DOTAP was dissolved in 1.12 mL of ethanol to create a stock lipid solution with a 20 mg / mL concentration. 7.0 mg of colominic acid was dissolved in 0.7 mL of Ultrapure distilled water to create a stock PSA solution with a 10 mg / mL concentration.

[0194] 100 pL of a 1 mg / mL Cre-mRNA solution was introduced to a 50-mL beaker containing 30 mL Ultrapure RNAse / DNase-free water. The beaker was equipped with a magnetic stir bar and was placed on a magnetic stirrer.

[0195] A total of 82.2 pL of the DOTAP stock solution was mixed with 3.0 mL of ethanol. The mixture was then introduced into a 5-mL syringe with a 22-G needle, which had been positioned above the 50-mL beaker using a clamp.

[0196] While the mRNA solution in the 50-mL beaker was magnetically stirred at 300 rpm, the plunger of the syringe was removed, and the DOTAP / ethanol solution was transferred to the syringe to allow the DOTAP solution to be added dropwise to the mRNA solution. After the addition was complete, stirring was continued for 5 minutes, followed by a repetition of the addition process with a 1-mL syringe with a 20-G needle filled with 84.7 pL of the PSA solution and 1.0 mL of Ultrapure RNAse / DNase-free water. After the second addition was completed, stirring was continued for 30 minutes.

[0197] The nanoparticle suspension was transferred into a Slide- A-Lyzer™ dialysis cassette with 20K MWCO, dialyzed against Ultrapure RNAse / DNase-free distilled water for 6 hours, concentrated with an Amicon Ultra-15 centrifugation at 2000 RCF. The resulting nanoparticles had a Z-average particle size of 188.7 nm, a zeta potential of -18.5 mV, and an RNA encapsulation efficiency (EE) measured by Ribogreen Assay of 86.6%. Encapsulated mRNA concentration in the final particle suspension was 56.2 pg / ml.

[0198] The final sample was collected and stored at -80° C in 15% w / v sucrose solution.

[0199] Example 3: Biodistribution study in BALB / c mice with luciferase-mRNA loaded Sigfinity NPs

[0200] 200 pL of a suspension of Sigfinity™ nanoparticles loaded with a luciferase-mRNA containing 23.5 pg / ml of mRNA (prepared in Example 1) was administered to a Balb / c female mouse via the lateral tail vein. Six hours after the injection, D-Luciferin substrate was administered intraperitoneally to the dosed mouse, followed by euthanasia. The whole body was then perfused with PBS. Lung, spleen, and liver were collected and imaged with IVIS. The collected IVIS image of the organs is reported in FIG. 1. Example 4: Biodistribution study in Ail4 mouse with Cre-recombinase-mRNA loaded Sigfinity NPs.

[0201] 178 pL of a suspension of Sigfinity ™ / nanoparticles loaded with a Cre-recombinase- mRNA containing 56.2 pg / ml of mRNA (prepared in Example 2) was administered to an Ail 4 mouse via the lateral tail vein. Six days after the injection, the animal was anesthetized via isoflurane and subjected to in-life imaging sessions for tdTomato fluorescence. The whole body was then perfused with PBS. Lung, spleen, and liver were collected and imaged with IVIS. The collected IVIS image of the organs is reported in FIG. 2.

[0202] Example 5: Preparation of Sigfinity nanoparticles with a Luciferase-mRNA

[0203] 145.4 pL of 1 mg / mL Luciferase-mRNA solution in Ultrapure RNAse / DNase-free water was introduced to a 50-mL beaker containing 30 mL Ultrapure RNAse / DNase-free water. The beaker was equipped with a magnetic stir bar and was placed on a magnetic stirrer.

[0204] 125.7 pL of 20 mg / mL DOTAP solution in ethanol was mixed with 3.0 mL of ethanol. The mixture was then introduced into a 5-mL syringe with a 22-G needle, which had been positioned above the 50-mL beaker using a clamp.

[0205] While the mRNA solution in the 50-mL beaker was magnetically stirred at 350 rpm, the plunger of the syringe was removed, and the DOTAP / ethanol solution was transferred to the syringe to allow the DOTAP solution to be added dropwise to the mRNA solution. After the addition was complete, stirring was continued for 5 minutes. The process was repeated with a 1-mL syringe with a 20-G needle filled with 129.5 pL of 10 mg / mL colominic acid solution diluted in 1.0 mL of Ultrapure RNAse / DNase-free water. After the second addition was completed, stirring was continued for 30 minutes. The nanoparticles were measured and had a Z-average particle size of 195.9 nm, poly dispersity a zeta potential of -22.5.

[0206] The nanoparticle suspension was transferred into a 30-mL Slide-A-Lyzer™ dialysis cassette with 20K MWCO, dialyzed against Ultrapure RNAse / DNase-free water in a 600-mL beaker for 6 hours, and concentrated with an Amicon Ultra- 15, 100K MWCO at 2000 RCF to 500 pL. The resulting nanoparticles had a Z-average particle size of 204 nm and an RNA encapsulation efficiency (EE) measured by Ribogreen Assay of 90.0%. Encapsulated mRNA concentration in the final particle suspension was 48.9 pg / ml.

[0207] The final sample was collected and stored at -80° C in 10% w / v sucrose solution. Example 6: Preparation of Sigfinity nanoparticles with a Cre-recombinase-mRNA

[0208] 150.0 pL of 1 mg / mL Cre-mRNA solution in Ultrapure RNAse / DNase-free water was introduced to a 50-mL beaker containing 35 mL Ultrapure RNAse / DNase-free water. The beaker was equipped with a magnetic stir bar and was placed on a magnetic stirrer.

[0209] 122.9 pL of 20 mg / mL DOTAP solution in ethanol was mixed with 2.9 mL of ethanol. The mixture was then introduced into a 3-mL syringe with a 22-G needle, which had been positioned above the 50-mL beaker using a clamp.

[0210] While the mRNA solution in the 50-mL beaker was magnetically stirred at 350 rpm, the plunger of the syringe was removed, and the DOTAP / ethanol solution was transferred to the syringe to allow the DOTAP solution to be added dropwise to the mRNA solution. After the addition was complete, stirring was continued for 5 minutes. The process was repeated with a 1-mL syringe with a 20-G needle filled with 126.7 pL of 10 mg / mL colominic acid solution and 1.0 mL of Ultrapure RNAse / DNase-free water. After the second addition was completed, stirring was continued for 30 minutes.

[0211] The nanoparticle suspension was transferred into a 70-mL Slide-A-Lyzer™ dialysis cassette with 20K MWCO, dialyzed against Ultrapure RNAse / DNase-free distilled water in a 1-L beaker for 6 hours, and concentrated with an Amicon Ultra-15, 100K MWCO at 2000 RCF to 1.2 mL. The resulting nanoparticles had a Z-average particle size of 203.7 nm, a zeta potential of -31.6 mV, and an RNA encapsulation efficiency (EE) measured by Ribogreen Assay of 91.1%. Encapsulated mRNA concentration in the final particle suspension was 74.6 pg / ml.

[0212] The final sample was collected and stored at -80° C in 15% w / v sucrose solution.

[0213] Example 7: Biodistribution study in BALB / c mice with Luciferase-mRNA loaded Sigfinity NPs prepared in Example 5

[0214] 112.5 pL of a suspension of Sigfinity™ nanoparticles loaded with a luciferase-mRNA containing 80.01 pg / ml of mRNA (prepared in Example 5) was administered to a Balb / c female mouse via the lateral tail vein. At 6h after the injection, D-Luciferin substrate was administered intraperitoneally to the dosed mouse, followed by euthanasia and subjected to in-life imaging sessions for luminescence (FIG. 3 A). The whole body was then perfused with PBS. Lung, spleen, and liver were collected and imaged with IVIS. The collected IVIS image of the organs is reported in FIG. 3B. Example 8: Biodistribution study in Ail4 mouse with Cre-recombinase-mRNA loaded Sigfinity NPs prepared in Example 6

[0215] 175 pL of a suspension of Sigfinity™ nanoparticles loaded with a Cre-recombinase- mRNA containing 57.1 or 114.2 pg / ml of mRNA (prepared in Example 6) was administered to an Ail 4 mouse via the lateral tail vein. Three days after the injection, mice were sacrificed. The whole body was then perfused with PBS. Lung, spleen, and liver were collected and imaged with IVIS. The collected IVIS image of the organs is reported in FIG. 4 A.

[0216] Lung tissues were harvested and finely minced using sterile scissors under aseptic conditions. The minced tissues were transferred into 5 mL of digestion buffer prepared in Dulbecco's Modified Eagle Medium (DMEM), supplemented with Liberase (0.1 mg / mL), DNase I (15 pg / mL), 2% fetal bovine serum (FBS), and 1% HEPES buffer. Tissue digestion was performed in a shaking incubator set to 200 revolutions per minute (RPM) at 37 °C for 30 minutes to ensure enzymatic dissociation into a single-cell suspension.

[0217] Following enzymatic digestion, the cell suspension was passed through a 70-pm nylon mesh cell strainer to remove undigested tissue fragments. The filtrate was washed with ice-cold phosphate-buffered saline (PBS) to a final volume of 20 mL and centrifuged at 1500 RPM for 5 minutes at 4 °C to pellet the cells.

[0218] The resulting cell pellet was resuspended in 1 mL of red blood cell (RBC) lysis buffer and incubated at room temperature (RT) for 5 minutes to lyse erythrocytes. The lysis reaction was then quenched by adding 5 mL of PBS, followed by centrifugation at 1500 RPM for 5 minutes. The supernatant was discarded, and the cell pellet was washed once with 1 mL of PBS. Cells were then transferred to a 1.5 mL microcentrifuge tube for subsequent processing.

[0219] Cell concentration was determined using a hemocytometer or automated cell counter. Approximately 2 * 106cells were used for flow cytometry analysis. Cells were first stained with a viability dye for 10 minutes at 4 °C to discriminate live and dead cells. After viability staining, cells were washed once with FACS staining buffer (PBS supplemented with 2% FBS and 2 mM EDTA), and incubated with 50 pL of Fc receptor blocking solution (diluted 1 :50) at 4 °C for 15 minutes to prevent non-specific antibody binding.

[0220] Following Fc blockade, cells were incubated with a panel of fluorophore-conjugated monoclonal antibodies targeting surface markers, including CD45, CD3, CD19, NKp46, CD1 lb, CD326 (EpCAM), CD31, and CD140a. Antibody incubation was carried out for 30 minutes at 4 °C in the dark. After staining, cells were washed once with FACS buffer and resuspended in an appropriate volume of FACS buffer for acquisition. Flow cytometric analysis was performed using the Attune NxT Flow Cytometer equipped with four lasers. Data acquisition and analysis were conducted by using Flow Jo. Representative results from this procedure are shown in FIG. 4B.

[0221] Example 9: Preparation of Sigfinity™ nanoparticles comprising an siRNA and ganglioside GQlb

[0222] 10.2 pL of 1 mg / mL GAPDH-siRNA solution in Ultrapure RNAse / DNase-free water was introduced to a 10-mL beaker containing 3 mL Ultrapure RNAse / DNase-free water. The beaker was equipped with a magnetic stir bar and was placed on a magnetic stirrer.

[0223] 8.4 pL of 20 mg / mL DOTAP solution in ethanol was diluted with 0.3 mL of ethanol. While the siRNA solution in the 10-mL beaker was magnetically stirred at 400 rpm, the DOTAP solution was added dropwise to the stirring aqueous siRNA using a pl 000 pipette. After the addition was complete, stirring was continued for 5 minutes.

[0224] 119.2 pL of 2.5 mg / mL ganglioside GQlb solution in methanol was mixed with 0.2 mL water. The molar ratio of DOTAP to GQlb lipids was 8:4. The GQlb solution was added dropwise to the stirring solution using a p200 pipette. Then, stirring was continued for 30 minutes at 400 rpm.

[0225] The nanoparticle suspension was transferred into a 3-mL Slide-A-Lyzer™ dialysis cassette with 20K MWCO, dialyzed against water in a 200-mL beaker for overnight, concentrated with an Amicon Ultra-15, 10K MWCO at 2000 RCF. 10X PBS was added 1 :9 for a final suspension in IX PBS. The resulting nanoparticles had a Z-average particle size of 87.45 nm, a zeta potential of -16.77 mV, and an RNA encapsulation efficiency (EE) measured by Ribogreen Assay of 67.5%. Encapsulated mRNA concentration in the final particle suspension was 3.46 pg / ml.

[0226] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

[0227] It should be understood that any preferred features of the invention described herein can be combined with any other preferred features, including preferred features described only under one aspect of the invention, and preferred features described only in the examples. Throughout the specification, any and all references to a publicly available document, including any U.S. patent or patent application publication, are specifically incorporated by reference.

Claims

1. CLAIMS1. A method of treating a lung-related disease or disorder, wherein the method comprises administering a nanoparticle to a subject in need, wherein the nanoparticle comprises a sialic acid (SA)-containing entity co-precipitated with an ionizable lipid or a cationic lipid (preferable, a cationic lipid), wherein the sialic acid groups reside on the surface of the nanoparticle.

2. The method of claim 1, wherein the nanoparticle has a negative charge.

3. The method of claim 1 or claim 2, wherein the nanoparticle has a zeta potential of from 0 to -100 mV.

4. The method of any one of the preceding claims, wherein the nanoparticle has a zeta potential from -15 to -50 mV.

5. The method of any one of the preceding claims, wherein the SA-contaning entity is water soluble.

6. The method of any one of the preceding claims, wherein the SA-contaning entity is a polysialic acid, or a ganglioside.

7. The method of any one of the preceding claims, wherein the SA-contaning entity is colominic acid.

8. The method of any one of claims 1-6, wherein the SA-contaning entity is GT1, GT2, GT3, or GQlb.

9. The method of any one of the preceding claims, wherein the nanoparticle comprises a cationic lipid.

10. The method of any one of the preceding claims, wherein the cationic lipid is DOTMA, ethyl PC’s, DDAB, di oleoyl-3 -trimethylammonium propane (DOTAP), DC-cholesterol, GL67, or DODMA.

11. The method of any one of the preceding claims, wherein the cationic lipid is DOTAP.

12. The method of any one of the preceding claims, wherein the nanoparticle further comprises an active pharmaceutical ingredient (API).

13. The method of claim 12, wherein the API is encapsulated in the nanoparticle.

14. The method of claim 12 or claim 13, wherein the API comprises a nucleic acid.

15. The method of claim 14, wherein the nucleic acid is DNA, mRNA, circular RNA, microRNA, siRNA, ASO, Cas9, gRNA.

16. The method of any one of the preceding claims, wherein the nanoparticle has an average size from about 20 nm to about 500 nm, or from about 50 nm to about 300 nm.

17. The method of any one of the preceding claims, wherein the nanoparticle is administered to the subject in need through systemic administration.

18. The method of any one of the preceding claims, wherein the lung-related disease or disorder is asthma, chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia (PCD), a-1 antitrypsin deficiency (AAT), pulmonary fibrosis, cystic fibrosis (CF), sarcoidosis, or lung cancer.

19. The method of any one of the preceding claims, wherein the method does not induce thrombosis.

20. A method for delivering an active pharmaceutical agent (or API) to the lung of a subject in need, wherein the method comprises administering a nanoparticle to a subject in need, wherein the nanoparticle comprises a sialic acid (SA)-containing entity co-precipitated with an ionizable lipid or a cationic lipid (preferable, a cationic lipid), wherein the sialic acid groups reside on the surface of the nanoparticle.

21. The method of claim 20, wherein the nanoparticle has a negative charge.

22. The method of claim 20 or claim 21, wherein the nanoparticle has a zeta potential from -15 to -50 mV.

23. The method of any one of claims 20-22, wherein the SA-contaning entity is water soluble.

24. The method of any one of claims 20-23, wherein the SA-contaning entity is a polysialic acid, or a ganglioside.

25. The method of any one of claims 20-24, wherein the SA-contaning entity is colominic acid.

26. The method of any one of claims 20-24, wherein the SA-contaning entity is GT1, GT2, GT3, or GQlb.

27. The method of any one of claims 20-26, wherein the nanoparticle comprises a cationic lipid.

28. The method of any one of claims 20-27, wherein the cationic lipid is DOTMA, ethyl PC’s, DDAB, di oleoyl-3 -trimethylammonium propane (DOTAP), DC-cholesterol, GL67, or DODMA.

29. The method of any one of claims 20-28, wherein the cationic lipid is DOTAP.

30. The method of any one of claims 20-29, wherein the API is encapsulated in the nanoparticle.

31. The method of any one of claims 20-30, wherein the API comprises a nucleic acid.

32. The method of any one of claims 20-31, wherein the nucleic acid is DNA, mRNA, circular RNA, microRNA, siRNA, ASO, Cas9, gRNA.

33. The method of any one of claims 20-32, wherein the nanoparticle has an average size from about 20 nm to about 500 nm, or from about 50 nm to about 300 nm.

34. The method of any one of claims 20-33, wherein the lung-related disease or disorder is asthma, chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia(PCD), a-1 antitrypsin deficiency (AAT), pulmonary fibrosis, cystic fibrosis (CF), sarcoidosis, and lung cancer.

35. The method of any one of claims 20-34, wherein the method does not induce thrombosis.

36. The method of any one of the preceding claims, wherein the nanoparticle is prepared by a process comprising a step of forming an intermediate lipoplex nanoparticle.

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