Novel particulate compositions containing sialic acid binding ligands

Abstract

The present invention provides polymeric particles that display non-conjugated sialic acid residues on the surface of the particle, compositions and methods of use thereof, as well as non-conjugation methods of making particles with sialic acid moieties on the surface of nanoparticles and microparticles.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 208,150, filed June 8, 2021. The entire teachings of the above application(s) are incorporated herein by reference. [Background technology]

[0002] 2. Background of the Invention Sialic acid, also known as N-acetylneuraminic acid, is a nine-carbon sugar that binds to sialic acid-binding immunoglobulin-like lectins (Siglecs). Sialic acid has three main derivatives, N-acetylneuraminic acid (Neu5Ac), N-acetylneuraminic acid hydroxyalkyl (Neu5Gc) and 3-deoxy-D-glycero-D-galacto-nonylketose (Kdn). There are other sialic acid derivatives that are further derived from these major derivatives. One important sialic acid derivative is the gangliosides found in the brain.

[0003] Siglecs expressed by various immune cells have intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that can mediate inhibitory signals upon binding to sialic acid and activate downstream inhibitory signaling via recruitment of tyrosine phosphatases SHP-1 and SHP-2. Sialic acid can also regulate alternative pathways of complement activation. The major serum protein complement factor H recognizes sialic acid as a "self" marker, which helps inhibit C1q / C3b fragment activation. Sialic acid also binds to carbohydrate-binding lectins that are overexpressed in several types of cancer.

[0004] Abnormal interactions between sialic acid and Siglecs are associated with several pathologies, such as infection, autoimmunity and cancer. Therefore, it may be therapeutically beneficial to bind chemical or biological entities that contain sialic acid residues to Siglecs on certain types of cells to regulate immune inhibition or activation for the treatment of pathologies, such as infection, autoimmunity and cancer. However, it is difficult to deliver such molecular entities of sialic acid to target cells in vivo. A common strategy is to attach molecules of sialic acid or polysialic acid onto the surface of nanoparticles, so that the nanoparticles can carry sialic acid entities to target cells.

[0005] Furthermore, when sialic acid binds to Siglec on certain types of immune cells, a chemical entity that contains a sialic acid moiety can be attached to the particle as a ligand to guide the particle to immune cells and bind the particle to Siglec on the cell. Such binding can facilitate the particle to enter the cell via receptor-mediated endocytosis. In this manner, nanoparticles that are loaded with therapeutic agents and have their surface coated with sialic acid or sialic acid-containing entities can target immune cells and deliver therapeutic agents to the cells.

[0006] Nanomedicine is an important tool in targeted drug delivery. It is desirable for drug-loaded nanoparticles administered systemically to have a long circulation time before they reach the targeted site. The current strategy to extend the in vivo circulation of nanoparticles is to PEGylate the surface of the particles to prevent them from being taken up by the reticuloendothelial system (RES). However, PEGylated nanoparticles may result in the production of PEG-specific antibodies, which may weaken drug release and target cell interactions, thereby impairing the therapeutic effect. Therefore, there is a need to develop novel strategies that can extend nanoparticle circulation in vivo while replacing polyethylene glycol (PEG).

[0007] The current method for attaching sialic acid to the surface of nanoparticles is by chemical conjugation. For example, sialic acid molecules can be functionalized with a reactive group that can form a covalent bond with another reactive group on the nanoparticle surface. However, this method suffers from low conjugation efficiency and side reactions that produce undesirable by-products in pharmaceutical formulations.

[0008] Therefore, there is an unmet need for new methods for attaching sialic acid-containing ligands to the surface of nanoparticles. Summary of the Invention

[0009] Summary of the Invention The present invention provides polymeric particles that display unconjugated sialic acid residues on their surface, compositions and methods of use thereof, as well as non-conjugation methods for making nano- and microparticles having sialic acid moieties on their surface.

[0010] The present invention includes compositions comprising polymeric particles presenting sialic acid residues on their surface, wherein the particles are microparticles or nanoparticles; each particle comprises a biodegradable polymer and a polysialic acid comprising sialic acid residues, wherein the sialic acid residues are not conjugated to the surface of the particle. The biodegradable polymer is preferably a pharma- ceutically acceptable biodegradable polymer. In some aspects, the biodegradable polymer may be selected from the group consisting of polylactide (PLA), poly(lactide-co-glycolide) (PLGA), copolymers of ethylene glycol and lactide / glycolide (PEG-PLGA), copolymers of ethylene glycol and lactide (PEG-PLA), copolymers of ethylene glycol and glycolide (PEG-PGA), poly(ethylene glycol) (PEG), polycaprolactone (PCL), polyanhydrides (PANH), poly(orthoesters), polycyanoacrylates, poly(hydroxyalkanoates) (PHAs), poly(sebasic acid), polyphosphazenes, polyphosphoesters, modified poly(saccharides), mixtures and copolymers thereof. In some embodiments, the biodegradable polymer is PLGA. In further aspects, the biodegradable polymer and polysialic acid form an interpenetrating network. The particles may further comprise an active agent, such as an active pharmaceutical ingredient.

[0011] The present invention further includes a method for administering an active agent to a subject in need thereof, comprising administering to the subject a composition comprising particles presenting sialic acid residues on their surface, the particles being microparticles or nanoparticles; each particle comprising a polysialic acid comprising a biodegradable polymer and sialic acid residues, the sialic acid residues not being conjugated to the surface of the particle; and the particles further comprising an active agent. The active agent may be an active pharmaceutical ingredient. In some aspects, the active agent is encapsulated within the particle.

[0012] The present invention further includes a method of treating a disease or disorder in a subject in need of such treatment comprising administering to the subject a particle described herein.

[0013] The present invention includes a method for the preparation of microparticles or nanoparticles presenting sialic acid residues on their surface, the method comprising: (1) dissolving a biodegradable polymer (and optionally an active agent, such as an active pharmaceutical ingredient (API) or a poorly water-soluble compound) in a first solvent to form a polymer solution; (2) emulsifying the polymer solution in a solution of a second solvent to form an emulsion, where the first solvent is immiscible or partially miscible with the second solvent, the second solvent solution comprising polysialic acid, the second solvent solution optionally further comprising a surfactant and / or an API soluble in the second solvent; and (3) removing the first solvent to form the microparticles or nanoparticles having surface sialic acid moieties.

[0014] The present invention also provides a method for the preparation of microparticles or nanoparticles presenting sialic acid moieties on their surface, the method comprising: (1) dissolving a biodegradable polymer (and optionally an active agent, API or poorly water-soluble compound) in a first solvent to form a polymer solution; (2) adding a first solution of a second solvent to the polymer solution to form a mixture, where the first solvent is immiscible or partially miscible with the second solvent, and the first solution of the second solvent optionally contains an active agent, which may be the same as or different from the API dissolved in the first solvent; (3) emulsifying the mixture to form a first emulsion; (4) emulsifying the first emulsion in a second solution of the second solvent to form a second emulsion, where the second solution of the second solvent contains polysialic acid and optionally further contains a surfactant; and (5) removing the first solvent to form microparticles or nanoparticles having surface sialic acid moieties.

[0015] In yet another aspect, the invention relates to particles made by the methods described herein.

[0016] Preferably, the microparticles or nanoparticles comprise an active agent, such as an active pharmaceutical ingredient (API).

[0017] Preferably, the API is encapsulated in a microparticle or nanoparticle. In one preferred aspect, the particle is a nanoparticle.

[0018] Alternatively or additionally, the API is covalently or ionically bound to the surface of the microparticle or nanoparticle. For example, the API can be covalently bound to the particle surface via a hydrolyzable bond that facilitates in vivo release.

[0019] Preferably, the second solvent solution further comprises or is saturated with the first solvent prior to adding the polymer solution in the first solvent to the first solution in the second solvent during emulsification. This can be beneficial in that the polymer in the first solvent is less likely to precipitate when added to the first solution in the second solvent for emulsification. Preferably, the first solvent is ethyl acetate and the second solvent solution (e.g., water or an aqueous solution) comprises about 7-8% v / v ethyl acetate.

[0020] Preferably, the microparticles and nanoparticles are based on a biodegradable polymer selected from the group consisting of: polylactide (PLA), poly(lactide-co-glycolide) (PLGA), copolymers of ethylene glycol and lactide / glycolide (PEG-PLGA), copolymers of ethylene glycol and lactide (PEG-PLA), copolymers of ethylene glycol and glycolide (PEG-PGA), poly(ethylene glycol) (PEG), polycaprolactone (PCL), polyanhydrides (PANH), poly(orthoesters), polycyanoacrylates, poly(hydroxyalkanoates) (PHAs), poly(sebacic acid), polyphosphazenes, polyphosphoesters, modified poly(saccharides), mixtures and copolymers thereof. In a further preferred aspect, the biodegradable polymer is PLGA. Optionally, the microparticles and nanoparticles include an active agent, such as a drug.

[0021] In certain preferred embodiments, the particles encapsulate an active agent.

[0022] Preferably, the polysialic acid is a pharma- ceutically acceptable polymer.

[0023] Preferably, the sialic acid is sialic acid, a salt, derivative or a mimetic thereof.

[0024] Preferably, the polysialic acid is attached to the surface of the microparticles and nanoparticles by non-chemical processes such as coating, absorption, adsorption and emulsification.

[0025] Preferably, the polysialic acid is durably attached to the surface of microparticles and nanoparticles and is capable of withstanding multiple washing cycles.

[0026] Preferably, the polysialic acid has a molecular weight of 500 to 50,000,000, 1,000 to 5,000,000, and 2,000 to 500,000 Da.

[0027] In some embodiments, the polysialic acid is a polysialic acid that contains only sialic acid repeat units. This type of polymer is often referred to as a "homopolymer". One example of such a homopolymer of polysialic acid is colominic acid, available commercially, for example, from Carbosynth, Oakbrook Terrace, IL, USA. Colominic acid, also referred to as polysialic acid, is a linear small polysaccharide containing α-2,8-linked sialic acid (neuraminic acid) with (n = 8 to > 100) residues.

[0028] In some embodiments, the polysialic acid is a "copolymer" comprising sialic acid repeat units and repeat units of at least one different chemical entity. Non-limiting examples of such copolymers include PLGA-PSia, PEG-PSia, PLGA-PEG-PSia, etc., where PLGA is poly(lactide-co-glycolide), PEG is polyethylene glycol, and PSia is polysialic acid.

[0029] In some embodiments, the polysialic acid is an oligomer of sialic acid available as N-acetylneuraminic acid oligomers, such as dimers, trimers, tetramers, pentamers or hexamers, or sodium salts thereof available from Nacalai USA, Inc., San Diego, Calif., USA.

[0030] In some embodiments, polysialic acid is a pharma- ceutically acceptable polymer with sialic acid moieties at the termini of its chemical structure. For example, PEG-Sia or PLGA-PEG-Sia, where Sia represents sialic acid moiety. Polysialic acid can also be a ganglioside. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Detailed Description of the Invention Overview Since abnormal interaction between sialic acid and Siglec is associated with several pathologies, such as infection, autoimmunity and cancer, providing particles that present sialic acid moieties that bind to Siglec on specific cells can be therapeutically useful.Compositions that include the particles can be used to treat pathologies, such as infection, autoimmunity and cancer.Also, the interaction between sialic acid and Siglec on specific immune cells can be used to direct particles that include sialic acid residues to immune cells.Thus, particles that include therapeutic agents and sialic acid moieties can be targeted to specific immune cells.

[0032] The present invention provides particles that display non-conjugated sialic acid residues on their surface, compositions and methods of use thereof, as well as non-conjugation methods for making nanoparticles and microparticles having sialic acid moieties on their surface. The non-conjugation methods described herein avoid the side reactions and by-products observed when using conjugation methods to attach sialic acid residues to the surface of particles.

[0033] The invention described herein provides pharmaceutical formulations comprising microparticles and nanoparticles having sialic acid residues on their surface (with or without drug / drug / API loading), as well as processes by which such pharmaceutical formulations comprising microparticles and / or nanoparticles may be made.

[0034] The present invention includes a method for preparing microparticles and nanoparticles that present sialic acid residues on their surface, which method comprises co-precipitation or coacervation of hydrophobic and / or neutral biocompatible polymers, such as PLGA or PLA, and polysialic acid.Without being bound by any theory, it is believed that the polymer backbones intertwine or interlock while in the organic phase of the emulsion.Using the method of the present invention, polysialic acid is tightly incorporated into the resulting microparticle or nanoparticle.Thus, preferably, polysialic acid is integrated on the microparticle or nanoparticle, presenting sialic acid residues on the surface of the microparticle or nanoparticle.

[0035] With the invention generally described above, particular aspects of the invention are further described in the following sections.

[0036] definition As used herein, "pharmacologically acceptable" includes those compounds, materials, compositions and / or dosage forms that are within the scope of sound medical judgment and suitable for medical or veterinary use without excessive toxicity, irritation, allergic response or other problem or complication when contacted with human and animal tissues at the concentrations, doses or amounts present in the product, commensurate with a reasonable benefit / risk ratio. Preferably, pharma-ceutically acceptable materials (e.g., polymers, excipients, surfactants, solvents or microparticles / nanoparticles made therefrom) are suitable or approved for human medical use.

[0037] As used herein, "microparticles" are preferably roughly round, spherical or sphere-like in shape and generally in the size range of, for example, about 1-1,000 μm or about 10-100 μm, as measured, for example, by laser diffraction. The subject microparticles can also include particles that are not prone to clump or aggregate into masses in vivo. However, it is understood that other particle morphologies are possible, such as rods, plates, sheets, and needles. Typically, it is understood that particle size reflects the volume median geometric size of the product sample being tested.

[0038] As used herein, a "nanoparticle" is preferably roughly round, spherical or sphere-like in shape and generally in the size range of, for example, about 1-1,000 nm, about 10-1,000 nm, or about 50-1,000 nm, or about 100-500 nm, as measured, for example, by laser diffraction. The subject nanoparticles can also include particles that do not tend to aggregate into clumps in vivo.

[0039] Particle size and size distribution can be measured by dynamic light scattering equipment, such as Malvern Zetasizer. Alternative techniques include, for example, sedimentation field flow fractionation, photon correlation spectroscopy, light scattering, dynamic light scattering, light diffraction, and disk centrifugation. The terms "microparticle" and "nanoparticle" are not intended to imply any particular shape restriction. Such particles include, but are not limited to, those with a generally polyhedral or spherical geometry. Preferred particles are characterized by a spherical geometry that is typically produced by emulsion-based encapsulation processes. It is understood that the terms "microparticle" and "nanoparticle" are used interchangeably herein unless a specific description of size is attached. For example, the term "microparticle" is also intended to encompass "nanoparticle" as well as when stated as "microparticle and / or nanoparticle" unless the context requires otherwise.

[0040] Although each microparticle or nanoparticle need not be of uniform size, they will generally be of a size sufficient to induce phagocytosis in antigen presenting cells (APCs) or other MPS cells. Preferably, the subject microparticles and nanoparticles have a diameter sufficient to induce phagocytosis in antigen presenting cells (APCs) or other MPS cells.

[0041] The term "particle" encompasses both nanoparticles and microparticles. As used herein, "a" or "an" means one or more, unless specified otherwise.

[0042] As used herein, "about" generally means up to plus or minus 10% of the particular term it modifies.

[0043] As used herein, the terms "encapsulates," "encapsulated," and the like, when referring to a drug or active agent that is encapsulated within a particle, mean that the drug or active agent is more likely to be found within the microparticle than on the surface of the microparticle.

[0044] "Polysialic acid" is a polymer that includes sialic acid monomers. Polysialic acid is described in more detail below.

[0045] The terms "sialic acid residue" and "sialic acid moiety," as well as their multiple referents, are used interchangeably herein.

[0046] As used herein, "conjugation" or "conjugated" and the like, in the context of sialic acid moieties on the surface of a particle(s), refers to the covalent attachment of a sialic acid moiety (e.g., a sialic acid moiety of a polysialic acid) to a particle or biodegradable polymer, for example, by forming a covalent bond via functionalization of a sialic acid residue with a reactive group that can form a covalent bond with a linker moiety or a reactive group on the nanoparticle surface (e.g., a reactive group of a biodegradable polymer). For example, conjugation has been described using thio derivatives of polysialic acid (Bondioli et. al (2010). PLGA nanoparticles surface decorated with the sialic acid, N-acetylneuraminic acid. Biomaterials. 31. 3395-403. 10.1016 / j.biomaterials.2010.01.049). Thus, "not conjugated" or "non-conjugated" and the like, in the context of sialic acid residues on the surface of a particle(s), means that the sialic acid residue(s) or polysialic acid containing sialic acid residues is not covalently attached to the particle or biodegradable polymer by forming a covalent bond therebetween. For example, polysialic acid is attached to the surface of microparticles and nanoparticles by processes such as coating, absorption, adsorption and / or emulsification. Without wishing to be bound by theory, it is believed that the biodegradable polymer, such as PLGA, and polysialic acid form an interpenetrating network that presents sialic acid residues on the surface of the formed particle.

[0047] As used herein, the term "subject" is used to mean an animal, preferably a mammal, such as a human or non-human. The terms "patient" and "subject" may be used interchangeably herein.

[0048] "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject or administration of an active agent to a subject with the purpose of reversing, alleviating, ameliorating, inhibiting, slowing or preventing the onset, progression, onset, severity or recurrence of symptoms, complications, conditions or biochemical signs associated with a disease. As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") includes clinical intervention to modify the natural course of a disease in the individual being treated, and may be performed either during the course of prophylaxis or clinical pathology. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of a disease, prevention of metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and remission or improved prognosis. In some embodiments, the combination of the present invention is used to delay the onset of a disease or to delay the progression of a disease.

[0049] Biodegradable Polymers A biodegradable polymer is a polymer that can be metabolized or broken down by a living thing. In some aspects, a biodegradable polymer is broken down or metabolized without causing substantial toxic effects. The biodegradable polymer of the present invention can be selected from the group consisting of: polylactide (PLA), poly(lactide-co-glycolide) (PLGA), copolymers of ethylene glycol and lactide / glycolide (PEG-PLGA), copolymers of ethylene glycol and lactide (PEG-PLA), copolymers of ethylene glycol and glycolide (PEG-PGA), poly(ethylene glycol) (PEG), polycaprolactone (PCL), polyanhydrides (PANH), poly(orthoesters), polycyanoacrylates, poly(hydroxyalkanoates) (PHAs), poly(sebacic acid), polyphosphazenes, polyphosphoesters, modified poly(saccharides), mixtures and copolymers thereof. PLGA is the preferred biodegradable polymer of the present invention.

[0050] PLGA PLGA is typically prepared by ring-opening polymerization of lactide and glycolide. Stannous octoate is usually used as a catalyst in this reaction, although other catalysts can also be used. Initiators such as alcohols are often used to start the polymerization reaction. If an initiator is not intentionally added, traces of polar compounds containing active protons, such as alcohols and water, can act as initiators. Polymerization usually proceeds as follows: R-OH + L (lactide monomer) + G (glycolide monomer) = PLGA-COOH This results in a PLGA polymer with carboxyl groups at the chain ends, as shown in Figure 1.

[0051] Therefore, each PLGA and / or PLA polymer molecule is typically linear and typically contains a single COOH group at the end of the chain.Also, there may not be a sufficient number of COOH groups to covalently attach API or other chemical moieties, such as protein ligands or other targeting agents, to the surface of the microparticles and nanoparticles.Such protein ligands or other targeting agents can bind to receptors or binding partners on the surface of target cells, tissues, organs or locations.

[0052] The present invention provides various methods or combinations thereof for making PLGA / PLA particles with polysialic acid. Such particles are particularly useful, for example, for treating certain diseases (e.g., inflammatory diseases, autoimmune diseases, and cancer) and for delivering active agents.

[0053] Preferably, the average molecular weight of the pharma- ceutically acceptable polymer PLGA is within a desirable range.

[0054] The lower limit of the range is preferably about 100 Da or more, 200 Da or more, 300 Da or more, 400 Da or more, 500 Da or more, 600 Da or more, 700 Da or more, 800 Da or more, 900 Da or more, 1000 Da or more, 1200 Da or more, 1500 Da or more, 2000 Da or more, 2500 Da or more, or 3000 Da or more. Desired ranges have a lower limit of any of the above values.

[0055] The upper limit of the range is preferably 50,000 Da or less, 40,000 Da or less, 35,000 Da or less, 30,000 Da or less, 25,000 Da or less, 20,000 Da or less, 15,000 Da or less, 10,000 Da or less, 7,500 Da or less, or 5,000 Da or less. Desired ranges have upper limits of any of the above values.

[0056] For example, the desired range can be from about 500 to about 50,000 Da, or from about 1,000 to about 30,000 Da.

[0057] Preferably, the PLGA has an average molecular weight of about 500 to about 1,000,000 Da, preferably about 1,000 to about 50,000 Da.

[0058] PLGA may contain multiple negatively charged end groups.

[0059] For PLGA, the average molecular weight can be expressed in terms of other physical properties, such as intrinsic viscosity. Intrinsic viscosity (IV) is a viscometric method for measuring molecular size. IV is based on the flow time of a polymer solution through a narrow capillary versus the flow time of a pure solvent through the capillary. For certainty measurements in this application, the solvent used is typically chloroform, and the polymer concentration is about 0.5% (w / v). The temperature at which the viscosity is measured is about 30°C. The units of IV are typically reported in deciliters per gram (dL / g). Thus, for example, the PLGA used in the present invention may have an intrinsic viscosity of about 0.01 to about 20 dL / g, or about 0.05 to about 2.0 dL / g.

[0060] The composition and biodegradability of the subject PLGA polymers are determined in part by the molar ratio of lactide (L) to glycolide (G) units in the polymer, or the L / G ratio. The L / G ratio of the PLGA polymers of the present invention can be 100 / 0 to 0 / 100. As used herein, an L / G ratio of "100 / 0" refers to polylactide or PLA, and an L / G ratio of "0 / 100" refers to polyglycolide or PGA. Preferably, the L / G ratio for PLGA polymers is about 100 / 0 to 0 / 100 or about 95 / 5 to 5 / 95, more preferably about 85 / 15 to 15 / 85. The most preferred L / G ratio of the present invention is about 50 / 50.

[0061] In the preparation of PLGA microparticles and nanoparticles, other polymers may be mixed with the PLGA polymer. For example, polyethylene glycol or PEG is often added to PLGA to increase performance. PEGylated particles are often useful because they have a high circulation time in the human or animal body.

[0062] Preferably, a copolymer of PEG and PLGA may also be used.

[0063] Microparticles and nanoparticles prepared from mixtures of PEG and PLGA or copolymers of PEG and PLGA are preferred as PEGylated PLGA microparticles and nanoparticles.

[0064] Such a "PEGylation" process can also be done after the microparticles and nanoparticles are formed, in which case PEG polymers or other polymers containing PEG units are coated onto the PLGA microparticles and nanoparticles via physical absorption.

[0065] PEG units can also be attached to the surface of PLGA microparticles or nanoparticles via covalent bonds. Such a process is often referred to as "conjugation." In the conjugation process, reactive entities including PEG units react with specific functional groups on the surface of microparticles and nanoparticles to form chemical bonds.

[0066] Thus, preferably, the pharma- ceutically acceptable polymer is PLGA, and the microparticle or nanoparticle is PEGylated. The microparticle or nanoparticle can be PEGylated by mixing polyethylene glycol (PEG) or a PEG-containing entity during the preparation of the microparticle and nanoparticle. The microparticle or nanoparticle can also be PEGylated using a copolymer of PEG and PLGA. The microparticle or nanoparticle can be further PEGylated by physically absorbing a PEG polymer or a polymer containing PEG units onto the PLGA microparticle and nanoparticle. The microparticle or nanoparticle can be further PEGylated by conjugating a PEG unit to the surface of the PLGA microparticle or nanoparticle via a covalent bond.

[0067] Preferably, the biodegradable polymer has an average molecular weight of about 500 to about 1,000,000 Da, preferably about 1,000 to about 200,000 Da.

[0068] Preferably, the biodegradable polymer is PLGA and has an L / G ratio of about 100 / 0 to 0 / 100, about 95 / 5 to 5 / 95, about 85 / 15 to 15 / 85 and about 50 / 50.

[0069] Polysialic acid Polysialic acid (PSia) comprises homopolymers of sialic acid. Naturally occurring PSia was first found in Escherichia coli and is one of the components of bacterial capsule materials such as Neisseria miningitidis B, Salmonella toucra 048 and Citrobacter freundii 05. PSia can be in α-2,8 (A in the figure below) or α-2,9 (B in the figure below) conformations or a mixture of α-2,8 and α-2,9. PSia composed of α-2,8 linkages is non-immunogenic, biodegradable and can reduce the immunogenicity of protein polypeptides. PSia has properties that evade phagocytes and extend circulation time in vivo. [ka]

[0070] Therefore, nanoparticles with sialic acid moieties on their surface may also facilitate RES evasion and allow nanoparticles and microparticles to have extended circulation in the bloodstream.Since sialic acid also binds to several receptors on tumor cells, sialic acid-coated nanoparticles and microparticles may have enhanced targeting to tumor sites via the high avidity binding of sialic acid to lectins.

[0071] As mentioned above, polysialic acid is a polymer comprising a chain of sialic acid monomers. In some aspects, the polymer is a homopolymer (e.g., all 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 still other aspects, the polymer comprises 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 monomers can be any derivative of 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). Sialic acid monomers have the formula (I): [ka] As an example,

[0072] 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.

[0073] As mentioned above, the sialic acid monomers can be linked with α-2,8-, α-2,9 or α-2,8 / α-2-9-ketosidic linkages, e.g., α-2,4-ketosidic and α-2,5-ketosidic linkages are also described (Janas et al. (2011), Biochimica et Biophysica Acta 1808: 2923-2932). The sialic acid monomers can be linked in any bond arrangement. In some embodiments, the polysialic acid includes 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 includes Neu5Ac monomers that are 2→8 linked, 2→9 linked, or a combination thereof. In yet another aspect, the polysialic acid comprises Neu5Gc monomers that are 2→8 linked, 2→9 linked, or a combination thereof. In a further embodiment, 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 two or three monomers selected from Neu5Ac, Neu5Gc, and Kdn. In certain embodiments, the homopolymer comprises Neu5Ac monomers. The homopolymer can be a poly(Neu5Ac)n, poly(Neu5Gc)n, or poly(Kdn)n polymer, where n is an integer greater than 10, greater than 15, or greater than 20; optionally, the monomers are 2→8 linked, 2→9 linked, or a combination thereof. In yet another particular embodiment, the heteropolymer comprises Neu5Ac and Neu5Gc monomers.

[0074] Polysialic acid can be a branched or unbranched polymer. An "unbranched" polymer is a linear polysialic acid polymer that contains a linear sequence of monomers. A "branched" polymer is a polysialic acid polymer that contains a backbone that has one or more substituent side chains or is branched. An example of a branched polymer is one that contains a sialic acid unit that is linked to three or more different sialic acid units, thereby creating a branch point within the polysialic acid.

[0075] The polysialic acid can have a molecular weight of, for example, at least 1 kDa, 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.

[0076] Preferably, the polysialic acid has a molecular weight of 500 to 50,000,000, 1,000 to 5,000,000, or 2,000 to 500,000 Da.

[0077] In some embodiments, the polysialic acid is a polysialic acid that contains only sialic acid repeat units. This type of polymer is often referred to as a "homopolymer". One example of such a homopolymer of polysialic acid is colominic acid, available from Carbosynth, Oakbrook Terrace, IL, USA. Colominic acid, also referred to as polysialic acid, is a linear small polysaccharide containing α-2,8 linked sialic acid (neuraminic acid) with (n = 8 to > 100) residues.

[0078] In some embodiments, the polysialic acid is a "copolymer" that includes sialic acid repeat units and repeat units of at least one different chemical entity. Non-limiting examples of such copolymers include PLGA-PSia, PEG-PSia, PLGA-PEG-PSia, etc., where PLGA is poly(lactide-co-glycolide), PEG is polyethylene glycol, and PSia is polysialic acid. Polysialic acid can also be a ganglioside.

[0079] In some embodiments, the polysialic acid is an oligomer of sialic acid available as N-acetylneuraminic acid oligomers, such as dimers, trimers, tetramers, pentamers and hexamers, or sodium salts thereof available from Nacalai USA, Inc., San Diego, CA, United States.

[0080] In some embodiments, the polysialic acid is a pharma- ceutically acceptable polymer having a sialic acid moiety at the terminus of its chemical structure, e.g., PEG-Sia or PLGA-PEG-Sia, where Sia represents a sialic acid moiety.

[0081] Sialic acid can also be a water-soluble salt and water-soluble derivative of sialic acid. For example, sialic acid salts can be sodium, potassium, magnesium, calcium or zinc salts. As mentioned above, polysialic acid can contain a combination of more than one type of sialic acid.

[0082] In one set of embodiments, one or more of the sialic acid monomers in the polysialic acid are modified. For example, one or more of the sialic acid units can be modified by attachment to polyethylene glycol or an alkyl group. In other embodiments, the polysialic acid is not modified.

[0083] Polysialic acid may also include other monomers or units in addition to sialic acid monomers. In some instances, polysialic acid is a conjugate of a polymer of sialic acid monomer units and another polymer, such as a synthetic polymer, such as, for example, polyethylene glycol (PEG) (e.g., polysialic acid-PEG copolymer). Examples of such conjugates are described, for example, in Zhang et al. (2018), Drug Delivery and Translational Research 8, 602-616. PEG may have the formula: H-(O-CH2-CH2)n-OH, where n is an integer representing the degree of PEG polymerization. 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 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 30 or less, or 10 or less.

[0084] The polysialic acids in the particles can be the same or different.

[0085] Polysialic acid can also be covalently or ionically substituted along the length of the chain or at the end of the chain. For example, one or more monomer units can be substituted with a targeting moiety, such as a cell ligand (or fragment), a peptide or a carbohydrate. The substitution or conjugation step of the targeting moiety can occur before or after the microparticle is formed.

[0086] In some examples, the amount of polysialic acid used in the present invention may be 0.01% to 30%, preferably 0.1% to 15%, based on the weight of the PLGA used in the formulation.

[0087] Activator The described particles may further include an active agent. The composition may include an API, which may be covalently or ionically bound to the surface of the microparticle or nanoparticle via a covalent bond, such as a bond formed between an amide group of the protein and a carboxyl group on the surface of the microparticle or nanoparticle. The API may also be encapsulated within the microparticle or nanoparticle. The amount of API may be about 0.01 to about 50% (w / w) of the microparticle or 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 microparticle or nanoparticle.

[0088] In some aspects, the active agent is advantageously a drug (also referred to herein as an active pharmaceutical ingredient or API). However, non-therapeutic active agents may also be included as part of the particles according to the method. For example, agents useful in diagnostics, agriculture, cosmetics, personal products, household products, industrial chemicals, dyes, fluorescent agents or colorants, etc. may be included. Preferred active agents include small molecules and macromolecules. For example, biomolecules such as peptides, peptidomimetics, oligonucleotides, nucleic acid molecules and their mimetics, such as DNA, RNA, PNA, siRNA, microRNA, antisense, proteins, antibodies and their antigen-binding fragments, enzymes, hormones, growth factors, antigens, neoantigens, saccharides, oligosaccharides, polysaccharides, and combinations thereof. The composition may not include other active pharmaceutical ingredients or APIs, such as conjugated peptides or antigenic moieties. It is understood that APIs may be replaced with non-therapeutic compounds, such as diagnostic, agricultural, or chemical agents. Thus, in each instance where the term API is used, it will be understood that the term "active agent", including diagnostic, agricultural, or chemical agents, may be used instead. The terms "API" and "drug" are used interchangeably herein.

[0089] The API may be water-soluble or may have relatively poor water solubility. For example, the poorly water-soluble API may be dissolved in the same first solvent used to dissolve PLGA, or may be dissolved in a suitable solvent (which may be the same as the first solvent or different) to form an API solution, and then the API solution is mixed with the first solvent containing PLGA, and both the API and PLGA remain in the resulting solution. The water-soluble API may first be dissolved in its own solvent (which may be the same as the second solvent or different) to form an API solution, and then the API solution is added to the second solvent.

[0090] The API or active agent can include a wide range of different compounds, such as compounds and mixtures of compounds, such as small organic or inorganic molecules; saccharin; oligosaccharides; polysaccharides; biological macromolecules, such as peptides, proteins and peptide analogs and derivatives; peptidomimetics; antibodies and antigen-binding fragments thereof; nucleic acids; nucleic acid analogs and derivatives; extracts made from biological materials such as bacteria, plants, fungi or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combination thereof. Preferably, the therapeutic agent is a small molecule.

[0091] As used herein, the term "small molecule" can refer to compounds that are "natural product-like", but the term "small molecule" is not limited to "natural product-like" compounds. Rather, small molecules are typically characterized as containing several carbon-carbon bonds and having a molecular weight of less than 5000 Daltons (5 kDa), preferably less than 3 kDa, even more preferably less than 2 kDa, and most preferably less than 1 kDa. In some cases, it is preferred that small molecules have a molecular weight of 700 Daltons or less.

[0092] As used herein, a "peptide" is an oligopeptide, e.g., a sequence of 2-25 amino acids. The term "peptide" includes within its scope peptides containing naturally occurring amino acids and known analogs of naturally occurring amino acids having a function, unless otherwise specified. A "protein" includes one or more peptide (polypeptide) chains and may contain more amino acids than a peptide. The terms "peptide", "polypeptide" and "protein" may be used interchangeably herein.

[0093] Exemplary therapeutic agents include, but are not limited to, those approved by the FDA that are subject to novel drug applications by the FDA in clinical trials or preclinical studies.

[0094] API includes the categories and specific examples disclosed herein. It is not intended that the categories are limited to the specific examples. Those skilled in the art will recognize many other compounds that fall within the category and are useful according to the present disclosure. Examples include radiosensitizers, steroids, xanthines, beta-2-agonist bronchodilators, anti-inflammatory agents, analgesics, calcium antagonists, angiotensin-converting enzyme inhibitors, beta-blockers, centrally active alpha agonists, alpha-1-antagonists, anticholinergic / anticonvulsants, vasopressin analogs, antiarrhythmic agents, antitremorants, antianginal / antihypertensive agents, anticoagulants, antiplatelet agents, sedatives, anxiolytic agents, peptide agents, biopolymer agents, antineoplastic agents, laxatives, antidiarrheals, antibacterial agents, antifungal agents, vaccines, proteins, or nucleic acids. In a further aspect, the pharma- ceutically active agent is a steroid, such as coumarin, albumin, betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone and pharma- ceutically acceptable hydrocortisone derivatives; a xanthine, such as theophylline and doxophylline; a beta-2-agonist bronchodilator, such as salbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol; an anti-inflammatory agent, such as an anti-asthma anti-inflammatory agent, an anti-arthritic anti-inflammatory agent and a non-steroidal anti-inflammatory agent, Examples of which include, but are not limited to, sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharma- ceutically acceptable diclofenac salts, nimesulide, naproxen, acetaminophen, ibuprofen, ketoprofen, and piroxicam; analgesics, 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;Beta-blockers (i.e. beta-adrenergic blockers), such as sotalol hydrochloride, timolol maleate, esmolol hydrochloride, carteolol, propanol 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 doxorubicin; zosin and prazosin; anticholinergic / anticonvulsant drugs such as dicyclomine hydrochloride, scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate and oxybutynin; vasopressin analogues such as vasopressin and desmopressin; antiarrhythmic drugs such as quinidine, lidocaine, tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamil hydrochloride, propafenone hydrochloride, flecainide acetate, procainamide hydrochloride, moricizine hydrochloride and disopyramide phosphate; Antitremorants, such as dopamine, L-dopa / carbidopa, selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine and bromocryptine; antianginals and antihypertensives, such as isosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol and verapamil; anticoagulants and antiplatelet agents, such as coumadin, warfarin, acetylsalicylic acid and ticlopidine; sedatives, such as benzodiazepines and barbiturates. turate; ansiolytic agents such as lorazepam, bromazepam and diazepam; peptidic and biopolymeric agents such as calcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporine, 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, casanthranole, bisacodyl and sodium picosulfate; antidiarrheal agents, such as difenoxin hydrochloride, loperamide hydrochloride, furazolidone, diphenoxylate hydrochloride and microorganisms; vaccines, such as bacterial and viral vaccines; antibacterial agents, such as penicillins, cephalosporins and macrolides, antifungal agents, such as imidazole and triazole derivatives; and nucleic acids, such as DNA sequences encoding biological proteins and antisense oligonucleotides.

[0095] 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; beta7 integrin blockers such as rhuMAb beta7; IgE pathway blockers such as Anti-M1 prime; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / .beta.2 blockers such as anti-lymphotoxin alpha (LTa) or anti-VEGF agents.

[0096] Drugs or APIs include proteins or peptides, including, but not limited to, 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, C1 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-1a (Plegridy®), recombinant factor VIII PEGylated (Adynovate®), humanized anti-HER2 / neu conjugated to emtansine (Kadcyla®), belimumab, ipilimumab, belatacept, brentuximab vedotin, aflibercept, asparaginase erwinia chrysanthemumchrsanthemi, glucarpidase, taliglucerase alfa, pertuzumab, ziv-aflibercept, tbo-filgrastim, ocriplasmin, raxibacumab, ado-trastuzumab emtansine, golimumab, tocilizumab, obinutuzumab, elosulfase alfa, metreleptin, albiglutide, ramucirumab, siltuximab, vedolizumab, peg interferon beta-1a, pembrolizumab, dulaglutide, bintumomab, ni Volumab, Secukinumab, Parathyroid hormone, Filgrastim-sndz, Dinutuximab, Alirocumab, Evolocumab, Idaracizumab, Asfotase-alpha, Mepolizumab, Dratumumab, Necitumumab, Elotuzumab, Sebelipase-alpha, Oviltoxaximab, Ixekizumab, Reslizumab, Infliximab-dyyb, Atezolizumab, Daclizumab, Etanercept-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, C1 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).

[0097] The present invention is particularly applicable to the administration of anti-cancer drugs, such as the DNA demethylating agents 5-azacytidine (azacitidine) or 5-aza-2'-deoxycytidine (decitabine), (cytarabine or ara-C); 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);N 4-octadecyl-cytarabine; or elaidic acid cytarabine. Cytidine analogs can also be structurally related to cytidine or deoxycytidine and functionally mimic and / or antagonize the action of cytidine or deoxycytidine. The agents also include 5-fluorouracil, afatinib, aplidine, 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, cyclospor ... Rabin, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, DM1, DM3, DM4, doxorubicin, 2-pyrrolinodoxorubicin (2-PDox), prodrug form of 2-PDox (pro-2-PDox), cyano-morpholinodoxorubicin, doxorubicin glucuronide, endostatin, epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor binding agent, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, 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 ZD1839 or pharma- ceutical acceptable salts thereof.

[0098] Anti-cancer agents include, but are not limited to, inhibitors, agonists, antagonists, ligands, modulators, stimulators, blockers, activators or suppressors of genes, ligands, receptors, proteins, factors, such as adenosine receptors (e.g., A2B, A2a, A3), Abelson murine leukemia viral oncogene homolog 1 gene (ABL, e.g., ABL1), acetyl-CoA carboxylase (e.g., ACC1 / 2), adrenocorticotropic hormone receptor (ACTH), activated CDC kinase (ACK, e.g., ACK1), adenosine deaminase, adenylate cyclase, ADP-ribosyl cyclase-1, aerolysin, angiotensinogen (AGT) gene, murine thymoma viral oncogene homolog 1 (AKT) protein kinase (e.g., AKT1, AKT2, AKT3), AKT1 gene, alkaline phosphatase, inflammatory bowel disease (IGPH), and the like. phosphatase, alpha 1 adrenergic receptor, alpha 2 adrenergic receptor, alpha-ketoglutarate dehydrogenase (KGDH), aminopeptidase N, arginine deaminase, beta adrenergic receptor, anaplastic lymphoma kinase receptor, anaplastic lymphoma kinase (ALK, e.g., ALK1), Alk-5 protein kinase, AMP-activated protein kinase, androgen receptor, angiopoietins (e.g., ligand-1, ligand-2), apolipoprotein AI (APOA1) gene, apoptosis signal-regulating kinase (ASK, e.g., ASK1), apoptosis inducer, apoptosis protein (e.g., 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 (e.g. 1, 2), Basigin, BCR (breakpoint cluster region) protein and gene, B-cell lymphoma 2 (BCL2) gene, Bc12 protein, Bc12 binding component 3, BCL2L11 gene, Baculovirus IAP repeat containing 5 (BIRCS) gene, B-Raf proto-oncogene (BRAF), Brc-Abl tyrosine kinase, beta-catenin, B-lymphocyte antigen CD19, B-lymphocyte antigen CD20, B-lymphocyte stimulator ligand, B-lymphocyte cell adhesion molecule,Bone morphogenetic protein-10 ligand, bone morphogenetic protein-9 ligand regulator, Brachyury proteins, bradykinin receptor, Bruton's tyrosine kinase (BTK), bromodomain and ectodomain (BET) bromodomain-containing proteins (e.g. BRD2, BRD3, BRD4), calmodulin, calmodulin-dependent protein kinase (CaMK, e.g. CAMKII), cancer testis antigen 2, cancer testis antigen NY-ESO-1, cannabinoid receptors (e.g. CB1, CB2), carbonic anhydrase, caspases 8 Apoptosis-related cysteine ​​peptidase CASP8-FADD-like regulator, caspases (e.g., caspase-3, caspase-7, caspase-9), caspase recruitment domain protein-15, cathepsin G, chemokine (CC motif) receptors (e.g., CCR2, CCR4, CCR5), CCR5 gene, chemokine CC21 ligand, cluster of differentiation (CD) receptors (e.g., CD4, CD27, CD29, CD30, CD33, CD37, CD40, CD40 ligand receptor, CD40 ligand, CD40LG gene, CD44, CD45, CD 47, CD49b, CD51, CD52, CD55, CD58, CD66e, CD70 gene, CD74, CD79, CD79b, CD79B gene, CD80, CD95, CD99, CD117, CD122, CDw123, CD134, CDw137, CD158a, CD158b1, CD158b2, CD223, CD276 antigen; chorionic gonadotropin, cyclin G1, cyclin D1, cyclin-dependent kinase (CDK, e.g., CDK1, CDK1B, CDK2-9), casein kinase (CK, e.g., CM, CMI), c-Kit ( tyrosine-protein kinase Kit or CD117), c-Met (hepatocyte growth factor receptor (HGFR)), CDK-activated kinase (CAK), checkpoint kinases (e.g. CHK1, CHK2), cholecystokinin CCK2 receptor, claudins (e.g. 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, cyclooxygenases (e.g. 1, 2), cancer / testis antigen 1B (CTAG1) gene,CTLA-4 (cytotoxic T-lymphocyte protein 4) receptor, CYP2B1 gene, cysteine ​​palmitoyltransferase porcupine, cytokine signaling-1, cytokine signaling-3, cytochrome P450 11B2, cytochrome P450 reductase, cytochrome P450 3A4, cytochrome P450 17A1, cytochrome P450 17, cytochrome P450 2D6, (provided the anti-cancer or cytochrome modifying agent is something other than cobicistat), cytoplasmic isocitrate dehydrogenase, cytosine deaminase, cytosine DNA methyltransferase, cytotoxic T lymphocyte protein-4, chemokine (C--X--C motif) receptors (e.g., CXCR4, CXCR1 and CXCR2), delta-like protein ligands (e.g., 3, 4), deoxyribonuclease, Dickkopf-1 ligand, dihydropyrimidine dehydrogenase, DNA binding proteins (e.g., HU-β), DNA-dependent protein kinase, DNA gyrase, DNA methyltransferase, DNA polymerase (e.g., α), DNA primase, discoidin domain receptors (DDR, e.g., DDR1), DDR2 gene, dihydrofolate reductase (DHFR), dipeptidyl peptidase IV, L-dopachrome tautomerase enzyme, dUTP pyrophosphatase, echinoderm microtubule-like protein 4, epidermal growth factor receptor (EGFR) gene, EGFR tyrosine kinase receptor, eukaryotic translation initiation factor 5A (EIFSA) gene, elastase, elongation factor 1α2, elongation factor 2, endoglin, endonuclease, endoplasmin, endosialin, endostatin, endothelin (e.g., ET-A, ET-B), enhancer of zeste homolog 2 - (EZH2), epidermal growth factor, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), ephrin (EPH) tyrosine kinase (e.g., 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 kinase (ERK),E-selectin, estradiol 17β dehydrogenase, estrogen receptors (e.g., α, β), estrogen-related receptors, exportin 1, extracellular signal-related kinases (e.g., 1, 2), factors (e.g., Xa, VIIa), Fas ligand, fatty acid synthase, ferritin, focal adhesion kinase (FAK, e.g., FAK2), fibroblast growth factors (FGFs, e.g., FGF1, FGF2, FGF4), FGF-2 ligand, FGF-5 ligand, fibronectin, Fms-related tyrosine kinase 3 (Flt3), farnesoid x receptor (FXR), folate, folate transporter 1, folate receptors (e.g., α), folate hydrolase, prostate-specific membrane antigen 1 (F OLH1), paired basic amino acid cleaving enzyme (FURIN), FYN tyrosine kinase, galactosyltransferase, galectin-3, glucocorticoid-induced TNFR-related protein GITR receptor, glucocorticoid, β-glucuronidase, glutamate carboxypeptidase II, glutaminase, glutathione S-transferase P, glypican 3 (GPC3), glycogen synthase kinase (GSK, e.g., 3-β), 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 (H19) gene, heat-stable enterotoxin receptor, heparanase, hepatocyte growth factor, heat shock protein genes, heat shock proteins (e.g. 27, 70, 90α, β), Hedgehog protein, HERV-H LTR-associated protein 2, hexose kinase, tyrosine-protein kinase HCK, histamine H2 receptor, histone deacetylase (HDAC, e.g. 1, 2, 3, 6, 10, 11), histone H1, histone H3, histone methyltransferase (DOT1L), human leukocyte antigen (HLA), HLA class I antigen (A-2α), HLA class II antigen, homeobox protein NANOG,Mitogen-activated protein kinase kinase 1 (MAP4K1, HPK1), HSPB1 gene, human papillomavirus (e.g. E6, E7) proteins, hyaluronidase, hyaluronic acid, hypoxia-inducible factor-1α, intracellular adhesion molecule 1 (ICAM-1), immunoglobulins (e.g. G, G1, G2, K, M), indoleamine 2,3-dioxygenase (IDO, e.g. IDO1), indoleamine pyrrole 2,3-dioxygenase 1 inhibitor, I-κ-B kinase (IKK, e.g. IKK.β..ε.), immunoglobulin Fc receptor, immunoglobulin gamma Fc receptor (e.g. I, III, IIIA), interleukin 1 ligand, interleukin 2 ligand, interleukin-2, IL-2 gene, IL-1α, IL-1β, IL-2, IL-2 receptor α subunit, IL-3 receptor, IL-4, IL-6, IL-7, IL-8, IL-12, IL-15, IL-12 gene, IL-17, interleukin-13 receptor α2, interleukin-29 ligand, interleukin-1 receptor-associated kinase 4 (IRAK4), insulin-like growth factors (e.g., 1, 2), insulin receptor, integrin α-V / β-3, integrin α-V / β-5, integrin α-V / β-6, integrin α-5 / β-1, integrin α-4 / β-1, integrin α-4 / β-7, interferon-inducible protein (IL-1) in melanoma (absent in melanoma) 2 (AIM2), interferons (e.g., alpha, alpha2, beta, gamma), interferon type I receptor, isocitrate dehydrogenase (e.g., IDH1, IDH2), Janus kinases (JAKs, e.g., 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), lysosome-associated membrane protein family (LAMP) genes,Lanosterol-14 demethylase, LDL receptor-related protein-1, leukotriene A4 hydrolase, listeorisin, L-selectin, luteinizing hormone receptor, lyase, lymphocyte antigen 75, lysine demethylase (e.g., 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, e.g., LOXL2), lysyl oxidase homolog 2, macrophage migration inhibitory factor (fact), melanoma antigen family A3 (MAGEA3) gene, MAGEC1 gene, MAGEC2 gene, major vault (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 metalloproteinase (MMP, e.g., MMP2, MMP9), myeloid cell leukemia 1 (MCL1) gene, Mcl-1 differentiation protein, macrophage colony-stimulating factor (MCSF) ligand, melanoma-associated antigen (e.g., 1, 2, 3, 6), melanocyte-stimulating hormone ligand, melanocyte protein Pmel 17, membrane copper amine oxidase, mesothelin, metabolite-derived glutamate receptor 1, mitogen-activated protein kinase (MEK, e.g., MEK1, MEK2), hepatocyte growth factor receptor (MET) gene, MET tyrosine kinase, methionine aminopeptidase-2, mitogen-activated protein kinase (MAPK), Mdm2 p53 binding proteins, Mdm4 protein, metalloreductase STEAP1 (six transmembrane epithelial antigen of the prostate 1), metastin, methyltransferase, mitochondrial 3-ketoacyl-CoA thiolase, MAPK-activated protein kinase (e.g. MK2), mTOR (mechanoactive target of rapamycin (serine / threonine kinase), mTOR complex (e.g. 1, 2), mucins (e.g. 1, 5A, 16), mut T homolog (MTH, e.g. 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) κB, NF κB activating protein, neurokinin 1 (NK1) receptor, NK cell receptor, NK3 receptor, NKG2 AB-activated NK receptor, NIMA-related kinase 9 (NEK9), norepinephrine transporter, Not, ch (e.g., 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 NR4A1, opioid receptors (e.g., δ), osteocalcin, osteoclast differentiation factor, osteopontin, OX-40 (tumor necrosis factor receptor superfamily member 4 TNFRSF4 or CD134) 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, e.g., PIM-1, PIM-2, PIM-3), poly ADP-ribose polymerase (PARP, e.g., PARP1, 2, and 3), p38 kinase, p38 MAP kinase, platelet-derived growth factor (PDGF, e.g., α, β), P-glycoprotein (e.g., 1), platelet-derived growth factor (PDGF, e.g., α, β), PKN3 gene, P-selectin, phosphatidylinositol 3-kinase (PI3K), phosphoinositide-3 kinase (PI3K, e.g., α, δ, γ), phosphorylase kinase (PK), placental growth factor, pleiotropic drug resistance transporter, Plexin B1, Polo-like kinase 1, peroxisome proliferator-activated receptor (PPAR, e.g., α, δ, γ), black Genes for preferentially expressed antigen in tumors (PRAME), likely transcription factor PML, programmed cell death ligand 1 inhibitor (PD-L1), progesterone receptor, prostate-specific antigen, prostatic acid phosphatase, prostanoid receptor (EP4), proteasome, protein farnesyltransferase, protein kinases (PK, e.g., 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 (e.g. 1, B), RAF1 gene, Ras GTPase, Ras gene, 5-alpha-reductase, RET gene, Ret tyrosine kinase receptor, retinoblastoma-associated protein, retinoic acid receptor (e.g. gamma), retinoid X receptor, Rheb (Ras homolog enriched in brain) GTPase, Rho (Ras homolog)-related protein kinase 2, ribonuclease, ribonucleotide reductase (e.g. M2 subunit), ribosomal protein S6 kinase, RNA polymerase (e.g. I, II), Ron (Recepteur d'Origine Nantais) tyrosine kinase, ROS1 (ROS proto-oncogene 1, receptor tyrosine kinase) gene, Ros1 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 lymphocyte 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, e.g., STAT-1, STAT-3, STAT-5), second mitochondrial-derived activator of caspases (SMAC) protein, smoothened (SMO) receptor, sodium phosphate cotransporter 2B, sodium iodide cotransporter, somatostatin receptors (e.g., 1, 2, 3, 4, 5), Sonic hedgehog protein, specific protein 1 (Sp1) transcription factor, sphingomyelin synthase, sphingosine-1-phosphate receptor-1, sphingosine kinase (e.g. 1, 2), SRC gene, STAT3 gene, gene for six-transmembrane epithelial antigen of the prostate (STEAP), 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 α, serine / threonine-protein kinase (TBK, e.g., TBK1), TATA box-binding protein-associated factor RNA polymerase I subunit B (TAF1B) gene, T cell surface glycoprotein CD8, T cell glycoprotein zeta chain, T cell differentiation antigen CD3 6, T cell surface glycoprotein CD28, Tec protein tyrosine kinase, Tek tyrosine kinase receptor, telomerase, tenascin, telomerase reverse transcriptase (TERT) gene, transforming growth factor (TGF, e.g., β) kinase, TGFβ2 ligand, T cell immunoglobulin and mucin-domain containing-3 (TIM-3), tissue factor, tumor necrosis factor (TNF, e.g., α, β), TNF-related apoptosis-inducing ligand, TNFR1-related death domain protein, TNFSF9 gene, TNFSF11 gene, trophoblast glycoprotein (TP BG) gene, transferrin, tropomyosin receptor kinase (Trk) receptors (e.g., TrkA, TrkB, TrkC), trophoblast glycoprotein, thymidylate synthase, tyrosine kinase (TIE) receptors with immunoglobulin-like and EGF-like domains, Toll-like receptors (TLRs, e.g., 1-13), topoisomerases (e.g., I, II, III), tumor protein 53 (TP53) gene, transcription factors, transferases, transforming growth factor TGF-β receptor kinase, transglutaminase, translocation-associated protein protein, transmembrane glycoprotein NMB, tumor necrosis factor 13C receptor, thymidine kinase, thymidine phosphorylase, thymidylate synthase, thymosin (e.g. α1), thyroid hormone receptor, Trop-2 calcium signal transducer, thyrotropin 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.

[0099] Anticancer agents include agents defined by their mechanism of action or class: antimetabolites / anticancer agents, such as pyrimidine analogs floxuridine, capecitabine, cytarabine, CPX-351 (liposomal cytarabine, daunorubicin), TAS-118; purine analogs, folate antagonists (e.g., pralatrexate) and related inhibitors; antiproliferative / antimitotic agents, such as natural products such as the vinca alkaloids (vinblastine, vincristine) and tacrolimus inhibitors. Microtubule inhibitors such as cyclosporine (paclitaxel, docetaxel), vinblastine, 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, ifosf iphosphamide, melphalan, mechlorethamine, mitomycin C, mitoxantrone, nitrosoureas, procarbazine, taxol, taxotere, teniposide, etoposide and triethylenethiophosphoramide; DNA hypomethylating agents such as guadecitabine (SGI-110) antibiotics such as dactinomycin, daunorubicin, doxorubicin, idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin cin (mithramycin) and; enzymes such as L-asparaginase that metabolize L-asparagine systemically to deplete cells that do not have the ability to synthesize their own asparagine; antiplatelet agents; DNAi oligonucleotides that target 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) inhibitor,antiproliferative / antimitotic alkylating agents such as nitrogen mustard cyclophosphamide and analogs (melphalan, chlorambucil, hexamethylmelamine and thiotepa), alkylnitrosoureas (carmustine) and analogs, streptozocin and triazenes (dacarbazine); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, oxiloplatinim and carboplatin), procarbazine hormones, hormone analogues (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. Dogrel; anti-migratory agents; anti-secretory agents (breveldin); immunosuppressants tacrolimus, sirolimus, azathioprine and mycophenolic acid; compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor inhibitors and fibroblast growth factor inhibitors, e.g. FPA14; angiotensin receptor blockers, nitric oxide donors; antisense oligonucleotides, e.g. AEG35156; DNA interference oligonucleotides, e.g. PNT2258, AZD-9150; antibodies, e.g. 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; anti-CD20 antibodies, such as obinutuzumab; anti-programmed cell death protein 1 (anti-PD-1) antibodies, 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 death-ligand 1 (anti-PD-L1) antibodies such as BMS-936559, atezolizumab (MPDL3280A), durvalumab (MEDI4736), avelumab (MSB0010718C) and MDX1105-01, CXCR4 antagonists such as BL-8040; CXCR2 antagonists such as AZD-5069; GM-CSF antibodies such as lenzilumab. Selective estrogen receptor downregulators (SERDs), such as Fulvestrant (Faslodex); Transforming growth factor-β (TGF-β) kinase antagonists, such as Galunisertib; Bispecific antibodies, such as MM-141 (IGF-1 / ErbB3), MM-111 (Erb2 / Erb3), JNJ-64052781 (CD19 / CD3). Mutant selective EGFR inhibitors, such as PF-06747775, EGF816, ASP8273, ACEA-0010, BI-1482694. α-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,β-catenin inhibitors, such as CWP-291; CD73 antagonists, such as MEDI-9447; c-PIM inhibitors, such as PIM447; BRAF inhibitors, such as dabrafenib, vemurafenib; Sphingosine kinase-2 (SK2) inhibitors, such as Yeliva. (ABC294640); Cell cycle inhibitors, such as selumetinib. (MEK1 / 2), sapacitabine, AKT inhibitors, e.g. MK-2206, ipatasertib, afuresertib, anti-CTLA-4 (cytotoxic T-lymphocyte protein-4) inhibitors, e.g. tremelimumab, c-MET inhibitors, e.g. AMG-337, savolitinib, tivantinib (ARQ-197), capmatinib, tepotinib inhibitors of CSF1R / KIT and FLT3, e.g. PLX3397, kinase inhibitors, e.g. vandetanib; E-selectin antagonists, e.g. GMI-1271, differentiation inducers, e.g. tretinoin; epidermal growth factor receptor (EGFR) inhibitors. anti-inflammatory agents, 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 signaling kinase inhibitors; dysfunction inducers; nucleoside analogues, 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,For example, peptide vaccine TG-01 (RAS), bacterial vector vaccines such as CRS-207 / GVAX, autologous Gp96 vaccines, dendritic cell vaccines, Oncoquest-L vaccines, DPX-Survivac, ProstAtak, DCVAC, ADXS31-142, demcizumab (anti-DLL4, delta-like ligand 4, Notch pathway), napabucasin (BBI-608) smoothened (SMO) receptor inhibitors such as Odomzo®. (sonidegib, formerly LDE-225, LEQ506, vismodegib (GDC-0449), BMS-833923, glasdegib (PF-04449913), LY2940680 and itraconazole; interferon alpha ligand modulators, such as interferon alpha-2b, interferon alpha-2a biosimilar (Biogenomics), ropeginterferon alpha-2b (AOP-2014, P-1101, PEG IFNα-2b), Multiferon (Alfanative, Viragen), interferon alpha 1b, Roferon-A (Canferon, Ro-25-3036), interferon alpha-2a successor biologics (Biosidus) (Inmutag, Inter 2A), interferon alpha-2b successor biologics (Biosidus - Bioferon, Citopheron, Ganapar) (Beijing Kawin Technology-Kaferon) (AXXO-Interferon alpha-2b), alfaferone, pegylated interferon alpha-1b, pegylated interferon alpha-2b successor biologics (Amega), recombinant human interferon alpha-1b, recombinant human interferon alpha-2a, recombinant human interferon alpha-2b, veltuzumab-IFNα2b conjugate, dynavax (SD-101) and interferon alpha-n1 (Humoferon, SM-10500, Sumiferon); interferon gamma ligand modulators, e.g. interferon gamma (OH-6000, Ogamma 100); IL-6 receptor modulators,for example tocilizumab, siltuximab, AS-101 (CB-06-02, IVX-Q-101); telomerase modulators, for example tertomotide (GV-1001, HR-2802, Riavax) and imetelstat (GRN-163, JNJ-63935937); DNA methyltransferase inhibitors, for example temozolomide (CCRG-81045), decitabine, guadecitabine (S-110, SGI-110), KRX-0402 and azacitidine; DNA gyrase inhibitors, for example pixantrone and sobuzoxane; Bcl-2 family protein inhibitors 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, γ-secretase inhibitors, such as PF-03084014, IDO Inhibitors such as indoximod, Grb-2 (growth factor receptor-bound protein-2) inhibitor BP1001 (liposomal Grb-2), TRAIL pathway inducer 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 inhibitors 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 agonists, such as urelumab, an anti-KIR monoclonal antibody;For example, lirilumab (IPH-2102). Antigen CD19 inhibitors, for example, MOR208, MEDI-551, AFM-11, CD44 binders, for example, A6, CYP17 inhibitors, for example, VT-464, ASN-001, ODM-204. RXR agonists, for example, IRX4204, TLR (Toll-like receptor) agonists, for example, IMO-8400 A hedgehog / smoothened (hh / Smo) antagonists, for example, taladegib. Immunomodulators, for example, complement C3 regulators, for example, Imprime PGG. Intratumoral immuno-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 plasmids, 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-(1-(but-2-ynoyl)pyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-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, selinexol, 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:These include, but are not limited to, abexinostat, ACY-241, AR-42, BEBT-908, belinostat, CKD-581, CS-055 (HBI-8000), CUDC-907, entinostat, givinostat, mocetinostat, panobinostat, pracinostat, xinostat (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 (SB1518), 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 Biologics). Matrix metalloproteinase (MMP) inhibitors: MMP inhibitors include inhibitors of MMP1-10. 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 Biologics). 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 of PI3K 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, OXY111A, PA799, PX-866, RG7604, rigosertib, RP5090, taselisib, TG100115, TGR-1202, TGX221, WX-037, X-339, X-414, XL147 (SAR245408), XL499, XL756, wortmannin, ZSTK474, and WO 2005 / 113556 (ICOS), WO 2013 / 052699 (Gilead Calistoga), WO 2013 / 116562 (Gilead Calistoga), WO 2014 / 100765 (Gilead Calistoga),These include compounds described in 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-(1H-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-alpyrazine-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 US 2015 / 0175616. Tyrosine-kinase inhibitors (TKIs): TKIs can target the epidermal growth factor receptor (EGFR) as well as the 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, brigutinib, 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); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodepa, carboquone, meturedepa, and uredepa; ethyleneimine and methylamelamine.such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; acetogenins, particularly bullatacin and bullatacinone; camptothecins, such as the synthetic analog topotecan; bryostatin, kallistatin; CC-1065, such as its adozelesin, carzelesin and bizelesin synthetic analogs; cryptophytes. cryptophycins, especially cryptophycin 1 and cryptophycin 8; dolastatins; duocarmycins, such as the synthetic analogues KW-2189 and CBI-TMI; eleutherobin; 5-azacytidine; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chromafazine, aphazine, 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; anti- Biological substances such as enediyne antibiotics (e.g., the calicheamicins, especially calicheamicin gamma II and calicheamicin phi I1), dynemicins, e.g., dynemicin A, bisphosphonates, e.g., clodronate, esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromomophores, aclacinomycins, actinomycin, autramycin, azaserine, bleomycin,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (e.g. morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin) ), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, s streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as demopterin, methotrexate, pteropterin, and trimetrexate; purine analogues pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane and testolactone; anti-adrenals such as aminoglutethimide,mitotane and trilostane; folic acid replinishers, such as folinic 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 analogues, such as cisplatin and carboplatin, NC-6004 nanoplatin; aceglatone; aldophosphamide glycosides glycoside);aminolevulinic acid;eniluracil;amsacrine;hestrabucil;bisantrene;edatrexate;defofamine;demecolcine;diaziquone;elformthine;elliptinium acetate acetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; leucovorin; lonidamine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; fluoropyrimidines; folinic acid; podophyllinic acid; 2-ethylhydrazide; procarbazine; polysaccharide-K (PSK); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazo, tenuazonic acid; trabectedin, triaziquone; 2,2',2''-trichlorotrimethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vancristine; vinorelbine (NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS2000; difluoromethylornithine (DFMO); retinoids, such as retinoic acid; capecitabine; FOLFIRI (fluorouracil, leucovorin and irinotecan); and pharma- ceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0100] Anti-hormonal agents, such as antiestrogens and selective estrogen receptor modulators (SERMs), inhibitors of the enzyme aromatase, anti-androgens and pharma- ceutically acceptable salts, acids or derivatives of any of the above that act to regulate or inhibit hormone action on tumors, are also included in the definition of anti-cancer agents. Examples of antiestrogens and SERMs include, for example, tamoxifen (e.g., NOLVADEX), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (FARESTON). Inhibitors of the enzyme aromatase regulate estrogen production in the adrenal gland. Examples include 4(5)-imidazole, aminoglutethimide, megestrol acetate (MEGACE), exemestane, formestane, fadrozole, vorozole (RIVISOR), letrozole (FEMARA), and anastrozole (ARIMIDEX). Examples of antiandrogens include apalutamide, abiraterone, enzalutamide, flutamide, galeterone, nilutamide, bicalutamide, leuprolide, goserelin, ODM-201, APC-100, ODM-204. Examples of progesterone receptor antagonists include onapristone.

[0101] Antiangiogenic agents include, but are not limited to, retinoic acid and its derivatives, 2-methoxyestradiol, angiostatin, endostatin, regorafenib, necuparanib, suramin, squalamine, tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase-2, plasminogen activator inhibitor-1, plasminogen activator inhibitor (inbibitor)-2, cartilage-derived inhibitor, paclitaxel (nab-paclitaxel), platelet factor 4, protamine sulfate (clupeine), sulfated chitin derivatives (queen crab shell), shell), sulfated polysaccharide peptidoglycan complex (sp-pg), staurosporine, regulators of matrix metabolism, such as proline analogues, e.g. 1-azetidine-2-carboxylic acid (LACA), cis-hydroxyproline, d,I-3,4-dehydroproline, thiaproline, α,α'-dipyridyl, β-aminopropionitrile fumarate, 4-propyl-5-(4-pyridinyl)-2(3h)-oxazolone, methotrexate, mitoxantrone, heparin, interferon, 2-macroglobulin-2-blood , chicken inhibitor of metalloproteinase-3 (ChIMP-3), chymostatin, β-cyclodextrin tetradecasulfate, eponemycin, fumagillin, sodium gold thiomalate, d-penicillamine, β-1-anticollagenase-serum, α-2-antiplasmin, bisantrene, lobenzarit disodium, disodium n-2-carboxyphenyl-4-chloroanthronilic acid or "CCA", thalidomide, angiostatic steroids, carboxyaminoimidazole, metalloproteinase inhibitors such as BB-94, inhibitors of S100A9 such as tasquinimod.Other anti-angiogenic agents include antibodies, preferably monoclonal antibodies against these angiogenic growth factors: β-FGF, α-FGF, FGF-5, VEGF isoforms, VEGF-C, HGF / SF and Ang-1 / Ang-2.

[0102] Antifibrotic agents include, but are not limited to, compounds such as β-aminoproprionitrile (BAPN) and those compounds disclosed in U.S. Pat. No. 4,965,288 relating to inhibitors of lysyl oxidase and their use in treating diseases and conditions associated with abnormal deposition of collagen, and U.S. Pat. No. 4,997,854 relating to compounds that inhibit LOX for the treatment of various pathological fibrotic conditions, which are incorporated herein by reference. Further exemplary inhibitors are described in U.S. Pat. No. 4,943,593 for compounds such as 2-isobutyl-3-fluoro-, chloro- or bromo-allylamine, U.S. Pat. No. 5,021,456 for 2-(1-naphthyloxymemyl)-3-fluoroallylamine, 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 and U.S. Pat. No. 2004-0248871, which are incorporated herein by reference. Exemplary antifibrotic agents also include primary amines that react with the carbonyl group of the active site of lysyl oxidase and more specifically those that produce resonance-stabilized products after binding with carbonyl, 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-halobenzylamine; and selenohomocysteine ​​lactone. Other antifibrotic agents are copper chelators that may or may not permeate cells. Exemplary compounds include indirect inhibitors that block the aldehyde derivatives resulting from the oxidative deamination of lysyl and hydroxylysyl residues by lysyl oxidase.Examples include thiolamine, in particular D-penicillamine and its analogues, 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 sulfate, and the like. sulphurate, 2-acetamidoethyl-2-acetamidoethanethiol sulfanate, and sodium-4-mercaptobutanesulfinate trihydrate.

[0103] The API can be an immunotherapeutic agent. Immunotherapeutic agents include, but 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, and the like. ntuzumab, catumaxomab, cetuximab, sitatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drotuzumab, durigotumab, dusigitumab, detumomab, dacetuzumab, darotuzumab, dinutuximab, ecromeximab , elotuzumab, emibetuzumab, ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab mab), 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, ocaratu- tumab, ofatumumab, olaratu- tumab, onartuzumab, oportuzumab, oregovomab, panitumumab, pal Parsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, ramucirumab (CYRAMZA®), rilotumumab, rituximab, robatumumab, samalizumab, satumomab, sibrotuzumab, siltuximab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumomab, tigatuzumab, These include tigatuzumab, tositumomab, trastuzumab, ABP-980, tucotuzumab, ubilituximab, veltuzumab, borsetuzumab, votumumab, zalutumumab, CC49, OBI-833 and 3F8. Rituximab can be used to treat indolent B-cell cancers such as marginal zone lymphoma, WM, CLL and small lymphocytic lymphoma. The combination of rituximab with chemotherapy agents is particularly effective.

[0104] Exemplary therapeutic antibodies may further be labeled with or combined with radioisotope particles, such as indium-111, yttrium-90 (90Y-clivatuzumab) or iodine-131.

[0105] The composition may contain a targeting moiety, such as a peptide or protein ligand or domain, covalently bound to the surface of the microparticle or nanoparticle, which specifically or preferentially binds to a target site (e.g., a cell surface receptor or binding partner of the targeting moiety), so that the microparticle or nanoparticle having such a targeting moiety is specifically or preferentially directed to the target site in vivo.The targeting moiety having a microparticle or nanoparticle may further contain an API that can be encapsulated or embedded in the microparticle or nanoparticle and released or otherwise available at the target site.In fact, sialic acid itself may be a targeting moiety for cancer cells.

[0106] By having a targeting moiety, the target-specific nanoparticle can effectively bind or otherwise associate with a biological entity, such as a membrane component or a cell surface receptor. Targeting of therapeutic agents (e.g., to a specific tissue or cell type, to a specific diseased tissue rather than normal tissue, etc.) is desirable for treating tissue-specific diseases such as cancer (e.g., prostate cancer). In contrast to the systemic delivery of, for example, cytotoxic anticancer agents, targeted delivery could prevent the agent from killing healthy cells.

[0107] Furthermore, targeted delivery allows for the administration of low doses of drugs, which could reduce the undesirable side effects usually associated with conventional chemotherapy. As mentioned above, the targeting specificity of the nanoparticles of the present invention is maximized by optimizing the ligand density on the nanoparticles. The targeting moiety can be covalently attached to the surface of the nanoparticle or microparticle. For example, the targeting moiety can be covalently attached to anionic polymers (e.g., by coupling one or more carboxylic acid or other functional moieties), PLGA / PLA (e.g., via the polymer termini) or by integrating further molecules or polymers into the interpenetrating network. For example, the targeting moiety can be covalently linked to polyethylene glycol (PEG) molecules or PLGA-PEG diblocks and added to the emulsion with the anionic polymer.

[0108] For example, the targeting moiety may be a moiety that can bind or otherwise associate with a biological entity, such as a membrane component, a cell surface receptor, a prostate specific membrane antigen, etc. In the present case, the targeting moiety is a low molecular weight PSMA ligand. The term "bind" or "binding" as used herein typically refers to the interaction between a corresponding pair of molecules or parts thereof that exhibit mutual affinity or binding capacity, due to specific or non-specific binding or interactions, including but not limited to biochemical, physiological and / or chemical interactions.

[0109] "Biological binding" defines a type of interaction that occurs between pairs of molecules, such as proteins, nucleic acids, glycoproteins, carbohydrates, hormones, etc.

[0110] The term "binding partner" refers to a molecule that can undergo binding with a specific molecule. "Specific binding" refers to a molecule, such as a polynucleotide, that can bind to or recognize a binding partner (or a limited number of binding partners) to a substantially greater extent than to other similar biological entities. In one set of embodiments, the targeting moiety has an affinity (as measured by dissociation constant) of less than about 1 micromolar, at least about 10 micromolar, or at least about 100 micromolar.

[0111] In a preferred embodiment, the targeting moiety of the present invention is a small molecule.In some embodiments, the term "small molecule" refers to an organic compound that has a relatively low molecular weight and is not a protein, polypeptide or nucleic acid, whether naturally occurring or artificially created (e.g., by chemical synthesis).Small molecules typically have multiple carbon-carbon bonds.In some 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.

[0112] In a particularly preferred embodiment, 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 the expression in normal tissue, the expression of prostate specific membrane antigen (PSMA) is at least 10 times overexpressed in malignant prostate compared with normal tissue, and the level of PSMA expression is further upregulated as disease progresses to metastatic stage, as described in US Patent Publication 2014 / 0235706 (Silver et al. 1997, Clin. Cancer Res., 3:81).

[0113] 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 their analogs and derivatives. In some embodiments, small molecule targeting moieties that can be used to target cells associated with prostate cancer tumors include thiol and indole thiol derivatives, such as 2-MPPA and 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid derivatives (Majer et al., 2003, J. Med. Chem., 46:1989; and US Patent Publication 2005 / 0080128). In some embodiments, small molecule targeting moieties that can 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 can be used to target cells associated with prostate cancer tumors include PBDA- and urea-based inhibitors, such as ZJ 43, ZJ 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 their analogs and derivatives. In some embodiments, small molecule targeting moieties that can be used to target cells associated with prostate cancer tumors include putrescine, spermine and spermidine, androgen receptor targeting agents (ARTA), such as those described in U.S. Patent Nos. 7,026,500; 7,022,870; 6,998,500; 6,995,284; 6,838,484; 6,569,896; 6,492,554; and U.S. Patent Publication Nos. 2006 / 0287547; 2006 / 0276540; 2006 / 0258628; 2006 / 0241180; 2006 / 0183931; 2006 / 0035966; 2006 / 0009529; 006 / 0004042;2005 / 0033074;2004 / 0260108;2004 / 0260092;2004 / 0167103;2004 / 0147550;2004 / 0147489;2004 / 0087810;2004 / 0067979;2004 / 0052727;2004 / 0029913;2 004 / 0014975;2003 / 0232792;2003 / 0232013;2003 / 0225040;2003 / 0162761;2004 / 0087810;2003 / 0022868;2002 / 0173495;2002 / 0099096;2002 / 0099036. A related aspect of the present invention provides a pharmaceutical composition comprising the subject composition and a pharma- ceutically acceptable carrier or excipient. The pharmaceutical composition is described in more detail below in another section.

[0114] The present invention also provides vehicles for delivery of nucleic acids to facilitate cellular uptake and transfection in vitro and in vivo. Examples of nucleic acids include DNA, RNA, PNA, siRNA, microRNA, antisense, etc.

[0115] Preparation of particles The invention described herein provides several basic methods for the preparation of particles that display sialic acid residues on their surface.

[0116] The particles can be made from the co-precipitation or coacervation of hydrophobic and / or neutral biocompatible polymers, such as PLGA or PLA, and polysialic acid. Without being bound by any theory, it is believed that the polymer backbones become entangled or intertwined while in the organic phase of the emulsion.

[0117] As used herein, "small amount" refers to a relatively small amount / volume of the first solution of the second solvent compared to the volume of the first solvent with the PLGA polymer, and emulsification of the first solution of the second solvent in the polymer solution in the first solvent forms an emulsion (i.e., a first emulsion) in which the continuous phase is the polymer solution. Typically, the volume ratio between the small amount of the first solution of the second solvent and the first solvent is at least about 1:n, where n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100.

[0118] As used herein, "large" refers to a relatively large amount / volume of the second solution of the second solvent compared to the volume of the first emulsion, and emulsification of the first emulsion in the second solution of the second solvent forms an emulsion (i.e., a second emulsion) in which the continuous phase is the second solution of the second solvent. Typically, the volume ratio between the first emulsion and the large amount of the second solution of the second solvent is at least about 1:m, where m can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100.

[0119] Using the preparative methods described herein, polysialic acid is intimately incorporated into the microparticles or nanoparticles that are produced.

[0120] The incorporation of polysialic acid into the microparticles or nanoparticles can be stable and dense, therefore preferably the method further comprises the steps of washing the microparticles or nanoparticles and / or concentrating the microparticles or nanoparticles to a desired volume.

[0121] Emulsion process can be used to prepare the particles described herein.The present invention includes a method for preparing microparticles or nanoparticles that present sialic acid moieties on their surface, which includes: (1) dissolving a biodegradable polymer (and optionally an active agent, such as an active pharmaceutical ingredient (API) or a poorly water-soluble compound) in a first solvent to form a polymer solution; (2) emulsifying the polymer solution in a second solvent solution to form an emulsion, where the first solvent is immiscible or partially miscible with the second solvent, the second solvent solution comprises polysialic acid, and the second solvent solution optionally further comprises a surfactant and / or API that are soluble in the second solvent; and (3) removing the first solvent to form the microparticles or nanoparticles with surface sialic acid moieties.

[0122] The present invention also provides a double emulsion method for the preparation of microparticles or nanoparticles having surface sialic acid moieties, the method comprising: (1) dissolving a biodegradable polymer (and optionally an active agent, API or poorly water soluble compound) in a first solvent to form a polymer solution; (2) adding a second solvent to the polymer solution to form a mixture, where the first solvent is immiscible or partially miscible with the second solvent, and the first solution of the second solvent optionally contains an active agent, which may be the same as or different from the API dissolved in the first solvent; (3) emulsifying the mixture to form a first emulsion; (4) emulsifying the first emulsion in a second solution of the second solvent to form a second emulsion, where the second solution of the second solvent contains polysialic acid and optionally further contains a surfactant; and (5) removing the first solvent to form microparticles or nanoparticles having surface sialic acid moieties.

[0123] The API in the present invention can be a nucleic acid therapeutic agent. Exemplary nucleic acid therapeutic agents include, but are not limited to, those approved by the FDA and subject to new drug applications by the FDA in clinical trials or preclinical studies. Nucleic acid molecules include molecules encoding therapeutic proteins, antigenic molecules and inhibitory molecules. For example, nucleic acid molecules that can mediate RNA interference include molecules active in RNA interference (RNAi molecules), such as double-stranded RNA, such as antisense, ribozymes, siRNAs (small interfering RNAs), miRNAs (microRNAs), shRNAs (short hairpin RNAs), shortmers, antagomirs, mRNAs, tRNAs, ddRNAs (DNA-guided RNAs), piRNAs (Piwi-interacting RNAs) or rasiRNAs (repeat-binding siRNAs) and modified forms thereof. The molecules can also include DNA, plasmids, vectors, hybrid oligonucleotides, catalytic DNAs or aptamers.

[0124] Coding nucleic acids and polynucleotides may include a region that encodes a polypeptide of interest (e.g., a coding region), a 5' end of a first region (e.g., a 5'-UTR), a 3' end of a first region (e.g., a 3'-UTR), at least one 5'-cap region and / or a 3'-stabilizing region. Nucleic acids may include a polyA region or a Kozak sequence (e.g., in the 5'-UTR), one or more intron nucleotide sequences that may be excised from the polynucleotide, a 5'-cap structure, a chain terminating nucleotide, a stem loop, a polyA sequence and / or a polyadenylation signal. Nucleic acids may include one or more alternative components (e.g., alternative nucleosides). For example, the 3'-stabilization region can include alternative nucleosides such as L-nucleosides, inverted thymidines or 2'-O-methyl nucleosides and / or the coding region, 5'-UTR, 3'-UTR, or the cap region can include alternative nucleosides such as 5-substituted uridines (e.g., 5-methoxyuridine), 1-substituted pseudouridines (e.g., 1-methyl-pseudouridine or 1-ethyl-pseudouridine), 5-substituted cytidines (e.g., 5-methyl-cytidine), and / or carnosine and anserine.

[0125] The length of the polynucleotide sequence is sufficient to encode a dipeptide or more, such as a tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In some cases, the polynucleotide is more than 30 nucleotides in length, such as more than 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, or more.

[0126] Nucleic acids and polynucleotides may contain one or more naturally occurring components, such as any of the canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine) or T (thymidine). In one embodiment, all or substantially all of the nucleotides comprising (a) the 5'-UTR, (b) the open reading frame (ORF), (c) the 3'-UTR, (d) the polyA tail and any combination of (a, b, c or d above) contain the naturally occurring canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine) or T (thymidine).

[0127] The nucleic acids and polynucleotides may contain one or more components to increase stability, reduce substantial induction of an innate immune response in a cell into which the polynucleotide is introduced, increase efficiency of protein production, intracellular retention of the polynucleotide, viability of contacted cells, and / or reduce immunogenicity.

[0128] Polynucleotides and nucleic acids may be naturally occurring or non-naturally occurring. Polynucleotides and nucleic acids may include one or more modified (e.g., altered or alternative) nucleobases, nucleosides, nucleotides, or combinations thereof. Nucleic acids and polynucleotides useful in nanoparticle compositions may include any useful modification or change, for example, to the nucleobase, sugar, or internucleoside linkage (e.g., to the phosphate linkage / to the phosphodiester linkage / to the phosphodiester backbone). The change (e.g., one or more changes) is present in each of the nucleobase, sugar, and internucleoside linkage. The change according to the present disclosure may be a change of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), for example, replacing the 2'-OH of the ribofuranosyl ring with 2'-H, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof.

[0129] Polynucleotides and nucleic acids can be uniformly or non-uniformly altered along the entire length of the molecule.For example, one or more or all types of nucleotides (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) can be uniformly or non-uniformly altered in polynucleotides or nucleic acids or in their predetermined sequence regions.In some examples, all nucleotides X in polynucleotides (or in their predetermined sequence regions) are altered, where X can be any one of nucleotides A, G, U, C, or combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0130] Different sugar modifications and / or internucleoside linkages (e.g., backbone structures) can be present at various positions in a polynucleotide. Those skilled in the art will appreciate that nucleotide analogs or other modifications can be placed at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially reduced. Modifications can also be at the 5'- or 3'-terminus. In some embodiments, the polynucleotide comprises a modification at the 3'-terminus.

[0131] In some instances, the nucleic acid does not substantially induce an innate immune response in a cell into which the polynucleotide (e.g., mRNA) has been introduced. Characteristics of an induced innate immune response include: 1) increased expression of proinflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc.), and / or 3) termination or reduction of protein translation.

[0132] The nucleic acid may optionally contain other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNAs, tRNAs, RNAs that induce triple helix formation, aptamers, vectors). In some embodiments, the nucleic acid may contain one or more messenger RNAs (mRNAs) with one or more alternative nucleosides or nucleotides (i.e., alternative mRNA molecules).

[0133] In some embodiments, cationic complexing agents may be used to complex the nucleic acid cargo prior to encapsulation.

[0134] The cationic complexing agent in the present invention may be a nitrogen-, sulfur- or phosphorus-containing molecule, such as a small molecule compound, lipid, polymer or dendrimer. The molecule is preferably amphiphilic, having one or more cationic moieties and one or more hydrophobic moieties. The cationic moiety may be a nitrogen-, sulfur- or phosphorus-containing group. Preferred cationic moieties include primary or secondary amines, ammonium or phosphonium. The hydrophobic moiety may be an organic or inorganic group. Preferred hydrophobic groups include substituted or unsubstituted, saturated or unsaturated higher alkyl, acyls or esters (C3-C20 or higher). The hydrophobic group may be linear, branched or cyclized (e.g., aryl groups or cholesterol and their analogs).

[0135] The small molecule complexing agent used in the present invention is typically a nitrogen-, sulfur- or phosphorus-containing compound or its salt.Non-limiting examples of small molecule complexing agents include ethyl lauroyl arginate HCl (LAE), tripropylamine, tributylamine, triphenylamine, hexadecylamine, hexylamine, didodecyldimethylammonium bromide, dodecyltrimethylammonium bromide (DTAB), cetrimonium bromide (CTAB), benzathine, dimethyldioctadecylammonium bromide, benethamine, hydrabamine, stearalkonium chloride, DC-cholesterol·HCl, cetylpyridinium chloride, 1,2-distearoyl-3-dimethylammonium-propane, DODMA, lipofectin, etc.

[0136] The lipids that may be used in the present invention as complexing agents may be cationic lipids or ionizable lipids.

[0137] Cationic lipids are amphipathic molecules with a cationic head group and a hydrophobic tail group linked by either a stable or degradable bond. Guanidine, imidazole, pyridinium, piperidine and amino acids (e.g., lysine, arginine, ornithine and tryptophan) are common head groups used in lipid modification. Lipids that may be used as complexing agents in the present invention include, but are not limited to, monovalent aliphatic lipids with one amine functional group in their head group, such as N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy-1-propanaminium bromide) (DMRIE), polyvalent aliphatic lipids with several amine functional groups in the head group, such as spermine groups, such as dioctadecylamidoglycylspermine (DO GS) or cationic cholesterol derivatives such as 3b-[N-(N0,N0-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), bis-guanidium-tren-cholesterol (BGTC) and neutral helper lipids such as 1,2-dioleyl-sn-glycerol-3-phosphoethanolamine (DOPE) or cholesterol added to complexes of DNA and RNA and cationic lipids to improve transfection efficiency.

[0138] Ionizable lipid is a class of lipid molecules that are neutral and non-ionic at physiological pH, but are protonated and positively charged at lower pH.Ionizable lipid can also form complexes with SA-containing entities, promoting endosomal escape and reducing toxicity.Commercially available examples of ionizable lipid include DLin-KC2-DMA, DLin-MC3-DMA, DLin-DMA, DODMA and DODAP.

[0139] Other chemical entities commonly used in lipid nanoparticle (LNP) formulations, such as structured lipids, PEGylated lipids, cholesterol, phospholipids, etc., may be added to the nanoparticle formulations of the invention to increase the stability, functionality and other performance characteristics of the RNA-lipid complexes.

[0140] The polymeric complexing agent can be a cationic polymer comprising one or more cationic monomers, and can include polylysine, cell penetrating peptides (eg, polyarginine), polyethyleneimine, chitosan, and poly(amino esters).

[0141] Polylysine is a cationic homopolypeptide and can be α-polylysine or ε-polylysine. Polylysine contains positively charged amino groups at neutral pH. α-polylysine is a synthetic polymer and can be in the form of poly-L-lysine (PLL) and poly-D-lysine (PDL), respectively. ε-polylysine (ε-poly-L-lysine, EPL) is typically made as a homopolypeptide of about 25-30 L-lysine residues. Polylysine used in the present invention can be a copolymer of lysine and other chemical entities. Polylysine can also be modified to have specific properties. For example, modified polylysine can be made more hydrophobic, for example by alkylating or acylating the amine groups on one or more lysines.

[0142] Cell-penetrating peptides (CPPs) have the ability to translocate the plasma membrane and facilitate the delivery of various molecular cargoes into the cytoplasm or organelles. Some cell-penetrating peptides, such as polyarginine, are cationic and suitable as complexing agents for nucleic acids.

[0143] Polyethylenimine (PEI) is a polymer with repeat units composed of one amine group and a 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 temperature. Branched PEIs can be liquid at room temperature. In contrast to branched PEIs, which contain primary, secondary, and tertiary amino groups, linear PEIs contain primarily secondary amines.

[0144] Chitosan is a linear polysaccharide composed of randomly distributed β-(1→4) linked D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine. The amino groups in chitosan have a pKa value of about 6.5, which causes significant protonation in neutral solution and positive charge. Therefore, chitosan can be used to form complexes with nucleic acids via ionic interactions.

[0145] Preferred poly(amino esters) are biodegradable, biocompatible polymers. One example of a poly(amino ester) is poly[α-(4-aminobutyl)-l-glycolic acid].

[0146] Poly(β-amino esters) (PBAEs) are a class of polymers derived from di-acrylates and functional amines, including primary and secondary amines, preferably formed via Michael addition reactions. PBAEs are pH-sensitive, biodegradable and biocompatible. The pH buffering ability of PBAEs, resulting from the presence of tertiary amines in the PBAE structure, facilitates endosomal escape and thereby intracellular delivery of therapeutic agents.

[0147] Cationic complexing agents can also be modified to impart other desired properties. For example, cationic complexing agents, such as PBAE, can be PEGylated to increase in vivo circulation time. Cationic complexing agents, particularly polymers, can be optimized for molecular weight, degradation profile, in vivo half-life, pH responsiveness, and other properties that may be desirable for a particular application.

[0148] Preferably, in the emulsification process, the weight ratio of the PLGA solution to the aqueous solution is typically 1:1,000-10:1, preferably 1:100-1:1.

[0149] As used herein, miscibility is defined as the property of solutions to mix together in all proportions to form a homogeneous solution. Substances / liquids are said to be immiscible or not miscible if they do not form a solution in some proportions.

[0150] Exemplary solvents that are miscible with water include acetone, tetrahydrofuran (THF), acetonitrile, dimethylsulfoxide (DMSO), and dimethylformamide (DMF).

[0151] The double emulsion process can be particularly useful when an active agent such as a drug or active pharmaceutical ingredient (API), e.g., a protein-based therapeutic, prepared in an aqueous solution, is first emulsified with a pharma- ceutically acceptable polymer solution to form a first emulsion, and the API is encapsulated in the polymer solution. The polymer and the encapsulated therapeutic are then re-emulsified with a larger volume of solvent to form a second emulsion (e.g., a water-in-oil-in-water or w / o / w double emulsion), after which the microparticles or nanoparticles are formed.

[0152] For example, in the w / o / w technique described above, a relatively small amount of a first solution of a second solvent (e.g., an aqueous protein solution) (e.g., about 20%, 15%, 10%, 5% v / v of the organic solvent) can be introduced into a relatively large amount of a first solvent (e.g., an organic solvent), such as methylene chloride or ethyl acetate, which dissolves the hydrophobic polymer PLGA. A first emulsion is then formed using a suitable method, such as probe sonication or homogenization. After the formation of the first emulsion, a second emulsion is formed by introducing the first emulsion into a larger volume of a second solution of a second solvent (e.g., approximately at least about 2, 3, 4, 5, 6, 10 times the volume of the first emulsion) containing an emulsifier, such as polyvinyl alcohol. Again, a homogenization method can be used to form a second emulsion. This is then followed by a period of solvent evaporation, typically by stirring for several hours, which results in hardening of the polymer. As a result, the protein solution becomes trapped in the relatively hydrophobic matrix of the PLGA polymer forming small inclusions. Finally, the microparticles or nanoparticles formed are collected and washed (e.g., with distilled water) via repeated centrifugation or filtration, and then dehydrated, typically by lyophilization.

[0153] In any of the above aspects, the first solvent is preferably methylene chloride, ethyl acetate or chloroform. Preferably, the second solution of the second solvent contains a surfactant, including organic or inorganic pharmaceutical excipients; various polymers; oligomers; natural products; non-ionic, cationic, zwitterionic or ionic surfactants; and mixtures thereof. The surfactant may include polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polysorbate (Tween series) surfactants, PEO-PPO-PEO polyethylene oxide polypropylene oxide triblock copolymer (Pluronic series or Poloxamer series) surfactants or t-octylphenyl-polyethylene glycol (Triton X-100) surfactants or salts, derivatives, copolymers or mixtures thereof. Preferably, the surfactant is PVA (see examples).

[0154] Preferably, the emulsification step involves homogenization, mechanical agitation and / or microfluidization.

[0155] Preferably, the first solvent is removed by solvent exchange and / or evaporation.

[0156] The solvent used in the dissolving step for polymer can be any kind of solvent that dissolves polymer (such as PLGA).However, for its removal, preferably volatile solvent is used.For example, the preferred solvent for forming PLGA solution includes methylene chloride, ethyl acetate and chloroform.

[0157] In the emulsification process, the (aqueous) solution may contain surfactants or surface stabilizers. Surfactants generally include compounds that lower the surface tension of a liquid, the interfacial tension between two liquids or between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents and dispersants. Surfactants are usually organic compounds that are amphiphilic, which contain both a hydrophobic group (usually a branched, linear or aromatic hydrocarbon chain(s), fluorocarbon chain(s) or siloxane chain(s) as the "tail(s)") and a hydrophilic group (usually the head). Surfactants are most commonly classified according to their polar head group: nonionic surfactants have no charged group in their head; ionic surfactants carry a net charge - if the charge is negative, the surfactant is anionic, if the charge is positive, it is cationic. If a surfactant contains a head with two oppositely charged groups, it is called zwitterionic. Anionic or zwitterionic surfactants, such as those containing a carboxyl group ("carboxylate"), are preferably used in the present invention. Carboxylate is the most common surfactant and includes alkyl carboxylates such as sodium stearate, sodium lauroyl sarcosinate, and carboxylate-based fluorosurfactants such as perfluorononanoate, perfluorooctanoate (PFOA or PFO).

[0158] Without wishing to be bound by any particular theory, surfactants may be useful in forming and stabilizing emulsion droplets. Surfactants may also include organic or inorganic pharmaceutical excipients, various polymers, oligomers, natural products, non-ionic, cationic, zwitterionic or ionic surfactants and mixtures thereof.

[0159] Surfactants that may be used in the preparation of the subject (PLGA) microparticles or nanoparticles include polyvinyl alcohol, polyvinylpyrrolidone, the Tween series, the Pluronic series, the poloxamer series, Triton X-100, etc. Further suitable surfactants are provided herein below.

[0160] The emulsification process can be carried out by any art-recognized means such as homogenization, sonication, mechanical stirring, microfluidization, or a combination thereof.

[0161] Solvent removal is typically accomplished by, for example, solvent exchange and evaporation.

[0162] The present invention can use more than one surfactant combination.The useful surfactant or surface stabilizer that can be used in the present invention can include, but is not limited to, known organic and inorganic pharmaceutical excipients.Such excipients include various polymers, low molecular weight oligomers, natural products and surfactants.The surfactant or surface stabilizer can include nonionic, cationic, zwitterionic and ionic surfactants.

[0163] Representative examples of other useful surfactants or surface stabilizers include hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, sodium lauryl sulfate, sodium dioctyl sulfosuccinate, gelatin, casein, lecithin (phosphatides), dextran, gum arabic, 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., commercially available TWEENS®, such as TWEEN 20® and TWEEN 80® (ICI Specialty Chemicals)); polyethylene glycols (e.g., CARBOWAXS 3550® and 934® (Union Carbide), polyoxyethylene stearate, colloidal silicon dioxide, phosphates, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose phthalate, amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), 4-(1,1,3,3-tetramethylbutyl)-phenol polymers with ethylene oxide and formaldehyde (also known as tyloxapol, superione and triton), poloxamers (e.g. PLURONICS F68® and F108®, which are block copolymers of ethylene oxide and propylene oxide);Poloxamines (e.g., TETRONIC 908®, also known as POLOXAMINE 908®, which is a tetrafunctional block copolymer derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Wyandotte Corporation, Parsippany, NJ); TETRONIC 1508® (T-1508) (BASF Wyandotte Corporation); TRITONS X-200®, an alkylaryl polyether sulfonate (Rohm and Haas); CRODESTAS F-110®, a mixture of sucrose stearates and sucrose distearates (Croda Inc.); p-isononylphenoxy poly-(glycidol), also known as OLIN-1OG® or SURFACTANT 10-G® (Olin Chemicals, Stamford, Conn.); Crodestas SL-40 (Croda, Inc.); and SA9OHCO (Eastman Kodak Co.), which is C18H37CH2(CON(CH3)-CH2(CHOH)4(CH2OH)2; decanoyl-N-methylglucamide; n-decyl β-D-glucopyranoside; n-decyl β-D-maltopyranoside; n-dodecyl β-D-glucopyranoside; n-dodecyl β-D-maltoside; heptanoyl-N-methylglucamide; n-heptyl-pD-glucopyranoside; n-heptyl β-D-thioglucoside; n-hexyl β-D-glucopyranoside; nonanoyl-N-methylglucamide; n-Nonyl β-D-glucopyranoside; Octanoyl-N-methylglucamide; n-Octyl β-D-glucopyranoside; Octyl β-D-thioglucopyranoside; PEG-derived phospholipids, PEG-derived cholesterol, PEG-derived cholesterol derivatives, PEG-derived vitamin A, PEG-derived vitamin E, lysozyme, random copolymers of vinylpyrrolidone and vinyl acetate, etc.;

[0164] Examples of useful cationic surfactants or surface stabilizers include, but are not limited to, polymers, biopolymers, polysaccharides, cellulose derivatives, alginates, phospholipids, and non-polymeric compounds such as zwitterionic stabilizers, poly-n-methylpyridinium, anthryl pyridinium chloride, cationic phospholipids, chitosan, polylysine, polyvinylimidazole, polybrene, polymethylmethacrylate trimethylammonium bromide bromide (PMMTMABr), hexyldecyltrimethylammonium 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, A1), poly(2-methacryloxyethyltrimethylammonium 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 the sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Wyandotte Corporation, Parsippany, NJ), lysozyme, long chain polymers such as alginic acid, carrageenan (FMC Corp.), and POLYOX (Dow, Midland, Mich.).

[0165] Other useful cationic stabilizers include, but are not limited to, cationic lipids, sulfonium, phosphonium and quaternary ammonium compounds such as stearyl trimethyl ammonium chloride, benzyl-di(2-chloroethyl)ethyl ammonium 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, C12-15 dimethyl hydroxyethyl ammonium chloride or bromide, coconut dimethyl hydroxyethyl ammonium chloride or bromide, myristyl trimethyl ammonium methyl sulfate, lauryl ammonium chloride, ethyl ... dimethylbenzyl 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-tetradecylidomethylbenzyl ammonium chloride monohydrate, dimethyldidecyl ammonium chloride, N-alkyl and (C12-14) dimethyl 1-naphthylmethyl ammonium chloride, trimethyl ammonium halides, alkyl-trimethyl ammonium salts and dialkyl-dimethyl ammonium salts, lauryl trimethyl ammonium chloride, ethoxylated alkyamidoalkyldialkylammonium saltssalt) and / or ethoxylated trialkylammonium salts, dialkylbenzene dialkyl ammonium chloride, N-didecyl dimethyl ammonium chloride, N-tetradecyl dimethyl benzyl ammonium, chloride monohydrate, N-alkyl (C12-14) dimethyl 1-naphthyl methyl ammonium chloride and dodecyl dimethyl benzyl ammonium chloride, dialkylbenzene alkyl ammonium chloride, lauryl trimethyl ammonium chloride, alkyl benzyl methyl ammonium chloride, alkyl benzyl dimethyl ammonium bromide, C12, C15, C17 trimethyl ammonium bromide, dodecyl benzyl triethyl ammonium chloride, poly-diallyl dimethyl ammonium chloride (DADMAC), dimethyl ammonium chloride, alkyl dimethyl ammonium halides, tricetyl methyl ammonium chloride, decyl trimethyl ammonium bromide, dodecyl triethyl ammonium bromide, tetradecyl trimethyl ammonium bromide, methyl trioctyl ammonium chloride (ALIQUAT 336TM), POLYQUAT 10TM, tetrabutylammonium bromide, benzyltrimethylammonium bromide, choline esters (e.g., choline esters of fatty acids), benzalkonium chloride, stearalkonium chloride compounds (e.g., stearyltrimonium chloride and di-stearyldimonium chloride), cetylpyridinium bromide or chloride, halide salts of quaternized polyoxyethylalkylamines, MIRAPOL TM and ALKAQUAT TM(Alkaril Chemical Company), alkylpyridinium salts; amines such as alkylamines, dialkylamines, alkanolamines, polyethylenepolyamines, N,N-dialkylaminoalkylacrylates and vinylpyridines, amine salts such as laurylamine acetate, stearylamine acetate, alkylpyridinium salts and alkylimidazolium salts, and amine oxides; imidoazolinium salts; protonated quaternary acrylamides; methylated quaternary polymers such as poly[diallyldimethylammonium chloride] and poly-[N-methylvinylpyridinium chloride]; and cationic guar.

[0166] Such exemplary cationic surfactants or surface stabilizers, as well as 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 herein by reference in its entirety.

[0167] The non-polymeric cationic surfactant or surface stabilizer is any non-polymeric compound, such as benzalkonium chloride of the formula NR1R2R3R4(+), carbonium compounds, phosphonium compounds, oxonium compounds, halonium compounds, cationic organometallic compounds, quaternary phosphorus compounds, pyridinium compounds, anilinium compounds, ammonium compounds, hydroxylammonium compounds, primary ammonium compounds, secondary ammonium compounds, tertiary ammonium compounds, and quaternary ammonium compounds. For compounds of formula NR1R2R3R4(+): (i) none of R1-R4 is 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 7 carbon atoms or less; (vi) two of R1-R4 are CH3, one of R1-R4 is C6H5CH2, and one of R1-R4 is an alkyl chain of 19 carbon atoms or more; (vii) two of R1-R4 are CH3 and one of R1-R4 is a group C6H5 (CH2)n, where n>1; (viii) two of R1-R4 are CH3, one of R1-R4 is C6H5CH2, and one of R1-R4 contains at least one heteroatom; (ix) two of R1-R4 are CH3, one of R1-R4 is C6H5CH2, and one of R1-R4 contains at least one halogen; (x) two of R1-R4 are CH3, one of R1-R4 is C6H5CH2, and one of R1-R4 contains 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.

[0168] Such compounds include, but are not limited to, behenalkonium chloride, benzethonium chloride, cetylpyridinium chloride, behentrimonium chloride, lauralkonium chloride, cetoalkonium chloride, cetrimonium bromide, cetrimonium chloride, cetylamine hydrofluoride, chloroallylmethenamine chloride (quaternium-15), distearyldimonium chloride (quaternium-5), dodecyldimethylethylbenzylammonium chloride (quaternium-14), quaternium-22, quaternium-26, quaternium-18 vectorite, dimethylaminoethyl chloride hydrochloride, cysteine ​​hydrochloride, diethanolammonium POE(10) oleyl ether phosphate, diethanolammonium POE(3) oleyl ether phosphate, diethanolammonium POE(4) oleyl ether phosphate, diethanolammonium POE(5) oleyl ether phosphate, diethanolammonium POE(6) oleyl ether phosphate, diethanolammonium POE(7) oleyl ether phosphate, diethanolammonium POE(8) oleyl ether phosphate, diethanolammonium POE(9) oleyl ether phosphate, diethanolammonium POE(10) oleyl ether phosphate, diethanolammonium POE(11) oleyl ether phosphate, diethanolammonium POE(12) oleyl ether phosphate, diethanolammonium POE(13) oleyl ether phosphate, diethanolammonium POE(14) oleyl ether phosphate, diethanolammonium POE(15) oleyl ether phosphate, diethanolammonium POE(16) oleyl ether phosphate, diethanolammonium POE(16) oleyl ether phosphate, diethanolammonium POE(17) oleyl ether phosphate, diethanolammonium POE(18) oleyl ether phosphate, diethanolammonium POE(18) oleyl ether phosphate, diethanolammonium POE Examples of suitable active ingredients include ethyl phosphate, tallowalkonium chloride, dimethyldioctadecylammonium bentonite, stearalkonium chloride, domiphen bromide, denatonium benzoate, myristalkonium chloride, laurotrimonium chloride, ethylenediamine dihydrochloride, guanidine hydrochloride, pyridoxine HCl, iofetamine hydrochloride, meglumine hydrochloride, methylbenzethonium chloride, myrtrimonium bromide, oleyltrimonium chloride, polyquaternium-1, procaine hydrochloride, cocobetaine, stearalkonium bentonite, stearalkonium hectonite, stearyl trihydroxyethyl propylenediamine dihydrofluoride, tallowtrimonium chloride and hexadecyltrimethylammonium bromide.

[0169] Most of these surfactants or surface stabilizers are well known pharmaceutical excipients and are described in detail in the Handbook of Pharmaceutical Excipients (The Pharmaceutical Press, 2000), jointly published by the American Pharmaceutical Association and The Pharmaceutical Society of Great Britain, which is specifically incorporated by reference.

[0170] The surfactants or surface stabilizers are commercially available and / or can be prepared by techniques known in the art.

[0171] Preferably, the surface of the subject microparticles or nanoparticles is composed of a material that minimizes non-specific or undesirable biological interactions between the particle surface and the interstitial spaces, e.g., the particle surface can be coated with a material to prevent or reduce non-specific interactions. Steric stabilization by coating the particles with a hydrophilic layer such as poly(ethylene glycol) (PEG) and its copolymers, e.g., PLURONICS (copolymers of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol)), can reduce non-specific interactions with interstitial proteins, as shown by improved lymphatic uptake after subcutaneous injection.

[0172] As used herein, "small amount" refers to a relatively small amount / volume of the first solution of the second solvent compared to the volume of the first solvent with the PLGA polymer, and emulsification of the first solution of the second solvent in the polymer solution in the first solvent forms an emulsion (i.e., a first emulsion) in which the continuous phase is the polymer solution. Typically, the volume ratio between the small amount of the first solution of the second solvent and the first solvent is at least about 1:n, where n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100.

[0173] As used herein, "large" refers to a relatively large amount / volume of the second solution of the second solvent compared to the volume of the first emulsion, and emulsification of the first emulsion in the second solution of the second solvent forms an emulsion (i.e., a second emulsion) in which the continuous phase is the second solution of the second solvent. Typically, the volume ratio between the first emulsion and the large amount of the second solution of the second solvent is at least about 1:m, where m can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100.

[0174] The incorporation of polysialic acid into the particles can be stable and dense, therefore preferably the method further comprises the step of washing the microparticles or nanoparticles and / or concentrating the microparticles or nanoparticles to a desired volume.

[0175] Microparticles and nanoparticles produced using the methods of the present invention may routinely undergo washing as part of a purification process to remove impurities and / or concentrate the microparticles and nanoparticles so produced.

[0176] Microparticles and nanoparticles produced using the methods of the present invention may also undergo more rigorous washing tests, for example as part of a quality control process to ensure that polysialic acid residues are stably incorporated into the microparticles and nanoparticles so produced.

[0177] Preferably, the washing test uses the same or similar conditions as exemplified below: Preferably, the polysialic acid is durably attached to the surface of microparticles and nanoparticles and can withstand multiple washing cycles.

[0178] Preferably, the polysialic acid on the particle surface is capable of withstanding certain washing tests, such as those exemplified herein, without significant loss in the amount of polysialic acid.

[0179] Preferably, after washing, the microparticles or nanoparticles retain at least about 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the amount of sialic acid moieties.

[0180] particle size The size of the subject microparticles and nanoparticles is from about 1 nm to about 1000 μm, preferably from about 10 nm to about 100 μm, most preferably from about 20 nm to about 5 μm, and most preferably from about 50 nm to about 2 μm. For example, the microparticles and nanoparticles can have an average size of about 100 to 900 nm, e.g., about 100, 300, 500, 700 or 900 nm.

[0181] As used herein, particle size may be determined by any conventional particle size measurement technique known to those of skill in the art, including, for example, sedimentation field flow fractionation, photon correlation spectroscopy, light scattering, dynamic light scattering, light diffraction, and disk centrifugation.

[0182] Further components The particles of the present invention can also include additional components. For example, the carrier can have an imaging agent incorporated or conjugated to the carrier. An example of the carrier nanosphere with imaging agent that is currently commercially available is Kodak X-sight nanosphere. Inorganic quantum-confined luminescent nanocrystals, known as quantum dots (QDs), have emerged as ideal donors in FRET applications; their high quantum yield and tunable size-dependent Stokes shift allow different sizes to emit blue to infrared light when excited with a single ultraviolet wavelength. (Bruchez et al., Science, 1998, 281:2013; Niemeyer, CM, Angew. Chem. Int. Ed., 2003, 42:5796; Waggoner, A. Methods Enzymol., 1995, 246:362; Brus, LE, J. Chem. Phys., 1993, 79, 5566).

[0183] Quantum dots, such as hybrid organic / inorganic quantum dots based on a class of polymers known as dendrimers, can be used in biological labeling, imaging and optical biosensing systems (Lemon et al., J. Am. Chem. Soc.,2000, 122:12886). Unlike the classical synthesis of inorganic quantum dots, the synthesis of these hybrid quantum dot nanoparticles does not require high temperatures or highly toxic, unstable reagents (Etienne et al., Appl. Phys. Lett., 87:181913, 2005).

[0184] Example Applications The particles and compositions thereof have many applications, including therapeutic methods.

[0185] Preferably, nanoparticles and microparticles or compositions comprising said particles can be used in a method of treating a disease or condition in a subject in need of treatment, or a method of reducing the duration or severity of a disease or condition in a subject in need of treatment, where the disease or condition can be treated with said particles (and optionally with a specific API), and said method comprises administering a composition or pharmaceutical composition comprising said particles to the subject, thereby treating the disease or condition.If the particles contain (e.g., encapsulate) an API, said particles can be used in a method of administering or delivering an API to a subject in need of administration or delivery of the API, and / or a method of treating a subject suffering from a disease or condition that can be treated by the API.For example, if the API is an anti-inflammatory agent, said particles can be administered to a subject from an inflammatory condition.

[0186] In a further aspect, the particles can include an immunotherapeutic agent and be used in immunotherapy.

[0187] The microparticles and nanoparticles described herein can be used to treat inflammatory conditions.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, graft 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 peritonitis, periodontal disease, arthritis, and juvenile chronic arthritis. (e.g. chronic iridocyclitis), psoriasis, osteoporosis, nephropathy in diabetes mellitus, asthma, pelvic peritonitis, chronic inflammatory liver disease, chronic inflammatory lung disease, pulmonary fibrosis, liver fibrosis, chronic inflammatory lung disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, peritonitis, cardiovascular disease, reperfusion injury, ischemic injury, stroke, burns and other acute and chronic inflammatory diseases of the central nervous system (CNS; e.g. multiple sclerosis), gastrointestinal system, skin and associated structures, immune system, hepatic-biliary system or any part of the body where pathology may result from an inflammatory component. Inflammatory diseases also include diseases involving the gastrointestinal tract and associated tissues (e.g., 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 localized inflammatory diseases and conditions (e.g., asthma, allergies, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxin shock, and chronic obstructive pulmonary disease) and / or chronic obstructive pulmonary disease (COPD). cough, cachexia, hyperthermia, eosinophilic granuloma, granulomatosis and sarcoidosis); diseases involving the genitourinary system and associated tissues (e.g. septic abortion, epididymitis, vaginitis, prostatitis and urethritis); diseases involving the respiratory system and associated tissues (e.g. bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonia, adult respiratory distress syndrome, pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis, pharyngitis, pleuritis and sinusitis);Diseases resulting from infection with various viruses (e.g. influenza, respiratory syncytial virus, HIV, hepatitis B virus, hepatitis C virus and herpes), bacteria (e.g. disseminated bacteremia, dengue fever), fungi (e.g. candidiasis) and protozoa and multicellular parasites (e.g. malaria, filariasis, amebiasis and hydatid cysts); dermatological diseases and skin conditions (e.g. burns, dermatitis, dermatomyositis, sunburn, urticaria warts and wheals); diseases involving the cardiovascular system and related tissues (e.g. stenosis, restenosis, vasulitis, vasculitis, 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 related tissues (e.g. Alzheimer's disease, Marr's disease, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis and uveitis; diseases of bone, joint, muscle and connective tissue (e.g. various arthritis and arthralgia, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis and synovitis); other autoimmune and inflammatory disorders (e.g. gravitational myasthenia, thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcet's syndrome, allograft rejection, graft versus host disease, type I diabetes, ankylosing spondylitis, Berger's disease and Retier's syndrome); and various cancers, tumors and proliferative disorders (e.g. Hodgkin's disease); and the inflammatory or immune host response to any primary disease in any case;

[0188] Diseases which may be treated also include allergic disorders or conditions such as allergic disease, allergies, eczema, asthma, allergic rhinitis or skin hypersensitivity.

[0189] The disease to be treated may also be a viral infection, such as, for example, a hepatitis virus infection, a West Nile virus infection, a flavivirus infection, 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 the subject. Preferably, the viral infection causes viral encephalitis or viral meningitis. In yet another aspect, the disease to be treated is a bacterial infection. Exemplary bacterial infections are Staphylococcus infection, Streptococcus infection, Mycobacterium infection, Bacillus infection, Salmonella infection, Vibrio infection, Spirochete infection, and Neisseria infection. Bacteria that infect the central nervous system of the subject are preferred. Bacteria that cause encephalitis or meningitis are most preferred.

[0190] The preferred condition for use in the claimed invention is to treat cancer.The cancer to be treated may include Burkitt's lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), indolent non-Hodgkin's lymphoma (iNHL), refractory iNHL, multiple myeloma (MM), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), B-cell ALL, acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldenstrom'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 a further embodiment, the cancer is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), indolent non-Hodgkin's lymphoma (iNHL) and refractory iNHL. In some embodiments, the cancer is indolent non-Hodgkin's lymphoma (iNHL). In some embodiments, the cancer is refractory iNHL. In one embodiment, the cancer is chronic lymphocytic leukemia (CLL). In other embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL).

[0191] In some embodiments, the cancer is pancreatic cancer; bladder cancer; colorectal cancer; breast cancer, e.g., metastatic breast cancer; prostate cancer, e.g., androgen-dependent and androgen-independent prostate cancer; kidney or renal cancer, e.g., metastatic renal cell carcinoma; hepatocellular carcinoma; lung cancer, e.g., non-small cell lung cancer (NSCLC), alveolar cell carcinoma (BAC) and adenocarcinoma of the lung; ovarian cancer, e.g., advanced epithelial or primary peritoneal carcinoma; cervical cancer; gastric cancer; esophageal cancer; e.g., squamous cell carcinoma of the head and neck, etc. head and neck cancer; melanoma; neuroendocrine cancers such as metastatic neuroendocrine tumors; brain tumors such as glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme and adult anaplastic astrocytoma; bone cancer; and soft tissue sarcoma, hepatic cancer, rectal cancer, penile cancer, vulvar cancer, thyroid cancer, salivary gland cancer, endometrial or uterine cancer, gastric or stomach cancer such as liver cancer, hepatocellular carcinoma, liver cancer, gastrointestinal cancer, cancer of the peritoneum, squamous cell carcinoma of the lung, gastroesophageal cancer, bile duct cancer, gallbladder cancer, colorectal / appendix cancer, squamous cell carcinoma (e.g. epithelial squamous cell carcinoma).

[0192] Any of the treatment methods provided can be used to treat various stages of cancer.

[0193] By way of example, cancer stages include, but are not limited to, early stage, advanced, locally advanced, in remission, refractory, recurrent after remission, and progressive.

[0194] Preferably, the microparticles or nanoparticles of the present invention may be used in combination with a second therapeutic agent that is effective to treat any one of the treatable conditions.

[0195] 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, cow, bull, cow, sheep, goat, pig, camel, etc.), a rodent (rabbit, hamster, mouse, rat, etc.) or a pet (cat, dog).

[0196] Preferably, the methods include administering a subject composition or pharmaceutical composition comprising a subject microparticle or nanoparticle by any suitable means or route, such as oral, nasal, intravenous, intramuscular, ophthalmic, transdermal, subcutaneous, intratumoral, intravesicular, intraarticular, intracranial and intraperitoneal.

[0197] Preferably, about 10 2 ~about 10 20 Preferably, about 10 particles are provided to the individual. 3 ~about 10 15 Preferably, about 10 particles are provided. 6 ~about 10 12 Preferably, about 10 particles are provided. 8 ~about 10 10 particles are provided. Preferably, the preferred dosage is 0.1% solids / ml. So for 0.5 μm beads, the preferred dosage is about 4×10 9 beads, for 0.05 μm beads the preferred dose is about 4×10 12 beads, for 3 μm beads the preferred dose is 2x10 7 However, any dose effective to treat the particular condition being treated is encompassed by the present invention.

[0198] The effectiveness of the microparticles and nanoparticles described herein against treatable diseases and conditions can be tested using a number of efficacy tests, such as appropriate animal models.

[0199] Pharmaceutical Compositions One aspect of the present invention provides pharmaceutical compositions comprising the subject microparticles and nanoparticles, and optionally comprising a pharma- ceutically acceptable carrier or excipient.Preferably, these compositions optionally further comprise one or more additional therapeutic agents.Alternatively, the subject particles of the present invention can be administered to a patient in need thereof in combination with the administration of one or more other therapeutic agents.For example, the additional therapeutic agent for co-administration with the compound of the present invention or inclusion in the pharmaceutical composition can be an approved anti-inflammatory agent, immunotherapeutic agent or chemotherapeutic agent, or any one of several drugs approved by the Food and Drug Administration.It is also understood that certain subject particles of the present invention can be present in free form for treatment, or as a pharma-ceutically acceptable derivative thereof, if appropriate.

[0200] Preferably, the pharmaceutical composition of the present invention further comprises a pharma- ceutically acceptable carrier, which as used herein includes any and all solvents, diluents or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular desired dosage form. Remington's Pharmaceutical Sciences, 16th Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation. Except insofar as any conventional carrier medium is incompatible with the compounds of the present invention, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated within the scope of the present invention.

[0201] Some examples of materials that may serve as pharma- ceutically 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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; 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; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol and phosphate buffer solutions; other non-toxic, compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening agents, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0202] Liquid dosage forms for oral administration include, but are not limited to, pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active compounds, liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, tuber, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof.In addition to inert diluents, oral compositions can also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and perfumes.

[0203] 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 pharma- ceutically 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 carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and gum arabic; 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 retarders, such as paraffin; f) absorption accelerators, such as quaternary ammonium compounds; g) humectants, such as 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 forms may also comprise buffering agents.

[0204] Similar types of solid compositions can also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and 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 art of pharmaceutical formulation. They can optionally contain opacifying agents and can also be of a composition that releases 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. Similar types of solid compositions can also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols and the like.

[0205] Particles can also be in microencapsulated form with one or more of the above-mentioned excipients.The solid dosage form of tablet, dragee, capsule, pill and granule can be prepared with coating and shell, such as enteric coating, release control coating and other coatings well known in the pharmaceutical formulation field.In such solid dosage form, active compound can be mixed with at least one inert diluent, such as sucrose, lactose and starch.

[0206] Such dosage forms can also contain, as in normal practice, other substances than inert diluents, such as tableting lubricants and other tableting aids, for example, magnesium stearate and microcrystalline cellulose.For capsules, tablets and pills, the dosage forms can also contain buffering agents.They can optionally contain opacifying agents, and can also be of a composition that releases modified particles 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.

[0207] It is also understood that the nanoparticles and microparticles of the present invention and pharmaceutical compositions can be formulated and used in combination therapy, i.e., the compounds and pharmaceutical compositions can be formulated together with one or more other desired therapeutic agents or medical procedures, or can be administered simultaneously, before or after.The specific combination of therapies (therapeutics or procedures) to be used in combination regimen takes into account the compatibility of the desired therapeutic agents and / or procedures and the desired therapeutic effect to be achieved.It is also understood that the treatments used can achieve the desired effect for the same disorder (e.g., the compounds of the present invention can be administered together with another anti-inflammatory agent), or the treatments can achieve different effects (e.g., control of any adverse effects).

[0208] Preferably, the pharmaceutical composition comprising the particles of the present invention further comprises one or more additional therapeutic active ingredients (e.g. anti-inflammatory and / or palliative).For the purpose of the present invention, the term "palliative" refers to a non-curative treatment that focuses on the relief of the symptoms of disease and / or the side effects of treatment regimen.For example, palliative treatment includes painkillers, antiemetic medicines and anti-disease drugs.

[0209] The following examples are given to illustrate the invention, however, it should be understood that the invention is not limited to the particular conditions or details described in these examples. EXAMPLES

[0210] Working Example Example 1. Preparation of PLGA nanoparticles with polysialic acid on the surface 200 mg of PLGA is dissolved in 8 ml of ethyl acetate to form a PLGA solution, which is mixed with 40 mL of 0.5% polyvinyl alcohol (PVA) solution containing 40 milligrams of colominic acid and homogenized using an IKA® DIGITAL ULTRA-TURRAX® T25 homogenizer at 25,000 rpm for 1 min. The resulting emulsion is poured into a glass container and magnetically stirred at 400 rpm for 3 h to evaporate the solvent. The nanoparticles are then washed three times with distilled water and subsequently lyophilized. The lyophilized particles are reconstituted in distilled water for measurements of particle size, size distribution, zeta potential and amount of polysialic acid on the surface.

[0211] Example 2. Preparation of protein-loaded PLGA nanoparticles with polysialic acid on the surface via a double emulsion process Dissolve 200 mg of PLGA in 4 mL of ethyl acetate to form a PLGA solution. Prepare a mixed solution consisting of 35 ml of 2% polyvinyl alcohol (PVA) solution (in water), 1.5 ml of ethyl acetate and 40 milligrams of colominic acid. Dissolve 4 mg of bovine serum albumin (BSA, a model therapeutic protein) in 0.4 mL of aqueous buffer to form a protein solution. Mix the BSA solution with the PLGA solution and homogenize the resulting mixture using a probe sonicator for 30 seconds. Mix the resulting emulsion with the PVA / polysialic acid solution and homogenize for 1 minute at 18,000 rpm using an IKA® DIGITAL ULTRA-TURRAX® T25 homogenizer. Pour the resulting final emulsion into a 250 mL glass flask and remove the solvent by rotor evaporation at a vacuum of 50 mbar. Wash the BSA-loaded particles three times with distilled water and freeze-dry them. The lyophilized particles are reconstituted in distilled water for measurement of particle size, size distribution, zeta potential, protein encapsulation efficiency and amount of polysialic acid on the surface.

[0212] Example 3. Preparation of paclitaxel-loaded PLGA nanoparticles with polysialic acid on the surface via a single emulsion process 200 mg of PLGA and 4 mg of paclitaxel are dissolved in 4 mL of ethyl acetate to form a PLGA-paclitaxel solution. The PLGA-paclitaxel solution is mixed with 16 mL of 2.5% polyvinyl alcohol solution containing 40 milligrams of polysialic acid and homogenized using an IKA® DIGITAL ULTRA-TURRAX® T25 homogenizer at 24,000 rpm for 1 minute. The resulting emulsion is poured into a glass container and magnetically stirred at 400 rpm for 4 hours to evaporate the solvent. The paclitaxel-loaded nanoparticles are then washed three times with distilled water and lyophilized. The BSA-loaded particles are washed three times with distilled water and lyophilized. The lyophilized particles are reconstituted in distilled water for measurements of particle size, size distribution, zeta potential, drug encapsulation efficiency and amount of polysialic acid on the surface.

[0213] Example 4. Preparation of polycaprolactone nanoparticles with polysialic acid on the surface 100 mg of polycaprolactone (PCL) is dissolved in 6 ml of dichloromethane (DCM) to form a PCL solution, which is mixed with 40 mL of a 5% polyvinyl alcohol (PVA) solution containing 40 milligrams of colominic acid and homogenized using an IKA® DIGITAL ULTRA-TURRAX® T25 homogenizer at 25,000 rpm for 1 min. The resulting emulsion is poured into a glass container and magnetically stirred at 400 rpm for 3 h to evaporate the DCM. The nanoparticles are then washed three times with distilled water and subsequently freeze-dried. The freeze-dried particles are reconstituted in distilled water for measurements of particle size, size distribution, zeta potential and quality of polysialic acid on the surface.

[0214] Example 5. Preparation of fluorescently labeled oligonucleotides Antisense oligonucleotides (ASOs) targeting the non-coding nuclear RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) with a primary amine appended at the 5' position (see FIG. 1) were obtained from Boston Open Labs, Cambridge, MA. Such MALAT1-ASO-5'-amine was reacted with an equimolar amount of Cy7 near-IR fluorescent dye functionalized with an NHS ester. The resulting reaction product is referred to as the ASO-Cy7 conjugate.

[0215] Example 6. Preparation of nanoparticles loaded with ASO-Cy7 conjugates and having polysialic acid displayed on the surface Approximately 50 mg of the ASO-Cy7 conjugate prepared in Example 5 was dissolved in 1 mL of distilled water to form an ASO solution. 100 mg of poly(lactide-co-glycolide) (PLGA, ester end-capped) was dissolved in 1 mL of ethyl acetate to form a polymer solution. 63.75 mg of ethyl lauroyl arginate (ELA) was dissolved in 1 mL of benzyl alcohol to form an ELA solution. 0.5 mL of the polymer solution, 0.2 mL of the ELA solution, 0.3 mL of ethyl acetate and 0.1 mL of the ASO solution were mixed in an 8 mL glass vial. The resulting mixture in an 8 mL vial was probe sonicated for 30 seconds at 90% amplitude to obtain a first emulsion, which was transferred to a 15 mL glass vial containing 5 mL of an aqueous solution consisting of 0.5% poly(vinyl alcohol) (PVA, 89% hydrolyzed), 0.2% Brij-S100-PA-SG (Brij-S100) and 0.5% polysialic acid, saturated with an appropriate amount of ethyl acetate. The entire mixture was immediately probe sonicated for 60 seconds at 90% amplitude to obtain a second emulsion, which was transferred to a 30 mL beaker and magnetically stirred for 2 hours in a chemical fume hood. Once the particles were formed and hardened, the suspension was washed twice with 50 mL of phosphate buffered saline, then 50 mL of distilled water using tangential flow filtration. After purification, the nanoparticles were lyophilized. The resulting nanoparticles were found to have an average particle size of 155.6 nm, a loading of 3.9% ASO-Cy7 and a surface zeta potential of -33.0 mV.

[0216] Example 7. Cleaning test 50 mg of PLGA nanoparticles prepared as described in Example 1 are reconstituted in 30 mL of deionized water. After a short sonication, the particles are fully suspended. A sample is taken for measurement of the zeta potential.

[0217] 300 mL of deionized water is then added to the nanoparticle suspension. The resulting mixture is concentrated to 30 mL using a tangential flow filtration (TFF) device and the ζ-potential is measured again. This washing step is repeated four more times and the ζ-potential obtained after each wash is measured and recorded.

[0218] While the present invention has been particularly shown and described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the scope of the invention encompassed by the appended claims.

[0219] It should be understood that any preferred feature of the invention described herein may be combined with any other preferred feature, including preferred features described in only one aspect of the invention and preferred features described in the examples only. Throughout the specification, any and all references to publicly available documents, including any U.S. patents or published patent applications, are specifically incorporated by reference.

Claims

1. A composition comprising particles presenting sialic acid residues on their surface, each particle comprising a polysialic acid comprising a biodegradable polymer and a sialic acid residue, the sialic acid residues being present on the surface of the particle and not conjugated to the particle; the particle being a microparticle or a nanoparticle.

2. 2. The composition of claim 1, wherein the biodegradable polymer is selected from the group consisting of polylactide (PLA), poly(lactide-co-glycolide) (PLGA), copolymers of ethylene glycol and lactide / glycolide (PEG-PLGA), copolymers of ethylene glycol and lactide (PEG-PLA), copolymers of ethylene glycol and glycolide (PEG-PGA), poly(ethylene glycol) (PEG), polycaprolactone (PCL), polyanhydrides (PANH), poly(orthoesters), polycyanoacrylates, poly(hydroxyalkanoates) (PHAs), poly(sebasic acid), polyphosphazenes, polyphosphoesters, modified poly(saccharides), mixtures and copolymers thereof.

3. The composition of claim 2 , wherein the biodegradable polymer is PLGA.

4. The composition of claim 1 or 3, wherein the particles are nanoparticles.

5. The composition of claim 1 , wherein the biodegradable polymer and the polysialic acid form an interpenetrating network.

6. The composition of claim 3, wherein the PLGA and polysialic acid form an interpenetrating network.

7. The composition of claim 1, wherein the sialic acid residue is selected from the group consisting of Neu5Ac, Neu5Gc and Kdn or a combination thereof.

8. The composition of claim 1 , wherein the polysialic acid is a homopolymer.

9. The composition of claim 1 , wherein the polysialic acid is colominic acid.

10. The composition of claim 1 , wherein the particles further comprise an active agent.

11. The composition of claim 10, wherein the active agent is an active pharmaceutical ingredient selected from the group consisting of small molecules, peptides, proteins and nucleic acids.

12. 12. The composition of claim 11, wherein the active agent is a nucleic acid selected from the group consisting of DNA, RNA and antisense oligonucleotides.

13. 13. The composition of claim 12, further comprising a cationic complexing agent selected from the group consisting of small molecule cationic agents, cationic or ionizable lipids, and cationic polymers.

14. The composition of claim 10 , wherein the active agent is encapsulated within the particle.

15. The composition of claim 1 further comprising a pharma- ceutically acceptable excipient.

16. 11. A method for administration of an active agent to a subject in need thereof, comprising administering to said subject the composition of claim 10.

17. 17. The method of claim 16, wherein the active agent is an active pharmaceutical ingredient.

18. The method of claim 16 , wherein the active agent is encapsulated within the particle.

19. 12. A method for the treatment of a disease or disorder in a subject in need of such treatment, comprising administering to the subject a composition according to claim 11.

20. 20. The method of claim 19, wherein the disease is cancer.

21. 20. The method of claim 19, wherein the active pharmaceutical ingredient is an anti-cancer agent or an immunotherapeutic agent.

22. 20. The method of claim 19, wherein the disease is an autoimmune disease.

23. 1. A method for the preparation of particles displaying sialic acid residues on a surface of the particle, each particle comprising a biodegradable polymer and polysialic acid, the sialic acid residues being present on the surface of the particle and not conjugated to the particle, and the particle being a microparticle or nanoparticle; the method comprising: i. dissolving a biodegradable polymer and optionally an active agent in a first solvent to form a polymer solution; ii. emulsifying the polymer solution in a solution of a second solvent to form an emulsion, where the first solvent is immiscible or partially miscible with the second solvent, the solution of the second solvent comprising polysialic acid, the solution of the second solvent optionally further comprising a surfactant and / or an active agent that is soluble in the second solvent; and iii. Removing the first solvent to form the particles. A method comprising:

24. 24. A particle prepared by the method of claim 23.

25. 1. A method for the preparation of particles displaying sialic acid residues on a surface of the particle, each particle comprising a biodegradable polymer and polysialic acid, the sialic acid residues being present on the surface of the particle and not conjugated to the particle, and the particle being a microparticle or nanoparticle; the method comprising: i. dissolving a biodegradable polymer and optionally an active agent, API, in a first solvent to form a polymer solution; ii. adding a first solution of a second solvent to the polymer solution to form a mixture, where the first solvent is immiscible or partially miscible with the second solvent, and the first solution of the second solvent optionally contains an active agent that is the same as or different from the API dissolved in the first solvent; emulsifying the mixture to form a first emulsion; iii. emulsifying the first emulsion in a second solution of a second solvent to form a second emulsion; wherein the second solution of the second solvent comprises polysialic acid and optionally further comprises a surfactant; and iv. Removing the first solvent to form particles. A method comprising:

26. 26. A particle prepared by the method of claim 25.