Dendrimer conjugates and compositions comprising the same

Abstract

This invention provides dendrite conjugates, compositions comprising dendrite conjugates, and methods of using dendrite conjugates and compositions thereof. In some embodiments, this invention provides dendrite conjugates comprising dendrites bound to at least one pharmaceutical agent. In some embodiments, the dendrite conjugates comprise one or more pharmaceutical agents suitable for therapeutic, imaging, and / or targeted delivery. In some embodiments, this invention provides a method of synthesizing functionalized dendrites, the method comprising reacting a first dendrite with one or more amines.

Description

[Technical Field]

[0001] This invention relates to dendritic compounds and their methods of use. [Previous Technology]

[0002] Advances in the field of nanotechnology have led to the development of many novel materials and devices with wide-ranging applications. Nanotechnology is embodied in a wide range of materials and particles, such as fullerenes and dendrites. However, due to the interaction between nanoparticles and biological systems, the in vivo application of nanoparticles faces the challenge of rapid self-circulation and elimination. Alternative strategies in nanoparticle development can allow for more effective utilization of the advantageous properties of nanoparticles in the context of clinical applications, diagnostics, and biomedical research. [Summary of the Invention]

[0003] In some embodiments, the present invention provides therapeutic and / or diagnostic compounds comprising dendrites bound to an agent via terminal ether or amide bonds. In some embodiments, the dendrites comprise terminal hydroxyl groups that are substituted by the agent as desired. In some embodiments, the agent is a therapeutic agent or an imaging agent.

[0004] In some embodiments, the present invention provides a dendritic complex of formula (I): (I), wherein: D is a dendrite; X is O or NH; Y1 is an alkyl group, an alkenyl group, an alkyne group, or a covalent bond, substituted as desired; Y2 is selected from the group consisting of: second amide, third amide, sulfonamide, second carbamate, third carbamate, carbonate, urea, methanol, disulfide, hydrazone, acetylhydrazine, ether, carbonyl, and combinations thereof; Z is a therapeutic agent or imaging agent; L is a linker; m is an integer from 16 to 4096, inclusive; and n is an integer from 1 to 100, inclusive. In some embodiments, the ratio of m to (m+n) is at least 0.5. In some embodiments, the ratio of m to (m+n) is between about 0.50 and about 0.99.

[0005] In some embodiments, the present invention provides a dendritic complex of formula (II): (II), wherein: D is a dendritic; each instance of X is independently O or NH; each instance of Y1 is independently a substituted alkyl group, an substituted alkenyl group, an substituted alkyne group, or a covalent bond as desired; each instance of Y2 is independently selected from the group consisting of: second amide, third amide, sulfonamide, second carbamate, third carbamate, carbonate, urea, methanol, disulfide, hydrazone, acetylhydrazine, ether, carbonyl, and combinations thereof; Z1 and Z2 are independently therapeutic agents, targeting agents, or imaging agents, provided that Z1 and Z2 are different; L1 and L2 are independently linkers; m is an integer from 16 to 4096, inclusive; and each instance of n is an integer from 1 to 100, inclusive.

[0006] In some embodiments, the dendrites or dendrite complexes of the therapeutic compounds of the present invention are selected from the group consisting of: poly(aminoamine) (PAMAM) polymers, polypropylene (POPAM) polymers, polyethylene imine polymers, polylysine polymers, polyester polymers, tungsten polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof.

[0007] In some embodiments, the linker of the therapeutic compound or dendritic conjugate comprises a polymer. In some embodiments, the polymer is a polymeric polyol, polypeptide, or unsubstituted alkyl chain. In some embodiments, the polymer is a polymeric polyol selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, and polyvinyl alcohol. In some embodiments, the polymer is a polypeptide comprising at least two and up to 25 amino acids. In some embodiments, the polymer is an unsubstituted C2-30 alkyl chain. In some embodiments, the linker comprises at least one portion selected from the group consisting of 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisooxazolyl, and 1,4-dihydropyridazinyl. In some embodiments, the linker comprises the polymer and at least one portion. In some embodiments, the linker is non-hydrolyzable under physiological conditions.

[0008] In some embodiments, the therapeutic agent of the therapeutic compound or dendritic complex is selected from the group consisting of: angiotensin II receptor blockers, farnesoid X receptor agonists, death receptor 5 agonists, sodium-glucose cotransporter type 2 inhibitors, lysophosphatidylcholine 1 receptor antagonists, endothelin-A receptor antagonists, PPARδ agonists, AT1 receptor antagonists, CCR5 / CCR2 antagonists, antifibrotic agents, anti-inflammatory agents, antioxidants, STING agonists, CSF1R inhibitors, AXL inhibitors, c-Met inhibitors, PARP inhibitors, receptor tyrosine kinase inhibitors, MEK inhibitors, group I p21 activated kinase (PAK1) inhibitors, glutamate inhibitors, TIE II antagonists, CXCR2 inhibitors, CD73 inhibitors, arginine kinase inhibitors, PI3K inhibitors, TLR4 agonists, TLR7 agonists, SHP2 inhibitors, chemotherapeutic agents, STING antagonists, and JAK1 inhibitors.

[0009] In some embodiments, the therapeutic agent of the therapeutic compound or dendritic complex is a MEK inhibitor. In some embodiments, the MEK inhibitor is selected from the group consisting of: trametinib, cobimetinib, binimetinib, selumetinib, PD325901, PD035901, PD032901, and TAK-733. In some embodiments, the therapeutic agent of the therapeutic compound or dendritic complex is a receptor tyrosine kinase inhibitor. In some embodiments, the receptor tyrosine kinase inhibitor is selected from the group consisting of: sunitinib, sorafenib, pazopanib, vandetanib, axitinib, cediranib, vatalanib, dasatinib, bemcentinib (R428), dubermatinib (TP-0903), nintedanib, cabozantinib, and motesanib. In some embodiments, the therapeutic agent of the therapeutic compound or dendritic complex is a PAK1 inhibitor. In some embodiments, the PAK1 inhibitor is Frax-1036 (6-[2-chloro-4-(6-methyl-2-pyrazinyl)phenyl]-8-ethyl-2-[[2-(1-methyl-4-piperidinyl)ethyl]amino]-pyrido[2,3-d]pyrimidin-7(8H)-one).

[0010] In some embodiments, the imaging agent of the therapeutic compound or dendritic complex is selected from the group consisting of dyes, fluorescent dyes, near-infrared dyes, single-photon emission computed tomography (SPECT) imaging agents, positron emission tomography (PET) imaging agents, magnetic resonance imaging (MRI) contrast agents, and radionuclides.

[0011] In some embodiments, the therapeutic compound or dendritic complex of the present invention comprises at least one targeting agent bound to the dendritic. In some embodiments, the targeting agent is trianthopterin-N-acetylglucosamine (GalNAc).

[0012] In some embodiments, the present invention provides a composition comprising a carrier and a functionalized dendrite of formula (IA): (IA), wherein: D is a dendrite selected from the group consisting of: poly(aminoamine) (PAMAM) polymer, polypropylene (POPAM) polymer, polyethyleneimine polymer, polylylamine polymer, polyester polymer, styrene polymer, aliphatic poly(ether) polymer, aromatic polyether polymer, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymer, and combinations thereof; X is NH; Y1 is an alkyl group, an alkenyl group, an alkyne group, or a covalent bond as desired; m is an integer from 16 to 4096, inclusive; and n is an integer from 1 to 100, inclusive, wherein the polydispersity value of the functionalized dendrite in the composition is less than or equal to 1.10.

[0013] In some embodiments, the present invention provides a method for synthesizing a functionalized dendrite of formula (IA): (IA), wherein: D is a dendrite selected from the group consisting of: poly(aminoamine) (PAMAM) polymers, polypropylene (POPAM) polymers, polyethylene imine polymers, polylylamine polymers, polyester polymers, acetonitrile polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; X is NH; Y1 is an alkyl group, an alkenyl group, an alkyne group, or a covalent bond as desired; m is an integer from 16 to 4096, inclusive; and n is an integer from 1 to 100, inclusive; the method comprises: making a dendrite of formula (II-A): (II-A), wherein: D is a dendrite selected from the group consisting of: poly(aminoamine) (PAMAM) polymers, polypropylene (POPAM) polymers, polyethylene imine polymers, polylylamine polymers, polyester polymers, tretinoin polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; t is an integer from 16 to 4096, inclusive; reacting with one or more amines under suitable conditions to form a functionalized dendrite of formula (IA), wherein each amine has the formula H2NR1, wherein R1 is a substituted alkyl group, a substituted alkenyl group, a substituted alkyne group, or a covalent bond, as desired. In some embodiments, the present invention provides a functionalized dendrite of the herein described, or a salt, solvate, hydrate, stereoisomer, polymorph, tautomer, or isotopically enriched form (e.g., isotopically labeled derivatives).

[0014] In some embodiments, n is about 3 or 10 (e.g., 3 or 10). In some embodiments, m is a value from 64 minus n. In some embodiments, n is 3 and m is 61. In some embodiments, n is 10 and m is 54. In some embodiments, m is about 61 or 54 (e.g., 61 or 54).

[0015] In some embodiments, the dendrite of formula (II-A) has the following formula: (PAMAM G3.5).

[0016] In some embodiments, the functionalized dendrite of formula (IA) has the following formula: or.

[0017] In yet another embodiment, the present invention provides a functionalized dendrite of formula (IA) synthesized by the method described herein. In some embodiments, the present invention provides a dendrite of formula (II-A) having the following formula: (PAMAM G3.5).

[0018] In some embodiments, the present invention provides a composition of a therapeutic compound comprising a dendrite bound to a therapeutic agent via a terminal ether or amide bond. In some embodiments, the dendrite comprises a high density of terminal hydroxyl groups, which may be replaced by the therapeutic agent as desired. In some embodiments, the therapeutic compound comprising the dendrite bound to the therapeutic agent is 10 to 20% by mass of the therapeutic agent. In some embodiments, the terminal ether or amide bond is bound to the therapeutic agent via a linker.

[0019] In some embodiments, the therapeutic compound is about 10% to about 15% by mass of the therapeutic agent. In some embodiments, the therapeutic compound is about 15% to about 20% by mass of the therapeutic agent. In some embodiments, at least 50% of the terminal sites on the dendrite contain terminal hydroxyl groups. In some embodiments, at least 50% and up to 99% (e.g., 50 to 95%, 50 to 90%, 50 to 80%, 50 to 70%, 50 to 60%, 60 to 80%, 70 to 90%) of the terminal sites on the dendrite contain terminal hydroxyl groups.

[0020] In some embodiments, the therapeutic agent has increased water solubility relative to an unbound compound containing the therapeutic agent but lacking the dendrite. In some embodiments, the water solubility is increased by at least 10% relative to the unbound compound. In some embodiments, the water solubility is increased by between about 10% and about 100% relative to the unbound compound. In some embodiments, the water solubility is increased by at least about two times relative to the unbound compound. In some embodiments, the water solubility is increased by between about two times and about ten times relative to the unbound compound. In some embodiments, the water solubility is solubility under physiological conditions. In some embodiments, the water solubility is solubility in water at a pH between about 7.0 and about 8.0. In some embodiments, the therapeutic agent is present at a concentration in which the unbound compound is insoluble under physiological conditions.

[0021] In some embodiments, the present invention provides a method for treating or imaging a disease or ailment of the brain or central nervous system in an individual in need, the method comprising administering to the individual a composition comprising the dendritic complex described herein in an amount effective for treating or imaging a disease or ailment of the brain or central nervous system in that individual.

[0022] In some embodiments, the dendritic complex is selectively absorbed by activated microglia and / or activated macrophages in the individual's brain or central nervous system. In some embodiments, the activated macrophages are resident macrophages of the central nervous system. In some embodiments, the dendritic complex crosses the blood-brain barrier of the individual. In some embodiments, the disease or ailment is a tumor of the brain or central nervous system. In some embodiments, the tumor is a benign or malignant tumor associated with neurofibromatosis. In some embodiments, the disease or ailment is neurofibromatosis (e.g., neurofibromatosis type 1 (NF1)). Therefore, in some embodiments, the individual has or is suspected of having neurofibromatosis (e.g., NF1). In some embodiments, the dendritic complex is selectively absorbed by tumor-associated macrophages of the tumor in the individual. In some embodiments, the tumor is brain cancer or cancer of the central nervous system. In some embodiments, the tumor is brain cancer selected from a group of lesion-forming and proliferating components. In some embodiments, the tumor is a cancer of the central nervous system selected from the group consisting of glioma, glioblastoma, astrocytoma, oligodendroglioma, meningioma, neuroblastoma, ganglioma, and Schwannoma. In some embodiments, the dendritic complex does not bind to the target site. In some embodiments, the composition is administered to the individual systemically, intravenously, or orally. In some embodiments, the dendritic complex comprises a therapeutic agent, and the composition has an increased therapeutic index relative to a composition comprising the therapeutic agent but lacking the dendritic complex. In some embodiments, the therapeutic agent is a MEK inhibitor (e.g., trametinib, cobimetinib, binimetinib, selumetinib, PD325901, PD035901, PD032901, or TAK-733). In some embodiments, the therapeutic agent is a receptor tyrosine kinase inhibitor (e.g., sunitinib, sorafenib, pazopanib, vandetanib, axitinib, cidinibub, vatalanib, dasatinib, bemtinib (R428), dubematinib (TP-0903), nintedanib, cabozantinib, or motesanib). In some embodiments, the therapeutic agent is a PAK1 inhibitor (e.g., Frax-1036 (6-[2-chloro-4-(6-methyl-2-pyrazinyl)phenyl]-8-ethyl-2-[[2-(1-methyl-4-piperidinyl)ethyl]amino]-pyrido[2,3-d]pyrimidin-7(8H)-one)).

[0023] In some embodiments, the present invention provides a method for treating or imaging an eye disease or ailment in an individual in need, the method comprising administering to the individual a composition comprising the dendritic complex described herein in an amount effective for treating or imaging the eye disease or ailment in that individual.

[0024] In some embodiments, the dendritic complex is selectively absorbed by activated microglia and / or activated macrophages in the individual's eye. In some embodiments, the dendritic complex crosses the blood-retinal barrier of the individual. In some embodiments, the dendritic complex does not bind to a target site. In some embodiments, the composition is administered to the individual systemically, intravenously, or orally. In some embodiments, the dendritic complex comprises a therapeutic agent, and the composition has an increased therapeutic index relative to a composition comprising the therapeutic agent but lacking the dendritic complex.

[0025] In some embodiments, the present invention provides a method for treating or imaging a proliferative disease in an individual in need, the method comprising administering to the individual a composition comprising the dendritic complex described herein in an amount effective for treating or imaging the proliferative disease in the individual.

[0026] In some embodiments, the proliferative disease is neurofibromatosis. In some embodiments, the proliferative disease is selected from neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2), and Schwann cell tumor. In some embodiments, the proliferative disease is NF1. In some embodiments, the dendritic complex does not bind to the target site. In some embodiments, the composition is administered to an individual systemically, intravenously, or orally. In some embodiments, the dendritic complex comprises a therapeutic agent, and the composition has an increased therapeutic index relative to a composition comprising the therapeutic agent but lacking the dendritic structure. In some embodiments, the therapeutic agent is a MEK inhibitor (e.g., trametinib, cobimetinib, binimetinib, selmetinib, PD325901, PD035901, PD032901, or TAK-733). In some embodiments, the therapeutic agent is a receptor tyrosine kinase inhibitor (e.g., sunitinib, sorafenib, pazopanib, vandetanib, axitinib, cidinibub, vatalanib, dasatinib, bemtinib (R428), dubematinib (TP-0903), nintedanib, cabozantinib, or motesanib). In some embodiments, the therapeutic agent is a PAK1 inhibitor (e.g., Frax-1036 (6-[2-chloro-4-(6-methyl-2-pyrazinyl)phenyl]-8-ethyl-2-[[2-(1-methyl-4-piperidinyl)ethyl]amino]-pyrido[2,3-d]pyrimidin-7(8H)-one)).

[0027] Details of certain embodiments of the present invention are set forth in the embodiments described below. Other features, objects, and advantages of the present invention will be apparent from the examples, drawings, and claims.

Implementation Method

[0087] Cross-reference to related applications

[0088] This application claims priority to U.S. Provisional Patent Application No. 63 / 327,301, filed April 4, 2022, and U.S. Provisional Patent Application No. 63 / 209,348, filed June 10, 2021, pursuant to 35 USC § 119(e), all of which are incorporated herein by reference in their entirety.

[0089] In other embodiments, the present invention provides dendritic compounds, compositions comprising dendritic compounds, and methods of using dendritic compounds and compositions thereof. In some embodiments, the dendritic compound comprises dendrites bound to at least one pharmaceutical agent. In some embodiments, the dendritic compound comprises one or more pharmaceutical agents suitable for therapeutic, imaging, and / or targeted delivery applications.

[0090] In some embodiments, the present invention relates to a therapeutic compound comprising a dendrite bound to a therapeutic agent. The inventors have recognized and understood that certain therapeutic agents with unfavorable in vivo profiles can be modified by binding to a dendrite to provide a therapeutic compound exhibiting reduced off-target toxicity, higher selective uptake, and sustained intracellular effects. The inventors have further recognized and understood that such therapeutic compounds are highly tunable in the dendritic portion, allowing the hydrophilicity of the therapeutic compound to be adjusted to allow targeted delivery of certain therapeutic agents to biological targets that would otherwise be difficult for the therapeutic agent to reach. In other embodiments, the present invention provides a method for synthesizing a functionalized dendrite of formula (IA), comprising: reacting a dendrite of formula (II-A) with one or more amines, wherein each amine has the formula H2NR1, under suitable conditions to form a functionalized dendrite of formula (IA). In other embodiments, the present invention provides a functionalized dendrite of formula (IA) synthesized by the methods described herein.

[0091] In some embodiments, the present invention provides a method for treating or imaging a disease or ailment in an individual in need, including administering to the individual a composition comprising the dendritic conjugate described herein. In some embodiments, the dendritic conjugate of the present invention can target reactive immune cells in the absence of any targeting portion. For example, in some embodiments, the dendritic conjugate described herein can cross the blood-brain barrier in the central nervous system of an individual, wherein the dendritic conjugate is selectively absorbed by activated microglia and / or activated macrophages. In some embodiments, the dendritic conjugate described herein can cross the blood-retinal barrier in the eye of an individual, wherein the dendritic conjugate is selectively absorbed by activated microglia and / or activated macrophages. Dendritic conjugate

[0092] In some embodiments, a dendritic complex refers to a dendrite that is bound to at least one pharmaceutical agent as described herein. In some embodiments, the dendritic system is covalently bound (e.g., covalently linked) to at least one pharmaceutical agent. In some embodiments, the dendritic complex comprises a dendrite that can be described as a molecular structure having an internal core and layers (or "generations") of repeating units connected to and extending from the internal core, each layer having one or more branch points, and the outermost layer having terminal functional groups.

[0093] In some embodiments, the terminal functional groups of the dendrite include one or more hydroxyl groups, one or more amino groups, and / or one or more carboxyl groups. In some embodiments, the terminal functional groups of the dendrite provide binding sites through which at least one agent binds to form a dendritic complex. Therefore, in some embodiments, the at least one agent is bound to the dendrite via an ether bond, amide bond, or ester bond formed by binding to the terminal functional groups of the dendrite. In some embodiments, the at least one agent is bound to the dendrite via an ether bond or an amide bond. In some embodiments, the at least one agent is bound to the dendrite via an ether bond.

[0094] In some embodiments, the number of terminal sites on the dendrite may depend on the specific dendritic scaffold and its generation. For example, in some embodiments, the dendritic system is based on generation 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 PAMAM dendritic scaffolds, which have 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, and 4096 terminal sites, respectively. However, it should be understood that different dendritic scaffolds with different numbers of terminal sites in each generation can be used according to the present invention.

[0095] In some embodiments, all terminal sites of the dendrite contain hydroxyl groups. In some embodiments, each terminal site of the dendrite contains a hydroxyl or an amino group. In some embodiments, each terminal site of the dendrite complex contains a hydroxyl, an amino group, or an agent bound to the dendrite via an ether or amide bond. In some embodiments, each terminal site of the dendrite complex contains a hydroxyl group or an agent bound to the dendrite via an ether bond.

[0096] In some embodiments, at least 50% of the terminal sites on the dendritic complex contain hydroxyl groups (e.g., at least 50% of the terminal sites do not contain amines or agents). For example, in some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the terminal sites on the dendritic complex contain hydroxyl groups. In some embodiments, about 50 to 99%, about 60 to 99%, about 70 to 99%, about 80 to 99%, about 90 to 99%, about 95 to 99%, about 98 to 99%, about 70 to 95%, about 70 to 90%, about 80 to 95%, or about 80 to 90% of the terminal sites on the dendritic complex contain hydroxyl groups.

[0097] In some embodiments, one or more terminal sites on the dendritic complex contain an agent. In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20 or more terminal sites on the dendritic complex contain an agent. In some embodiments, at least 1% of the terminal sites on the dendritic complex contain an agent. For example, in some embodiments, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% of the terminal sites on the dendritic complex contain an agent. In some embodiments, about 1 to 50%, about 1 to 40%, about 1 to 25%, about 1 to 10%, about 5 to 50%, about 5 to 40%, about 5 to 25%, about 5 to 10%, about 10 to 50%, about 10 to 40%, or about 10 to 25% of the terminal sites on the dendrite contain an agent. In some embodiments, about 1%, about 2%, about 3%, about 4%, or about 5% of the terminal sites on the dendrite contain an agent. In some embodiments, less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, and less than 75% of the terminal sites on the dendrites contain an agent. In some embodiments, as described herein, the dendrite conjugate has an effective amount for targeting terminal functional groups (e.g., terminal hydroxyl groups) of a specific cell type, and also has an effective amount of an agent for therapeutic and / or imaging purposes. In some embodiments, the terminal sites of the dendrite conjugate can be assessed using proton nuclear magnetic resonance (1H NMR) or other analytical methods known in the art to determine the percentage of terminal sites containing an agent and / or terminal functional groups.

[0098] In some embodiments, the required drug loading may depend on factors including the selection of the drug, dendritic structure and size, and the cells or tissues to be treated. In some embodiments, the dendritic conjugate (e.g., a therapeutic compound) is about 0.01% by mass to about 45% by mass (m / m) of the drug (e.g., a therapeutic agent). In some embodiments, the dendritic conjugate (e.g., a therapeutic compound) is about 10% by mass to about 20% by mass of the drug (e.g., a therapeutic agent). In some embodiments, the dendritic conjugate is about 0.1% by mass to about 30% by mass, about 0.1% by mass to about 20% by mass, about 0.1% by mass to about 10% by mass, about 1% by mass to about 10% by mass, about 1% by mass to about 5% by mass, about 3% by mass to about 20% by mass, and about 3% by mass to about 10% by mass of the drug.

[0099] As described herein, in some embodiments, the dendritic conjugate may be characterized by a mass percentage of the agent (e.g., mass % (m / m)). In some embodiments, the mass percentage refers to the percentage of the molecular weight (Da) of the agent in the dendritic conjugate. In some embodiments, the mass percentage may be determined by the following general formula: (agent MW) / (conjugate MW) × 100. For example, in some embodiments, (agent MW) may be determined by calculating or approximating the molecular weight of the agent as a single molecule or compound (bound or unbound) and multiplying this value by the number of terminal sites of the agent present in the dendritic conjugate. In some embodiments, (agent MW) may be determined by calculating or approximating the sum of the atomic masses of all atoms forming the agent in the dendritic conjugate. The value of (agent MW) may be a fraction of the total molecular weight (conjugate MW) of the dendritic conjugate and multiplied by 100 to provide the mass percentage. In some embodiments, the mass percentage may be determined by experimental or empirical methods. For example, in some embodiments, the mass percentage can be determined using proton nuclear magnetic resonance (1H NMR) or other analytical methods known in this art.

[0100] In some embodiments, the dendrites have a diameter between about 1 nm and about 50 nm. For example, in some embodiments, the diameter is between about 1 nm and about 20 nm, between about 1 nm and about 10 nm, or between about 1 nm and about 5 nm. In some embodiments, the diameter is between about 1 nm and about 2 nm. In some embodiments, dendrites bound to relatively large agents (e.g., large proteins, such as antibodies) may have a diameter that increases this value by about 5 to 15 nm relative to unbound dendrites. In some embodiments, the dendrites have a molecular weight between about 500 Daltons (Da) and about 100,000 Da (e.g., between about 500 Da and about 50,000 Da, or between about 1,000 Da and about 20,000 Da).

[0101] In some embodiments, the dendritic systems of the combinations described herein include poly(aminoamine) (PAMAM) dendrites, polypropylene (POPAM) dendrites, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) dendrites, polyethyleneimine dendrites, polyisocyanate dendrites, polyester dendrites, iridium dendrites, aliphatic poly(ether) dendrites, aromatic polyether dendrites, or combinations thereof.

[0102] In some embodiments, the dendritic complex comprises PAMAM dendrites. In some embodiments, the PAMAM dendrites comprise different cores having amide-amine building blocks. In some embodiments, the PAMAM dendrites comprise any generation of carboxyl, amine, and / or hydroxyl terminal groups, including (but not limited to) first-generation, second-generation, third-generation, fourth-generation, fifth-generation, sixth-generation, seventh-generation, eighth-generation, ninth-generation, or tenth-generation PAMAM dendrites. In some embodiments, the PAMAM dendrite system comprises fourth-generation, fifth-generation, sixth-generation, seventh-generation, or eighth-generation hydroxyl-terminated PAMAM dendrites.

[0103] In some embodiments, the dendrites comprise a plurality of hydroxyl groups. Some exemplary high-density hydroxyl-containing dendrites include commercially available polyester dendritic polymers, such as hyperbranched 2,2-bis(hydroxy-methyl)propionic acid polyester polymers (e.g., hyperbranched bis-MPA polyester-64-hydroxy, fourth generation), and dendritic polyglycerol. In some embodiments, these high-density hydroxyl-containing dendritic systems are oligoethylene glycol (OEG)-like dendrites. For example, second-generation OEG dendrites (D2-OH-60) can be synthesized using efficient, robust, and atom-economical chemical reactions (such as Cu(I)-catalyzed alkyne-azide click chemistry and photocatalyzed thiol-ene click chemistry). High-density polyol dendrites can be achieved with very low yields in minimal reaction steps by using orthogonal supermonomers and supercore strategies, such as those described in WO 2019094952. In some embodiments, the dendritic backbone has non-breakable polyether bonds throughout the structure to prevent dendritic decomposition within the dendritic structure and allows such dendritic structures to be eliminated as a single entity within the structure (e.g., non-biodegradable).

[0104] In some embodiments, the dendritic complex comprises dendrites bound to one or more therapeutic agents, one or more imaging agents, and / or one or more targeting agents. It should be understood that in some embodiments, the terms "at least one" agent, "one or more" agents, and similar terms refer to a specific agent and not necessarily an amount of a specific agent bound to a dendrite. For example, in some embodiments, a dendritic complex comprising two agents refers to a dendrite having a first agent at one or more terminal positions and a second agent at one or more different terminal positions, wherein the first and second agents are different (e.g., chemically different). In some embodiments, the first and second agents may be suitable for similar purposes (e.g., both agents are therapeutic agents), or the first and second agents may be suitable for different purposes (e.g., the first agent is a therapeutic agent, and the second agent is a targeting agent). When used for similar purposes, the first and second agents are chemically different and therefore can provide different functions, such as different therapeutic agents targeting different receptors or biological pathways, or different imaging agents with different spectral properties.

[0105] In some embodiments, the agent of the dendritic complex (e.g., a therapeutic agent, imaging agent, targeting agent) is a peptide, protein, sugar, carbohydrate, oligonucleotide, nucleic acid, lipid, small molecule compound, or a combination thereof. In some embodiments, the agent is an antibody or an antigen-binding fragment of an antibody. In some embodiments, the agent is a nucleic acid or oligonucleotide encoding a protein, such as a DNA expression vector or mRNA. In some embodiments, the agent is an RNA silencing agent, such as siRNA, shRNA, or microRNA.

[0106] In some embodiments, the agent is a small molecule compound, such as a small molecule organic, organometallic, or inorganic compound. In some embodiments, the agent is a small molecule compound having a molecular weight of less than 2,000 Da, less than 1,500 Da, less than 1,000 Da, or less than 500 Da. In some embodiments, the agent is a small molecule compound having a molecular weight between about 100 and about 2,000 Da. For example, in some embodiments, the small molecule compound has a molecular weight between about 100 and about 1,500 Da, between about 100 and about 1,000 Da, between about 500 and about 2,000 Da, or between about 300 and about 700 Da.

[0107] In some embodiments, the non-released form of the dendritic conjugate described herein provides enhanced therapeutic efficacy compared to the releaseable form of the same conjugate. Therefore, in some embodiments, the agent is bound to the dendrite via a linker that is non-released (e.g., via ether and / or amide bonds) connected to both the dendrite and the agent. In some embodiments, the linker has a composition that is minimally releaseable (e.g., minimally cleavable) under physiological conditions.

[0108] In some embodiments, the dendritic system is bound to the drug via covalent bonds that are stable under in vivo conditions. In some embodiments, these covalent bonds are minimally cleavable upon administration to an individual and / or complete elimination from the body. For example, in some embodiments, less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or less than 0.1% of the total dendritic complex has cleaved the drug within 24, 48, or 72 hours after administration to an individual. In some embodiments, these covalent bonds comprise ether bonds. In some embodiments, the covalent bonds between the dendrites and the drug are not hydrolyzable or enzymatically cleavable bonds, such as ester bonds.

[0109] In some embodiments, the present invention provides a dendritic complex of formula (I): (I), wherein: D is a dendritic; X is O or NH; Y1 is a first group; Y2 is a second group; Z is a reagent; L is a linker; m is an integer from 16 to 4096, including the number itself; and n is an integer from 1 to 100, including the number itself.

[0110] In some embodiments, the D-system is selected from dendrites of the group consisting of: poly(aminoamine) (PAMAM) polymers, polypropylene (POPAM) polymers, polyethylene imine polymers, polyisocyanate polymers, polyester polymers, styrene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof.

[0111] In some embodiments, Y1 is not hydrolyzable under physiological conditions. In some embodiments, Y1 is a substituted alkyl group, a substituted alkenyl group, a substituted alkynyl group, or a covalent group, as desired. In some embodiments, Y1 is a substituted C1-20 alkyl group, as desired. In some embodiments, Y1 is an unsubstituted C1-10 alkyl group.

[0112] In some embodiments, the Y2 group is selected from the group consisting of: second amide, third amide, sulfonamide, second carbamate, third carbamate, carbonate, urea, methanol, disulfide, hydrazone, acetylhydrazine, ether, carbonyl, and combinations thereof. In some embodiments, the Y2 group is selected from the group consisting of: -CONH-, -CONRA-, -SO2NRA-, -OCONH-, -NHCOO-, -OCONRA-, -NRACOO-, -OC(=O)O-, -NHCONH-, -NRACONH-, -NHCONRA-, -NRCONRA-, -CHOH-, -CRAOH-, -C(=O)-, and -C(=O)RA-, wherein RA is an alkyl group, an aryl group, or a heterocyclic group that is substituted as desired.

[0113] In some embodiments, Z is a therapeutic agent, imaging agent, or targeting agent as described herein. In some embodiments, Z is a therapeutic agent or imaging agent. In some embodiments, the dendritic complex of formula (I) further comprises at least one targeting agent bound to the dendrite.

[0114] In some embodiments, the L-chain comprises a polymer and at least one linker. In some embodiments, the polymer is a polymeric polyol, peptide, or unsubstituted alkyl chain. In some embodiments, the polymer is a polymeric polyol selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, and polyvinyl alcohol. In some embodiments, the polymer is a peptide having at least two amino acids. In some embodiments, the polymer is a peptide having about 2 to about 40 amino acids (e.g., 2 to 25, 5 to 30, 10 to 25, or 5 to 15 amino acids). In some embodiments, the polymer is an unsubstituted alkyl chain. In some embodiments, the polymer is an unsubstituted C2-50 alkyl chain. In some embodiments, the polymer is an unsubstituted C2-30 alkyl chain. In some embodiments, the polymer is an unsubstituted C5-25 alkyl chain. In some embodiments, the polymer is a polymer as described elsewhere herein.

[0115] In some embodiments, at least one portion of L is a portion generated by a click reaction. In some embodiments, the at least one portion is a 5-membered heterocycle generated by an electrocyclization reaction (e.g., 3+2 cycloaddition or 4+2 cycloaddition) between reactive click chemistries (e.g., azides and terminal or cycloalkynes, dienes and dienophiles, thiols and alkenes) used to generate the complex. In some embodiments, the at least one portion is a diradical comprising 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisooxazolyl, or 1,4-dihydropyridazinyl.

[0116] In some embodiments, the present invention provides a dendritic complex of formula (II): (II), wherein: each instance of D, m, n, each instance of X, each instance of Y1 and each instance of Y2 are independently as defined with respect to formula (I); L1 and L2 are independently connectors as defined with respect to formula (I); and Z1 and Z2 are different agents.

[0117] In some embodiments, Z1 and Z2 are independently therapeutic agents, targeting agents, or imaging agents, provided that Z1 and Z2 are different (e.g., chemically different). In some embodiments, Z1 and Z2 are different therapeutic agents. In some embodiments, Z1 and Z2 are different therapeutic agents targeting different biological pathways involving a common pathology. In some embodiments, Z1 and Z2 are different therapeutic agents, and the dendritic complex of formula (II) further comprises at least one targeting agent bound to the dendrite. In some embodiments, Z1 and Z2 are different imaging agents. In some embodiments, Z1 is a therapeutic agent, and Z2 is a targeting agent. In some embodiments, Z1 is an imaging agent, and Z2 is a targeting agent. Synthesis Method

[0118] In some embodiments, the present invention relates to the discovery of novel techniques for synthesizing dendrites (e.g., functionalized dendrites, such as dendrites functionalized with a specified amount of PEG-alkyne to allow for further loading of agents via click-binding chemistry, such as azide-alkyne cycloaddition). Advantageously, the methods and compositions provided herein allow for large-scale synthesis (e.g., more than 10 grams, more than 40 to 50 grams, more than 100 grams) of dendrites (e.g., dendrites functionalized with a specified amount of PEG-alkyne) with high product uniformity (low polydispersity, e.g., less than 1.05) under milder conditions with adjusted stoichiometry compared to prior art.

[0119] In some embodiments, the present invention provides a method for synthesizing a functionalized dendrite of formula (IA): (IA), wherein: D refers to a dendrite selected from the group consisting of: poly(aminoamine) (PAMAM) polymers, polypropylene (POPAM) polymers, polyethylene imine polymers, polylylamine polymers, polyester polymers, acetic acid polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; X refers to NH; Y1 refers to an alkyl group, an alkenyl group, an alkyne group, or a covalent bond as desired; m refers to an integer from 16 to 4096, inclusive; and n refers to an integer from 1 to 100, inclusive; The method comprises: making a dendrite of formula (II-A): (II-A), wherein: D is a dendrite selected from the group consisting of: poly(aminoamine) (PAMAM) polymers, polypropylene (POPAM) polymers, polyethylene imine polymers, polyisocyanate polymers, polyester polymers, t-polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; t is an integer from 16 to 4096, inclusive; reacts with one or more amines under suitable conditions to form a functionalized dendrite of formula (IA), wherein each amine has the formula H2NR1, wherein R1 is a substituted alkyl group, a substituted alkenyl group, a substituted alkyne group, or a covalent bond, as desired.

[0120] In some embodiments, in dendrites of formula (IA) or (II-A), the D series is selected from dendrites of the group consisting of: poly(aminoamine) (PAMAM) polymers, polypropylene (POPAM) polymers, polyethylene imine polymers, polyisocyanate polymers, polyester polymers, styrene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof.

[0121] In some embodiments, Y1 is a substituted alkyl group, a substituted alkenyl group, a substituted alkyne group, or a covalent bond. In some embodiments, Y1 is a substituted alkyl group (e.g., a substituted C1-20 alkyl group). In some embodiments, X-NH and Y1 are substituted ethyl groups. In some embodiments, X-NH and Y1 are PEG-alkynyl-substituted ethyl groups. In some embodiments, X-NH and Y1 have the following formula: , where q is 1, 2, 3, 4, 5, or 6 (e.g., where q is 1, 2, or 3). In some embodiments, X-NH and Y1 have the following formula: , where q is 2. In some embodiments, Y1 is an unsubstituted alkyl group (e.g., an unsubstituted C1-10 alkyl group).

[0122] In some embodiments, m is an integer from 16 to 4096, including the stated number. In some embodiments, m is 16 to 20, 20 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 120 to 130, 130 to 140, 140 to 160, 160 to 180, 180 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, 450 to 500, 500 Integers up to 550, 550 to 600, 600 to 650, 650 to 700, 700 to 750, 750 to 800, 800 to 850, 850 to 900, 900 to 950, 950 to 1000, 1000 to 1500, 1500 to 2000, 2000 to 2500, 2500 to 3000, 3000 to 3500, 3500 to 4000, 4000 to 4100, or 4100 to 4200, including the number itself. In some embodiments, m is approximately 61 or 54 (e.g., 61 or 54).

[0123] In some embodiments, n is an integer from 1 to 100, including the stated number. In some embodiments, n is 1 to 3, 3 to 5, 1 to 5, 5 to 7, 7 to 9, 9 to 10, 10 to 12, 12 to 14, 14 to 16, 16 to 18, 18 to 20, 20 to 22, 22 to 24, 24 to 26, 26 to 28, 28 to 30, 30 to 32, 32 to 34, 34 to 36, 36 to 38, 38 to 40, 40 to 42, 42 to 44, 44 to 46, 46 to 48, 48 to 50, 50 Integers up to 52, 52 to 54, 54 to 56, 56 to 58, 58 to 60, 60 to 62, 62 to 64, 64 to 66, 66 to 68, 68 to 70, 70 to 72, 72 to 74, 74 to 76, 76 to 78, 78 to 80, 80 to 82, 82 to 84, 84 to 86, 86 to 88, 88 to 90, 90 to 92, 92 to 94, 94 to 96, 96 to 98, or 98 to 100, including the number itself. In some embodiments, n is about 3 or 10 (e.g., 3 or 10). In some embodiments, m is a value from 64 minus n. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In some embodiments, n is between about 1 to 2, 2 to 4, 4 to 5, 5 to 6, 6 to 8, 8 to 10, or 10 to 12; or between about 2 to 11 (e.g., 3, 4, 5, 8, 10). In some embodiments, n is about 3 or 10 (e.g., 3 or 10). In some embodiments, n is 3 and m is 61. In some embodiments, n is 10 and m is 54. In some embodiments, m is about 61 or 54 (e.g., 61 or 54).

[0124] In some embodiments, in the synthetic method described herein, a dendrite of formula (II-A) is provided: (II-A), wherein D is a dendrite selected from the group consisting of: poly(aminoamine) (PAMAM) polymers, polypropylene (POPAM) polymers, polyethyleneimine polymers, polylylamine polymers, polyester polymers, t-polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; t is an integer from 16 to 4096, inclusive; and one or more amines, wherein each amine has the formula H2NR1, wherein R1 is a substituted alkyl group, a substituted alkenyl group, a substituted alkynyl group, or a covalent bond, as desired.

[0125] In some embodiments, in formula (II-A), D refers to a dendrite as described herein. In some embodiments, the synthetic method includes reacting a dendrite of formula (II-A), or a salt, solvate, hydrate, stereoisomer, polymorph, tautomer, or isotopically enriched form (e.g., an isotopically labeled derivative). Formula (II-A) includes a substituent t. In some embodiments, t is an integer from 16 to 4096, inclusive. In some embodiments, t is 16 to 20, 20 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 120 to 130, 130 to 140, 140 to 160, 160 to 180, 180 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, 450 to 500, 500 Integers up to 550, 550 to 600, 600 to 650, 650 to 700, 700 to 750, 750 to 800, 800 to 850, 850 to 900, 900 to 950, 950 to 1000, 1000 to 1500, 1500 to 2000, 2000 to 2500, 2500 to 3000, 3000 to 3500, 3500 to 4000, 4000 to 4100, or 4100 to 4200, including the number itself. In some embodiments, n refers to t. In some embodiments, t refers to n. In some embodiments, in the dendrite of formula (II-A), t is the same as n. In some embodiments, in the dendrite of formula (II-A), t refers to 64. In some embodiments, t is an integer from 20 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, including the stated number.

[0126] In some embodiments, the synthesis method includes reacting a dendrite of formula (II-A) with one or more amines. In some embodiments, the synthetic method includes reacting a dendrite of formula (II-A) with one or more amines, wherein at least one example of the one or more amines is a compound of formula (Z), (Z), or a salt, solvate, hydrate, stereoisomer, polymorph, tautomer, or isotopically enriched form (e.g., an isotopically labeled derivative), wherein LA is a linker, and R1A is a halogen, a substituted acetyl group, a substituted alkenyl group, a substituted alynyl group, a substituted acetylene group, a substituted carbocyclic group, a substituted heterocyclic group, a substituted aryl group, a substituted heteroaryl group, -CH(=N)(OH)RD1, -CN, -NO2, -ORD1, -N(RD1a)2, -SO2ORD1, or -SRD1, and RD1 and RD1a are as defined herein. In some embodiments, the linker LA comprises a polymer. In some embodiments, the linker LA comprises an alkyl chain (e.g., C1-100,000 alkyl groups) wherein the chain is the shortest path between -NH2 and R1A, excluding hydrogen atoms and substituents. In some embodiments, the linker LA chain comprises up to 5,000 to 7,000; 7,000 to 9,000; 9,000 to 10,000; 10,000 to 12,000; 100,000 to 120,000; 120,000 to 150,000; or 150,000 to 200,000 consecutive covalently bonded atoms or lengths, excluding hydrogen atoms and substituents. In some embodiments, LA is an all-carbon, substituted or unsubstituted C1-200,000 hydrocarbon chain, which, as the shortest path between -NH2 and R1A, excludes hydrogen atoms and substituents. In some embodiments, any atom in LA may be substituted. In some embodiments, all atoms in the linker LA are unsubstituted. In some embodiments, all carbon atoms in the linker are unsubstituted. In some embodiments, at least one chain atom of the LA hydrocarbon chain is independently substituted with -C(=O)-, -O-, -NRb-, -S-, or a cyclic portion, wherein Rb is independently hydrogen, a substituted or unsubstituted C1-6 alkyl group, or a nitrogen-protecting group. In some embodiments, at least one chain atom of the LA hydrocarbon chain is independently substituted with amide, isohydroxamic acid, ether, n-alkyl, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, ethyl carbamate, oxime, a carbamocyclic group as desired, a heterocyclic group as desired, an aryl group as desired, and / or a heteroaryl group as desired. In some embodiments, at least one chain atom of the LA hydrocarbon chain is independently replaced by amide, isohydroxamic acid, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, ethyl carbamate and / or oxime.In some embodiments, the linker LA comprises a polyethylene glycol portion (e.g., having the formula where q is an integer between 1 and 100,000, inclusive), and at least one chain atom of the LA hydrocarbon chain is independently substituted with an amide, isohydroxamic acid, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, ethyl carbamate, and / or oxime. In some embodiments, the linker LA comprises a portion generated by a click reaction. In some embodiments, at least one portion generated by a click reaction is a 5-membered heterocycle generated by an electrocyclization reaction (e.g., 3+2 cycloaddition or 4+2 cycloaddition) between reactive click chemistries (e.g., azides and terminal or cycloalkynes, dienes and dienophiles, thiols and alkenes) to generate the complex. In some embodiments, at least one portion generated by the click reaction comprises a diradical of 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisooxazolyl, or 1,4-dihydropyridazinyl. In some embodiments, the linker LA has the following formula: , wherein ln indicates the link to -NH2 in the compound of formula (Z), and l1 indicates the link to R1A. In some embodiments, the synthetic method comprises reacting a dendrite of formula (II-A) with one or more amines, wherein at least one example of the one or more amines is a compound of formula (A): (A), or a salt, solvate, hydrate, stereoisomer, polymorph, tautomer, or isotopically enriched form (e.g., isotopically labeled derivative), wherein p, q, r, W, and R1A are as defined herein.In some embodiments, the synthetic method includes reacting a dendrite of formula (II-A) with one or more amines, wherein at least one example of the one or more amines is a compound of formula (A): (A), wherein: R1A is a halogen, a substituted amide group, a substituted alkenyl group, a substituted alynyl group, a substituted acetylene group, a substituted carbocyclic group, a substituted heterocyclic group, a substituted aryl group, a substituted heteroaryl group, or -CH (=N). (OH)RD1, -CN, -NO2, -ORD1, -N(RD1a)2, -SO2ORD1, or -SRD1, wherein RD1 is independently hydrogen-containing, optionally substituted with an amide group, optionally substituted with an alkyl group, optionally substituted with an alkenyl group, optionally substituted with an alkynyl group, optionally substituted with an alkynyl group, optionally substituted with a carbocyclic group, optionally substituted with a heterocyclic group, optionally substituted with an aryl group, optionally substituted with a heteroaryl group, or when attached to an oxygen atom, is an oxygen-protecting group, or when attached to a sulfur atom, is a sulfur-protecting group; wherein RD1a is independently hydrogen-containing, optionally substituted with an amide group, optionally substituted with an alkenyl group, optionally substituted with a sulfur atom, or when attached to an oxygen atom, is a sulfur-protecting group; wherein RD1a is independently hydrogen-containing, optionally substituted with an amide group, optionally substituted with an alkenyl group, optionally substituted with an alkenyl group, optionally substituted with a sulfur atom, or optionally substituted with a sulfur atom. The alkyl group to be substituted, the alkenyl group to be substituted, the alkynyl group to be substituted, the carbocyclic group to be substituted, the heterocyclic group to be substituted, the aryl group to be substituted, the heteroaryl group to be substituted, or the nitrogen-protecting group; or, if desired, two instances of RD1a together with their intermediate atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring; where valence permits, W is -O- or -CH2-; p is 0, 1, 2, or 3; q is an integer from 1 to 100,000, inclusive; and r is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, R1A is a halogen (e.g., F, Cl, Br, or I). In some embodiments, R1A is a acetyl group to be substituted (e.g., -C(=O)Me). In some embodiments, R1A is an alkyl group to be substituted (e.g., substituted or unsubstituted C1-6 alkyl). In some embodiments, R1A is a substituted or unsubstituted methyl group. In some embodiments, R1A is a substituted or unsubstituted ethyl group. In some embodiments, R1A is a substituted or unsubstituted propyl group. In some embodiments, R1A is an alkenyl group that needs to be substituted (e.g., a substituted or unsubstituted C2-6 alkenyl group). In some embodiments, R1A is an alkynyl group that needs to be substituted (e.g., a substituted or unsubstituted C2-6 alkynyl group). In some embodiments, R1A is a carbocyclic group that needs to be substituted (e.g., a substituted or unsubstituted 3- to 7-membered monocyclic carbocyclic group comprising zero, one, or two double bonds in the carbocyclic system). In some embodiments, R1A is a heterocyclic group that needs to be substituted (e.g., a substituted or unsubstituted 5- to 10-membered monocyclic or bicyclic heterocycle, wherein one or two atoms in the heterocycle are independently nitrogen, oxygen, or sulfur).In some embodiments, R1A is a substituted aryl group (e.g., a substituted or unsubstituted 6- to 10-membered aryl group). In some embodiments, R1A is benzyl. In some embodiments, R1A is a substituted or unsubstituted phenyl group. In some embodiments, R1A is a substituted heteroaryl group (e.g., a substituted or unsubstituted 5- to 6-membered monocyclic heteroaryl group, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or a substituted or unsubstituted 9- to 10-membered bicyclic heteroaryl group, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, R1A is -CH(=N)(OH)RD1, -CN, -NO2, -ORD1, -N(RD1a)2, -SO2ORD1, or -SRD1, wherein RD1 and RD1a are as defined herein. In some embodiments, R1A-based -CH(=N)(OH)RD1 (e.g., -CH(=N)(OH) (C1-6 alkyl group substituted as desired)). In some embodiments, R1A-based -CH(=N)(OH) (C1-6 alkyl group substituted as desired with a polyethylene glycol linker). In some embodiments, R1A-based -CN. In some embodiments, R1A-based -NO2. In some embodiments, R1A-based -ORD1 (e.g., -OH or -OMe). In some embodiments, R1A-based -N(RD1a)2 (e.g., -NMe2). In some embodiments, R1A-based -SO2ORD1 (e.g., -SO2O (alkyl group substituted as desired)). In some embodiments, R1A-based -SRD1 (e.g., -SMe). In some embodiments, R1A-based -CH(=N) (OH)RD1, -CN, -NO2, -ORD1, -N(RD1a)2, -SO2ORD1, or -SRD1, wherein RD1 is independently a hydrogen atom, an amide group as required, an alkyl group as required, an alkenyl group as required, an alkynyl group as required, a carbocyclic group as required, a heterocyclic group as required, an aryl group as required, a heteroaryl group as required, or an oxygen protecting group when attached to an oxygen atom, or when attached to a sulfur atom. The RD1a is a sulfur-protected group; and each occurrence of RD1a is independently hydrogen, substituted acetyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted carbocyclic, substituted heterocyclic, substituted aryl, substituted heteroaryl, or nitrogen-protected group; or, if desired, two instances of RD1a together with their intermediate atoms form a substituted or unsubstituted heterocycle or a substituted or unsubstituted heteroaryl ring. In some embodiments, R1A is bromine, alkyne, acetylene, olefin, aldehyde, amine, COOH, hydroxyl, carboxyl (e.g., dibenzocyclooctylene or DBCO), thiol, sulfonate, or -CN.In some embodiments, R1A is a click reaction handle partner (e.g., the click chemical handle shown in Table A, or in schemes 1 to 19 of Example 6). In some embodiments, the click handle, click partner, and click reaction handle partner are used interchangeably. In some embodiments, the click reaction is: Cu (I)-catalyzed alkyne-azide click and photocatalyzed thiol-ene click chemistry; or electrocyclization click reaction (e.g., 3+2 cycloaddition or 4+2 cycloaddition). Table A: Exemplary click chemistry reactions and click chemical handles (a) ketone / aldehyde condensation; (b) Staudinger reaction; (c) 1,3 bipolar cycloaddition (top: copper-catalyzed alkyne-azide cycloaddition (CuAAC); bottom: strain-promoted [3+2] azide-alkyne cycloaddition (SPAAC)); (d) anti-electron-demanding Diels-Alder reaction. In some embodiments, one of R1A and R2 is -N3 and the other of R1A and R2 is dibenzocyclooctylene. In some embodiments, one of R1A and R2 and the other of R1A and R2 are -SH. In some embodiments, one of R1A and R2 is tetrazine and the other of R1A and R2 is transcyclooctene. In some embodiments, one of R1A and R2 and the other of R1A and R2 are... In some embodiments, one of R1A and R2 is -SH and the other of R1A and R2 are... In some embodiments, one of R1A and R2 is -SH and the other of R1A and R2 are...

[0127] In some embodiments, RD1 is hydrogen. In some embodiments, RD1 is a amide group that needs to be substituted (e.g., -C(=O)Me). In some embodiments, RD1 is an alkyl group that needs to be substituted (e.g., substituted or unsubstituted C1-6 alkyl). In some embodiments, RD1 is a methyl group that needs to be substituted. In some embodiments, RD1 is an ethyl group that needs to be substituted. In some embodiments, RD1 is a propyl group that needs to be substituted. In some embodiments, RD1 is an alkenyl group that needs to be substituted (e.g., substituted or unsubstituted C2-6 alkenyl). In some embodiments, RD1 is an alkynyl group that needs to be substituted (e.g., a substituted or unsubstituted 3- to 7-member monocyclic carbocyclic group comprising zero, one, or two double bonds in the carbocyclic system). In some embodiments, RD1 is a heterocyclic group that needs to be substituted (e.g., a substituted or unsubstituted 5- to 10-member monocyclic or bicyclic heterocycle, wherein one or two atoms in the heterocycle are independently nitrogen, oxygen, or sulfur). In some embodiments, RD1 is an aryl group that needs to be substituted (e.g., a substituted or unsubstituted 6- to 10-member aryl group). In some embodiments, RD1 is benzyl. In some embodiments, RD1 is a substituted or unsubstituted phenyl group. In some embodiments, RD1 is a heteroaryl group that needs to be substituted (e.g., a substituted or unsubstituted 5- to 6-member monocyclic heteroaryl group, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or a substituted or unsubstituted 9- to 10-member bicyclic heteroaryl group, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, RD1 is an oxygen protecting group when attached to an oxygen atom. In some embodiments, RD1 is a sulfur protecting group when attached to a sulfur atom.

[0128] In some embodiments, at least one example of RD1a is hydrogen. In some embodiments, at least one example of RD1a is a substituted acetyl group (e.g., -C(=O)Me). In some embodiments, at least one RD1a is an alkyl group (e.g., substituted or unsubstituted C1-6 alkyl). In some embodiments, at least one example of RD1a is a substituted or unsubstituted methyl group. In some embodiments, at least one example of RD1a is a substituted or unsubstituted ethyl group. In some embodiments, at least one example of RD1a is a substituted or unsubstituted propyl group. In some embodiments, at least one example of RD1a is an alkenyl group (e.g., substituted or unsubstituted C2-6 alkenyl). In some embodiments, at least one example of RD1a is an alkyne group (e.g., substituted or unsubstituted C2-6 alkyne). In some embodiments, at least one example of RD1a is a carbocyclic group that requires substitution (e.g., a substituted or unsubstituted 3- to 7-member monocyclic carbocyclic group comprising zero, one, or two double bonds in a carbocyclic system). In some embodiments, at least one example of RD1a is a heterocyclic group that requires substitution (e.g., a substituted or unsubstituted 5- to 10-member monocyclic or bicyclic heterocycle, wherein one or two atoms in the heterocycle are independently nitrogen, oxygen, or sulfur). In some embodiments, at least one example of RD1a is an aryl group that requires substitution (e.g., a substituted or unsubstituted 6- to 10-member aryl group). In some embodiments, at least one example of RD1a is benzyl. In some embodiments, at least one example of RD1a is a substituted or unsubstituted phenyl group. In some embodiments, at least one instance of RD1a is a heteroaryl group that is substituted as required (e.g., a substituted or unsubstituted 5- to 6-membered monocyclic heteroaryl group, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or a substituted or unsubstituted 9- to 10-membered bicyclic heteroaryl group, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, at least one instance of RD1a is a nitrogen protecting group (e.g., benzyl (Bn), tributyl carbonate (BOC or Boc), benzoyl carbamate (Cbz), methyl 9-methyl carbonate (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl, or p-toluenesulfonamide (Ts)).In some embodiments, two instances of RD1a, together with their intermediate atoms, form a heterocycle that is substituted as needed (e.g., a substituted or unsubstituted 5- to 10-member monocyclic or bicyclic heterocycle, wherein one or two atoms in the heterocycle are independently nitrogen, oxygen, or sulfur) or a heteroaryl ring that is substituted as needed (e.g., a substituted or unsubstituted 5- to 6-member monocyclic heteroaryl ring, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or a substituted or unsubstituted 9- to 10-member bicyclic heteroaryl ring, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur).

[0129] In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3.

[0130] In some embodiments, W is -O-. In some embodiments, W-O- and q- are integers between 1 and 100,000, including the number itself. In some embodiments, W is -CH2-. In some embodiments, W-CH2- and q- are integers between 1 and 10,000, including the number itself.

[0131] In some embodiments, q is an integer between 1 and 50, including the number itself. In some embodiments, q is an integer between 1 and 100, including the number itself. In some embodiments, q is an integer between 1 and 5,000, including the number itself. In some embodiments, q is an integer between 1 and 10,000, including the number itself. In some embodiments, q is an integer between 1 and 50,000, including the number itself. In some embodiments, q is an integer between 1 and 100,000, including the number itself. In some embodiments, q is between 1 and 25, 25 and 50, 50 and 75, 75 and 100, 100 and 125, 125 and 150, 150 and 175, 175 and 200, 200 and 225, 225 and 250, 250 and 275, 275 and 300, 300 and 325, 325 and 350, 350 and 375, 375 and 400, 400 and 425, 400 and 450, 450 and 500, 500 and 550, 550 and 600, 600 and 650, 650 and 700, 700 and 750, 750 and 800, 800 and 850, 850 and 900. Integers between 0 and 1000, 1000 and 1500, 1500 and 2000, 2000 and 2500, 2500 and 3000, 3000 and 3500, 3500 and 4000, 4000 and 4500, 4500 and 5000, 5000 and 6000, 6000 and 7000, 7000 and 8000, 8000 and 9000; 9000 and 10,000; 10,000 and 12,000; 100,000 and 120,000; 120,000 and 150,000; 150,000 and 175,000; or 175,000 and 200,000.

[0132] In some embodiments, the synthetic method includes reacting a dendrite of formula (II-A) with one or more amines, wherein each amine has the formula H2NR1, and R1 is as defined herein. In some embodiments, at least one example of R1 is a substituted alkyl group, a substituted alkenyl group, a substituted alkyne group, or a covalent bond, as desired. In some embodiments, at least one example of R1 is a substituted alkyl group (e.g., a substituted C1-20 alkyl group, as desired). In some embodiments, each example of R1 is Y1 of one or more amines, wherein each amine has the formula H2NR1. In some embodiments, at least one example of R1 is an ethyl group, as desired. In some embodiments, at least one example of R1 is an ethyl group substituted with a PEG-alkynyl group. In some embodiments, at least one example of R1 has the following formula: , wherein q is 1, 2, 3, 4, 5, or 6 (e.g., wherein q is 1, 2, or 3). In some embodiments, at least one example of R1 has the following formula: , wherein q is 2. In some embodiments, at least one instance of R1 is an unsubstituted alkylene group (e.g., an unsubstituted C1-10 alkylene group).

[0133] In some embodiments, the amine is of the following formula: PEG-alkyne: , wherein q is 1, 2, 3, 4, 5, or 6. In some embodiments, the amine is of the following formula: PEG-alkyne: , wherein q is 1, 2, or 3. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, q is 4. In some embodiments, q is 5. In some embodiments, q is 6. In some embodiments, at least one example of the one or more amines is of the following formula: PEG-alkyne: . In some embodiments, at least one example of the one or more amines is of the following formula: ethanolamine: . In some embodiments, the synthetic method described herein includes two different amines. In some embodiments, one of the one or more amines is of the following formula: PEG-alkyne: , wherein q is 1, 2, 3, 4, 5, or 6; and the other amine is an ethanolamine (e.g., ). In some embodiments, one of the one or more amines is of the following formula: PEG-alkyne: ; and the other amine is an ethanolamine (e.g., ). In some embodiments, in the synthetic method described herein, the dendritic system of formula (II-A) reacts with both ethanolamine and the following PEG-alkyne:

[0134] In some embodiments, the ratio of the first amine (e.g., ethanolamine) to the second amine (e.g., PEG-acetylene of the following formula: , where q is 1, 2, 3, 4, 5, or 6) is about 10:1, 9.8:1, 9.5:1, 9.4:1, 9.3:1, 9.2:1, 9.1:1, or 9:1. In some embodiments, the ratio of the ethanolamine to the PEG-acetylene of the following formula: (e.g., ) is 9.4:1. In some embodiments, the ratio of the ethanolamine to the PEG-acetylene of the following formula: is 9.42:1.

[0135] In some embodiments, the ratio of the first amine (e.g., ethanolamine) to the second amine (e.g., PEG-acetylene of the following formula: , wherein q is 1, 2, 3, 4, 5, or 6) is about 3:1, 3.0:1, 2.8:1, 2.6:1, 2.4:1, 2.3:1, 2.2:1, 2.1:1, or 2.0:1. In some embodiments, the ratio of the ethanolamine to the PEG-acetylene of the following formula: (e.g., ) is 2.2:1. In some embodiments, the ratio of the ethanolamine to the PEG-acetylene of the following formula: 2.23:1. In some embodiments, the ratio of the ethanolamine to the PEG-acetylene of the following formula: , wherein q is 1, 2, 3, 4, 5, or 6 (e.g., the PEG-acetylene has ) is 2.2:1 or 9.4:1.

[0136] In some embodiments, the ratio of an amine (e.g., the following PEG-acetylene:) to a dendrite of formula (II-A) is about 150:1. In some embodiments, the ratio of an amine (e.g., the following PEG-acetylene:) to a dendrite of formula (II-A) (where D is PAMAM and t is about 50 to 60, 60 to 70, or 70 to 80, inclusive) is about 150:1. In some embodiments, the ratio of an amine (e.g., the following PEG-acetylene:) to a dendrite of formula (II-A) is about 150:1; the ratio of the ethanolamine to the PEG-acetylene is 9.42:1; and the n-series is 3.

[0137] In some embodiments, the ratio of an amine (e.g., the following PEG-alkynylene:) to a dendritic of formula (II-A), wherein D is PAMAM and t is an integer of about 50 to 60, 60 to 70, or 70 to 80, inclusive) is about 152:1, 150:1, 149:1, 148:1, 145:1, 142:1, 140:1, or 138:1. In some embodiments, the ratio of an amine (e.g., the following PEG-alkynylene:) to a dendritic of formula (II-A), wherein D is PAMAM and t is an integer of about 50 to 60, 60 to 70, or 70 to 80, inclusive) is about 152:1, 150:1, 149:1, 148:1, 145:1, 142:1, 140:1, or 138:1; and n is 3. In some embodiments, the ratio of an amine (e.g., the following PEG-acetylene:) to a dendritic of formula (II-A), wherein D is PAMAM and t is an integer of about 50 to 60, 60 to 70, or 70 to 80, inclusive) is about 152:1, 150:1, 149:1, 148:1, 145:1, 142:1, 140:1, or 138:1; the ratio of the ethanolamine to the PEG-acetylene is 9.42:1; and n is 3. In some embodiments, the ratio of an amine (e.g., the following PEG-acetylene:) to a dendritic of formula (II-A), wherein D is PAMAM and t is an integer of about 50 to 60, 60 to 70, or 70 to 80, inclusive) is about 150:1; the ratio of the ethanolamine to the PEG-acetylene is 9.42:1; and n is 3.

[0138] In some embodiments, D-series PAMAM. In some embodiments, the D-series is derived from 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, or 9.5 generation PAMAM, wherein the PAMAM dendrites carry carboxymethyl groups. In some embodiments, the ratio of an amine (e.g., the following PEG-acetylene:) to the (II-A) dendrite is about 500:1. In some embodiments, the ratio of an amine (e.g., the following PEG-acetylene:) to the (II-A) dendrite is about 500:1; the ratio of the ethanolamine to the PEG-acetylene is 2.2:1; and n-series 10. In some embodiments, the ratio of an amine (e.g., the following PEG-acetylene:) to the (II-A) dendrite is 495:1. In some embodiments, the ratio of an amine (e.g., the following PEG-alkyne:) to a dendritic of formula (II-A) (where D is PAMAM and t is an integer of about 50 to 60, 60 to 70, or 70 to 80, inclusive) is about 505:1, 503:1, 502:1, 501:1, 500:1, 499:1, 498:1, 497:1, 496:1, 495:1, 494:1, 493:1, 492:1, 491:1, or 490:1. In some embodiments, the ratio of an amine (e.g., the following PEG-alkyne:) to a dendritic of formula (II-A) (where D is PAMAM and t is an integer of about 50 to 60, 60 to 70, or 70 to 80, inclusive) is about 495:1. In some embodiments, the ratio of an amine (e.g., the following PEG-alkynylene:) to a dendrite of formula (II-A) (where D is PAMAM and t is an integer from about 50 to 60, 60 to 70 or 70 to 80, inclusive) is about 505:1, 503:1, 502:1, 501:1, 500:1, 499:1, 498:1, 497:1, 496:1, 495:1, 494:1, 493:1, 492:1, 491:1 or 490:1 (e.g., about 495:1); and n is 10. In some embodiments, the ratio of an amine (e.g., the following PEG-acetylene:) to a dendritic of formula (II-A), wherein D is PAMAM and t is an integer of about 50 to 60, 60 to 70, or 70 to 80, inclusive) is about 495:1; and n is 10. In some embodiments, the ratio of an amine (e.g., the following PEG-acetylene:) to a dendritic of formula (II-A), wherein D is PAMAM and t is an integer of about 50 to 60, 60 to 70, or 70 to 80, inclusive) is about 500:1; the ratio of the ethanolamine to the PEG-acetylene is 2.2:1; and n is 10.

[0139] In some embodiments, the molecular weight of the dendrite of formula (II-A) is about 12,500 g / mol, about 12,425 g / mol, about 12,420 g / mol, or about 12,418 g / mol or about 12,415 g / mol. In some embodiments, the molecular weight of the dendrite of formula (II-A) is between about 12,000 and 12,500 g / mol.

[0140] In some embodiments, the dendrite of formula (II-A) has the following formula: (PAMAM G3.5).

[0141] In some embodiments, the polydispersity value of the functionalized dendrites of formula (IA) is low, for example, from about 1.00 to about 1.05 (e.g., about 1.03). In some embodiments, the polydispersity value of the functionalized dendrites of formula (IA) is about 1.03.

[0142] In some embodiments, functionalized dendritic systems of formula (IA) in quantities exceeding 10 to 20 grams, 20 to 30 grams, 30 to 40 grams, 40 to 50 grams, 50 to 60 grams, 60 to 70 grams, 70 to 80 grams, 80 to 90 grams, 90 to 100 grams, 100 to 110 grams, 110 to 120 grams, 120 to 130 grams, 130 to 140 grams, 150 to 160 grams, 160 to 180 grams, and 180 to 200 grams are synthesized by the synthetic methods described herein. In some embodiments, functionalized dendritic systems of formula (IA) in quantities exceeding 30 grams or exceeding 50 to 100 grams (e.g., 40 grams, 160 grams) are synthesized by the synthetic methods described herein.

[0143] In some embodiments, in a method for synthesizing a functionalized dendrite of formula (IA), the method includes reacting a dendrite of formula (II-A): (II-A) with one or more amines, wherein each amine has the formula H2NR1; under suitable conditions to form a functionalized dendrite of formula (IA); such suitable conditions include a reaction solvent and reacting the reactants at room temperature (e.g., from about 19°C to about 23°C). In some embodiments, such suitable conditions include a reaction solvent that is a protic solvent. For example, the reaction solvent may be a protic solvent, or a mixture of protic and non-protic solvents. In some embodiments, the protic solvent is an alcohol, such as methanol, ethanol, or isopropanol. In some embodiments, the protic solvent is methanol. In some embodiments, the alcohol is anhydrous, or may contain water. In some embodiments, the reaction solvent may include dichloromethane.

[0144] In some embodiments, suitable conditions for the functionalized dendrite of the synthetic formula (IA) include reacting the reactants at about 19°C to about 23°C (e.g., about 20°C to about 22°C). In some embodiments, suitable conditions include reacting the reactants at about 18 to 19°C, 19 to 20°C, 20 to 21°C, 21 to 22°C, or 22 to 23°C. Suitable conditions include a methanol reaction solvent and reacting the reactants at about 20°C.

[0145] In some embodiments, the conditions suitable for the functionalized dendrite of synthetic formula (IA) include a first step of stirring one or more amines, each amine having the formula H2NR1, together with a dendrite of formula (II-A) and a reaction solvent (e.g., an alcohol, such as methanol) at 0°C for about 1 to 5 hours (e.g., 2 hours); followed by a second step of stirring at about 19°C to about 23°C (e.g., 20°C) for about 3 to 8 days (e.g., 6 days). In some embodiments, the functionalized dendrite of synthetic formula (IA) has the following formula: or.

[0146] In some embodiments, the method for synthesizing the functionalized dendrite of formula (IA) further comprises reacting with a compound of formula (B): (B), wherein: R2 is a halogen, an acetyl group to be substituted as desired, an alkenyl group to be substituted as desired, an alkynyl group to be substituted as desired, an acetylated group to be substituted as desired, a carbocyclic group to be substituted as desired, a heterocyclic group to be substituted as desired, an aryl group to be substituted as desired, a heteroaryl group to be substituted as desired, -N3, -CH(=N) (OH)RD1, -CN, -NO2, -ORD1, -N(RD1a)2, -SO2ORD1, or -SRD1, wherein RD1 is independently hydrogen-based, and optionally substituted with an amide group, an alkyl group, an alkenyl group, an alkynyl group, a carbocyclic group, a heterocyclic group, an aryl group, or a heteroaryl group, or an oxygen-protecting group when attached to an oxygen atom, or a sulfur-protecting group when attached to a sulfur atom; wherein RD1a is independently hydrogen-based, optionally substituted with an amide group, an alkyl group, or an alkenyl group, each occurrence being optionally hydrogen-based. Substituted alkynyl groups, substituted carbocyclic groups, substituted heterocyclic groups, substituted aryl groups, substituted heteroaryl groups, or nitrogen-protecting groups as required; or, as required, two instances of RD1a together with their equivalent intermediate atoms to form substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl rings; provided that R1A and R2 are reaction partners; LB is an alkyl linker in which one or more chain atoms of the hydrocarbon chain are independently replaced by amides, esters, isohydroxamic acids, ethers, carbonates, carbamates, hydrazones, thioethers, thioesters, disulfides, orthoesters, ethyl carbamates, or oximes or cyclic components; and T is a therapeutic agent.

[0147] In some embodiments, the linker LB comprises a polymer. In some embodiments, the linker LB comprises an alkyl chain (e.g., C1-100,000 alkyl), wherein the chain is the shortest path between R2 and T, excluding hydrogen atoms and substituents. In some embodiments, the linker LB chain comprises up to 5,000 to 7,000; 7,000 to 9,000; 9,000 to 10,000; 10,000 to 12,000; 100,000 to 120,000; 120,000 to 150,000; or 150,000 to 200,000 consecutive covalently bonded atoms or lengths, excluding hydrogen atoms and substituents. In some embodiments, the LB is an all-carbon, substituted or unsubstituted C1-200,000 hydrocarbon chain, which, as the shortest path between R2 and T, excludes hydrogen atoms and substituents. In some embodiments, any atom in the LB may be substituted. In some embodiments, none of the atoms in the linker LB are substituted. In some embodiments, none of the carbon atoms in the linker are substituted. In some embodiments, at least one chain atom of the LB hydrocarbon chain is independently substituted with -C(=O)-, -O-, -NRb-, -S-, or a cyclic moiety, wherein Rb is independently hydrogen, a substituted or unsubstituted C1-6 alkyl group, or a nitrogen-protecting group. In some embodiments, at least one chain atom of the LB hydrocarbon chain is independently substituted with amide, isohydroxamic acid, ether, n-alkyl, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, ethyl carbamate, oxime, a carbamocyclic group as desired, a heterocyclic group as desired, an aryl group as desired, and / or a heteroaryl group as desired. In some embodiments, at least one chain atom of the LB hydrocarbon chain is independently substituted with amide, isohydroxamic acid, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, ethyl carbamate, and / or oxime. In some embodiments, the linker LB comprises an alkyl group (e.g., having the formula where q is an integer between 1 and 10,000 or between 1 and 100,000, inclusive). In some embodiments, the linker LB comprises a polyethylene glycol group (e.g., having the formula where q is an integer between 1 and 100,000, inclusive), and at least one chain atom of the LB hydrocarbon chain is independently substituted with amide, isohydroxamic acid, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, ethyl carbamate, and / or oxime. In some embodiments, the linker LB comprises a portion generated by a click reaction. In some embodiments, at least one portion generated by the click reaction is a 5-membered heterocycle produced by an electrocyclization reaction (e.g., 3+2 cycloaddition or 4+2 cycloaddition) between reactive click chemistries (e.g., azides and terminal or cycloalkynes, dienes and dienophiles, thiols and alkenes) used to generate the complex.In some embodiments, at least one portion generated by the click reaction is a diradical comprising 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisooxazolyl, or 1,4-dihydropyridazinyl.

[0148] In some embodiments, R2 is a halogen (e.g., F, Cl, Br, or I). In some embodiments, R2 is an acetyl group that needs to be substituted (e.g., -C(=O)Me). In some embodiments, R2 is an alkyl group that needs to be substituted (e.g., substituted or unsubstituted C1-6 alkyl). In some embodiments, R2 is a substituted or unsubstituted methyl group. In some embodiments, R2 is a substituted or unsubstituted ethyl group. In some embodiments, R2 is a substituted or unsubstituted propyl group. In some embodiments, R2 is an alkenyl group that needs to be substituted (e.g., substituted or unsubstituted C2-6 alkenyl). In some embodiments, R2 is an acetyl group that needs to be substituted. In some embodiments, R2 is a carbocyclic group that requires substitution (e.g., a substituted or unsubstituted 3- to 7-membered monocyclic carbocyclic group comprising zero, one, or two double bonds in the carbocyclic system). In some embodiments, R2 is a heterocyclic group that requires substitution (e.g., a substituted or unsubstituted 5- to 10-membered monocyclic or bicyclic heterocycle, wherein one or two atoms in the heterocycle are independently nitrogen, oxygen, or sulfur). In some embodiments, R2 is an aryl group that requires substitution (e.g., a substituted or unsubstituted 6- to 10-membered aryl group). In some embodiments, R1A is benzyl. In some embodiments, R2 is a substituted or unsubstituted phenyl group. In some embodiments, R2 is a heteroaryl group that is substituted as required (e.g., a substituted or unsubstituted 5- to 6-membered monocyclic heteroaryl group, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or a substituted or unsubstituted 9- to 10-membered bicyclic heteroaryl group, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, R2 is -CH(=N)(OH)RD1, -CN, -NO2, -ORD1, -N(RD1a)2, -SO2ORD1, or -SRD1, wherein RD1 and RD1a are as defined herein. In some embodiments, R2 is -CH(=N)(OH)RD1 (e.g., -CH(=N)(OH) (C1-6 alkyl group substituted as desired). In some embodiments, R2 is -CH(=N)(OH) (C1-6 alkyl group substituted as desired with a polyethylene glycol linker). In some embodiments, R2 is -CN. In some embodiments, R2 is -NO2. In some embodiments, R2 is -ORD1 (e.g., -OH or -OMe). In some embodiments, R1A is -N(RD1a)2 (e.g., -NMe2). In some embodiments, R2 is -SO2ORD1 (e.g., -SO2O (alkyl group substituted as desired)). In some embodiments, R2 is -SRD1 (e.g., -SMe).In some embodiments, R2 is -CH(=N)(OH)RD1, -CN, -NO2, -ORD1, -N(RD1a)2, -SO2ORD1, or -SRD1, wherein RD1 is independently hydrogen, a substituted acetyl group, a substituted alkyl group, a substituted alkenyl group, a substituted alkynyl group, a substituted carbocyclic group, a substituted heterocyclic group, a substituted aryl group, a substituted heteroaryl group, or an oxygen-protecting group when attached to an oxygen atom, or a sulfur-protecting group when attached to an oxygen atom. The RD1a is a sulfur-protected group; and each occurrence of RD1a is independently hydrogen, a substituted acetyl group, a substituted alkyl group, a substituted alkenyl group, a substituted alkynyl group, a substituted carbocyclic group, a substituted heterocyclic group, a substituted aryl group, a substituted heteroaryl group, or a nitrogen-protected group; or, if desired, two instances of RD1a together with their intermediate atoms form a substituted or unsubstituted heterocycle or a substituted or unsubstituted heteroaryl ring. In some embodiments, R2 is a bromine, alkyne, acetylene, alkene, aldehyde, amine, COOH, hydroxyl, carboxyl (e.g., dibenzocyclooctylene or DBCO), thiol, sulfonate, or -CN. In some embodiments, R2 is a click chemist (e.g., the click chemist shown in Table A, or in Schemes 1 to 19 of Examples 6). In some embodiments, R1A and R2 are reaction partners (e.g., bio-binding reaction partners). In some embodiments, R1A and R2 are bio-binding reaction couplers (e.g., click reaction couplers). In some embodiments, R1A and R2 are click reaction couplers derived from Table A. In some embodiments, LB is an alkyl linker, wherein one or more chain atoms of the hydrocarbon chain are independently substituted with an oxime, ester, isohydroxamic acid, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, ethyl carbamate, or oxime portion or cyclic portion.

[0149] In some embodiments, at least one instance of T is a therapeutic agent, targeting agent, or imaging agent as defined herein. In some embodiments, at least one instance of T is a therapeutic agent as defined herein. In some embodiments, at least one instance of T is a biomolecule. In some embodiments, at least one instance of T is a protein hydrolysis-targeting chimeric (PROTAC) drug. In some embodiments, at least one instance of T is a biological therapeutic agent (e.g., protein, peptide, nucleic acid, or antibody). In some embodiments, at least one instance of T is a peptide, nucleic acid, or antibody. In some embodiments, the nucleic acid is an oligonucleotide, DNA, or RNA (e.g., siRNA, mRNA). In some embodiments, at least one instance of T is a gene, protein, peptide, oligonucleotide, carbohydrate, DNA, or RNA. In some embodiments, the instances of T are different. In some embodiments, the instances of T are the same.

[0150] In some embodiments, this document provides an intermediate dendrite of formula (II-A): (II-A), wherein D is a dendrite selected from the group consisting of: poly(aminoamine) (PAMAM) polymers, polypropylene (POPAM) polymers, polyethylene imine polymers, polyisocyanate polymers, polyester polymers, t-polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; and t is an integer from 16 to 4096, including the stated number.

[0151] In some embodiments, this document provides an intermediate dendrite in the synthetic method described herein, wherein the intermediate dendrite has the following formula: (PAMAM G3.5). Compositions comprising functionalized dendrites

[0152] In some embodiments, the present invention provides a composition comprising a functionalized dendrite of formula (IA): (IA), wherein: D is a dendrite selected from the group consisting of: poly(aminoamine) (PAMAM) polymer, polypropylene (POPAM) polymer, polyethyleneimine polymer, polylylamine polymer, polyester polymer, styrene polymer, aliphatic poly(ether) polymer, aromatic polyether polymer, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymer, and combinations thereof; X is NH; Y1 is an alkyl group, an alkenyl group, an alkyne group, or a covalent bond as desired; m is an integer from 16 to 4096, inclusive; and n is an integer from 1 to 100, inclusive.

[0153] In some embodiments, the polydispersity value of the functionalized dendrite in the composition is less than or equal to 1.10. In some embodiments, the present invention provides a composition comprising a carrier and a functionalized dendrite of formula (IA): (IA), wherein: D is a dendrite selected from the group consisting of: poly(aminoamine) (PAMAM) polymer, polypropylene (POPAM) polymer, polyethylene imine polymer, polylylamine polymer, polyester polymer, styrene polymer, aliphatic poly(ether) polymer, aromatic polyether polymer, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymer, and combinations thereof; X is NH; Y1 is an alkyl group, an alkenyl group, an alkyne group, or a covalent bond as desired; m is an integer from 16 to 4096, inclusive; and n is an integer from 1 to 100, inclusive.

[0154] In some embodiments, the polydispersity value of the functionalized dendrites in the composition is less than or equal to 1.10. In some embodiments, the substituents D, X, Y1, m, and n in the functionalized dendrites of formula (IA) are defined as described above. In some embodiments, the polydispersity value of the functionalized dendrites in the composition is less than or equal to 1.10 (e.g., less than or equal to 1.09, less than or equal to 1.08, less than or equal to 1.07, less than or equal to 1.06, less than or equal to 1.05, less than or equal to 1.04, less than or equal to 1.03, less than or equal to 1.02). In some embodiments, the polydispersity value of the functionalized dendrites in the composition is less than or equal to 1.05, less than or equal to 1.04, less than or equal to 1.03, less than or equal to 1.02, less than or equal to 1.01, or less than or equal to 1.00. In some embodiments, the composition comprises at least 10 grams of functionalized dendrite. In some embodiments, the composition comprises at least 10 grams, at least 25 grams, at least 50 grams, at least 150 grams, or between 10 and 100 grams, 100 and 150 grams, 150 and 200 grams, 20 and 200 grams, 50 and 200 grams, or 100 and 200 grams of functionalized dendrite (e.g., the functionalized dendrite of formula (IA)). In some embodiments, the composition comprises more than 10 to 20 grams, 20 to 30 grams, 30 to 40 grams, 40 to 50 grams, 50 to 60 grams, 60 to 70 grams, 70 to 80 grams, 80 to 90 grams, 90 to 100 grams, 100 to 110 grams, 110 to 120 grams, 120 to 130 grams, 130 to 140 grams, 150 to 160 grams, 160 to 180 grams, or 180 to 200 grams of the functionalized dendritic (e.g., the functionalized dendritic of formula (IA)). In some embodiments, the composition comprises more than 30 grams or more than 50 to 100 grams (e.g., 40 grams, 160 grams) of the functionalized dendritic (e.g., the functionalized dendritic of formula (IA)).

[0155] In some embodiments, the composition comprises a suitable carrier (e.g., a solid or liquid carrier) as described herein. Therapeutic agent

[0156] In some embodiments, the present invention provides a dendritic complex comprising a dendrite having at least one therapeutic agent at one or more terminal sites of the dendrite. In some embodiments, such dendritic-drug complexes have an increased therapeutic index relative to an unbound drug (e.g., a therapeutic agent lacking the dendrite). In some embodiments, the dendritic-drug complex has a therapeutic index greater than 10% of the therapeutic index of the unbound drug. In some embodiments, the dendritic-drug complex has a therapeutic index greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the therapeutic index of the unbound drug.

[0157] The therapeutic index (TI) of a therapeutic agent is a comparison between the amount of therapeutic agent that produces a therapeutic effect and the amount that produces toxicity. In some embodiments, the therapeutic index may be expressed as a ratio LD50 / ED50, where ED50 corresponds to the dose that is therapeutically effective in 50% of the population, and LD50 corresponds to the dose that is lethal to 50% of the population. In some embodiments, the therapeutic efficacy and toxicity of the drug and dendritic-drug conjugate may be determined in cell cultures or laboratory animals using standard pharmaceutical procedures.

[0158] In some embodiments, the dendritic system is combined or conjugated with two or more different classes of therapeutic agents to provide simultaneous delivery at the target site with different or independent release kinetics. For example, in some embodiments, a STING agonist and a CSF1R inhibitor are conjugated to dendrites for delivery to target cells or tissues. In some embodiments, dendritic conjugates carrying different classes of therapeutic agents are simultaneously administered for combination therapy. In some embodiments, a fourth- or sixth-generation PAMAM dendritic system is conjugated to sunitinib and a CXCR2 inhibitor, or the like. In some embodiments, a fourth- or sixth-generation PAMAM dendritic system is conjugated to vincristine and sunitinib, or the like.

[0159] In some embodiments, the therapeutic agent is a pharmaceutically acceptable derivative, analog, or prodrug of any of the compounds described below or any of the compounds described below. A prodrug is a compound that, when metabolized in vivo, is converted into a compound having the desired pharmacological activity. A prodrug can be prepared by replacing the appropriate functional group present in the therapeutic agent with the “pro-part” as described in this art (see, for example, H. Bundgaar, Design of Prodrugs (1985)). Examples of prodrugs include esters, ethers, or amide derivatives of the therapeutic agents described herein; polyethylene glycol derivatives of the therapeutic agents described herein; N-amide derivatives; dihydropyridine-pyridine derivatives; amino-containing derivatives bound to peptides; 2-hydroxybenzoamide derivatives; carbamate derivatives; N-oxide derivatives bioreduced to active amines; and N-Mannich base derivatives. For further discussion of the prodrug, see, for example, Rautio, J. et al., Nature Reviews Drug Discovery. 7:255-270 (2008).

[0160] In some embodiments, the dendritic complex of the present invention comprises a therapeutic agent selected from the group consisting of: angiotensin II receptor blockers, farnesoid X receptor (FXR) agonists, death receptor 5 agonists, sodium-glucose cotransporter type 2 (SGLT2) inhibitors, lysophosphatidylcholine 1 receptor antagonists, endothelin-A receptor antagonists, peroxisome proliferator-activated receptor δ (PPARδ) agonists, AT1 receptor antagonists, CCR5 / CCR2 antagonists, antifibrotic agents, anti-inflammatory agents, antioxidants, interferon gene-stimulating factor (STING) agonists, colony-stimulating factor 1 receptor (CSF1R) inhibitors, AXL inhibitors, c-Met inhibitors, poly(ADP-ribose) polymerase (PARP) inhibitors, receptor tyrosine kinase inhibitors, MEK inhibitors, PAK1 inhibitors, glutamate inhibitors, TIE II antagonists, and chemokine receptor 2. (CXCR2) inhibitors, CD73 inhibitors, arginase inhibitors, phosphatidylinositol-3-kinase (PI3K) inhibitors, TLR4 agonists, TLR7 agonists, protein tyrosine phosphatase-2 (SHP2) inhibitors containing the Src homology-2 domain, chemotherapeutic agents, STING antagonists, and JAK1 inhibitors.

[0161] In some embodiments, the therapeutic agent is an immunomodulator. In some embodiments, an immunomodulator refers to an agent that produces a specific effect on the recipient's immune system. In some embodiments, immunomodulation may include, compared to a control, inhibiting, reducing, enhancing, prolonging, or stimulating one or more physiological processes of an innate or adaptive immune response to an antigen. In some embodiments, the immunomodulator may modulate the immune microenvironment by targeting one or more immune cells or cell types at a target site to obtain a desired immune response (e.g., increasing antitumor activity, or increasing desired anti-inflammatory activity sites in autoimmune diseases). In some embodiments, the immunomodulator is delivered to kill, inhibit, or reduce the activity or amount of suppressive immune cells (such as tumor-associated macrophages) to enhance the antitumor response at the tumor site. In other embodiments, the immunomodulator is delivered to kill, inhibit, or reduce the activity or amount of pro-inflammatory immune cells (e.g., M1 macrophages) to reduce the pro-inflammatory immune environment at pathogenic sites associated with autoimmune diseases.

[0162] Examples of immunomodulators used according to the present invention include (but are not limited to) STING agonists, STING antagonists, Janus kinase 1 (JAK1) inhibitors, CSF1R inhibitors, AXL inhibitors, c-Met inhibitors, PARP inhibitors, receptor tyrosine kinase inhibitors, MEK inhibitors, PAK1 inhibitors, glutamate inhibitors, TIE II antagonists, CXCR2 inhibitors, CD73 inhibitors, arginine inhibitors, PI3K inhibitors, TLR4 agonists, TLR7 agonists, SHP2 inhibitors, anti-inflammatory agents, and combinations thereof.

[0163] In some embodiments, the therapeutic agent is selected from the STING agonists of cyclic dinucleotide GMP-AMP and DMXAA. In some embodiments, the therapeutic agent is selected from the STING antagonists of C-178, C-176, C18, Astin C, NO2-CLA, H-151 and α-azin. In some embodiments, the therapeutic agent is selected from tofacitinib, ruxolitinib, baricitinib, peficitinib, decernotiniba, filgotinib, solcitinib, itacitinib, SHR0302, upadacitinib, PF-04965842, Target-007, and Target-006 JAK1 inhibitors. In some embodiments, the therapeutic agent is a CSF1R inhibitor selected from PLX3397, PLX108-01, ARRY-382, PLX7486, BLZ945, JNJ-40346527, and GW 2580. In some embodiments, the therapeutic agent is a PARP inhibitor selected from olaparib, veliparib, niraparib, and rucaparib. In some embodiments, the therapeutic agent is a receptor tyrosine kinase inhibitor of vascular endothelial growth factor receptor (VEGFR) or epidermal growth factor receptor (EGFR). In some embodiments, the therapeutic agent is an AXL inhibitor (e.g., bemtinib (R428), dubematinib (TP-0903)). In some embodiments, the therapeutic agent is a c-Met inhibitor (e.g., cabozantinib). In some embodiments, the therapeutic agent is selected from receptor tyrosine kinase (e.g., VEGFR, CSF1R, AXL, and / or c-Met) inhibitors of sunitinib, sorafenib, pazopanib, vandetanib, axitinib, cidinibub, vatalanib, dasatinib, bemtinib (R428), dubematinib (TP-0903), nintedanib, cabozantinib, and motsarib. In some embodiments, the therapeutic agent is selected from trametinib, cobimetinib, binimetinib, selumetinib, PD325901, PD035901, PD032901, and TAK-733. In some embodiments, the therapeutic agent is a PAK1 inhibitor. In some embodiments, the PAK1 inhibitor is Frax-1036 (6-[2-chloro-4-(6-methyl-2-pyrazinyl)phenyl]-8-ethyl-2-[[2-(1-methyl-4-piperidinyl)ethyl]amino]-pyrido[2,3-d]pyrimidin-7(8H)-one).In some embodiments, the therapeutic agent is selected from bis-2-(5-phenylacetimino-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), azaserine, acivicin, and CB-839, which are glutamate inhibitors. In some embodiments, the therapeutic agent is selected from navarixin, SB225002, and SB332235, which are CXCR2 inhibitors. In some embodiments, the therapeutic agent is selected from APCP, quercetin, and tenofovir, which are CD73 inhibitors. In some embodiments, the therapeutic agent is an arginase inhibitor, such as 2-(S)-amino-6-boronocic acid. In some embodiments, the therapeutic agent is a PI3K inhibitor selected from alpelisib, serabelisib, pilaralisib, WX-037, dactolisib, prexasertib, voxtalisib, PX-866, ZSTK474, buparlisib, pictilisib, and copanlisib.

[0164] In some embodiments, the therapeutic agent is an anti-inflammatory agent. In some embodiments, the anti-inflammatory agent reduces inflammation and may include steroid-type and non-steroid-type drugs. Examples of steroid-type drugs include (but are not limited to) glucocorticoids, progestins, mineralocorticoids, and corticosteroids. Examples of non-steroid-type anti-inflammatory drugs (NSAIDs) include (but are not limited to) mefenamic acid. acid), aspirin, diflunisal, salicylate, ibuprofen, naproxen, fenoprofen, ketoprofen, deacketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, sulindac, etodolac, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, Isoxicam, meclofenamic acid The following are listed: acid, flufenamic acid, tolfenamic acid, celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, firocoxib, sulphonanilide, nimesulide, niflumic acid, and licofelone.Other examples of anti-inflammatory agents include (but are not limited to) triamcinolone acetonide, fluocinolone acetonide, methylprednisolone, prednisone, dexamethasone, loteprendol, fluorometholone, ibuprofen, aspirin, naproxen, cyclosporine, tacrolimus, rapamycin, and metformin. In some embodiments, the therapeutic agent is triamcinolone, prednisone, or dexamethasone.

[0165] In some embodiments, the therapeutic agent is a cytotoxic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of cytotoxic agents used according to the present invention include (but are not limited to) amsacrine, bevacizumab, bleomycin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cristaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, and doxorubicin. Podophyllotoxin, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, innotecan, leucovorin, daunorubicin, lomustine, mechlorethamine, melphalan, mecaptopurine, mertansine, mesna, methotrexate, mitomycin, mitoxantrone, monomethyl auristatin EE), oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, taxol, trichostatin A A) Trastuzumab, Cetuximab, and Rituximab.

[0166] In some embodiments, the therapeutic agent is an anticancer agent, such as the cytotoxic agents described herein. In some embodiments, the therapeutic agent is a histone deacetase (HDAC) inhibitor, such as vorinostat. In some embodiments, the therapeutic agent is a topoisomerase I and / or II inhibitor, such as etoposide or camptothecin. Other examples of anticancer agents include (but are not limited to) irinotecan, exemestane, octreotide, carmofur, clarithromycin, zinostatin, tamoxifen, tegafur, toremifene, doxifluridine, nimustine, vindensine, nedaplatin, pirarubicin, flutamide, fadrozole, prednisone, medroxyprogesterone, mitotane, mycophenolate mofetil, and mizoribine.

[0167] In some embodiments, the therapeutic agent is an anti-angiogenic agent. Examples of anti-angiogenic agents include (but are not limited to) bevacizumab (AVASTIN®), rhuFAb V2 (ranibizumab, LUCENTIS®), aflibercept (EYLEA®), MACUGEN® (pegaptanim sodium, anti-VEGF aptamer or EYE001), pigment epithelial-derived factor (PEDF), celecoxib (CELEBREX®), rofecoxib (VIOXX®), interferon-alpha, interleukin-12 (IL-12), thalidomide (THALOMID®), lenalidomide (REVLIMID®), squalamine, endostatin, angiostatin, ANGIOZYME® (Sirna Therapeutics), and NEOVASTAT® (AE-941) (Aeterna Laboratories, Quebec). (City, Canada), sunitinib (SUTENT®), sorafenib (Nexavar®), erlotinib (Tarceva®), panitumumab (VECTIBIX®), and cetuximab (ERBITUX®). Other examples of anti-angiogenic agents include those targeting the platelet-derived growth factor family, epidermal growth factor family, fibroblast growth factor family, transforming growth factor-β superfamily (TGF-β1, activin, follistatin, and bone morphogenetic protein), angiopoietin-like family, hemolymphocyte-derived growth factor family, integrin superfamily, pigment epithelial-derived factor, hepatocyte growth factor, angiopoietin, endothelin, hypoxia-inducible factor, insulin-like growth factor, cytokines, matrix metalloproteinases and their inhibitors, and glycosylated protein members.

[0168] In some embodiments, the therapeutic agent is an agent that can be used to treat one or more liver-related conditions or diseases and / or related diseases or conditions such as infection, sepsis, diabetic complications, hypertension, obesity, high blood pressure, heart failure, kidney disease and cancer. Examples of such therapeutic agents include (but are not limited to) the angiotensin II receptor blockers, FXR agonists, death receptor 5 agonists, SGLT2 inhibitors, lysophosphatidylcholine 1 receptor antagonists, endothelin-A receptor antagonists, PPARδ agonists, AT1 receptor antagonists, CCR5 / CCR2 antagonists, insulin sensitizers, pioglitazone, antifibrotic agents, antioxidants, antiangiogenic agents, anti-stimulants (e.g., valproic acid, D-aminophosphovalerate, D-aminophosphoheptane), inhibitors of glutamate formation / release (e.g., baclofen, NMDA receptor antagonists, ranibizumab, antiVEGF agents), and immunomodulatory and cytotoxic agents described herein.

[0169] In some embodiments, the therapeutic agent is an angiotensin II receptor blocker, such as telmisartan, telmisartan-acetamide derivatives, or telmisartan-ester derivatives. In some embodiments, the therapeutic agent is an FXR agonist, such as chenodeoxycholic acid, chenodeoxycholic acid-acetamide derivatives, or chenodeoxycholic acid-ester derivatives. In some embodiments, the therapeutic agent is an SGLT2 inhibitor selected from phlorizin, T-1095, canagliflozin, dapagliflozin, ipragliflozin, tofogliflozin, empagliflozin, luseogliflozin, ertugliflozin, and remogliflozin etabonate. In some embodiments, the therapeutic agent is a PPARδ agonist, such as GW0742, GW0742-acetamide derivative, or GW0742-ester derivative. In some embodiments, the therapeutic agent is an antioxidant, such as vitamin E.

[0170] In some embodiments, the therapeutic agent is N-acetylglucosamine. In some embodiments, N-acetylglucosamine is bound to a hydroxyl-terminated dendrite via a non-cleavable bond to minimize the release of free N-acetylglucosamine in vivo after administration. In some embodiments, the non-cleavable form of the dendritic / N-acetylglucosamine conjugate provides enhanced therapeutic efficacy compared to a releasable or cleavable form of the dendritic / N-acetylglucosamine complex.

[0171] In some embodiments, the therapeutic agent is polysialic acid (e.g., low molecular weight polySia (polySia avDP20) with an average degree of polymerization of 20), a transporter ligand (e.g., a diazoxide binding inhibitor (DBI)), interferon-β (IFN-β), or minocycline.

[0172] In some embodiments, the therapeutic agent is an anti-infective agent. Examples of anti-infective agents include (but are not limited to) antiviral agents, antibacterial agents, antiparasitic agents, and antifungal agents. In some embodiments, the therapeutic agent is selected from moxifloxacin, ciprofloxacin, erythromycin, levofloxacin, cefazolin, vancomycin, tigecycline, gentamycin, tobramycin, ceftazidime, ofloxacin, gatifloxacin, amphotericin B, voriconazole, and natamycin.

[0173] More detailed descriptions of therapeutic agents applicable to use according to the present invention are found in co-pending international applications PCT / US2020 / 063332, PCT / US2020 / 063347, PCT / US2020 / 063342 and PCT / US2021 / 029139, the relevant contents of which are incorporated herein by reference in their entirety. Imaging agent

[0174] In some embodiments, the present invention provides a dendritic complex comprising dendrites having at least one imaging agent at one or more of the terminal sites of the dendrites. In some embodiments, the dendritic complex comprising the imaging agent can be used for diagnostic, therapeutic, or labeling purposes. In some embodiments, the imaging agent is a paramagnetic molecule, a fluorescent compound, a magnetic molecule, a radionuclide, an X-ray imaging agent, or a contrast agent. In some embodiments, the contrast agent is a radiopaque gas or exothermic compound. In some embodiments, the dendritic complex comprising the imaging agent can be used to determine the location of a composition to be administered. Imaging agents suitable for this purpose include (but are not limited to) fluorescent tags, radionuclides, and contrast agents. Examples of imaging agents suitable for diagnostic purposes include (but are not limited to) dyes, fluorescent dyes, near-infrared dyes, SPECT imaging agents, PET imaging agents, and radioisotopes. Examples of dyes include (but are not limited to) carbocyanine, indole carbocyanine, oxacarbocyanine, thiocarbocyanine, and cyanine, polymethine, coumarin, rhodamine, xanthracene, fluorescein, boron dipyrrolemethane (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, DyLight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, and IRDye. 700DX, ADS780WS, ADS830WS and ADS832WS.

[0175] In some embodiments, the dendrite conjugate includes a radionuclide reporter suitable for imaging by scintillation, single-photon emission computed tomography (SPECT), or positron emission tomography (PET). In some embodiments, the dendrite conjugate includes a radionuclide suitable for radiotherapy. In some embodiments, the dendrite conjugate includes a contrast agent for imaging by magnetic resonance imaging (MRI). In some embodiments, the dendrite conjugate includes a chelating agent for the radionuclide or an MRI contrast agent suitable for diagnostic imaging, and a chelating agent suitable for radiotherapy. Thus, in some embodiments, a single dendrite / imaging agent conjugate can simultaneously treat and diagnose a disease or condition at one or more sites in the body. In some embodiments, the dendrite conjugate includes a radiolabeled SPECT or a scintillation imaging agent having an appropriate amount of radioactivity.

[0176] A suitable imaging agent may be selected based on a specific imaging method. For example, in some embodiments, the imaging agent is a near-infrared fluorescent dye for optical imaging, a zirconia chelate for MRI imaging, a radionuclide for PET or SPECT imaging, or gold nanoparticles for CT imaging.

[0177] In some embodiments, the dendritic complex contains one or more imaging agents for PET imaging, such as one or more radionuclides. PET is a technique that uses a special camera and computer to detect small amounts of radioactive tracers or radiopharmaceuticals in the body to assess organ and tissue function (e.g., to detect the early onset of disease).

[0178] PET involves the use of coupling detection to detect gamma rays in the form of annihilated photons from short-lived positron-emitting radioisotopes, including (but not limited to) 18F with a half-life of about 110 minutes, 11C with a half-life of about 20 minutes, 13N with a half-life of about 10 minutes, and 15O with a half-life of about 2 minutes. Therefore, in some embodiments, examples of developers used for PET imaging include (but are not limited to) one or more of various positron-emitting metal ions such as 51Mn, 52Fe, 60Cu, 68Ga, 72As, 94mTc, or 110In. In some embodiments, the developer is selected from radionuclides of 18F, 124I, 125I, 131I, 123I, 77Br, and 76Br. Examples of metallic radionuclides used in scintillation or radiotherapy include (but are not limited to) 99mTc, 51Cr, 67Ga, 68Ga, 47Sc, 51Cr, 167Tm, 141Ce, 111In, 168Yb, 175Yb, 140La, 90Y, 88Y, 153Sm, 166Ho, 165Dy, 166Dy, 62Cu, 64Cu, 67Cu, 97Ru, 103Ru, 186Re, 188Re, 203Pb, 211Bi, 212Bi, 213Bi, 214Bi, 105Rh, 109Pd, 117mSn, 149Pm, 161Tb, 177Lu, 225Ac, 198Au, and 199Au. The choice of metal will be determined based on the desired therapeutic or diagnostic application. For example, for diagnostic purposes, in some embodiments, useful radionuclides include 64Cu, 67Ga, 68Ga, 99mTc, and 111In. For therapeutic purposes, in some embodiments, useful radionuclides include 64Cu, 90Y, 105Rh, 111In, 117mSn, 149Pm, 153Sm, 161Tb, 166Tb, 166Dy, 166Ho, 175Yb, 177Lu, 225Ac, 186 / 188Re, and 199Au.

[0179] In some embodiments, the imaging agent is 99m-99m (99mTc). In some embodiments, 99mTc is suitable for diagnostic applications due to its low cost, availability, imaging properties, and high specific activity. The nuclear and radioactive properties of 99mTc make this isotope suitable for scintillation imaging. This isotope has a single-photon energy of 140 keV and a radioactive half-life of approximately 6 hours, and is readily available from 99Mo-99mTc generators. In some embodiments, radionuclides suitable for PET imaging include 18F, 4-[18F]fluorobenzaldehyde (18FB), Al[18F]-NOTA, 68Ga-DOTA, and 68Ga-NOTA. In some embodiments, 153Sm may be used as an imaging agent together with a chelating agent such as ethylenediaminetetramethylenephosphonic acid (EDTMP) or 1,4,7,10-tetraazacyclododecanetetramethylenephosphonic acid (DOTMP)).

[0180] MRI can be used to evaluate brain diseases, spinal diseases, angiography, cardiac function, and musculoskeletal injuries. MRI does not use ionizing radiation and can scan in any selected orientation. MRI provides full three-dimensional capability, high soft tissue contrast, high spatial resolution, and excels at morphological and functional imaging. Therefore, in some embodiments, the dendrite contains one or more imaging agents for MRI, such as one or more MRI contrast agents. Examples of MRI contrast agents are known in the art and include (but are not limited to) Gd, Mn, BaSO4, iron oxide, and iron platinum. Targeting agents

[0181] In some embodiments, the dendrite includes one or more tissue-targeting or tissue-binding portions for targeting the dendrite to a specific location in the body and / or for enhancing the residence time at the desired location in the body. For example, in some embodiments, after local or systemic administration, the dendritic system is isolated or bound to one or more different tissues or organs. Therefore, the presence of a targeting or binding portion can enhance the delivery of the agent to the target site, compared to the dendrite and agent in the absence of a targeting or binding portion. The binding of the dendrite to one or more targeting or binding portions may be via a spacer and a bond between the spacer and the dendrite, and / or the spacer and the targeting agent may be designed to provide a releaseable or non-releaseable form of the dendrite-targeting agent complex.

[0182] An exemplary targeting agent is alendronate (alendronate), which binds to hydroxyapatite on the bone surface and enhances the residence time of the dendritic complex on bone. Alendronate is a small molecule targeting moiety that selectively binds to hydroxyapatite (a component of bone). Thus, in some embodiments, the dendritic system is bound to alendronate to selectively target the dendritic to bone. In some embodiments, the binding between alendronate and the dendritic is via a reversible (non-covalent) linker. In other embodiments, the binding between alendronate and the dendritic is via a non-cleavable or minimally cleavable linker. In some embodiments, the targeting agent also has a therapeutic effect at the target site. In some embodiments, the dendritic system is bound to alendronate to target the dendritic complex to bone and provide a therapeutic effect at the site of bone inflammation. In some embodiments, the dendritic system bound to alendronate is bound to one or more agents to selectively deliver such agents to the site of bone inflammation.

[0183] Dendritic structures that are bound to or complexed with the carbohydrate tri-antennae N-acetylglucosamine (GalNAc) have been found to selectively accumulate in hepatocytes. This invention describes a composition of dendritic structures modified by adding tri-antennae N-acetylglucosamine (GalNAc) to the surface of the dendritic structures.

[0184] The desialylate glycoprotein receptor (ASGPR), which is abundantly expressed on hepatocytes, selectively recognizes galactose and N-acetylglucosamine (GalNAc) via its carbohydrate recognition domain (CRD) and binds tightly to the receptor. The efficient binding of the carbohydrate portion to these ASGPR receptors allows for selective internalization within the hepatocyte via receptor-mediated endocytosis. The low pH in the endosome disrupts the tetravalent calcium chelate between the glycoligand and the ASGPR receptor, thereby releasing the ligand within the hepatocyte. Once the ligand is released, the receptor complex circulates, allowing for the internalization of a large amount of ligand into the hepatocyte without a saturation effect. The binding of GalNAc to ASGPR occurs on the sinusoidal surface of hepatocytes; each cell contains approximately 500,000 ASGPR receptors, of which about 5% to 10% are always present on the cell surface. Previous studies have shown that the binding of ligands to ASGPR depends on the sugar type (GalNAc > Gal) and the number of sugars in the order 4 > 3 > 2 > 1. X-ray crystallography of the extracellular domain of ASGPR reveals shallow carbohydrate-binding pockets, explaining the requirement for multivalent binding. Therefore, multivalent binding has been explored, and due to the glycocluster effect, trivalent and tetravalent carbohydrate constructs exhibit 100 to 1000 times stronger affinity for ASGPR than monovalent ligands.

[0185] The biantennary and triantennary GalNAc ligands bound to siRNA showed significantly higher levels of GalNAc-siRNA in the livers of C57BL / 6 mice administered subcutaneously, with 94% of the GalNAc-siRNA located in hepatocytes. Furthermore, these siRNA conjugates mediated highly efficient gene silencing. Other studies have reported that, in mouse models, antisense oligonucleotides (ASOs) linked to triantennary GalNAc are up to 10 times more potent than parental ASOs.

[0186] Carbohydrate-protein interactions play a crucial role in biological processes, such as receptor-mediated endocytosis, and have been applied in cell recognition research and the design of biomedical materials. Enhancing the binding affinity of carbohydrate-terminated dendrites (glycated dendrites) with receptor-like structures allows them to interact with specific cell types that exhibit strong binding affinity and selectivity for targeted drug delivery. Introducing the carbohydrate portion into drug delivery platforms also provides biocompatibility and increases the water solubility of these dendritic complexes.

[0187] Trianthopter-GalNAc provides efficient multivalent binding to ASGPR on hepatocytes. Therefore, in some embodiments, the dendrite is modified with one or more trianthopter-GalNAc groups at one or more surface terminal groups (e.g., -OH).

[0188] The three-antennae GalNAc of the dendrites are modified with terminal groups on each surface to produce a set of three GalNAcs. In some embodiments, the three β-GalNAc molecules are grafted to the building blocks via one or more linkers to produce AB3 building blocks (i.e., three-antennae GalNAc dendrites) suitable for binding to the surface functional groups of the dendrites.

[0189] In some embodiments, three β-GalNAc molecular systems are grafted to propargylated neopentyl tertrol building blocks via one or more linkers to produce AB3 building blocks suitable for binding to the surface functional groups of the dendrites shown below.

[0190] In some embodiments, prior to binding, tritendinous-GalNAc binds to one or more surface groups via about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or 30% of the total available surface functional groups (such as hydroxyl groups) of the dendrites. In other embodiments, prior to binding, the binding of tritendinous-β-GalNAc occurs on less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, or less than 50% of the total available surface functional groups of the dendrites. In some embodiments, the dendritic system binds to an effective amount of tritendinous-β-GalNAc to bind to ASGPR and / or target hepatocytes, while simultaneously binding to an effective amount of an agent to treat, prevent, and / or image liver diseases or disorders. Composition

[0191] In some embodiments, the present invention provides a composition comprising one or more dendritic conjugates described herein. In some embodiments, the composition is a pharmaceutical composition. Pharmaceutical compositions comprising one or more dendritic conjugates may be formulated in a conventional manner using one or more physiologically acceptable carriers (including excipients and adjuvants) that facilitate the processing of the active compound into a pharmaceutically usable formulation. A suitable formulation depends on the chosen route of administration. In some embodiments, the compositions are formulated for non-enteral delivery. In some embodiments, the compositions are formulated for intratumoral injection. In some embodiments, the compositions may be formulated in sterile saline or buffer solutions for injection into the tissue or cells to be treated. The compositions may be lyophilized and stored in single-use vials for immediate rehydration prior to use.

[0192] In some embodiments, the pharmaceutical composition comprises one or more dendritic compounds and one or more pharmaceutically acceptable excipients. Examples of excipients include solvents, diluents, pH modifiers, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tension agents, stabilizers, and combinations thereof. Suitable pharmaceutically acceptable excipients may be selected from materials that are generally recognized as safe (GRAS) and can be administered to an individual without causing adverse biological side effects or unwanted interactions.

[0193] In some embodiments, pharmaceutically acceptable salts may be prepared by reacting the free acid or base form of a compound with a stoichiometric amount of a suitable base or acid in water or an organic solvent (e.g., a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile). Pharmaceutically acceptable salts may include salts of compounds derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts, as well as salts formed by reacting the compound with a suitable organic ligand (e.g., a quaternary amine salt). A list of suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 20th edition, Lippincott Williams & Wilkins, Baltimore, MD, 2000, page 704.

[0194] In some embodiments, the composition is formulated into dose units to facilitate dosing and uniformity of dosage. In some embodiments, dose units refer to physically discrete units of the conjugate suitable for the individual to be treated. The therapeutically effective dose can initially be estimated in cell culture analysis or animal models (such as mice, rabbits, dogs, or pigs). Animal models can also be used to achieve the desired concentration range and route of administration. This information can be used to determine the appropriate dose and route of administration for humans. The therapeutic efficacy and toxicity of the conjugate can be determined in cell cultures or laboratory animals using standard pharmaceutical procedures, for example, ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal in 50% of the population). The dose-to-toxicity ratio is a therapeutic index and can be expressed as the ratio LD50 / ED50.

[0195] In some embodiments, the composition is administered topically, for example, by direct injection into the site of treatment. In some embodiments, the composition is administered by injection, topical application, or otherwise directly into the vascular system of the vascular tissue at or near the site of injury, surgery, or implantation. For example, in some embodiments, the composition is topically applied to exposed vascular tissue during surgery, implantation, or transplantation procedures. The present invention describes pharmaceutical compositions formulated for administration via non-enteric (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous) injection, enteral, and topical routes.

[0196] In some embodiments, the dendritic complex is prepared for parenteral administration. The phrase "parenteral administration" is a recognized term in this art and includes administration methods other than enteric and local administration, such as injection, and may include intravenous, intramuscular, intrapleural, intravascular, intracardiac, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, and intrasternal injections and infusions. In some embodiments, the composition is administered parenterally, for example, via subdural, intravenous, intrasheath, intravenous, intraarticular, intra-articular, intrasynovial, intra-amniotic, intraperitoneal, or subcutaneous routes.

[0197] For liquid formulations, pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Non-enteric carriers (for subcutaneous, intravenous, intra-arterial, or intramuscular injection) include, for example, sodium chloride solutions, Ringer's glucose, dextran and sodium chloride, lactated Ringer's, and non-volatile oils. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and injectable organic esters (such as ethyl oleate). Aqueous carriers include, for example, water, alcohol / water solutions, cyclodextrins, emulsions, or suspensions, including saline and buffer media. The composition may also be administered as an emulsion (e.g., water-in-oil). Examples of oils include those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, cod liver oil, sesame oil, cottonseed oil, and corn oil. Fatty acids suitable for use in non-enteric formulations include, for example, oleic acid, stearic acid, isostearic acid, ethyl oleate, and isopropyl myristate.

[0198] In some embodiments, compositions suitable for non-enteral administration may include antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Intravenous carriers may include fluids and nutritional supplements, and electrolyte supplements, such as those based on Ringer's glucose. Generally, water, saline, aqueous dextran and related sugar solutions, and glycols (such as propylene glycol or polyethylene glycol) are preferred liquid carriers, particularly for injectable solutions. Injectable pharmaceutical carriers for use in injectable compositions are known in this art (see, for example, Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 15th edition, pp. 622-630 (2009)).

[0199] In some embodiments, the dendritic complex is formulated for intestinal administration. The carrier or diluent may be a solid carrier or diluent for solid formulations, a liquid carrier or diluent for liquid formulations, or a mixture thereof. For liquid formulations, pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters (such as ethyl oleate). Aqueous carriers include, for example, water, alcohol / water solutions, cyclodextrins, emulsions, or suspensions, including saline and buffer media. Examples of oils and fatty acids are as described with respect to the formulation of compositions for non-intestinal administration.

[0200] Carriers include, for example, sodium chloride solution, Ringer's dextran, dextran and sodium chloride, lactated Ringer's, and non-volatile oils. Formulations include, for example, aqueous and non-aqueous, isotonic sterile injectable solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes to make the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Generally, water, saline, aqueous dextran, and related sugar solutions can be used as liquid carriers. These can also be formulated with proteins, fats, sugars, and other components of infant formula.

[0201] In some embodiments, the dendritic compound is formulated for oral administration. The oral formulation may be in the form of chewing gum, gel strips, tablets, capsules, or lozenges. Encapsulating materials used to prepare enteric-coated oral formulations include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and methacrylate copolymers. Solid oral formulations (such as capsules or tablets) are preferred. Elixirs and syrups are also well-known oral formulations.

[0202] In some embodiments, the dendritic complex is formulated for local administration. Local administration may include direct application to exposed tissue, vascular system, mucosa, or application to tissue or prosthesis, for example, during surgery. A tumor is a preferred tissue type for local administration. Therapeutic use.

[0203] In some embodiments, these dendritic complexes are used to treat cancer. In other embodiments, these dendritic complexes are used to treat autoimmune diseases. These methods typically involve administering an effective amount of a composition comprising dendritic cells and one or more therapeutic agents to an individual in need to modulate the immune microenvironment, reduce autoimmune responses, or increase antitumor responses.

[0204] Generally, these compositions and their treatment methods are applicable to the context of cancer (including oncology therapy). These compositions may also be used to treat other diseases, ailments and injuries, including inflammatory diseases, including (but not limited to) ulcerative colitis, Crohn's disease and rheumatoid arthritis.

[0205] In some embodiments, the individual to be treated is a human. All methods described in this invention may include the steps of identifying and selecting an individual to be treated, or an individual who would benefit from being administered the composition. Thus, in some embodiments, in the treatment of cancer, or other diseases, disorders, and injuries, including inflammatory diseases (such as ulcerative colitis, Crohn's disease, rheumatoid arthritis) and bone diseases, an individual requiring immunomodulation is administered a dendritic composition in combination with or in combination with one or more immunomodulators and / or other therapeutic or diagnostic agents.

[0206] In some embodiments, an individual suffering from a proliferative disorder, such as a benign or malignant tumor, is administered a dendritic compound combination with one or more immunomodulatory agents and / or other therapeutic or diagnostic agents. In some embodiments, the individual to be treated has been diagnosed with stage I, II, III, or IV cancer. In some embodiments, the proliferative disorder is neurofibromatosis. Neurofibromatosis refers to a group of genetic disorders that cause the formation of tumors in nerve tissue. These tumors can develop anywhere in the nervous system, including the brain, spinal cord, and nerves. There are three types of neurofibromatosis: neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2), and Schwann cell tumor. NF1 is usually diagnosed in childhood, while NF2 and Schwann cell tumor are usually diagnosed in early adulthood. The tumors in these disorders are usually non-cancerous (benign) but can sometimes become cancerous (malignant). Therefore, in some embodiments, the individual to be treated has or is suspected of having a proliferative disorder, such as NF1, NF2, and / or Schwann cell tumor. In some embodiments, the individual to be treated has or is suspected of having NF1.

[0207] Neurofibromatosis type 1 (NF1) is a common cancer-predisposing syndrome characterized by the progressive development of slowly growing tumors (called plexiform neurofibromas). These tumors involve the cephalic and greater peripheral nerves, are caused by the loss of NF1 heterozygosity in Schwann cells, contain high levels of collagen, and are infiltrated by inflammatory cells. Due to the highly unmanageable nature of neurofibromas, drug therapy is the primary strategy for targeting these tumors. Selmetinib (Koselugo™) (a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor) is the only FDA-approved drug for NF1, and although this important inhibitor promotes partial responses in children and adults, greater shrinkage and durability are still needed. However, the overall toxicity of combination therapies is a limiting factor in the treatment of neurofibromas, thereby reducing the drug's ability to reduce tumor shrinkage or progression.

[0208] These compositions and methods are suitable for treating individuals with benign or malignant tumors by delaying or inhibiting tumor growth, reducing tumor growth or size, inhibiting or reducing tumor metastasis, and / or inhibiting or reducing symptoms associated with tumor development or growth. For example, in some embodiments, the present invention provides compositions and methods for treating individuals with tumors associated with (e.g., caused by) proliferative disorders such as neurofibromatosis (e.g., NF1).

[0209] The types of cancer that can be treated with these compositions and methods include (but are not limited to) cancers of the bone, bladder, brain, breast, cervix, colorectal, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, and uterus, such as vascular cancers, multiple myeloma, adenocarcinoma, and sarcoma. In some embodiments, these compositions are used to treat multiple types of cancer simultaneously. These compositions can also be used to treat metastases or tumors in multiple locations.

[0210] In some embodiments, an individual suffering from an autoimmune or inflammatory disease or ailment is administered a composition of dendritic cells combined with or in combination with one or more immunomodulatory agents and / or other therapeutic or diagnostic agents. Autoimmune diseases occur when the body's natural defense system is unable to effectively distinguish between its own cells and foreign cells, causing the body to mistakenly attack normal cells. There are more than 80 types of autoimmune diseases affecting a wide range of parts of the body. Common autoimmune diseases include rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), type 1 diabetes, inflammatory bowel disease, and thyroid disease.

[0211] In some embodiments, these compositions may also be used to treat autoimmune or inflammatory diseases or disorders, such as rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), and Behçet's disease. Diseases including bullous pemphigoid, cardiomyopathy, celiac stomatitis, chronic fatigue syndrome, immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, Crohn's disease, Dego's disease, dermatomyositis, juvenile dermatomyositis, discoid lupus, primary mixed cryoglobulinemia, fibromyalgia-fibromyositis, Grave's disease, guillain-barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), Iga nephropathy, insulin-dependent diabetes mellitus (type I), juvenile arthritis, and Meniere's disease. Diseases, mixed connective tissue diseases, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polygonadotropin syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-person syndrome, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0212] In some embodiments, these compositions and methods can also be used to treat autoimmune or inflammatory diseases or disorders involving bones and joints, including infection and immune-mediated local and systemic diseases.

[0213] These compositions and methods are suitable for treating one or more eye diseases or conditions. These compositions and methods are suitable for relieving one or more symptoms associated with one or more eye diseases or conditions, such as discomfort, pain, dryness, excessive tearing, injury, infection, burns, and progressive vision loss.

[0214] In some embodiments, the ocular disease to be treated is a posterior ocular disease, such as diabetic retinopathy, symptomatic vitreomacular adhesion / vitreous traction (sVMA / VMT), and wet (neovascular) or dry AMD (age-related macular degeneration). In some embodiments, the ocular disease to be treated is one or more retinal and choroidal vascular diseases (e.g., AMD, retinopathy of prematurity, diabetic macular edema, retinal vein occlusion, retinopathy associated with chemotherapy toxicity, such as MEK retinopathy). In some embodiments, the ocular disease to be treated is age-related macular degeneration (AMD). Age-related macular degeneration (AMD) is a neurodegenerative and neuroinflammatory disease of the macula that causes central vision loss. The pathogenesis of age-related macular degeneration involves chronic neuroinflammation of the choroid (the vascular layer under the retina), retinal pigment epithelium (RPE), the cellular layer under the sensory retina, Bruch's membrane, and the sensory retina itself.

[0215] In other embodiments, the ocular disease to be treated is an inflammatory disease of the eye, that is, an ocular disease associated with inflammation of ocular tissues, including, for example, AMD, retinitis pigmentosa, optic neuritis, sarcoma, retinal detachment, temporal arteritis, retinal ischemia, arteriosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion, diabetic retinopathy, macular edema, Stargardt's disease (also known as Stargardt's macular atrophy or juvenile macular degeneration), geographic atrophy, neuromyelitis optica, and also includes angiogenic diseases, including, for example, retinal neovascularization and choroidal neovascularization. Other conditions can also lead to inflammation and / or angiogenesis of the eye, such as infection, sickle cell disease, hypotension, etc.

[0216] Other examples of treatable eye diseases include amebic keratitis, fungal keratitis, bacterial keratitis, viral keratitis, onchorcercal keratitis, bacterial keratoconjunctivitis, viral keratoconjunctivitis, corneal dystrophy, Fuchs' endothelial dystrophy, meibomian gland dysfunction, anterior and posterior blepharitis, conjunctival hyperemia, conjunctival necrosis, cicatricial scarring and fibrosis, punctate epithelial keratopathy, filamentous keratitis, corneal erosion, thinning, ulceration and perforation, Doe's syndrome, Stevens-Johnson syndrome, autoimmune dry eye, environmental dry eye, corneal neovascularization, and corneal... Prevention and treatment of transplant rejection; autoimmune uveitis; infectious uveitis; anterior uveitis; posterior uveitis (including toxoplasmosis); panuveitis; inflammatory diseases of the vitreous or retina; prevention and treatment of endophthalmitis; macular edema; macular degeneration; age-related macular degeneration; proliferative and non-proliferative diabetic retinopathy; hypertensive retinopathy; autoimmune diseases of the retina; primary and metastatic intraocular melanoma; other metastatic intraocular tumors; open-angle glaucoma; angle-closure glaucoma; pigmentary glaucoma and combinations thereof. Other conditions include corneal injury, burns or abrasions, cataracts, and age-related or associated visual impairment.

[0217] In some embodiments, an individual is given a dendritic compound modified with Trianthus-GalNAc containing one or more pharmaceutical agents for treating, preventing and / or diagnosing one or more liver diseases and / or conditions to treat, prevent and / or diagnose one or more symptoms of one or more liver diseases and / or conditions in that individual.

[0218] The dendritic-trianthrax-β-GalNAc composition is effective in treating or alleviating one or more symptoms of liver diseases or disorders (such as acute or chronic liver diseases). Exemplary indications for treatment include (but are not limited to) acute liver failure (acute hepatitis, fulminant hepatitis) caused by lesion infiltration, acute Budd-Chiari syndrome, heatstroke, mushroom ingestion, metabolic disorders such as Wilson's disease, or those associated with viral hepatitis such as those caused by herpes simplex virus, cytomegalovirus, Epstein-Barr virus, parvovirus, hepatitis viruses (e.g., hepatitis A, hepatitis E, hepatitis D + hepatitis B infection), or drug-induced liver injury, including rifampicin-induced hepatotoxicity, acetaminophen-induced hepatotoxicity, recreational drug-induced toxicity such as that induced by 3,4-methylenedioxy-N-methylamphetamine (MDMA, also known as ecstasy), or cocaine-induced toxicity, acute ischemic hepatocellular injury, or hypoxic hepatitis, or those caused by traumatic liver injury. These methods can treat and prevent any hyperacute, acute, and subacute liver disease defined as encephalopathy, coagulation disorders, and jaundice in individuals with a previously normal liver.

[0219] Symptoms and clinical manifestations of acute liver disease include jaundice and encephalopathy, and impaired liver function (e.g., loss of metabolic function, reduced glucose production leading to hypoglycemia, decreased lactate clearance leading to lactic acidosis, decreased ammonia clearance leading to hyperammonemia, and decreased synthetic capacity leading to coagulopathy). Acute liver disease and disorders are often associated with a variety of systemic manifestations, including immunodeficiency paralysis leading to high risk of sepsis; systemic inflammatory response with high energy expenditure or catabolism; portal hypertension; renal insufficiency; myocardial injury; pancreatitis (particularly in acetaminophen-related diseases); adrenal insufficiency leading to hypotension; and acute lung injury leading to acute respiratory distress syndrome.

[0220] All methods described in this invention may also include the steps of identifying and selecting an individual to be treated, or an individual to whom the composition would benefit. In some embodiments, the individual has been medically diagnosed with an acute liver disease or condition by exhibiting clinical (e.g., physical) symptoms of the disease. In other embodiments, the individual has been medically diagnosed with a subacute or chronic liver disease or condition by exhibiting clinical (e.g., physical) symptoms that indicate an increased risk or likelihood of developing an acute liver disease. Therefore, in some embodiments, the individual is given a formulation of the dendritic composition disclosed in this invention prior to a clinical diagnosis of an acute liver disease.

[0221] In some embodiments, these methods treat or prevent non-alcoholic steatohepatitis, liver fibrosis associated with non-alcoholic steatohepatitis, and primary biliary cholangitis.

[0222] In some embodiments, the dendritic complex may be administered in combination with one or more other therapeutically active agents known to treat one or more of the conditions or diseases discussed above. Example 1: Didesethylsunitinib via non-cleavable bond binding

[0223] Overexpression of vascular endothelial growth factor (VEGF) has been involved in many diseases associated with angiogenesis. Sunitinib is a receptor tyrosine kinase inhibitor that blocks VEGF receptors and has excellent anti-angiogenic activity, and is FDA-approved for use in various types of cancer. Didesethylsunitinib is the active metabolite of sunitinib. Although sunitinib and its analogues have excellent therapeutic value, their clinical development has been hampered by related toxicities. The dendritic-didesethylsunitinib conjugate aims to overcome the dose-related toxicities of sunitinib by linking it to a hydroxyl-terminated dendrite. The chemical structure of the dendritic conjugate synthesized in this example is shown in Figure 1.

[0224] Synthesis and Characterization of N,N-Didesethylsunitinib Acetamide Azide

[0225] The reaction scheme for synthesizing N,N-didesethylsunitinib azide with amide linkage is shown in Figure 2.

[0226] Step 1: Synthesis of 5-fluoro-2,3-dihydro-1H-indol-2-one (compound 2)

[0227] Triethylamine (6.12 mL, 1.2 equivalents) was added to a stirred solution of 5-fluoro-2,3-dihydro-1H-indole-2,3-dione (6.0 gm, 1.0 equivalents) in n-butanol (10 V), followed by the addition of hydrazine hydrate (3.56 mL, 2.0 equivalents) at room temperature. The resulting solution was stirred at 100 °C for 16 h. The reaction progress was monitored by TLC (50% ethyl acetate in hexane). If the reaction was determined to be complete, the reactants were evaporated to dryness under vacuum at 45 °C to obtain a dark brown solid. The resulting solid was quenched with water (20 V) and extracted with ethyl acetate (30 V), and the organic layer was washed with water. The organic layer was concentrated to dryness on a rotary evaporator. The crude product was purified by recrystallization with ethyl acetate to obtain a gray, fluffy solid (4.0 g, 72% yield). Compound 2 shown in Figure 2 was confirmed by 1H NMR, liquid chromatography and mass spectrometry.

[0228] Step 2: Synthesis of 5-{[(3Z)-5-fluoro-2-sideoxy-2,3-dihydro-1H-indole-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (compound 4)

[0229] Pyrrolidine (4.42 mL, 2.0 equivalent) was added to a stirred solution of 5-fluoro-2,3-dihydro-1H-indol-2-one (compound 2) (4.0 gm, 1.0 equivalent) and 5-methoxy-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (compound 3) (4.41 gm, 1.0 equivalent) in ethanol (10 V). The resulting solution was stirred at 80 °C for 3 h. The reaction progress was monitored by TLC (10% methanol in DCM). If the reaction was determined to be complete, the reactants were cooled to room temperature and 2 M HCl solution was added to pH=3. A brownish-red precipitate formed and was filtered. The resulting solid was washed with ethanol (20 V) followed by hexane (30 V) and filtered to obtain a reddish-orange solid (6.6 g, 82% yield). Compound 4 shown in Figure 2 was confirmed by 1H NMR.

[0230] Step 3: Synthesis of N-{2-[(5-{[(3Z)-5-fluoro-2-sideoxy-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrolo-3-yl)methamido]ethyl}aminocarbamate tributyl ester (compound 6)

[0231] A solution of 5-{[(3Z)-5-fluoro-2-sideoxy-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (compound 4) (6.5 g, 1.0 equivalent) in DMF was added to triethylamine (6.08 mL, 2.0 equivalent), EDC.HCl (8.68 g, 2.1 equivalent), HOBT (3.94 g, 1.35 equivalent), and N-(2-aminoethyl)aminocarboxylic acid tributyl ester (4.16 g, 1.2 equivalent). The reaction mixture was stirred for 16 hours at room temperature. The reaction mixture was diluted with water (20.0 V), stirred for 10 min until a precipitate formed, and filtered to obtain a brown solid. The obtained solid was dilute with ethyl acetate (15.0 V), followed by hexane (15.0 V). V) The sample was washed, filtered, and dried to obtain N-{2-[(5-{[(3Z)-5-fluoro-2-sideoxy-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrolo-3-yl)methamido]ethyl}aminocarbamate (compound 6) (7.5 g, 78% yield) as a brownish-orange solid. Compound 6 shown in Figure 2 was confirmed by 1H NMR.

[0232] Step 4: Synthesis of N-(2-aminoethyl)-5-{[(3Z)-5-fluoro-2-sideoxy-2,3-dihydro-1H-indole-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-methylamine (compound 7):

[0233] Trifluoroacetic acid (3.0 V) was added to a solution of N-{2-[(5-{[(3Z)-5-fluoro-2-sideoxy-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrolo-3-yl)methamido]ethyl}aminocarbamate (compound 6) (9.0 g, 1.0 equivalent) in DCM (10.0 V) at 0 to 5 °C. The reaction was stirred for 12 hours at room temperature. The reactants were then evaporated to dryness under vacuum at 45 °C to obtain a dark brown solid. The resulting solid was washed with diethyl ether (15.0 V), filtered, and dried to obtain an orange-yellow solid (6.0 g crude). Compound 7 shown in Figure 2 was confirmed by 1H NMR, liquid chromatography, and mass spectrometry.

[0234] Step 5: Synthesis of N-{2-[(5-{[(3Z)-5-fluoro-2-sideoxy-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrolo-3-yl)methamido]ethyl}-3-[2-(2-propoxyethoxy)ethoxy]propionic acid (compound 9):

[0235] A solution of 3-[2-(2-propoxyethoxy)ethoxy]propionic acid (8) (5.95 g, 1.0 equivalent) in DMF (10.0 V) was added to DIPEA (8.40 mL, 2.0 equivalent), EDC.HCl (6.90 g, 1.5 equivalent), HOBT (0.65 g, 0.2 equivalent), N-(2-aminoethyl)-5-{[(3Z)-5-fluoro-2-sideoxy-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-methylamine (compound 7) (11.0 g, 1.0 equivalent) and DMAP (0.294 g, 0.1 equivalent) at 0 to 5 °C. The reaction was stirred for 3 hours at room temperature. The reaction progress was monitored by TLC (10% MeOH in DCM). The reaction mixture was diluted with water (20.0 V), stirred for 10 min to form a brown precipitate, and filtered. The resulting solid was purified by reversed-phase column chromatography to obtain an orange solid, N,N-didesethylsunitinib azidide (5.2 g, 37% yield). Compound 9 was identified by 1H NMR, liquid chromatography, and mass spectrometry.

[0236] A dendritic complex was synthesized by linking it to a dendritic structure via an indestructible ether.

[0237] The synthesis began with the construction of a bifunctional dendrite. In the 3.5th generation dendrite, seven alkyne functional groups were introduced using a polyethylene glycol (PEG) linker with one end being an amine and the other end being hexyne to generate a 4th generation bifunctional dendrite with seven alkyne arms and 57 hydroxyl groups on the surface (compound 1 in Figure 3). The structure of this dendrite was confirmed by 1H NMR spectroscopy.

[0238] A clickable didesethylsunitinib analog (compound 2 in Figure 3, VT-4517) was synthesized, comprising didesethylsunitinib, a triethylene glycol (PEG3) spacer, and a terminal azide, to participate in the click reaction of alkyne groups on the dendritic surface. The active agent compound 2 was synthesized using the 5-step synthesis shown in Figure 2 and described above.

[0239] AVT-4517 (compound 2 in Figure 3) was finally reacted with a bifunctional dendrite with a hexyne group (compound 1 in Figure 3) via copper (I)-catalyzed alkyne-azide click chemistry to produce D-4517.2 (compound 3 in Figure 3) having the complete structure shown in Figure 1. After the analogue was bound to the dendrite, D-4517.2 was purified by tangential flow filtration (TFF) to remove any impurities and was purified to the final formulation.

[0240] 1H-NMR Analysis of D-4517.2 Conjugate

[0241] The formation of product D-4517.2 was confirmed by 1H NMR. The 1H NMR spectrum of the conjugate clearly shows peaks corresponding to the dendrite, the drug, and the linkers attached to it, and the drug loading was calculated by comparing these peaks with the aid of proton integration. When the spectrum was recorded in deuterated DMSO, the internal amide protons from the dendrite were present between δ 8.5 and 7.5 ppm. These amide peaks are reference standards for the residual peaks. The -NH peaks from the drug appeared at δ 13.6 and 10.8 ppm. Four protons from the drug and one triazole proton formed and appeared between δ 8.5 and 7.5 ppm after the click reaction and merging with the internal amide peaks. In addition, two aromatic protons from sunitinib, located next to the fluorine group, appeared between δ 6.95 and 6.85 ppm. When the NMR solvent was switched from deuterated DMSO to CD3OD, a sharp triazole peak was observed at δ 7.7 ppm, which is a characteristic peak of click conversion. After click, CH2, present next to the azide, was masked and could be observed at δ 4.4 ppm. NMR is also used to quantify the number of drug molecules bound to the hydroxyl dendrites. Drug loading is calculated by comparing the protons of amide protons within the dendrites with the protons of the drug using proton integration.

[0242] HPLC analysis for evaluating the purity of D-4517.2

[0243] The purity of the dendritic drug conjugate, intermediate, and drug linker was evaluated using HPLC. The final conjugate was >99% pure according to HPLC. In HPLC, the dendritic G4-OH and dendritic hexyne intermediates were visible in the 210 nm channel, and didesethylsunitinib was visible in the 430 nm channel. The residence time of compound 2 was approximately 16.9 min, but once the hydrophobic drug molecule was attached to the dendrite, the peak of the final conjugate shifted to the right and reached approximately 27 min, confirming that the hydrophobic drug was attached to the dendritic building block. Once the drug was attached to the dendrite, peaks corresponding to it were observed in both the 210 nm (dendritic absorption wavelength) and 430 nm (drug absorption wavelength) channels, further confirming product formation. The drug loading of the dendritic conjugate was approximately 12.6% wt / wt, corresponding to 7 drug molecules attached to each dendritic molecule.

[0244] Dimensions and ζ-potential

[0245] The size and zeta potential distribution of D-4517.2 were determined using a Zsizer Nano ZS instrument. For size measurements, the sample was prepared by dissolving the dendrites in deionized water (18.2 Ω) to prepare a solution with a final concentration of 0.5 mg / mL. This solution was then filtered directly into the cell (UV transparent disposable colorimetric tube, size: 12.5 x 12.5 x 45 mm) through a 0.2 μm syringe filter (Pall, 0.2 μm HT Tuffryn membrane). For zeta potential measurements, the sample was prepared at a concentration of 0.2 mg / mL in 10 mM NaCl using the procedure mentioned above. A Malvern Zsizer Nanosizer disposable folded capillary cell was used for the measurements. The size of D-4517 was 5.5 ± 0.5 nm and the zeta potential was slightly positive (+5.4 ± 0.4 mV).

[0246] Size-size exclusion chromatography with multi-angle laser scattering (SEC-MALS)

[0247] The molar mass of D-4517.2 will be determined by size exclusion chromatography multi-angle laser scattering (SEC-MALS).

[0248] Result

[0249] D-4517 exhibits nanomolar affinity for VEGFR2 without the need for drug release, AVT-4517. As observed in D-4517 buffer and plasma stability studies, to further increase the stability of the conjugate under physiological conditions and further reduce drug release from the conjugate, as confirmed in the structure of D-4517.2, the cleavable ester bonds on the dendritic surface are replaced by non-cleavable bonds (Figure 1). No cleavable bonds exist in the structure of D-4517.2.

[0250] D-4517.2 is a covalently coupled fourth-generation hydroxyl-terminated PAMAM dendrite containing an ethylenediamine (EDA) core, a amide repeating unit [CH2CH2CONHCH2CH2N], and 64 hydroxyl terminal groups (chemical formula: C622H1184N186O188), and a didesethylsunitinib analog (AVT-4517) bound to the dendrite via a highly efficient click chemistry method. The hydroxyl, fourth-generation, PAMAM dendritic system is monodisperse and produced with high component purity (>95%). For the preparation of D-4517.2, seven of the 64 hydroxyl groups on the dendrite are modified to connect to AVT-4517 (~12.6% of total mass).

[0251] Stability studies in human, mouse and rat plasma

[0252] The in vitro stability of dendritic didesethylsunitinib conjugates D-4517 and D-4517.2 in human, mouse, and rat plasma was evaluated under physiological conditions. The results are presented in Figure 4. The plasma stability of D4517.2 was significantly improved compared to D4517 (2% (w / w) release in human plasma and 4% (w / w) release in rat plasma. At 48 hours, less than 0.5% (w / w) of the drug was released from the dendritic drug conjugate in all three plasmas.

[0253] Combining affinity

[0254] The kinase binding affinity of D-4517 and D-4517.2 was evaluated, and the results are presented in Table 1. Table 1: Binding analysis of dendritic compounds D-4517 and D-4517.2 Compound Name Gene symbol Decorations Kd (nM) D4517 VEGFR2 = 27 D4517 VEGFR1 = 1100 D4517 CSF1R = 82 D4517 KIT = 3.4 D4517 PDGFRA = 16 D4517 PDGFRB = 11 D-4517.2 CSF1R = 41 D-4517.2 VEGFR1 = 890 D-4517.2 KIT = 3 D-4517.2 PDGFRA = 11 D-4517.2 PDGFRB = 7.5 D-4517.2 VEGFR2 = 14

[0255] In all test analyses, the IC50 result of D-4517.2 was lower than that of D-4517, indicating a stronger binding between D4517.2 and the tyrosine kinase receptor. Example 2: Binding of N-acetylglucosamine via a non-cleavable bond.

[0256] Synthesis of dendrites bound to N-acetylglucosamine via a non-cleavable linker. The synthetic pathway for the non-release (or non-cleavable) form of the dendrite / N-acetylglucosamine conjugate is shown in Figure 5. As shown, N-acetylglucosamine is bound to a hydroxyl-terminated PAMAM dendrite via a non-cleavable linker to minimize the release of free N-acetylglucosamine in vivo after administration. Compared to the release or cleavable forms of the dendrite / N-acetylglucosamine complex, the non-release form of the dendrite / N-acetylglucosamine complex provides enhanced therapeutic efficacy. Example 3: Targeting agent bound via a non-cleavable linker

[0257] To evaluate the site-specific targeting and delivery of drugs to hepatocytes via hydroxyl dendrites based on tri-antennae GalNAc. It has been shown that in a STAM model of non-alcoholic steatohepatitis, surface GalNAc sugars exhibit multivalent binding to ASGPR, allowing these dendrites to selectively target and internalize in hepatocytes in vivo.

[0258] The synthesis scheme of β-GalNAc-trianthracite-PEG3-azide (AB3 building block) is shown in Figure 6. Reagents and conditions: (i) scandium trifluoromethanesulfonate, DCE, 3 h, 80 °C; (ii) propargyl bromide, toluene, sodium hydroxide, water, TBAB; (iii) pyridine, thionyl chloride, chloroform, 65 °C, 2 h; (iv) tetrabutylammonium hydrogen sulfate, 50% NaOH, 16 h, room temperature; (v) (iii) CuSO4·5H2O, sodium ascorbate, THF, water, 10 h; (vi) DMF, tetrabutylammonium iodide, NaN3, 80 °C, 5 h; (vii) sodium methoxide, anhydrous methanol, 30 °C, 3 h.

[0259] A three-armed construct was prepared by grafting three β-GALNAc-PEG3 azide molecules onto a propargylated neopentyl tetrol construct to generate an AB3-type orthogonal construct. The synthesis was initiated by the glycosylation of β-D-GalNAc pentaacetate (1, Fig. 6) with 2-[2-(2-azidoethoxy)ethoxy] ethylene-1-ol (2) in the presence of scandium trifluoromethanesulfonate in dichloroethane to generate peracetylated β-GalNAc-PEG3-azide (3). On the other hand, neopentyl tetrol 4 was selectively modified with three propargyl arms in the presence of sodium hydroxide and tetrabutylammonium bromide in DMSO according to a literature method to generate triargylneopentyl tetrol (5). Using sodium hydroxide and TBAB in DMSO, a residual hydroxyl group on compound (5) was reacted with tetraethylene glycol dichloroethylene (7) to provide intermediate compound (8). During the next synthetic step, AB3 building block (8) was clicked through acetylated β-GalNAc-PEG3-azide using familiar CuAAC click reaction conditions (copper sulfate pentahydrate (II) and sodium ascorbate in THF:water) to produce compound (9). The success of the click reaction was confirmed by 1H NMR, HRMS and HPLC. In 1H NMR, a characteristic sharp singlet of triazole was observed at δ 7.9 ppm. Other characteristic peaks were acetate peaks between δ 2.0 and 1.74 ppm, from GalNAc protons at δ 5.2 to 3.2 ppm and NH at GalNAc at δ 7.78 ppm. In the next synthetic step, in the presence of sodium azide and tetrabutylammonium iodide in DMF, the terminal chlorine group of compound (9) was replaced with an azide by nucleophilic substitution to produce compound (10). The final step was a transesterification reaction under zemplen conditions, in which the reaction was carried out in methanol using sodium methoxide to provide the building blocks of deacetylated β-GalNAc-trianthracite-PEG3 azide (11).

[0260] Successful completion of the reaction was confirmed by 1H NMR, in which the peak corresponding to O-acetate completely disappeared and all sugar protons shifted to a higher field. The entire synthetic sequence was characterized by 1H NMR, HPLC and HRMS to confirm the desired compound.

[0261] Preparation of dendritic-β-GalNAc conjugates as described in Examples 1 and 2. Example 4: Preparation of PEG-alkyne functionalized PAMAM-G4-OH.

[0262] PAMAM G4-OH-acetylene 7-8 is produced by a multi-step process. This process has been successfully scaled up to provide large quantities of 1 kg and 0.5 kg of this material.

[0263] A method for manufacturing a poly(aminoamine) fourth-generation hydroxyl-terminated dendrite (PAMAM-G4-OH) for targeted drug delivery applications. The PAMAM G4-OH is modified with an azide to bind the drug using an azide-alkyne cycloaddition (click) binding technique. One PAMAM G4-OH synthetic route follows different strategies for preparing PAMAM dendrites. The final step of this method involves the addition of ethanolamine and PEG-alkyne amine, resulting in a G4 dendrite having 56 to 57 alcohol functional groups and 7 to 8 PEG-alkyne functional groups.

[0264] Analytical Methods

[0265] Various analytical methods were developed for higher-generation PAMAM dendrites (≥G4). A summary of each analytical method is described below.

[0266] UPLC method (ASHV001O)

[0267] A reversed-phase UPLC method for resolving the dendritic structures and starting materials of each PAMAM was developed using modifications to the methods described in the literature (Table 1-1). See Cason, CA et al., Journal of Nanomaterials. 2008, 1-7. DOI:10.1155 / 2008 / 456082. Table 1-1: Summary of UPLC conditions Reverse-phase tubing ACE Excel 3 Super C18, LC column, 50 x 3 x 3 µm Column temperature 40℃ Mobile phase A H2O (0.05% TFA) Mobile phase B MeCN (0.05% TFA) Flow rate 1.27 mL / min Observation wavelength 210 nm

[0268] This method was developed using PAMAM cores obtained through synthesis and G0 to G4 samples obtained from Sigma Aldrich, Inc. A summary of the residence times and UPLC reports for the last two generations of samples is provided in Table 2 below. Table 2: Residence times and UPLC reports for PAMAM generations and starting materials compound Dwell time (min) Relative residence time (min) PAMAM G3.5 2.805 1.462 PAMAM G4-OH-Alkyne 7.8 1.918 1

[0269] SEC-MALLS Method

[0270] A size exclusion chromatography method with multi-angle laser light scattering (MALLS) and differential refractive index (dRI) detectors was developed using the method described in the literature. Mullen, DG et al., Macromolecules. 2012, 45, 5316-5320. A summary of this method is provided in the appendix. Peaks of all generations of PAMAM can be resolved using this method. A summary of the dissolution times and polydispersity measurements for the last two generations is provided in Table 3 below. Table 3: SEC dissolution times of PAMAM dendrites compound Dissolution time (min) Ð (M w / M n ) a PAMAM G3.5 17.9 1.036 PAMAM G4-OH-Alkyne 7.8 17.8 1.031

[0271] Quantitative 1H NMR

[0272] Quantitative 1H NMR spectroscopy was performed using Sigma Aldrich TraceCERT® grade internal standard. This method has been used to quantify the potency of PAMAM dendritic solutions and separated products. This method has also been used to analyze the quality of PEG-alkyne materials (2-[2-(propoxy)ethoxy]ethylamine). 1,3,5-Trimethoxybenzene was used as an internal standard in d4-methanol solvent.

[0273] Scheme for analyzing the average number of PEG-acetylene arms

[0274] The degree of PEG-alkyne functionalization of PAMAM G4-OH was determined by 1H-NMR spectroscopy using the average values ​​provided by two methods (denoted as Method A and Method B). Each method utilized the integral observed with PAMAM-G4-64-OH and the theoretical integral observed with 100% PEG-alkyne functionalization of PAMAM-G4-64-PEG-alkyne. The respective integrals for these two materials are provided in Tables 4 and 5 below. Table 4: Proton chemical shifts and integrals of PAMAM-G4-64-OH proton Chemical shift (ppm) Proton# H a 2.38 ppm 248 H H b 2.58 ppm 124 H H c 2.79 ppm 248 H H d 3.26 to 3.33 ppm 248 H H e 3.61 ppm 128 H Table 5: Proton chemical shifts and integrals (theoretical) of PAMAM-G4-64-PEG-alkynes proton Chemical shift (ppm) Proton# H f 2.38 ppm 258 H H g 2.58 ppm 124 H H h 2.79 ppm 248 H H i 3.27 ppm 120 H H j 3.37 ppm 128 H H k 3.55 ppm 128 H H l 3.65 ppm (multiple peak) 256 H H m 4.2 ppm

[0275] Method A: Method A is based on the calculation of the percentage of the actual integral of proton Hm relative to the Hm integral of 100% alkyne-functionalized PAMAM-G4. This is determined by setting the signal integrals corresponding to protons Ha and Hf to their theoretical value of 248 (for both PAMAM-G4-64-OH and PAMAM-G4-64-PEG-alkyne, this signal is expected to be 248). An example calculation of this method is shown in Figure 7.

[0276] Method B: Method B is based on the calculation of the ratio of protons He from the ethanolamine terminal arm to protons Hm from the PEG-alkyne terminal arm. Due to the overlap between the protons He from the ethanolamine arm and the Hk and HCl protons from the PEG-alkyne terminal arm, the integral of these PEG-alkyne protons must be subtracted (the integral is determined using the integral of the Hm protons). This ratio can then be used to calculate the average number of alkyne functionalized arms. The calculation is shown in the example in Figure 8.

[0277] Manufacturing Summary

[0278] PAMAM Core Synthesis reagents MW (g / mol) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume concentration (weight%) methyl acrylate solution 602.4699 Methyl acrylate 86.09 0.95 409.6795 431.24 4758.74 11 68 MeOH 0.792 192.7904 243.42 32 ethylenediamine solution 173.3333 ethylenediamine 60.1 0.9 26.0000 28.89 432.61 1 15 MeOH 0.792 147.3333 186.03 85

[0279] As shown in the above protocol, the synthesis of PAMAM dendritic cores was performed according to previous descriptions in the literature. A methanol solution of ethylenediamine was added dropwise to an excess methanol solution of methyl acrylate at 0°C. After 1 hour, the solution was heated to room temperature and stirred for 24 hours. The solution was then concentrated under reduced pressure at 20°C. The resulting oil was then diluted with MeOH (1 L) and concentrated again under reduced pressure. This process was repeated once more to provide a quantitative yield (17.5 g) of PAMAM cores in a colorless oil. ¹H-NMR and GC analyses showed that the PAMAM cores were produced with high purity. A summary of the metrics used in this method is shown in Table 6 below. Table 6: Summary of metrics used in experiment AA08-055 Crude product (g) 304.5 g Product potency (%w / w) 55.12% Analyze the yield (g) 167.8 grams Analyze yield (%) 97%

[0280] Synthesis of PAMAM dendrites capped with methylamine

[0281] Full-generation PAMAM dendrites were prepared according to literature conditions. Under these conditions, an ethylenediamine solution (86% w / w in methanol, 25 equivalents per ester of PAMAM starting material) was cooled to 0°C under a nitrogen atmosphere. Then, a semi-generation PAMAM dendrite or PAMAM core solution (10% w / w in methanol) was added over a 2-hour period. After addition, the reaction mixture was stirred at 0°C for 1 hour. Subsequently, the reaction mixture was heated to 20°C and stirred for 5 days. The crude product was then concentrated by vacuum distillation while maintaining the crude product temperature < 25°C. Residual ethylenediamine was then removed by one of the following two methods: Method 1: 1. Dissolve the crude product residue in methanol (2.5 volumes). 2. Dilute with toluene (24 volumes). 3. Concentrate by vacuum distillation. This method was repeated 4 times or until residual ethylenediamine was no longer detectable in the crude product or distillate by GC and 1H-NMR analysis. Method 2: 1. Dissolve the crude product residue in pentanol to produce a 10% by weight dendritic solution. 2. Concentrate by distillation using a stirred membrane evaporator. Repeat this method >4 times or until no residual ethylenediamine is observed in the crude product or distillate by GC and 1H-NMR analysis. Method 1 was used for PAMAM G0 and G1, and Method 2 was used for PAMAM G2 and G3. A summary of the results during the synthesis of PAMAM dendritices with each fully amine-terminated group is provided in Table 7 below. Table 7: Summary of Results for Fully Amine-Terminated PAMAM Dendritices experiment PAMAM Yield (g) Yield (%) Polydispersity (M) w / M n ) AA08-063 PAMAM G0 213.80 99.7 1.015 HA04-029 PAMAM G1 563 95 1.012 HA04-036 PAMAM G2.0 652 -* 1.056 AA08-086 PAMAM G3.0 1322 95 1.14

[0282] Synthesis of PAMAM dendrites capped with semi-ester

[0283] Semi-generation PAMAM dendrites were prepared according to literature conditions. Under these conditions, a methyl acrylate solution (55% w / w in methanol, 3 equivalents of each terminal amine on the dendritic starting material) was cooled to 0°C under a nitrogen atmosphere. A full-generation PAMAM dendrite solution (10% w / w in methanol) was then added over a 2-hour period. After the addition, the reaction mixture was stirred at 0°C for 1 hour. Subsequently, the reaction mixture was heated to 20°C and stirred for 3 to 4 days until 1H-NMR analysis of the crude product indicated complete conversion of the starting material. The crude product was then concentrated by vacuum distillation while maintaining the temperature of the crude product < 25°C. Residual methyl acrylate was then removed by vacuum distillation using a 3x azeotropic reaction with methanol (the removal of methyl acrylate was indicated by 1H-NMR, GC, and / or UPLC analysis). A summary of the results during the synthesis of each semi-generation PAMAM dendrite is shown in Table 8 below. Table 8: Summary of Results for Semi-Ester-Terminated PAMAM Dendrites experiment PAMAM Yield (g) Yield (%) Polydispersity (M)w / M n ) HA04-024 PAMAM G0.5 499.4 Quantitative 1.020 HA04-33 PAMAM G1.5 1025 91 1.020 HA04-64 PAMAM G2.5 1215 Quantitative 1.034 AA10-062 PAMAM G3.5 2360 99 1.1

[0284] PAMAM G4-OH-Alkyne 7-8 production

[0285] A jacketed container was loaded with ethanolamine and PEG-alkyne in a 3:1 molar ratio, followed by methanol. The container was purged with N2(g) and cooled to 0°C. Then, 10 wt% PAMAM G3.5 was added to the reactor in methanol over 2 hours. The solution was then stirred at 0°C for 4 hours, followed by heating to 20°C. The solution was then allowed to be stirred at 20°C for 6 days. Afterward, the solution was diluted with water to half its concentration and purified by ultrafiltration to provide an aqueous product. For production batch AA10-065, the aqueous solution was then purified using a larger pore size membrane to remove dimer impurities. The solvent was then replaced with methanol by ultrafiltration using 10 permeate volumes of methanol. A summary of the results for each scale-up is provided in Table 9 below. Table 9: Summary of Results for Experiment AA10-007 PAMAM G4-OH-Alkyne 7-8 batch Batch number AA10-064 AA10-065 PAMAM G3.5 source Dendritech Snapdragon (Batch AA10-062) Mass of methanol solution (kg) 3.25 8.21 kg Valence (% w / w) 16.6%w / w 19.5% w / w Yield 539.5 g 1.6 kg Yield (%) 93% 71.8% Polydispersity (M) w / M n ) 1.030 1.045 Average value of PEG-alkyne arm# 8.1 7.8 -OH / -NH ratio 0.39 ND Residual ethanolamine (dry basis, GC) 0.2% 0.4% Residual PEG-alkyne (dry basis, GC) 0.02% 0.01%

[0286] Conclusion

[0287] A method for producing PAMAM G4-OH-acetylene 7-8 in quantities >1 kg has been successfully scaled up and developed. This method is carried out on 500-gram and 2-kg scales, producing the product with reproducible quality and yield. This demonstrates increased product quality and the ability to remove dimer impurities downstream via ultrafiltration purification. This allows for the removal of significant amounts of dimer impurities generated upstream in the pathway of batch AA10-065.

[0288] Experiment

[0289] PAMAM Core Synthesis reagents MW (g / mol) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume concentration (weight%) methyl acrylate solution 602.4699 Methyl acrylate 86.09 0.95 409.6795 431.24 4758.74 11 68 MeOH 0.792 192.7904 243.42 32 ethylenediamine solution 173.3333 ethylenediamine 60.1 0.9 26.0000 28.89 432.61 1 15 MeOH 0.792 147.3333 186.03 85

[0290] The synthesis of PAMAM dendritic cores was performed according to previous descriptions in the literature. A methanol solution of ethylenediamine was added dropwise to an excess methanol solution of methyl acrylate at 0°C. After 1 hour, the solution was heated to room temperature and stirred for 24 hours. The solution was then concentrated under reduced pressure at 20°C. The resulting oil was then diluted with MeOH (1 L) and concentrated again under reduced pressure. This was repeated once more to provide a quantitative yield (17.5 g) of PAMAM cores in a colorless oil. ¹H-NMR and GC analyses showed that the PAMAM cores were produced with high purity. A summary of the metrics used in this method is shown in Table 10. Table 10: Summary of metrics used in experiment AA08-055 Crude product (g) 304.5 g Product potency (%w / w) 55.12% Analyze the yield (g) 167.8 grams Analyze yield (%) 97%

[0291] PAMAM G0.0 Synthesis reagents MW (g / mol) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume ethylenediamine solution 1501.9406 ethylenediamine 60.1 0.9 1296.5647 1440.63 21573.46 52 MeOH 0.792 205.3758 259.31 0.2 core solution 1762.5712 core 404.46 167.8000 414.87 1 MeOH 0.792 1594.7712 2013.60 12

[0292] A methanol solution of ethylenediamine was charged into a 5 L jacketed vessel under a nitrogen atmosphere. The reaction solution was cooled to 0°C and the PAMAM core solution was added over a period of 2 hours. After the addition, the reaction mixture was stirred at this temperature for 1 hour. Subsequently, the reaction mixture was heated to room temperature and stirred for 5 days. Methanol and most of the ethylenediamine were then removed by vacuum distillation while maintaining the reactor contents below 25°C. Residual ethylenediamine was then removed by 5x azeotropic distillation with 21 volumes of 1:9 methanol / toluene. Complete removal was confirmed by 1H-NMR analysis. After complete removal of ethylenediamine, residual toluene was removed by 3x azeotropic distillation from 3 volumes of methanol to provide a 35.61% w / w solution of 600 g of PAMAM G0.0 in methanol.

[0293] PAMAM G0.5 Synthesis Table 11. reagents MW (g / mol) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume valence (weight%) concentration (weight%) methyl acrylate solution 2316.2707 Methyl acrylate 86.09 0.95 534.3509 562.47 6206.89 15 MeOH 0.792 1781.9197 2249.90 4 PAMAM Core Solution 2138.0244 PAMAM Core 516.69 0.9 600.4000 667.11 413.79 1 35.61 10 MeOH 0.792 1537.6244 1941.44 90

[0294] A methanol solution of methacrylate and a methanol solution containing PAMAM cores were added to a 10 L reactor at 0°C for 2 hours. The solution was then stirred at 0°C for another hour, followed by heating to 20°C and stirring for 3 days. The completion of the reaction was determined by Kaiser assay for residual primary amines and further verified by 1H-NMR spectroscopy. Volatiles were then removed by distillation under reduced pressure, and the reactor jacket was maintained at 20°C. Residual methyl acrylate was then removed by azeotropic distillation with methanol (3 volumes) at 2x. GC and NMR analysis of the crude material showed complete removal of methyl acrylate. A product of 2359.9 g viscous oil was obtained. Quantitative 1H-NMR analysis showed that the oil was 21.16 wt% PAMAM G0.5, with an analytical yield of 499.35 g (100.11%). A summary of the metrics of this method is shown in Table 12. Table 12: Method Measurements Used in Experiment HA04-24 reaction time 72 h crude yield 2359.9 g Product valence 21.16% w / w Analyze yield (g) 499.35 g Analyze yield (%) Quantitative Polydispersity (Ð (M) w / M n )) 1.025

[0295] PAMAM G1.0 Synthesis Table 13. reagents MW (g / mol) valence (% w / w) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume concentration (weight%) ethylenediamine solution 6086.83 6930.93 ethylenediamine 60.1 100 0.9 4979.46 5532.74 82852.97 200 - MeOH 100 0.792 1107.37 1398.19 2.8 - PAMAM G 0.5 solution 4993.55 PAMAM G0.5 1205.4 21.16 2359.9 414.26 1 10 MeOH 100 0.792 2633.65 3325.31 90

[0296] A 20 L jacketed reactor was purged with N2 (g) and loaded with an ethylenediamine solution. PAMAM G0.5 solution was added dropwise at 0°C for ~2 hours. After stirring at 0°C for ~4 hours, the solution was heated to 20°C and stirred for 5 days. 1H-NMR analysis showed complete consumption of the methyl ester of PAMAM G0.5. Volatiles were removed under reduced pressure, and the reactor jacket was maintained at 20°C. Residual ethylenediamine was then removed by azeotropic distillation with 1:9 MeOH / toluene (23 volumes) at 7x. Ethylenediamine removal was monitored by NMR analysis of the crude reaction mixture. Residual toluene was then removed from methanol at 3x concentration under reduced pressure. An oil product was obtained. Quantitative 1H-NMR analysis showed the oil to be 23.5 wt% PAMAM G1.0, analyzed in a yield of 563.03 g (95% yield). A summary of the metrics of this method is shown in Table 14. Table 14: Method Measurements Used in Experiment HA04-29 reaction time 120 h crude yield 2395 g Product valence 23.5% w / w Analyze yield (g) 563 g Analyze yield (%) 95% Polydispersity (Ð (M) w / M n )) 1.012

[0297] PAMAM G1.5 Synthesis Table 15. reagents MW (g / mol) valence (weight%) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume concentration (weight%) methyl acrylate solution 1523.60 Methyl acrylate 86.09 0.95 830.89 874.63 9561.46 twenty four MeOH 0.792 692.70 874.63 1.5 ethylenediamine solution 5750.16 PAMAM G1.0 1429.876 twenty four 0.9 2395.90 2662.11 402.14 1 10 MeOH 0.792 3354.26 4235.18 90

[0298] A methanol solution of methyl acrylate and a methanol solution of PAMAM G1.0 were added to a 10 L reactor at 0°C for 2 hours. The solution was stirred at 0°C for another 2 hours, then heated to 20°C and stirred for 3 days. The reaction was verified by 1H-NMR spectroscopy. Volatiles were then removed by distillation under reduced pressure and the reactor jacket was maintained at 20°C. Residual methyl acrylate was then removed by azeotropic distillation with methanol (3 volumes) at 3x. GC and NMR of the crude material showed complete removal of methyl acrylate. A product of 2359.9 g viscous oil was obtained. Quantitative 1H-NMR analysis showed that the oil was 32.89 wt% PAMAM G1.5, which was provided in an analytical yield of 1025 g (91% yield). The metric of this method is summarized in Table 16 below. Table 16: Metrics of the Method Used in Experiment HA04-33 reaction time 72 h crude yield 3116.1 Product valence 32.89% w / w Analyze yield (g) 1024.9 g Analyze yield (%) 91% Polydispersity (Ð (M) w / M n )) 1.020

[0299] PAMAM G2.0 Synthesis Table 17. reagents MW (g / mol) valence (% w / w) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume concentration (weight%) ethylenediamine solution 10708.31 12190.83 ethylenediamine 60.1 100 0.9 8776.44 9751.60 146030.63 400 - MeOH 100 0.792 1931.87 2439.23 2.38 - PAMAM G1.5 solution 10248.85 PAMAM G1.5 2807.316 32.89 3116.10 365.08 1 10 MeOH 100 0.792 7132.75 9006.00 90

[0300] The jacketed reactor was purged with N2(g) and filled with an ethylenediamine solution. A PAMAM G1.5 solution was added dropwise at 0°C for ~2 hours. After stirring at 0°C for ~4 hours, the solution was heated to 20°C and stirred for 5 days. 1H-NMR analysis showed complete consumption of the methyl ester of PAMAM G1.5. Volatiles were removed under reduced pressure, and residual ethylenediamine was removed by distillation. Ethylenediamine removal was monitored by NMR analysis of the crude reaction mixture. An oily product was obtained. Quantitative 1H-NMR analysis showed that the oil was 9.5 wt% PAMAM G2.0. A summary of the method metrics is shown in Table 18. Table 18: Method metrics used in experiment HA04-36 reaction time 120 h crude yield 6832.5 Product valence 9.55% w / w Analyze yield (g) 652.5 g Analyze yield (%) - Polydispersity (Ð (M) w / M n )) 1.056

[0301] PAMAM G2.5 Synthesis Table 19. reagents MW (g / mol) valence (weight%) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume concentration (weight%) methyl acrylate solution 1518.3962 Methyl acrylate 86.09 0.95 828.0576 871.64 9618.51 48 MeOH 32.04 0.792 690.3386 871.64 1.34 PAMAM G2.0 solution 6832.5000 PAMAM G2.0 3256.24 9.55 0.9 6832.5000 7591.67 200.39 1 9.55 MeOH 0.792 0.0000 0.00

[0302] A methanol solution of methyl acrylate and a methanol solution of PAMAM G2.0 were added to a 20 L jacketed reactor at 0°C for 2 hours. The solution was stirred at 0°C for another 2 hours, then heated to 20°C and stirred for 4 days. The reaction was verified by 1H-NMR spectroscopy. Volatiles were then removed by distillation under reduced pressure and the reactor jacket was maintained at 20°C. Residual methyl acrylate was then removed by azeotropic distillation with methanol (3 volumes) at 3x. GC and NMR of the crude material showed complete removal of methyl acrylate. A product of 7610 g viscous oil was obtained. Quantitative 1H-NMR analysis showed that the oil was 15.97 wt% PAMAM G2.5, which was provided in an analytical yield of 1215.3 g (100% yield). A summary of the method metrics is shown in Table 20. Table 20: Method metrics used in experiment HA04-64 reaction time 96 h crude yield 7610 g Product valence 16% w / w Analyze yield (g) 1215.3 g Analyze yield (%) Quantitative Polydispersity (Ð (M) w / M n )) 1.024

[0303] PAMAM G3.0 Synthesis Table 21. reagents MW (g / mol) valence (% w / w) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume ethylenediamine solution 10708.31 12190.83 ethylenediamine 60.1 100 0.9 9739 10821 162.044 800 MeOH 100 0.792 2143 2705 0.36 PAMAM G2.5 solution 12176 PAMAM G2.5 6011.2 16 7610 202.56 1 MeOH 100 0.792 4566 5765 11.37

[0304] The jacketed reactor was purged with N2(g) and filled with ethylenediamine solution. PAMAM G2.5 solution (HA04-064) was added via a diaphragm pump at 0°C for ~2 hours. After stirring at 0°C for ~4 hours, the solution was heated to 20°C and stirred for 5 days. 1H-NMR analysis showed complete consumption of the methyl ester of PAMAM G2.5. Volatile substances were removed under reduced pressure. Residual ethylenediamine was then removed by distillation using a stirred membrane evaporator. The removal of ethylenediamine was monitored by NMR analysis of the crude reaction mixture. An oily product was obtained. Quantitative 1H-NMR analysis showed that the oil was 21.5 wt% PAMAM G3.0. A summary of the metric of this method is shown in Table 22. Table 22: Method Measurements Used in Experiment AA08-086 Response time 120 h crude yield 6162.5 g Product efficacy 21.5% w / w Analyze yield (g) 1322 g Analyze yield (%) 95% Polydispersity (Ð (M) w / M n )) 1.14

[0305] PAMAM G3.5 Synthesis Table 23. reagents MW (g / mol) valence (weight%) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume methyl acrylate solution 1518.3962 Methyl acrylate 86.09 0.95 1740.1 1831.7 20212.81 96 MeOH 32.04 0.792 1450.71 1831.7 0.30 PAMAM G2.0 solution 13219 PAMAM G2.0 3256.24 21.45 0.9 6162.5 191.32 1 MeOH 0.792 7056 8909

[0306] A methanol solution of methyl acrylate and a methanol solution of PAMAM G3.0 (AA08-086) were added to a 20 L jacketed reactor at 0°C for 2 hours. The solution was stirred at 0°C for another 2 hours, then heated to 20°C and stirred for 4 days. The reaction was verified by 1H-NMR spectroscopy. Volatiles were then removed by distillation under reduced pressure and the reactor jacket was maintained at 20°C. Residual methyl acrylate was then removed by 3x azeotropic distillation with methanol (3 volumes). GC and NMR of the crude material showed complete removal of methyl acrylate. A product of 7610 g viscous oil was obtained. Quantitative 1H-NMR analysis showed that the oil was 15.97 wt% PAMAM G2.5, provided with an analytical yield of 2360 g (99% yield). A summary of the method metrics is shown in Table 24. Table 24: Method metrics used in experiment AA10-062 reaction time 96 h crude yield 13800 g Product valence 17.1% w / w Analytical quantity (g) 2359.8 g Analyze yield (%) 99% Polydispersity (Ð (M) w / M n )) 1.1

[0307] PAMAM G4-OH-Alkyne 7-8 Synthesis - Batch AA10-064 Table 25. reagents MW (g / mol) valence (% w / w) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume amine solution 6233 ethanolamine 61.08 100 1.012 2853 2821 46623 1200 PEG-Alkynes 143.19 100 2355 15541 400 MeOH 100 0.792 1025 1294 2.59 PAMAM G3.5 solution 4825 PAMAM G2.5 6011.2 96.5 500 38.85 1 MeOH 100 0.792 4325 5461 11.32

[0308] The jacketed reactor was purged with N2 (g) and loaded with ethylenediamine solution. PAMAM G3.5 solution was added via a diaphragm pump at 0°C for approximately 2 hours. After stirring at 0°C for approximately 4 hours, the solution was heated to 20°C and stirred for 6 days. The solution was then diluted with H2O (11 kg). The solution was then purified by ultrafiltration using a regenerated cellulose membrane with a 5 kDa MWCO, providing an aqueous product with complete removal of ethanolamine and PEG-alkyne as shown by 1H-NMR. The solvent was exchanged with methanol via continuous percolation to provide a methanol solution (3230.9 g) of PAMAM G4. Quantitative 1H-NMR analysis showed that the solution contained 16.6 wt% PAMAM G4-OH-alkyne 7-8 (93% yield). A summary of the metrics of this method is shown in Table 26. Table 26: Summary of Results AA10-064 Batch number AA10-064 PAMAM G3.5 source Dendritech Mass of methanol solution (kg) 3.25 Valence (% w / w) 16.6%w / w Yield 539.5 g Yield (%) 93% Polydispersity (M) w / M n ) 1.030 Average value of PEG-alkyne arm# 7.8 -OH / -NH ratio 0.39 Residual ethanolamine (dry basis, GC) 0.2% Residual PEG-alkyne (dry basis, GC) 0.02%

[0309] PAMAM G4-OH-Alkyne 7-8 Synthesis - Batch AA10-065 reagents MW (g / mol) valence (% w / w) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume amine solution 23457 ethanolamine 61.08 100 1.012 11002 10876 180139 1200 PEG-Alkynes 143.19 100 8598 60046 400 MeOH 100 0.792 3856 4868 2.61 PAMAM G3.5 solution 18642 PAMAM G2.5 6011.2 25.9 10902 150.12 1 MeOH 100 0.792 7740 9773 5.24

[0310] The jacketed reactor was purged with N2 (g) and loaded with an ethylenediamine solution. PAMAM G3.5 solution was added via a diaphragm pump at 0°C for approximately 2 hours. After stirring at 0°C for approximately 4 hours, the solution was heated to 20°C and stirred for 6 days. The solution was then diluted with H2O (42 kg). The solution was then purified by ultrafiltration using a regenerated cellulose membrane with a 5 kDa MWCO, providing an aqueous product with complete removal of ethanolamine and PEG-alkyne as shown by 1H-NMR. Dimeric impurities were then removed by ultrafiltration using a regenerated cellulose membrane with a 30 kDa MWCO, and the desired product was permeated. Continuous permeation was used to exchange the solvent with methanol to provide a methanol solution (8205 g) of product PAMAM G4. Quantitative 1H-NMR analysis showed that the solution contained 19.5 wt% PAMAM G4-OH-alkyne 7-8 (71.8% yield). The results of this method are summarized in Table 27 below. Table 27: Summary of Results for AA10-065 Batch number AA10-065 PAMAM G3.5 source Snapdragon (Batch AA10-062) Mass of methanol solution (kg) 8.21 kg Valence (% w / w) 19.5% w / w Yield 1.6 kg Yield (%) 71.8% Polydispersity (M) w / M n ) 1.045 Average value of PEG-alkyne arm# 7.8 -OH / -NH ratio ND Residual ethanolamine (dry basis, GC) 0.4% Residual PEG-alkyne (dry basis, GC) 0.01%

[0311] Analytical Methods

[0312] UPLC Method (ASHV001O) A. Reagents 1. Water: HPLC grade 2. Acetonitrile: HPLC grade 3. Trifluoroacetic acid: HPLC grade B. Solutions 1. Diluent: MiliQ water 2. Mobile phase: A. 0.05% trifluoroacetic acid in water B. 0.05% trifluoroacetic acid in acetonitrile 3. Sample solution: Sample solution at a target concentration of approximately 2 mg / mL. 4. Blank: Diluent. C. Chromatographic conditions Column: ACE Excel 3 Super C18, LC column, 50 x 3 x 3 µm Temperature: 40℃ Flow rate: 1.27 mL / min Detection: A: UV at 210 nm; 4.8 nm bandwidth B: UV at 220 nm; 4.8 nm bandwidth Injection volume: 10 µL Run time: 12.3 min Gradient Time (min) %A %B 0 98 2 10.00 0 100 12.02 98 2 12.30 98 2 D. Calculate Ab = Total area count from blank; Ac = Area count of components in sample injection; Ta = Total area from sample injection. This method can also be used for weight percentage analysis using calibration standards. Typical standard concentrations are 0.025 to 1.0 mg / mL.

[0313] GC Method (ASV003F) A. Chromatographic Conditions Column: Agilent CP-Volamine (Part No. CP7447) Inlet Temperature: 250℃ Inlet Pressure: 14.3 psi Total Inlet Flow Rate: 37.3 mL / min Inlet Split Ratio: 10:1 Injection Volume: 5 µL Column Flow Rate: 3.1 mL / min FID Temperature: 300℃ FID H2 Flow Rate: 40.0 mL / min FID Air Flow Rate: 360.0 mL / min FID Make-up Flow Rate: 25 mL / min FID Ignition Compensation: 0.5 pA Run Time: 20.33 min Oven Temperature Gradient Rate (°C / min) Temperature (°C) Duration Runtime N / A 65 4 4 15 100 3 9.33 30 250 6 20.33 D. Calculate Ab = Total area count from blank Ac = Area count of component in sample injection Ra = Component response factor = Sample concentration

[0314] To determine impurities using this method, standards of 14.928 and 10.332 mg / mL ethanolamine and PEG-acetylene in isopropanol were prepared and injected onto a GC. This resulted in a residence time of approximately 6.88 minutes for ethanolamine and 14.1 minutes for PEG-acetylene. These solutions were combined in a 1:1 ratio. This combined solution was used as a stock solution for a ½ series of dilutions, achieving concentrations of 0.007 and 0.005 mg for ethanolamine and PEG-acetylene, respectively, using isopropanol. Calibration results are shown below. GC calibration data for ethanolamine and PEG-acetylene. Concentration of ethanolamine (mg / mL) Concentration of PEG-alkyne (mg / mL) ethanolamine signal region PEG-Alkyne Signal Region 14.928 10.332 22543.5 22341 7.4640 5.1660 11862.6 11688.7 3.7320 2.5830 5755.5 5656.6 1.8660 1.2915 2962 2966 0.93300 0.64575 1423.5 1486.3 0.46650 0.32288 708.2 758.3 0.23325 0.16144 323.2 360.1 0.11663 0.080719 153.4 177.7 0.058313 0.040359 72.9 91 0.029156 0.020180 30.5 42.3 0.014578 0.010090 13 twenty one 0.0072891 0.0050449 4.7 11.5

[0315] The standard curve of ethanolamine was used to calculate the concentration of ethanolamine in the sample. The signal of PEG-acetylenes was below LOD, so the lowest value achieved in this calibration was reported as the concentration.

[0316] SEC-MALLS Method A. Reagents 1. Water: HPLC grade 2. Citric acid: ACS reagent, ≥99.5% B. Solutions 1. Diluent: Water, 0.1 M citric acid, pH 2.7 2. Mobile phase: A. Water, 0.1 M citric acid, pH 2.7 3. Sample solution: Sample solution at a target concentration of approximately 2 mg / mL. 4. Blank: Diluent. C. Chromatographic Conditions: Columns: TosoHaas TSK-Gel Guard PHW 0662 (75 mm x 7.5 mm, 12 µm), G2000 PW 05761 (300 mm x 7.5 mm, 10 µm, 125 Å), G 3000 PW 05762 (300 mm x 7.5 mm, 10 µm, 200 Å), G4000 PW (300 mm x 7.5 mm, 17 µm, 500 Å) Temperature: 25℃ Flow Rate: 1 mL / min Detection: A: Wyatt Optilab differential refractive index detector operating at 658 nm B: Wyatt miniDAWN multi-angle laser light scattering detector with a 120 mW laser operating at 658 nm Injection Volume: 100 µL Run Time: 40 min

[0317] References for Example 4: 1. Cason, C. A et al., Journal of Nanomaterials. 2008, 1-7. DOI:10.1155 / 2008 / 456082 2. Mullen, DG et al., Macromolecules. 2012, 45, 5316-5320. Example 5: Preparation of PEG-Alkyne Functionalized PAMAM-G4-OH

[0318] A method was developed for loading G4.0 PAMAM dendrites with 3 PEG-alkynes (PAMAM G4-OH-alkyne 3) and 10 PEG-alkynes (PAMAM G4-OH-alkyne 3) as connecting arms. These functionalized dendritic systems are responsible for the difference in reactivity by adjusting the stoichiometry of the PEG-alkynes relative to ethanolamine. This method was successfully scaled up to provide 40 g of PAMAM G4-OH-alkyne 3 and 160 g of PAMAM G4.0-50-OH-10-PEG-alkyne. In both cases, the products obtained had a low polydispersity of approximately 1.03.

[0319] Additionally, a method for manufacturing a poly(aminoamine) fourth-generation hydroxyl-terminated dendrite (PAMAM-G4-OH) for targeted drug delivery applications is developed. The PAMAM G4-OH is modified with an azide to enable the binding of active substances using an azide-alkyne cycloaddition (click) binding technique. A method for preparing PAMAM G4 having an average of seven alkyne functional groups is developed. Current PAMAM G4-OH synthetic routes follow different strategies for preparing PAMAM dendrites. The final step of this method involves the addition of ethanolamine, resulting in a G4 dendrite with 64 alcohol functional groups. For binding, a method for preparing two G4-OH analogs is developed, where n is approximately 3 and 10, respectively.

[0320] Analytical Methods

[0321] Due to the complexity of PAMAM dendrites, the quality properties of the product are characterized by many analytical methods, including SEC-MALLS (polydispersity), 1H NMR (identity, potency, average linker load), 13C NMR (identity), and GC (residual ethanolamine and PEG alkyne monomers).

[0322] SEC-MALLS Method

[0323] A size exclusion chromatography method using multi-angle laser light scattering (MALLS) and differential refractive index (dRI) detectors was developed using the method described in the literature. See Mullen, DG et al., Macromolecules. 2012, 45, 5316-5320. This method can be used to resolve peaks of all generations of PAMAM. A summary of the dissolution times and polydispersity measurements for each generation is provided in Table 28. Table 28: SEC Dissolution Time of PAMAM Dendrites compound batch# Dissolution time (min) Ð (M w / M n ) a PAMAM G3.5 ---- 17.9 1.036 PAMAM G4 MKCL7410 17.8 1.031

[0324] Quantitative 1H NMR

[0325] Quantitative 1H NMR spectroscopy was performed according to Sigma Aldrich TraceCERT® grade internal standards. These methods have been used to quantify the potency of PAMAM dendritic solutions and separated products. This method has also been used to analyze the quality of PEG-alkyne materials (2-[2-(propoxy)ethoxy]ethylamine). 1,3,5-Trimethoxybenzene was used as an internal standard in d4-methanol solvent.

[0326] Method Development

[0327] PAMAM 3.5 Generation Source Material

[0328] A methanol solution (29.4% w / w) of PAMAM G3.5 was prepared prior to use. Quality properties of this input material are provided in Table 29. Table 29: PAMAM G3.5 Reagent Solution Product Information PAMAM G3.5 manufacturer Dentritech Manufacturer batch # 0121-02-E3.5-LD-A Total solution mass 1648 g Solution potency (% w / w) 29.4 PAMAM G3.5 (g) 494.3 g Polydispersity (M) w / M n ) 1.036

[0329] Reaction of PAMAM G3.5 with PEG-alkynyl / ethanolamine under high amine excess conditions

[0330] A series of experiments were conducted to explore the variation of the PEG-alkynyl / ethanolamine molar ratio under high amine excess conditions (1600 equivalents of total amine, 25 equivalents per terminal ester). First, a methanol solution of PEG-alkynyl and ethanolamine (~1600 equivalents) was cooled to 0°C. Then, a methanol solution of PAMAM G3.5 (10% w / w) was added dropwise over 2 hours. After stirring at 0°C for another 2 hours, the solution was heated to 20°C and stirred for 6 days. For purification, the material was treated with TFF to provide an aqueous solution of the product. For analysis, a fraction of this solution was taken and then water was removed under vacuum, followed by analysis by 1H-NMR. Following the same protocol, three other reactions were set up to explore PEG-alkynyl / ethanolamine ratios achieving 10 and 14 alkyne chains. A similar study is currently underway to optimize conditions for the average functionalization of the three alkyne arms.

[0331] Scheme for analyzing the average number of PEG-acetylene arms

[0332] The degree of functionalization of PAMAM G4-OH with PEG-alkyne was determined by 1H-NMR spectroscopy using the average values ​​provided by two methods (referred to as Method A and Method B). Each method utilized the integral observed with PAMAM-G4-64-OH and the theoretical integral observed with 100% PEG-alkyne functionalization of PAMAM-G4-64-PEG-alkyne. The respective integrals for these two materials are provided in Tables 30 and 31. Table 30: Proton chemical shift and integral of PAMAM-G4-64-OH proton Chemical shift (ppm) Proton# H a 2.38 ppm 248 H H b 2.58 ppm 124 H H c 2.79 ppm 248 H H d 3.26 to 3.33 ppm 248 H H e 3.61 ppm 128 H Table 31: Proton chemical shifts and integrals of PAMAM-G4-64-PEG-alkynes (theoretical) proton Chemical shift (ppm) Proton# H f 2.38 ppm 258 H H g 2.58 ppm 124 H H h 2.79 ppm 248 H H i 3.27 ppm 120 H H j 3.37 ppm 128 H H k 3.55 ppm 128 H H l 3.65 ppm (multiple peak) 256 H H m 4.2 ppm 128 H

[0333] Experimental results of different ratios of PEG-acetylane / ethanolamine

[0334] Methods A and B were used to calculate the average number of PEG-alkyne arms on dendrites obtained using 1600 equivalents of total amine relative to the dendrite (25 equivalents relative to the terminal group) in experiment DP07-51. These results are summarized in Table 32. The relationship between the average number of PEG-alkyne arms and the molar percentage of PEG-alkyne relative to total amine content is shown in Figure 10. Table 32: Summary of Results of PEG-Alkyne Functionalization of PAMAM G-4 Using 1600 Equivalents of Amine sample PEG-Alkynes (in moles relative to total amine content) Average value of PEG-alkyne arm# Polydispersity (M) w / Mn ) Method A Method B average value DP07-51-1 25 mol% 7.80 7.96 7.88 1.034 DP07-51-2 35 mol% 11.84 11.36 11.60 1.053 DP07-51-3 40 mol% 14.01 13.28 13.65 1.030 DP07-51-4 45 mol% 15.61 15.15 15.38 1.056

[0335] Data and previously obtained development data are combined and listed in Table 33 and plotted in Figure 11. This data further validates the reproducible linear correlation between the average number of PEG-acetylenic arms and the molar percentage of PEG-acetylenic hydrocarbons relative to the total amine content. Table 33: Summary of results from all experiments using 1600 equivalents of amine for PEG-acetylenic hydrocarbon functionalization of PAMAM G-4. LNB# Morphine Average acetylene hydrocarbon arm AA08-88-2 8.33 2.33 AA08-88-1 10.00 2.93 AA08-88-3 20.00 6.62 DP07-51-1 25.00 7.78 AA08-88-3 26.30 8.03 DP07-51-2 35.00 11.6 DP07-51-3 40.00 13.28 DP07-51-4 45.00 15.38

[0336] Experimental results for different ratios of PEG-acetylene / ethanolamine with n=3

[0337] A series of experiments were conducted to fine-tune the PEG-alkynyl / ethanolamine molar ratio to achieve a target average linker n=3 (1600 equivalents of total amine, 25 equivalents per terminal ester). First, a methanol solution of PEG-alkynyl and ethanolamine (~1600 equivalents) was cooled to 0°C. Then, a methanol solution of PAMAM G3.5 (10% w / w) was added dropwise over 2 hours. After stirring at 0°C for another 2 hours, the solution was heated to 20°C and stirred for 6 days. For purification, the material was treated with TFF to provide an aqueous solution of the product. For analysis, a portion of this solution was taken and then water was removed under vacuum, followed by analysis by 1H-NMR.

[0338] These results are summarized in Table 34. The relationship between the average number of PEG-alkyne arms and the mole % of PEG-alkyne relative to total amine content is shown in Figure 12 below. Based on the linear equation, 9.6 mole % of PEG-alkyne is required for n = 3 and 31 mole % for n = 10. Table 34: Summary of Results of PEG-Alkyne Functionalization of PAMAM G-4 Using 1600 Equivalent Amines sample PEG-Alkynes (in moles relative to total amine content) Average value of PEG-alkyne arm# Polydispersity (M) w / M n ) Method A Method B average value DP07-60-1 8 mol% 2.67 2.54 2.60 1.026 DP07-60-2 9 mol% 2.95 2.74 2.85 1.029 DP07-60-3 10 mol% 3.18 3.12 3.15 1.025 DP07-60-4 11 mol% 3.58 3.44 3.51 1.026 Table 35: Summary of the results of PEG-alkyne functionalization of PAMAM G-4 using 1600 equivalent amines for DP07-60 and DP07-51 experiment Morphine Average alkyne arm DP07-60-1 8 2.61 DP07-60-2 9 2.85 DP07-60-3 10 3.15 DP07-60-4 11 3.51

[0339] 5 g validation run to produce PAMAM G4.0 (DP07-74-1) with n=3 alkyne arms.

[0340] Experiment DP07-74-1 was conducted on a 5-gram scale to allow for further verification of the PEG-alkyne / ethanolamine ratio previously established through small-scale reaction screening. As described above, the reaction was established with 9.6 moles of PEG-alkyne and stirred at 20°C for 6 days. The crude reactant was diluted 2x with water and purified by TFF using a 5 kDa ultrafiltration membrane to allow for the removal of impurities. After this purification, water was removed by lyophilization. Quantitative 1H-NMR analysis indicated that this material was 97% w / w PAMAM G-4 material with an adjusted separation yield of 74%. A summary of the analysis for this method is provided in Table 36 below. The reduced separation yield compared to the previous working range is currently unclear and can only be attributed to mechanical losses during TFF purification. Table 36: Summary of the analysis of DP07-74-1 with 3 alkyne arms, PAMAM G4.0 parameter value Separation yield (g) 4.5 g valence ( 1 H-NMR, %w / w) 97% Adjust production volume (g) 4.34 g Separation yield (%) 74% Polydispersity (M) w / M n ) 1.029 Average # Alkyne Arm 3.18 (Method A: 3.20 and Method B: 3.15) GC data GC impurity calibration showed: 0.21% ethanolamine and 0.14% PEG-alkyne (assuming 100% by weight). GC impurity calibration showed: 0.21% ethanolamine and 0.14% PEG-alkyne (assuming 100% by weight).

[0341] 5 g verification to produce PAMAM G4.0 (DP07-68-1) with n=10 alkyne arms.

[0342] Experiment DP07-68-1 was conducted on a 5-gram scale to allow for further validation of the PEG-alkyne / ethanolamine ratio obtained from reaction screening to synthesize n=10 alkyne arms. As described above, the reaction was established with 31 moles of PEG-alkyne and stirred at 20°C for 6 days. The crude reactants were diluted 2x with water and purified by TFF using a 5 kDa ultrafiltration membrane to allow for the removal of impurities. After this purification, water was removed by lyophilization. A summary of the analysis for this method is provided in Table 37. As mentioned above, the reduced separation yield compared to previous development work is believed to be due to mechanical losses in the filtration equipment. Table 37: Summary of analysis of DP07-68-1 with 10 alkyne arms, PAMAM G4.0 parameter value Separation yield (g) 3.8 g valence ( 1 H-NMR, %w / w) 100% Separation yield (%) 63% Polydispersity (M) w / M n ) 1.038 Average # Alkyne Arm 10.03 (Method A: 10.34 and Method B: 9.72)

[0343] Scale up to produce 40 grams of PAMAM G4.0 (DP07-82-1) with 3.0 alkyne arms.

[0344] Based on the 5 g validation run results, the method was further scaled up to produce 40 g of dendritic structures functionalized with n = 3 PEG-alkyne groups. A 9.42:1 molar ratio of ethanolamine and PEG-alkyne was loaded into a 1-liter jacketed vessel, followed by methanol. The vessel was purged with N2(g) and cooled to 0°C. Then, 10 wt% PAMAM G3.5 was added to the reactor in methanol over 2 hours. The solution was then stirred at 0°C for 2 hours, followed by heating to 20°C. The solution was then allowed to be stirred at 20°C for 6 days. Subsequently, the solution was diluted with water to half its concentration and purified by ultrafiltration to provide an aqueous product. The solution was frozen and water was removed by lyophilization to provide a pale yellow foam product. A small amount of the foam sample was taken for analysis using 1H-NMR and 13C-NMR to determine the degree of alkyne functionalization using methods A and B described above. The residual solids were dissolved in methanol to produce a 15.3 wt% methanol solution (batch number DP07-82-2). A summary of the results for scaling up is provided in Table 38. Table 38: Summary of Results for Experiment DP07-82-1 (Batch Number DP07-82-2) Batch number DP07-82-2 PAMAM G4 methanol solution (g) 300.4 Valence (% w / w) 15.3% Yield (g) 46.0 Yield (%) 79% Polydispersity (M) w / M n ) 1.034 Average value of PEG-acetylene arm # (from methods A and B) 3.02 -OH / -NH ratio 0.46 Residual ethanolamine (dry basis, GC) Below LOD Residual PEG-alkyne (dry basis, GC) Below LOD 13 C-NMR

[0345] Scale up to produce 160 grams of PAMAM G4.0 (DP07-85-1) with 10.0 alkyne arms.

[0346] Similarly, as described above, the synthesis of PAMAM G4.0 was scaled up to produce dendritic structures functionalized with n = 10 PEG-alkyne units. A 2.23:1 molar ratio of ethanolamine and PEG-alkyne was loaded into a 5-liter jacketed vessel, followed by methanol. The vessel was purged with N2(g) and cooled to 0°C. 10 wt% PAMAM G3.5 was added to the reactor in methanol over 2 hours. The solution was then stirred at 0°C for 2 hours, followed by heating to 20°C. The solution was then allowed to stir at 20°C for 6 days. Subsequently, the solution was diluted with water to half its concentration and purified by ultrafiltration. The solution was frozen and water was removed by lyophilization to provide a product exhibiting pale yellow foam. A small amount of foam sample was taken for analysis using 1H-NMR and 13C-NMR to determine the degree of alkyne functionalization using methods A and B described in Example 4. The residual solids were dissolved in methanol to produce a 15.3 wt% methanol solution (batch number DP07-85-3). A summary of the results for scaling up is provided in Table 39. Table 39: Summary of Results for Experiment DP07-85-1 (Batch Number DP07-85-3) Batch number DP07-85-3 PAMAM G4 methanol solution (g) 1469.6 Valence (% w / w) 15.3% Yield (g) 224.85 Yield (%) 98% Polydispersity (M) w / M n ) 1.032 Average value of PEG-acetylene arm # (from methods A and B) 10.01 -OH / -NH ratio 0.34 Residual ethanolamine (dry basis, GC) 0.1% Residual PEG-alkyne (dry basis, GC) 0.1% 13 C-NMR

[0347] Conclusion

[0348] A method for producing >40 g of PAMAM G4-OH-acetylene 3 and >224 g of PAMAM G4-OH-acetylene 10 has been successfully developed. The quality properties of the products are within the expected control range.

[0349] Experiment

[0350] PAMAM G4-OH-Alkyne 3 Synthesis - Batch DP07-82-2 reagents MW (g / mol) valence (% w / w) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume amine solution 536.07 6441.96 1600 ethanolamine 61.08 100.00 1.01 355.74 5823.73 2-[2-(2-propynoxy)ethoxy]ethylamine 143.19 96.5% 1.00 91.74 618.23 MeOH 100.00 0.79 88.60 111.87 0.25 PAMAM G3.5 solution (10% by weight) 500.01 PAMAM G3.5 solution (29.4% by weight) 12418.72 29.40 0.80 170.07 212.59 4.026 1 MeOH 100.00 0.79 329.94 416.59

[0351] A 9.42:1 molar ratio of ethanolamine and PEG-alkyne (Ambeed, batch number 100902012-00223 0BFY) was charged into a 1-liter jacketed container, followed by methanol. The container was purged with N2(g) and cooled to 0°C. Then, 10 wt% PAMAM G3.5 (Dentritech, batch number 0121-02-E3.5-LD-A) was added to the reactor in methanol over 2 hours. The solution was then stirred at 0°C for 2 hours, followed by heating to 20°C. The solution was then allowed to be stirred at 20°C for 6 days. Subsequently, the solution was diluted with water to half its concentration and purified by ultrafiltration to provide an aqueous product. The solution was frozen and the water was removed by lyophilization to provide a pale yellow foamy product. A small amount of foam sample was taken for analysis using 1H-NMR and 13C-NMR to determine the degree of alkyne functionalization using methods A and B described in Example 4. The residual solid was dissolved in methanol to produce a 15.3 wt% methanol solution (batch number DP07-82-2). A summary of the results for the scaled-up experiment is provided in Table 40. Table 40: Summary of results for experiment DP07-82-1 (batch number DP07-82-2) Batch number DP07-82-2 PAMAM G4 methanol solution (g) 300.4 Valence (% w / w) 15.3% Yield (g) 46.0 Yield (%) 79% Polydispersity (M) w / M n ) 1.034 Average value of PEG-acetylene arm # (from methods A and B) 3.02 -OH / -NH ratio 0.46 Residual ethanolamine (dry basis, GC) Below LOD Residual PEG-alkyne (dry basis, GC) Below LOD 13 C-NMR

[0352] PAMAM G4-OH-Alkyne 10 Synthesis - Batch DP07-85-3 reagents MW (g / mol) valence (% w / w) density (g / mL) quantity (g) quantity (mL) mmol Equivalent / volume amine solution 2558.94 24242.11 1600 ethanolamine 61.084 100.00 1.0117 1022.35 16736.81 2-[2-(2-propynoxy)ethoxy]ethylamine 143.19 97% 1.00 1113.66 7505.30 MeOH 100.00 0.792 422.93 534.00 0.25 PAMAM G3.5 solution (10% by weight) 1881.60 PAMAM G3.5 solution (29.4% by weight) 12418.72 29.40 0.8 640.00 800.00 15.151 1 MeOH 100.00 0.792 1241.60 1567.68

[0353] A 2.23:1 molar ratio of ethanolamine and PEG-alkyne (Ambeed, batch number 100902012-00223 0BFY) was loaded into a 5-liter jacketed container, followed by methanol. The container was purged with N2(g) and cooled to 0°C. Then, 10 wt% PAMAM G3.5 (Dentritech, batch number 0121-02-E3.5-LD-A) was added to the reactor in methanol over 2 hours. The solution was then stirred at 0°C for 2 hours, followed by heating to 20°C. The solution was then allowed to be stirred at 20°C for 6 days. Subsequently, the solution was diluted with water to half its concentration and purified by ultrafiltration. The solution was frozen and water was removed by lyophilization to provide a product that was a pale yellow foam. A small amount of foam sample was taken for analysis using 1H-NMR and 13C-NMR to determine the degree of alkyne functionalization using methods A and B described above. The residual solids were dissolved in methanol to produce a 15.3 wt% methanol solution (batch number DP07-85-3). A summary of the results for scaling up is provided in Table 41. Table 41: Summary of Results for Experiment DP07-85-1 (Batch Number DP07-85-3) Batch number DP07-85-3 PAMAM G4 methanol solution (g) 1469.6 Valence (% w / w) 15.3% Yield (g) 224.85 Yield (%) 98% Polydispersity (M) w / M n ) 1.032 Average value of PEG-acetylene arm # (from methods A and B) 10.01 -OH / -NH ratio 0.34 Residual ethanolamine (dry basis, GC) 0.1% Residual PEG-alkyne (dry basis, GC) 0.1% 13 C-NMR

[0354] Analytical Methods

[0355] GC Method (ASV003F) A. Chromatographic Conditions Column: Agilent CP-Volamine (Part No. CP7447) Inlet Temperature: 250℃ Inlet Pressure: 14.3 psi Total Inlet Flow Rate: 37.3 mL / min Inlet Split Ratio: 10:1 Injection Volume: 5 µL Column Flow Rate: 3.1 mL / min FID Temperature: 300℃ FID H2 Flow Rate: 40.0 mL / min FID Air Flow Rate: 360.0 mL / min FID Make-up Flow Rate: 25 mL / min FID Ignition Compensation: 0.5 pA Run Time: 20.33 min Oven Temperature Gradient Rate (°C / min) Temperature (°C) Duration Runtime N / A 65 4 4 15 100 3 9.33 30 250 6 20.33 D. Calculate Ab = Total area count from blank Ac = Area count of component in sample injection Ra = Component response factor = Sample concentration

[0356] To determine impurities using this method, standards of ethanolamine and PEG-acetylene at concentrations of 14.928 and 10.332 mg / mL in isopropanol were prepared and injected onto a GC. This resulted in a residence time of approximately 6.88 minutes for ethanolamine and 14.1 minutes for PEG-acetylene. These solutions were combined in a 1:1 ratio. This combined solution was used as a stock solution for a ½ series of dilutions, achieving concentrations of 0.007 mg for ethanolamine and 0.005 mg for PEG-acetylene using isopropanol. The calibration results are shown in Table 42 below. Table 42: GC Calibration Data for Ethanolamine and PEG-acetylene Concentration of ethanolamine (mg / mL) Concentration of PEG-alkyne (mg / mL) ethanolamine signal region PEG-Alkyne Signal Region 14.928 10.332 22543.5 22341 7.4640 5.1660 11862.6 11688.7 3.7320 2.5830 5755.5 5656.6 1.8660 1.2915 2962 2966 0.93300 0.64575 1423.5 1486.3 0.46650 0.32288 708.2 758.3 0.23325 0.16144 323.2 360.1 0.11663 0.080719 153.4 177.7 0.058313 0.040359 72.9 91 0.029156 0.020180 30.5 42.3 0.014578 0.010090 13 twenty one 0.0072891 0.0050449 4.7 11.5

[0357] Using the continuously diluted data described above, a standard curve was plotted (Figure 13).

[0358] The standard curve of ethanolamine was used to calculate the concentration of ethanolamine in the sample. The signal of PEG-acetylenes was below LOD, so the lowest value achieved during calibration was reported as the concentration.

[0359] SEC-MALLS Method A. Reagents 1. Water: HPLC grade 2. Citric acid: ACS reagent, ≥99.5% B. Solutions 1. Diluent: Water, 0.1 M citric acid, pH 2.7 2. Mobile phase: A. Water, 0.1 M citric acid, pH 2.7 3. Sample solution: Sample solution at a target concentration of approximately 2 mg / mL. 4. Blank: Diluent. C. Chromatographic Conditions: Columns: TosoHaas TSK-Gel Guard PHW 0662 (75 mm x 7.5 mm, 12 µm), G2000 PW 05761 (300 mm x 7.5 mm, 10 µm, 125 Å), G 3000 PW 05762 (300 mm x 7.5 mm, 10 µm, 200 Å), G4000 PW (300 mm x 7.5 mm, 17 µm, 500 Å) Temperature: 25℃ Flow Rate: 1 mL / min Detection: A: Wyatt Optilab differential refractive index detector operating at 658 nm B: Wyatt miniDAWN multi-angle laser light scattering detector with a 120 mW laser operating at 658 nm Injection Volume: 100 µL Run Time: 40 min Example 6: Exemplary PAMAM dendrites bound to drugs

[0360] Schemes 1 to 19 below show exemplary reactions for functionalizing PAMAM dendrites (e.g., PAMAM carboxymethyl-functionalized dendrites), wherein a linker is connected to a functional group R, wherein R is an amine, alkyne, acetylene, COOH, hydroxyl, bromine, DBCO, thiol, olefin, aldehyde, hydroxyl, sulfonate, or nitrile. In Schemes 1 to 19, the polyethylene glycol (PEG) linker may be replaced by linkers such as: , or . The exemplary reaction pair in step 1 of Schemes 1 to 19 includes: or , wherein the linker length may vary from 1 to 100,000 CH2 and PEG units, and the linker may include PEG linkers with different molecular weights.

[0361] Other exemplary connection subsystems:

[0362] Option 1:

[0363] Option 2:

[0364] Other types of click reactions used for the functionalization of dendrites include the following (shown in schemes 3 to 19):

[0365] Thiol maleimide (click:)

[0366] Option 3:

[0367] Tetraazine linkage:

[0368] Option 4:

[0369] Isooxazole is formed from alkynes and oximes:

[0370] Option 5:

[0371] Thiol-Alkyne:

[0372] Option 6:

[0373] Thiol-ene:

[0374] Option 7:

[0375] Thiols - Michael Click:

[0376] Option 8:

[0377] Option 9:

[0378] Option 10:

[0379] Option 11:

[0380] The method of use is taught in three different parts of a dendrite in a pot:

[0381] Option 12:

[0382] The methodology for using two drugs on a dendrite in a single pot:

[0383] Option 13:

[0384] A drug and antibody on a dendrite:

[0385] Option 14:

[0386] An antibody and oligonucleotide on a dendrite:

[0387] Option 15:

[0388] A peptide and drug on a dendritic structure in a pot:

[0389] Option 16:

[0390] Targeting peptide and PROTAC on a dendritic structure in a pot:

[0391] Option 17:

[0392] A drug and RNA or DNA on a dendrite:

[0393] Option 18:

[0394] Three different drugs on a dendritic structure in a pot:

[0395] Scheme 19: Example 7: Dendritic-PAK1 inhibitor conjugate

[0396] Synthesis and characterization of dendrites bound to a PAK1 inhibitor (Frax-1036 analogue) (Figures 56A to 56C). Figure 56A shows an example synthetic protocol for preparing the dendritic-PAK1 conjugate D4-5100 (D4-Frax-1036 analogue). The binding of the PAK1 kinase group to PAK1 was evaluated, and the results are shown in Figure 56A (insert table). Figures 56B to 56C show the characterization results from D4-5100. D4-5100 was determined to be >99% pure by HPLC (Figure 56B, top), and the linkage of 6 molecules was confirmed by 1H NMR (Figure 56B, bottom), indicating a loading of 15%. D4-5100 was further evaluated through plasma stability studies in human plasma (Fig. 56C, left), mouse plasma (Fig. 56C, middle), and rat plasma (Fig. 56C, right), which confirmed that D4-5100 appeared to be stable for 72 hours in various plasma sources. Example 8: Dendritic-MEK Inhibitor Conjugates

[0397] Synthesize and characterize dendrites that bind to MEK inhibitors (selmetinib, trametinib or cobimetinib) (Figs. 57A to 57T).

[0398] Figure 57A shows an example synthetic scheme for preparing dendritic-selmetinib conjugate D4-5111 (D4-selmetinib). Figures 57B to 57D show the characterization results of D4-5111.

[0399] Figures 57E to 57H show an example synthetic scheme for preparing dendritic-trametinib conjugate D4-5116 (D4-trametinib-acetamide analog), and the characterization results of D4-5116. Figure 57I shows an example synthetic scheme for preparing dendritic-trametinib conjugate D4-5119 (D4-trametinib-disulfide analog), and the characterization results of D4-5119. Figures 57J to 57M show an example synthetic scheme for preparing dendritic-trametinib conjugate D4-5121 (D4-trametinib-disulfide), and the characterization results of D4-5121. Figures 57N to 57P show an example synthetic scheme for preparing dendritic-trametinib conjugate D4-5124 (D4-trametinib-ester), and the characterization results of D4-5124.

[0400] Figure 57Q shows an example synthetic regimen for preparing dendritic-cobimetinib conjugate D4-5123. Figures 57R to 57T show an example synthetic regimen for preparing dendritic-cobimetinib conjugate D4-5120, and the characterization results of D4-5120. Example 9: Conjugate of dendritic-receptor tyrosine kinase inhibitor

[0401] Synthesize and characterize dendrites that bind to receptor tyrosine kinase inhibitors (dasatinib, bemtinib (R428), dobematinib (TP-0903) or cabozantinib) (Figs. 58A to 58T).

[0402] Figure 58A shows an example synthetic scheme for preparing dendritic-dasatinib conjugate D4-5113 (D4-dasatinib-thiolation). Figures 58B to 58E show the characterization results from D4-5113. Figures 58F to 58G show the chemical structure of the synthesized dendritic-dasatinib conjugate D4-4531 (D4-dasatinib ester analog) and the results from its characterization. Figure 58H shows an example chemical structure of a dasatinib analog chemically bonded via a terminal azide group.

[0403] Figure 58I shows an example synthetic scheme for preparing the dendritic-bemtinib (R428) conjugate D4-R428. Figures 58J to 58L show the characterization results of D4-R428. Figure 58M shows an example synthetic scheme for preparing the dendritic-bemtinib (R428) conjugate D4-R428-thiolation. Figure 58N shows the characterization results of D4-R428-thiolation.

[0404] Figure 58O shows an example synthetic scheme for preparing dendritic-dobematinib (TP-0903) conjugate D4-5132 (D4-TP-0903 analogue). Figure 58P shows the characterization results from D4-5132.

[0405] Figure 58Q shows an example synthetic scheme for preparing dendritic-cazozidinib conjugates D4-4595 and D4-4598. Figures 58R to 58T show the characterization results of D4-4595 and D4-4598. (Equivalents and scope)

[0406] In the claims, unless indicated herein or otherwise obvious from the text, articles such as "a," "an," and "the" may mean one or more. Unless indicated herein or otherwise obvious from the text, the inclusion of "or" in the claims or descriptions of a group of members is considered to satisfy the presence, use, or other relatedness of one, more, or all of the members in that group in or in a given product or method. The invention includes embodiments in which exactly one member of that group is present in or in a given product or method, or otherwise related. The invention includes embodiments in which more than one or all of those members are present in or in a given product or method, or otherwise related.

[0407] Furthermore, the present invention includes all variations, combinations, and arrangements of one or more of the limitations, elements, clauses, and descriptive terms introduced into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations appearing in any other claim dependent on the same basic claim. Where elements are presented in enumerated form (e.g., in Markush group form), the subgroups of such elements are also disclosed, and any element may be removed from that group. Generally, it should be understood that where the invention or a morphology of the invention is referred to as containing specific elements and / or features, certain embodiments of the invention or a morphology of the invention are constituted by, or substantially constituted by, such elements and / or features. For simplicity, such embodiments have not been explicitly described herein in the same words.

[0408] It should be understood that the phrase "and / or" as used herein in the specification and claims means "any one or both" of the elements so combined, that is, elements that exist together in some cases and separately in others. Multiple elements listed using "and / or" should be interpreted in the same way, that is, "one or more" of the elements so combined. Other elements besides those explicitly identified by the "and / or" clause may also exist as needed, whether or not they are related to or unrelated to those explicitly identified elements. Therefore, as a non-limiting example, when used in conjunction with open-ended language (such as "comprising"), reference to "A and / or B" may in one embodiment refer only to A (including elements other than B as needed); in another embodiment, only to B (including elements other than A as needed); in yet another embodiment, to both A and B (including other elements as needed); and so on.

[0409] As used herein in the specification and accompanying claims, it should be understood that "or" has the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including many elements or at least one of the list of elements, but also including more than one, and, if necessary, additional unlisted items. Only explicitly indicating the opposite terms, such as "only one of..." or "exact one of...", or when used in the claims, "consisting of..." will mean including many elements or exactly one of the list of elements. Generally, when preceded by exclusive terms such as "any one", "one of...", "only one of..." or "exact one of...", the term "or" as used herein should only be interpreted as indicating an exclusive alternative (i.e., "one or the other, but not both"). When used in the scope of a patent application, "consisting essentially of..." should have the same general meaning as it is used in the field of patent law.

[0410] As used herein in the specification and the claims, it should be understood that the phrase "at least one" when referring to a list of one or more elements means at least one element selected from any one or more of the elements in the list, but does not necessarily include at least one of each element expressly listed in the list, nor excludes any combination of elements in the list. This definition also allows for, and may be used as needed, the presence of elements other than those expressly identified in the list of elements referred to by the phrase "at least one," whether or not they are related to or unrelated to those expressly identified elements. Therefore, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B") in one embodiment may refer to at least one, including more than one A as needed but without B (and including elements other than B as needed); in another embodiment, it refers to at least one, including more than one B as needed but without A (and including elements other than A as needed); in yet another embodiment, it refers to at least one, including more than one A as needed, and at least one, including more than one B as needed (and including other elements as needed); etc.

[0411] It should be understood that, unless expressly indicated otherwise, in any method which is claimed herein to include more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are listed herein.

[0412] In the claims and in the foregoing description, it should be understood that all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “consisting of,” and similar terms are open-ended, meaning including but not limited to. As described in Chapter 2111.03 of the U.S. Patent Examination Procedure Manual, only the transitional phrases “consisting of” and “substantially constituted of” should be closed or semi-closed transitional phrases, respectively. It should be recognized that embodiments described using open-ended transitional phrases (e.g., “comprising”) in this document have also been carefully considered as alternative embodiments “consisting of the features described by the open-ended transitional phrase” and “substantially constituted thereof.” For example, if the application describes “a composition comprising A and B,” then the application has also carefully considered as alternative embodiments “a composition comprising A and B” and “a composition substantially comprising A and B.”

[0413] In the case of a given range, the number is inclusive. Furthermore, unless otherwise indicated or apparent from the text and to those skilled in the art, values ​​expressed as ranges may be assumed to be any particular value or subrange within the specified range in different embodiments of the invention, up to one-tenth of the unit of the lower limit of the range unless otherwise expressly stated in the text.

[0414] This application refers to all issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of any conflict between any of the incorporated references and this specification, this specification shall prevail. Furthermore, any particular embodiment of the invention falling within the prior art may be expressly excluded from any or more of the claims. Because such embodiments are considered known to those skilled in the art, they may be excluded even if not expressly stated herein. Any particular embodiment of the invention may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0415] Those skilled in the art will recognize that many equivalents of the particular embodiments described herein can be determined using only routine experiments. The scope of the embodiments described herein is not intended to be limited to the foregoing description, but is as set forth in the appended claims. Those skilled in the art will recognize that various changes and modifications can be made to this description without departing from the spirit or scope of the invention as defined in the following claims.

[0416] The description of the list of chemical groups in any definition of the variables herein includes the definition of the variable as any single group or a combination of the listed groups. The description of embodiments of the variables herein includes the embodiment as any single embodiment or in combination with any other embodiment or part thereof. [Simplified Explanation of the Diagram]

[0028] Several embodiments of the invention are illustrated in the accompanying drawings, which form part of this specification, and together with the description, are used to explain the principles of the invention.

[0029] Figure 1 is a schematic diagram showing the chemical structure of the dendritic complex (compound D-4517.2).

[0030] Figure 2 shows a reaction scheme for a synthetic strategy of N,N-didesethylsunitinib azide with amide linkage.

[0031] Figure 3 shows the reaction scheme for the synthesis of the dendritic complex (D-4517.2), in which N,N-didesethylsunitinib is linked to the dendritic structure by an ether bond to enhance its in vivo stability.

[0032] Figure 4 is a bar graph showing the percentage of drug release (0.0% to 0.50%) of D-didesethylsunitinib conjugate D-4517.2 in human, mouse and rat plasma at 4, 24 and 48 hours respectively.

[0033] Figure 5 shows the reaction scheme for the synthesis of dendritic-N-acetylglucosamine methyl ester conjugate.

[0034] Figure 6 shows the reaction scheme for the synthesis of β-GalNAc-trianthracite-PEG3-azide building blocks that are bound to dendrites.

[0035] Figure 7 is a tomographic diagram showing an exemplary integral performed using method A. Example calculation:

[0036] Figure 8 is a tomographic diagram showing an exemplary integral performed using method A. Example calculation:

[0037] Figure 9 shows the calibration data of FID compared to ethanolamine (mg / mL) and FID compared to PEG-alkyne (mg / mL).

[0038] Figure 10 shows the average alkyne arm of experiment DP07-55-1 compared to PEG-alkyne loading.

[0039] Figure 11 shows the average alkyne arm of experiments AA08-85, AA08-88 and DP07-55-1 compared to the PEG-alkyne loading.

[0040] Figure 12 shows the average alkyne arm of experiments DP07-51 and DP07-60 compared to the PEG-alkyne loading.

[0041] Figure 13 shows the calibration data of FID compared to ethanolamine (mg / mL) and FID compared to PEG-alkyne (mg / mL).

[0042] Figures 14A to 14C are ASTRA reports on PAMAM G3.5 dendrites. Concentration: 7.080 mg / mL.

[0043] Figures 15A to 15C are ASTRA reports on PAMAM G4. Concentration: 7.080 mg / mL.

[0044] Figures 16A to 16C are ASTRA reports on PAMAM G3.5. Concentration: 7.080 mg / mL.

[0045] Figure 17 shows the 1H-NMR data for DP07-51-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br t, J=5.78 Hz, 248 H) 3.50 - 3.74 (m, 191 H) 4.20 (d, J=2.49 Hz, 27 H) 4.18 -4.20 (m, 1 H).

[0046] Figure 18 shows the 1H-NMR data for DP07-51-2. 1H NMR (300 MHz, methanol-d4) δ ppm 2.23 - 2.49 (m, 248 H) 3.61 (s, 194 H) 4.16 - 4.24 (m, 31 H).

[0047] Figure 19 shows the 1H-NMR data for DP07-51-3. 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br t, J=5.78 Hz, 248 H) 3.50 - 3.74 (m, 191 H) 4.20 (d, J=2.49 Hz, 27 H) 4.18 -4.20 (m, 1 H).

[0048] Figure 20 shows the 1H-NMR data for DP07-51-4. 1H NMR (300 MHz, methanol-d4) δ ppm 2.23 - 2.49 (m, 248 H) 3.61 (s, 194 H) 4.16 - 4.24 (m, 31 H).

[0049] Figures 21A to 21C are ASTRA reports of DP07-51-1.

[0050] Figures 22A to 22C are ASTRA reports of DP07-51-2.

[0051] Figures 23A to 23C are ASTRA reports of DP07-51-3.

[0052] Figures 24A to 24C are ASTRA reports of DP07-51-4.

[0053] Figure 25 shows the 1H-NMR data for DP07-60-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.25 - 2.49 (m, 248 H) 3.50 - 3.74 (m, 145 H) 4.20 (d, J=2.34 Hz, 6 H).

[0054] Figure 26 shows the 1H-NMR data for DP07-60-2. 1H NMR (300 MHz, methanol-d4) δ ppm 2.26 - 2.50 (m, 248 H) 3.61 (t, J=5.63 Hz, 150 H) 4.20 (d, J=2.49 Hz, 6 H).

[0055] Figure 27 shows the 1H-NMR data for DP07-60-3. 1H NMR (300 MHz, methanol-d4) δ ppm 2.26 - 2.49 (m, 248 H) 3.61 (t, J=5.63 Hz, 143 H) 4.18 - 4.22 (m, 6 H).

[0056] Figure 28 shows the 1H-NMR data for DP07-60-4. 1H NMR (300 MHz, methanol-d4) δ ppm 2.28 - 2.48 (m, 248 H) 3.61 (t, J=5.63 Hz, 148 H) 4.18 - 4.22 (m, 7 H).

[0057] Figures 29A to 29C are ASTRA reports of DP07-60-1.

[0058] Figures 30A to 30C are ASTRA reports of DP07-60-2.

[0059] Figures 31A to 31C are ASTRA reports for DP07-60-3.

[0060] Figures 32A to 32C are ASTRA reports of DP07-60-4.

[0061] Figures 33A to 33C are ASTRA reports of DP07-74-1.

[0062] Figure 34 shows the 1H-NMR data for DP07-74-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.27 - 2.48 (m, 243 H) 2.53 - 2.67 (m, 121 H) 2.68 - 2.95 (m, 255 H) 3.31 (s, 46 H) 3.61 (t, J=5.71 Hz, 143 H) 4.20 (d, J=2.34 Hz, 6 H).

[0063] Figures 35A to 35B are GC data from DP07-74-1.

[0064] Figures 36A to 36B are GC data for IPA blanks.

[0065] Figures 37A to 37C are ASTRA reports of DP07-68-1.

[0066] Figures 38A to 38B are 1H-NMR data for DP07-68-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br t, J=6.07 Hz, 248 H) 2.50 - 2.67 (m, 138 H) 2.71 - 2.87 (m, 241 H) 2.94 (s, 36 H) 3.18 - 3.44 (m, 39 H) 3.49 - 3.73 (m, 178 H) 4.20 (d, J=2.34 Hz, 21 H).

[0067] Figures 39A to 39B are GC data from DP07-68-1.

[0068] Figure 40 shows the 1H-NMR data for DP07-82-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.37 (br s, 248 H) 3.71 - 3.74 (m, 84 H) 6.05 - 6.08 (m, 198 H).

[0069] Figures 41A to 41C are ASTRA reports of DP07-82-1.

[0070] Figure 42 shows the 1H-NMR data for the alkyne DP07-82-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.25 - 2.48 (m, 247 H) 2.36 - 2.36 (m, 5 H) 3.50 - 3.71 (m, 140 H) 3.60 - 3.61 (m, 3 H) 4.20 (d, J=2.49 Hz, 5 H) 4.20 - 4.20 (m, 1 H).

[0071] Figure 43 shows the 13C-NMR data for DP07-82-1. 13C NMR (75 MHz, methanol-d4) δ ppm 33.32 (s, 1 C) 41.61 (s, 1 C) 47.87 (s, 1 C) 49.80 (s, 1 C) 60.23 (s, 1 C) 173.75 (s, 1 C).

[0072] Figure 44 shows the 1H-NMR data for DP07-85-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br s, 242 H) 6.04 - 6.08 (m, 218 H).

[0073] Figures 45A to 45C are ASTRA reports of DP07-85-1.

[0074] Figure 46 shows the 1H-NMR data for the alkyne DP07-85-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br t, J=6.14 Hz, 244 H) 2.36 - 2.36 (m, 9 H) 3.49 - 3.73 (m, 167 H) 4.16 - 4.22 (m, 20 H).

[0075] Figures 47A to 47B are 13C-NMR data for DP07-85-1. 13C NMR (75 MHz, methanol-d4) δ ppm 33.31 (s, 1 C) 41.63 (s, 1 C) 47.87 (s, 1 C) 60.23 (s, 1 C) 173.74 (s, 1 C).

[0076] Figure 48 shows the 1H-NMR data for DP07-82-1 (batch number DP07-82-2). 1H NMR (300 MHz, methanol-d4) δ ppm 2.37 (br s, 248 H) 3.71 - 3.74 (m, 84 H) 6.05 - 6.08 (m, 198 H).

[0077] Figures 49A to 49C are ASTRA reports for DP07-82-1 (batch number DP07-82-2).

[0078] Figure 50 shows the 1H-NMR data of alkynes from series DP07-82-1 (batch number DP07-82-2). 1H NMR (300 MHz, methanol-d4) δ ppm 2.25 - 2.48 (m, 247 H) 2.36 - 2.36 (m, 5 H) 3.50 - 3.71 (m, 140 H) 3.60 - 3.61 (m, 3 H) 4.20 (d, J=2.49 Hz, 5 H) 4.20 - 4.20 (m, 1 H).

[0079] Figures 51A to 51B are 13C-NMR data for DP07-82-1 (batch number DP07-82-2). 13C NMR (75 MHz, methanol-d4) δ ppm 33.32 (s, 1 C) 41.61 (s, 1 C) 47.87 (s, 1 C) 49.80 (s, 1 C) 60.23 (s, 1 C) 173.75 (s, 1 C).

[0080] Figure 52 shows the 1H-NMR data for DP07-85-1 (batch number DP07-85-3). 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br s, 242 H) 6.04 - 6.08 (m, 218 H).

[0081] Figures 53A to 53C are ASTRA reports for DP07-85-1 (batch number DP07-85-3).

[0082] Figure 54 shows the 1H-NMR data of alkynes from DP07-85-1 (batch number DP07-85-3). 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br t, J=6.14 Hz, 244 H) 2.36 - 2.36 (m, 9 H) 3.49 - 3.73 (m, 167 H) 4.16 - 4.22 (m, 20 H).

[0083] Figures 55A to 55B are 13C-NMR data for DP07-85-1 (batch number DP07-85-3). 13C NMR (75 MHz, methanol-d4) δ ppm 33.31 (s, 1 C) 41.63 (s, 1 C) 47.87 (s, 1 C) 60.23 (s, 1 C) 173.74 (s, 1 C).

[0084] Figures 56A to 56C show the results of the synthesis and characterization of dendrites bound to the following PAK1 inhibitor: Frax-1036.

[0085] Figures 57A to 57T show the results of synthesis and characterization of dendrites bound to the following MEK inhibitors: selumetinib (Figures 57A to 57D), trametinib (Figures 57E to 57P), and cobimetinib (Figures 57Q to 57T).

[0086] Figures 58A to 58T show the results of synthesis and characterization of dendrites from the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A to 58H), betatinib (Figures 58I to 58N), dubematinib (Figures 58O to 58P) and cabozantinib (Figures 58Q to 58T).

Claims

1. A composition comprising a carrier and a functionalized dendrite having the following formula: , wherein: G3.5 refers to 3.5-generation poly(amidoamine) (PAMAM) dendrites; n is an integer from 1 to 64, including the number itself, wherein the polydispersity value of the functionalized dendrites in the composition is from 1.00 to 1.

10.

2. The composition of claim 1, wherein the polydispersity value of the functionalized dendrites in the composition is between 1.00 and 1.

05.

3. The composition of claim 1 or 2, wherein the polydispersity value of the functionalized dendrites in the composition is between 1.00 and 1.

04.

4. The composition of claim 1 or 2, wherein the polydispersity value of the functionalized dendrites in the composition is between 1.00 and 1.

03.

5. The composition of claim 1 or 2, wherein the composition comprises 20 to 200 grams of the functionalized dendrite.

6. The composition of claim 1 or 2, wherein the composition comprises 50 to 200 grams of the functionalized dendrite.

7. The composition of claim 1 or 2, wherein the composition comprises 100 to 200 grams of the functionalized dendrite.

8. The composition of claim 1 or 2, wherein the functionalized dendrite has the following formula: .

9. The composition of claim 1 or 2, wherein the functionalized dendrite has the following formula: .

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