JP7525981B2 - Nanoparticles stabilized by nitrophenylboronic acid compositions - Google Patents

Description

(Government Rights)
This invention was made with Government support under Grant CA 119347 awarded to the National Cancer Institute. The Government has certain rights in this invention.

FIELD OF THEINVENTION
The present disclosure relates to carrier nanoparticles, particularly nanoparticles suitable for delivering compounds of interest, as well as related compositions, methods and systems.

Effective delivery of compounds of interest to cells, tissues, organs and organisms is a challenge in the biomedical, imaging and other fields where delivery of molecules of various sizes and dimensions to a given target is desired.

Several methods are known and used to deliver various classes of biomaterials and biomolecules, typically with biological and/or chemical activity of interest, for either pathology testing, therapeutic treatment, or basic biological research.

As the number of molecules suitable for use as chemical or biological agents (e.g., drugs, biologics, therapeutic agents, or imaging agents) increases, the development of delivery systems suitable for use with compounds of various complexities, sizes, and chemical properties has proven particularly challenging.

Nanoparticles are useful structures as carriers for the delivery of drugs by a variety of delivery methods. Several nanoparticle delivery systems exist that use a number of different strategies to package, transport, and deliver drugs to specific targets.

Provided herein are nanoparticles and related compositions, methods, and systems that provide multifunctional tools for effective and specific delivery of compounds of interest. In particular, in some embodiments, the nanoparticles described herein can be used as flexible systems for the transport and delivery of a wide range of molecules of various sizes, dimensions, and chemistries to a given target.

According to one aspect, a nanoparticle is described that includes a polyol-containing polymer and a nitrophenylboronic acid-containing polymer. In the nanoparticle, the boronic acid-containing polymer is coupled to the polyol-containing polymer, and the nanoparticle is configured such that the boronic acid-containing polymer is present in the external environment of the nanoparticle. One or more compounds of interest can be carried by the nanoparticle as part of or attached to the polyol-containing polymer and/or the boronic acid-containing polymer.

According to another aspect, a composition is described. The composition includes the nanoparticles described herein and a suitable vehicle and/or excipient.

According to another aspect, a method of delivering a compound to a target is described. The method includes contacting the target with a nanoparticle described herein, where the compound is contained within a polyol-containing polymer or a boronic acid-containing polymer of the nanoparticle described herein.

According to another aspect, a system for delivering a compound to a target is described. The system includes at least a polyol-containing polymer and a boronic acid-containing polymer that can be interconnected via reversible covalent bonds and assembled into a nanoparticle as described herein that includes the compound.

According to another aspect, a method of administering a compound to an individual is described. The method includes administering to the individual an effective amount of a nanoparticle described herein, where the compound is contained within a polyol-containing polymer and/or a boronic acid-containing polymer.

According to another aspect, a system for administering a compound to an individual is described. The system includes at least a polyol-containing polymer and a boronic acid-containing polymer that can be interconnected via reversible covalent bonds and assembled into nanoparticles as described herein that are coupled with a compound that is administered to an individual according to the methods described herein.

According to another aspect, a method of preparing nanoparticles comprising a polyol-containing polymer and a nitrophenylboronic acid-containing polymer is described. The method includes contacting the polyol-containing polymer with the boronic acid-containing polymer for a time and under conditions that allow coupling of the polyol-containing polymer with the boronic acid-containing polymer.

According to another aspect, several boronic acid-containing polymers are described, which are described in detail in the following sections of this disclosure.

According to another aspect, several polyol-containing polymers are described, which are described in detail in the following sections of this disclosure.

Described herein are nanoparticles having polyol-containing polymers conjugated to polymers having nitrophenylboronic acid groups, which lower the pKa of the nanoparticles and increase their stability.

Another aspect of the present disclosure provides a description of targeted nanoparticles, which in some embodiments may have only one single targeting ligand. This can facilitate delivery of the nanoparticle to a specific target, such as a cell that displays a binding partner for the particle's targeting ligand. This class of targeted nanoparticles has advantages over nanoparticles with multiple targeting ligands, such as smaller overall size by having fewer surface ligands, as well as fewer ligands mediating non-specific binding via avidity-based interactions than affinity-based interactions. Thus, nanoparticles containing or carrying therapeutic agents can be successfully targeted to a desired location (e.g., a cell or tissue) using only one targeting ligand, and the therapeutic agent can be delivered to the target at a very high targeting ligand to therapeutic agent ratio. This aspect of the described nanoparticles significantly increases the efficiency of manufacturing such therapeutic agents while also reducing the need to use multiple expensive antibodies to mediate targeting.

The nanoparticles and related compositions, methods and systems described herein can be used in some embodiments as flexible molecular structures suitable for carrying compounds of various sizes, dimensions and chemical properties.

The nanoparticles and related compositions, methods and systems described herein can, in some embodiments, be used as delivery systems that can provide protection of the compounds they carry from degradation, recognition by the immune system, and loss due to binding to serum proteins or blood cells.

The nanoparticles and related compositions, methods and systems described herein can be used, in some embodiments, as delivery systems characterized by steric stability and by the ability to deliver compounds to specific targets, such as tissues, specific cell types within tissues, and even specific intercellular locations within specific cell types.

The nanoparticles and related compositions, methods and systems described herein, in some embodiments, can be designed for release of carried compounds in a controllable manner, including controlled release of multiple compounds at different rates and/or times within the same nanoparticle.

The nanoparticles and related compositions, methods and systems described herein can be used in some embodiments to deliver compounds with increased specificity and/or selectivity during targeting and/or with increased recognition of the compound by the target compared to certain systems in the art.

The nanoparticles and related compositions, methods and systems described herein may, in some embodiments, be used in conjunction with applications in which controlled delivery of a compound of interest is desirable, including, but not limited to, medical applications such as therapeutic, diagnostic and clinical applications. Further applications include biological analysis, veterinary applications, and delivery of a compound of interest to non-animal organs, particularly plant organs.

The details of one or more embodiments of the present disclosure are set forth in the accompanying drawings and the following detailed description and examples. Other features, objects and advantages will become apparent from the detailed description, examples and drawings, and from the appended claims.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the disclosure and, together with the detailed description and examples, serve to explain the principles and practice of the disclosure.
1 shows a schematic diagram of a related method for the formation of nanoparticles and related associations in the absence of boronic acid containing compounds. Panel A shows a schematic diagram of a polyol-containing polymer (MAP, 4) and a compound of interest (nucleic acid) according to an embodiment described herein. Panel B shows nanoparticles formed by assembly of the polymer containing the polyol and compound shown in Panel A. 1 shows a schematic diagram of nanoparticles and associated manufacturing methods according to embodiments of the present disclosure. Panel A shows a polyol (MAP, 4)-containing polymer and a boronic acid (BA-PEG, 6)-containing polymer according to embodiments of the present disclosure along with a molecule of interest (nucleic acid). Panel B shows a BA-PEGylated stabilized nanoparticle formed by assembly of the polymers and compounds shown in Panel A. FIG. 3 shows the formation of a complex comprising a polyol-containing polymer and a compound of interest according to an embodiment described herein. In particular, FIG. 3 shows the results of a MAP gel shift assay using plasmid DNA according to an embodiment of the present disclosure. A DNA ladder is loaded in lane 1. Lanes 2-8 show plasmid DNA combined with MAPs of increasing charge ratios. The charge ratio is defined as the amount of positive charge on the MAP divided by the amount of negative charge on the nucleic acid. FIG. 4 shows the formation of a complex comprising a polyol-containing polymer and a compound of interest according to embodiments described herein. In particular, FIG. 4 shows the results of a MAP gel shift assay using siRNA according to embodiments of the present disclosure. A DNA ladder is loaded in lane 1. Lanes 2-8 show siRNA combined with MAPs of increasing charge ratios. FIG. 5 shows characteristics of nanoparticles according to some embodiments described herein. In particular, FIG. 5 shows a diagram illustrating plots of particle size (as measured by dynamic light scattering (DLS)) versus charge ratio as well as zeta potential (a property related to the surface charge of a nanoparticle) versus charge ratio for MAP-plasmid nanoparticles according to embodiments of the present disclosure. 6 shows properties of nanoparticles according to some embodiments described herein. In particular, FIG. 6 shows a diagram illustrating plots of particle size (DLS) vs. charge ratio and zeta potential vs. charge ratio for BA-PEGylated MAP-plasmid nanoparticles according to embodiments of the present disclosure. 1 shows the salt stability of BA-PEGylated MAP-plasmid nanoparticles according to an embodiment of the present disclosure. Plot A: 5:1 BA-PEG+np+1X PBS after 5 min; Plot B: 5:1 BA-PEG+np, dialysis 3X w/100 kDa+1X PBS after 5 min; Plot C: 5:1 pre-PEGylated w/BA-PEG+1X PBS after 5 min; Plot D: 5:1 pre-PEGylated w/BA-PEG, dialysis 3X w/100 kDa+PBS after 5 min. 8 shows the delivery of drugs to human cells in vitro using nanoparticles according to embodiments described herein. In particular, FIG. 8 shows a diagram illustrating a plot of relative light units (RLU) versus charge ratio, which is a measure of the amount of luciferase protein expressed from pGL3, for MAP/pGL3 transfection into HeLa cells according to embodiments of the present disclosure. 9 shows targeted drug delivery using nanoparticles according to embodiments described herein. In particular, FIG. 9 shows a diagram illustrating a plot of cell survival versus charge ratio following MAP/pGL3 transfection according to an embodiment of the present disclosure. The survival data is for the experiment shown in FIG. Figure 10 shows the delivery of multiple compounds to a target using nanoparticles according to embodiments described herein. In particular, Figure 10 shows a diagram illustrating a plot of relative light units (RLU) versus particle species for co-transfection of MAP particles containing pGL3 and siGL3 into HeLa cells at a charge ratio of 5+/- according to embodiments described herein. The term siCON refers to siRNA with a control sequence. 11 shows the delivery of compounds to targets using nanoparticles according to embodiments described herein. In particular, FIG. 11 shows a diagram illustrating a plot of relative light units (RLU) versus siGL3 concentration for the delivery of MAP/siGL3 into HeLa-Luc cells at a charge ratio of 5+/- according to embodiments described herein. 12 shows a schematic for the synthesis of boronic acid-containing polymers displaying targeting ligands according to some embodiments of the present disclosure. In particular, FIG. 12 shows a schematic for the synthesis of boronic acid-PEG disulfide-transferrin according to embodiments of the present disclosure. 13 shows a schematic diagram for the synthesis of nanoparticles according to some embodiments described herein. In particular, FIG. 13 shows a schematic diagram for the formation of nanoparticles using Campothecin mucic acid polymer (CPT-mucic acid polymer) in water according to an embodiment of the present disclosure. 1 shows a table summarizing the particle size and zeta potential of nanoparticles formed from CPT-mucin acid polymers conjugated in water, prepared according to embodiments of the present disclosure. 15 shows a schematic diagram of the synthesis of nanoparticles according to some embodiments described herein. In particular, FIG. 15 shows the formation of boronic acid-PEGylated nanoparticles using CPT-mucinic acid polymer and boronic acid-disulfide-PEG ₅₀₀₀ in water according to an embodiment of the present disclosure. Salt stability of MAP-4/siRNA stabilized by no PEG, nitroPEG-PEG and 0.25 mol% Herceptin-PEG-nitroPBA prepared at charge ratio 3 (+/-) and 10x PBS was added at 5 minutes such that the resulting solution was in 1x PBS. A and B show (A) the cellular uptake of targeted, medium or targeted MAP-CPT nanoparticles at increasing ratios of Herceptin®-PEG-nitroPBA to nanoparticles in BT-474 and (B) the cellular uptake of medium or targeted MAP-CPT nanoparticles with or without free Herceptin® (10 mg/ml) in BT-474 (solid bars) and MCF-7 (open bars) cell lines. A and B show the plasma pharmacokinetics of short, medium and long MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles in BALB/c mice at 10 mg CPT/kg injection. Free CPT injected into CD2F1 mice at 10 mg/kg is shown for comparison. (A) Plasma concentration of polymer-conjugated CPT as a function of time. (B) Plasma concentration of unconjugated CPT as a function of time. Biodistribution of MAP-CPT (5 mg CPT/kg) and targeted MAP-CPT (5 mg CPT/kg, 29 mg Herceptin®/kg) nanoparticles 4 and 24 hours after treatment. Percentage of injected dose (ID) of total CPT per gram in tumor, heart, liver, spleen, kidney or lung. Biodistribution of MAP-CPT (5 mg CPT/kg) and targeted MAP-CPT (5 mg CPT/kg, 29 mg Herceptin®/kg) nanoparticles 4 and 24 hours after treatment. Percentage of total unconjugated CPT in each organ in tumor, heart, liver, spleen, kidney and lung. Figure 1 shows the biodistribution of MAP-CPT (5 mg CPT/kg) and targeted MAP-CPT (5 mg CPT/kg, 29 mg Herceptin®/kg) nanoparticles 4 and 24 hours after treatment. Total concentration of CPT in plasma. Biodistribution of MAP-CPT (5 mg CPT/kg) and targeted MAP-CPT (5 mg CPT/kg, 29 mg Herceptin®/kg) nanoparticles 4 and 24 hours after treatment. Percentage of total unconjugated CPT in plasma. Confocal immunofluorescence micrographs of BT-474 tumor sections taken from NCr nude mice treated with (A) MAP-CPT (5 mg CPT/kg) at 4 hours, (B) MAP-CPT (5 mg CPT/kg) at 24 hours, (C) targeted MAP-CPT (5 mg CPT/kg, 29 mg Herceptin®/kg) at 4 hours, (D) targeted MAP-CPT (5 mg CPT/kg, 29 mg Herceptin®/kg) at 24 hours are shown. Left panel, emission at 440 nm (CPT, pink), center left panel, emission at 519 nm (MAP, green), center right panel, emission at 647 nm (Herceptin®, blue), right panel, image overlay. Figure 1 shows antitumor efficacy in NCr nude mice bearing BT-474 xenograft tumors. Mean tumor volume as a function of time, with groups receiving Herceptin dosing once every 2 weeks, all other groups receiving dosing once every 3 weeks. Antitumor efficacy is shown for NCr nude mice treated with (A) 5.9 mg Herceptin®/kg and (B) targeted MAP-CPT nanoparticles (5.9 mg Herceptin®/kg and 1 mg CPT/kg).

Provided herein are nanoparticles, as well as compositions, methods and systems that may be used in conjunction with the nanoparticles to deliver compounds contained therein, methods of making the described nanoparticles, methods of treatment using the described nanoparticles, and kits for assembling the described nanoparticles.

The term "nanoparticle" as used herein refers to a composite structure of nanoscale dimensions. In particular, nanoparticles are typically particles ranging in size from about 1 to about 1000 nm, usually spherical, although different morphologies are possible depending on the nanoparticle composition. The portion of the nanoparticle that contacts the external environment of the nanoparticle is generally defined as the surface of the nanoparticle. In the nanoparticles described herein, the size limitations are limited in two dimensions, and thus the nanoparticles described herein include composite structures having diameters of about 1 to about 1000 nm, where the particular diameter depends on the nanoparticle composition and the application of the nanoparticle according to the experimental design. For example, nanoparticles used in some therapeutic applications typically have a size of about 200 nm or less, and in particular those used for delivery related to cancer treatment typically have a diameter of about 1 to about 100 nm. The term "targeted nanoparticle" refers to a nanoparticle conjugated to a targeting agent or a targeting ligand.

Further desirable properties of nanoparticles, such as surface charge and steric stabilization, may vary in view of the particular application of interest. Some examples of properties that may be desirable in clinical applications, such as cancer treatment, are described in the scientific literature. Further properties will be understood by those skilled in the art upon reading this disclosure. The dimensions and properties of nanoparticles can be detected by techniques known in the art. Examples of techniques for detecting particle dimensions include, but are not limited to, dynamic light scattering (DLS) and various microscopy such as transmission electron microscopy (TEM) and atomic force microscopy (AFM). Examples of techniques for detecting particle morphology include, but are not limited to, TEM and AFM. Examples of techniques for detecting surface charge include, but are not limited to, zeta potential zeta potential methods. Further techniques are suitable for detecting other chemical properties, including ¹ H, ¹¹ B, ¹³ C and ¹⁹ F NMR, UV/visible and infrared/Raman microscopy and fluorescence microscopy (when nanoparticles are used in combination with fluorescent labels) and further techniques understood by those skilled in the art.

The nanoparticles and related compositions, methods and systems described herein can be used to deliver compounds of interest, and in particular drugs, to a given target.

The terms "deliver" and "delivery", as used herein, refer to an activity that affects the spatial location of a compound, and in particular, controls such location. Thus, delivery of a compound within the meaning of this disclosure refers to the ability to affect the positioning and movement of a compound at a particular time and under a particular set of conditions, such that the positioning and movement of the compound under those conditions is altered relative to the positioning and movement that the compound would otherwise have.

In particular, with respect to a reference endpoint, delivery of a compound indicates the ability to control the positioning and movement of the compound so that the compound is ultimately located at the selected reference endpoint. In in vitro systems, delivery of a compound is usually accompanied by a corresponding modification of the chemical and/or biological detectable properties and activity of the compound. In in vitro systems, delivery of a compound is also typically accompanied by a modification of the pharmacokinetics of the compound, and may also be accompanied by a modification of the pharmacodynamics.

The pharmacokinetics of a compound refers to the absorption, distribution, metabolism and excretion of the compound from a system, typically effected by the body of an individual. In particular, the term "absorption" refers to the process by which a substance enters the body, the term "distribution" refers to the dispersion or seeding of a substance through the body's fluids and tissues, the term "metabolism" refers to the irreversible conversion of a parent compound to a daughter metabolite, and the term "excretion" refers to the elimination of a substance from the body. If the compound is in a formulation, pharmacokinetics also includes the release of the compound from the formulation, which refers to the process of release of the compound, typically a drug, from the formulation. The term "pharmacodynamics" refers to the physical effect of the compound on the body or on a microorganism or a parasite in or on the body, the mechanism of drug action, and the relationship between drug concentration and effect. A person skilled in the art can identify techniques and procedures that are suitable for the pharmacokinetic and pharmacodynamic characteristics and properties of a compound of interest and in particular an agent of interest, e.g., a drug.

The term "drug" as used herein refers to a compound capable of exhibiting chemical or biological activity relative to a target. The term "chemical activity" as used herein refers to the ability of a molecule to undergo a chemical reaction. The term "biological activity" as used herein refers to the ability of a molecule to affect an organism. Examples of chemical activity of a drug include covalent bond formation or electrostatic interactions. Examples of biological activity of a drug include production and secretion of endogenous molecules, absorption and metabolism of endogenous or exogenous molecules, and activation or inactivation of gene expression, including transcription and translation of a gene of interest.

The term "target" as used herein includes a biological system of interest, including a single-celled or multicellular organism or any portion thereof, as well as an in vitro or in vivo biological system or any portion thereof.

In the nanoparticles described herein, the boronic acid-containing polymer bound to the polyol-containing polymer is positioned on the nanoparticle so as to be presented to the environmental exterior of the nanoparticle.

The term "polymer" as used herein refers to a large molecule composed of repeating structural units typically linked by covalent chemical bonds. Suitable polymers may be linear and/or branched and may be in the form of homopolymers or copolymers. When copolymers are used, they may be random or branched copolymers. Examples of polymers include water-dispersible polymers, and in particular water-soluble polymers. For example, suitable polymers include, but are not limited to, polysaccharides, polyesters, polyamides, polyethers, polycarbonates, polyacrylates, and the like. For therapeutic and/or pharmaceutical uses and applications, the polymer must have a low toxicity profile, in particular low toxicity or cytotoxicity. Suitable polymers include those having a molecular weight of about 500,000 or less. In particular, suitable polymers may have a molecular weight of about 100,000 or less.

The term "polyol-containing polymer" or "polyol polymer" as used herein refers to a polymer that exhibits multiple hydroxyl functional groups. In particular, polyol-containing polymers suitable for forming the nanoparticles described herein include polymers that exhibit at least a portion of hydroxyl functional groups for coupling interaction with at least one boronic acid of the boronic acid-containing polymer.

The term "presented," as used herein with respect to a compound or functional group, refers to a bond that is made to maintain the chemical reactivity of the attached compound or functional group. Thus, a functional group presented on a surface is capable, under appropriate conditions, of undergoing one or more chemical reactions that chemically characterize the functional group.

Structural units forming the polymer include polyol monomers, such as pentaerythritol, ethylene glycol, and glycerin. Examples of polymers containing polyols include polyesters, polyethers, and polysaccharides. Examples of suitable polyesters include, but are not limited to, diols, and in particular diols of the general formula HO-(CH ₂ CH ₂ O) _p -H, with p≧1, such as polyethylene glycol, polypropylene glycol, and poly(tetramethylene ether) glycol. Examples of suitable polysaccharides include, but are not limited to, cyclodextrin, starch, glycogen, cellulose, chitin, and β-glucan. Examples of suitable polyesters include, but are not limited to, polycarbonate, polybutyrate, and polyethylene terephthalate, all terminated with hydroxyl end groups. Examples of polyol-containing polymers include polymers with a molecular weight of about 500,000 or less, and in particular a molecular weight of about 300 to about 100,000.

Some polyol-containing polymers are commercially available and/or can be produced using techniques and procedures that can be identified by one of skill in the art. Exemplary procedures for the synthesis of example polyol polymers have been previously described in the scientific literature, and others are described in Examples 1-4. Additional procedures for making polyol-containing polymers can be identified by one of skill in the art in view of this disclosure.

The term "boronic acid-containing polymer" or "BA polymer" as used herein refers to a polymer that includes at least one boronic acid group presented for bonding to a hydroxyl group of a polyol-containing polymer. In particular, the boronic acid-containing polymers of the nanoparticles described herein include polymers that include at least one structural unit of an alkyl or aryl substituted boronic acid that includes a chemical bond between carbon and boron. Suitable BA polymers include those in which the boronic acid is present at a terminal structural unit or any other suitable moiety, imparting hydrophilic properties to the resulting polymer. Examples of polyol-containing polymers include polymers with a molecular weight of about 40,000 or less, particularly a molecular weight of about 20,000 to about 10,000.

Some boronic acid-containing polymers are commercially available and/or can be produced using techniques and procedures that can be identified by one of skill in the art. Exemplary procedures for the synthesis of example polyol polymers have been previously described in the scientific literature, and others are described in Examples 5-8. Additional procedures for producing BA polymers can be identified by one of skill in the art in view of this disclosure.

In the nanoparticles described herein, the polyol polymer is coupled to the BA polymer. The term "coupled" or "coupling" as used herein with respect to the bond between two molecules refers to an interaction that forms a reversible covalent bond. Specifically, in the presence of a suitable medium, the boronic acid presented on the BA polymer interacts with the hydroxyl group of the polyol through a rapid and reversible pairing covalent interaction to form a boronic acid ester in the suitable medium. Suitable media include water and some aqueous solutions as well as additional organic media that may be identified by one of skill in the art. Specifically, when contacted in an aqueous medium, the BA polymer and the polyol react, producing water as a by-product. This boronic acid polyol interaction is generally more preferred in aqueous solutions, but is also known to proceed in organic media. Additionally, the cyclic esters formed by 1,2 diols and 1,3 diols are generally more stable than their non-cyclic ester counterparts.

Thus, in the nanoparticles described herein, at least the boronic acid of the boronic acid-containing polymer is bound to the hydroxyl group of the polyol-containing polymer by a reversible covalent bond. The boronic acid ester between the BA polymer and the polyol polymer can be detected by methods and techniques identified by those skilled in the art, such as, for example, boron-11 nuclear magnetic resonance ( ¹¹ B NMR), potentiometric titration, selected UV/visible and fluorescence detection techniques, depending on the particular chemistry and properties of the boronic acid and polyol that make up the nanoparticle.

Nanoparticles resulting from the coupling interaction of the BA polymers described herein with the polyol polymers described herein present the BA polymer on the surface of the particle. In some embodiments, the nanoparticles can have a diameter of about 1 to about 1000 nm and a spherical morphology, although the particle size and morphology are determined primarily by the BA polymer and polyol polymer used to form the nanoparticles, as well as by the compounds carried in the nanoparticles in accordance with the present disclosure.

In some embodiments, the compound of interest carried by the nanoparticle forms part of the BA polymer and/or the polyol polymer. An example of such an embodiment is provided by a nanoparticle in which one or more atoms of the polymer have been replaced with a particular isotope, such as, in particular, ¹⁹ F and ¹⁰ B, and thus is suitable as an agent for imaging a target and/or for providing radiation treatment to a target.

In some embodiments, the compound of interest carried by the nanoparticle is bound to a polymer, typically a polyol polymer, via covalent or non-covalent bonds. An example of such an embodiment is provided by a nanoparticle in which one or more moieties of at least one of the compound polyol polymer and the BA polymer are bound to one or more targets.

The terms "bond," "bonded," or "bonding," as used herein, refer to a connection or integration by a bond, connection, force, or association that holds two or more components together, including direct or indirect bonding, and thus including, for example, where a first compound is directly bonded to a second compound, as well as embodiments where one or more intermediate compounds, particularly intermediate molecules, are interposed between the first and second compounds.

In particular, in some embodiments, a compound may be attached to a polyol polymer or a BA polymer via covalent attachment of the compound to a suitable portion of the polymer. Examples of covalent attachment are illustrated in Example 19, where the attachment of the drug camptothecin to a mucic acid polymer is via a biodegradable ester bond linkage, and in Example 9, where the attachment of transferrin to BA-PEG ₅₀₀₀ is via pegylation of transferrin.

In some embodiments, the polymer can be designed or modified (e.g., by the addition of one or more functional groups that can specifically bind to corresponding functional groups on the compound of interest) to allow for the attachment of a particular compound of interest. For example, in some embodiments, the nanoparticles can be pegylated with BA-PEG-X, where X can be a maleimide group or an iodoacetyl group or a free radical that reacts specifically with thiols or non-specifically with amines. The compound to be attached can then be reacted with the maleimide group or the iodoacetyl group after modification to reveal the thiol functionality. The compound to be attached can also be modified with an aldehyde or ketone group, which can react via a condensation reaction with a diol on a polyol to give an acetal or ketal.

In some embodiments, the compound of interest can be attached to the polyol polymer or BA polymer via non-covalent bonds, such as ionic bonds and intermolecular interactions, between the compound to be attached and a suitable portion of the polymer. Examples of non-covalent bonds are described in Example 10.

The compound of interest may be attached to the nanoparticles prior to, during, or after the formation of the nanoparticles, for example, via modification of the polymer and/or modification of any attached compounds in the particle composite. Exemplary procedures for the attachment of compounds to nanoparticles are described in the Examples section. Additional procedures for the attachment of compounds to the BA polymer, polyol polymer, or other components of the nanoparticles described herein (e.g., pre-introduced compounds of interest) may be identified by one of skill in the art upon reading this disclosure.

In some embodiments, at least one compound of interest bound to the BA polymer presented on the nanoparticles described herein is a drug that can be used as a targeting ligand. In particular, in some embodiments, the nanoparticles are bound to one or more drugs on the BA polymer that are used as targeting ligands, and to one or more drugs on the polyol polymer and/or the BA polymer that are delivered to a selected target.

The term "targeting ligand" or "targeting agent" as used in this disclosure refers to any molecule that can be presented on the surface of a nanoparticle for the purpose of engaging a specific target and, in particular, for the purpose of specific cell recognition, for example by enabling cell receptor binding of the nanoparticle. Examples of suitable ligands include, but are not limited to, vitamins (e.g., folic acid), proteins (e.g., transferrin and monoclonal antibodies), peptides and polysaccharides. In particular, targeting ligands may be antibodies against specific surface cell receptors, small molecules such as anti-VEGF, folic acid, as well as other proteins, such as holo-transferrin.

The ligand selected will be understood by one of skill in the art and may vary depending on the type of delivery desired. As another example, the ligand may be a membrane permeabilizing or permeabilizing agent, such as the TAT protein from HIV-1. The TAT protein is a viral transcription activator that actively enters the cell nucleus. Torchilin, V. P. et al, PNAS. 98, 8786 8791, (2001). Suitable targeting ligands attached to BA polymers typically include a flexible spacer such as poly(ethylene oxide) with a boronic acid attached to its terminus (see Example 9).

In some embodiments, at least one compound of the polyol polymer and/or the BA polymer (including the targeting ligand) may be a pharmaceutical agent, particularly a drug, that is delivered to a target, e.g., an individual, where a chemical or biological activity, e.g., a therapeutic activity, is exerted.

The section on polyol polymers and BA polymers for forming nanoparticles described herein can be carried out in terms of compounds of interest and targets. In particular, the section on suitable polyol-containing polymers and suitable BA polymers for forming nanoparticles described herein can be carried out by providing candidate polyol polymers and BA polymers and selecting polyol polymers and BA polymers capable of forming coupling interactions in view of the present disclosure, where the selected BA polymers and polyol polymers have a chemical composition such that the polyol polymer is more hydrophilic than the BA polymer in terms of the compounds of interest and targeting ligands contained in or attached to the polyol polymer and/or BA polymer. Detection of the presentation of the BA polymer on the surface nanoparticles and related in the external environment to the nanoparticles can be carried out by detection of the zeta potential, which can carry out modification of the surface of the nanoparticles, as described in Example 12. (See FIG. 6.) Further procedures for detecting the surface charge of the particles and the stability of the particles in salt solutions include detection of the charge of the particle size, such as those exemplified in Example 12 (see FIG. 7 in detail), as well as further procedures that can be identified by those skilled in the art.

In some embodiments, the polyol-containing polymer comprises at least one of the following structural units:

式中、
Ａは、以下の式：

In the formula,
A has the following formula:

ここで、
Ｒ_１およびＲ_２は、約１０ｋＤａ以下の分子量を有する任意の炭素ベースの基又は有機基から独立して選択され；
Ｘは、－Ｈ、－Ｆ、－Ｃ、－Ｎ又は－Ｏの１つ以上を含む脂肪族基から選択され；且つ
Ｙは、－ＯＨであるか、又はヒドロキシル（－ＯＨ）基を有する有機部分（－ＣＨ_２ＯＨ、－ＣＨ_２ＣＨ_２ＯＨ、－ＣＦ_２ＯＨ、－ＣＦ_２ＣＦ_２ＯＨおよびＣ（Ｒ_１Ｇ_１）（ＲＧ_２）（Ｒ_１Ｇ_３）ＯＨが挙げられるがこれらに限定されず、ここでＲ_１Ｇ_１、Ｒ_１Ｇ_２およびＲ_１Ｇ_３は、独立して有機ベースの官能基である）から独立して選択され、
且つ
Ｂは、第１のＡ部分のＲ_１およびＲ_２の１つを第２のＡ部分のＲ_１およびＲ_２の１つに連結する有機部分である。

here,
_R1 and _R2 are independently selected from any carbon-based or organic group having a molecular weight of about 10 kDa or less;
X is selected from an aliphatic group containing one or more of -H, -F, -C, -N, or -O; and Y is -OH or is independently selected from an organic moiety having a hydroxyl (-OH) group, including but not limited to, -CH ₂ OH, -CH ₂ CH ₂ OH, -CF ₂ OH, -CF ₂ CF ₂ OH, and C(R ₁ G ₁ )(RG ₂ )(R ₁ G ₃ )OH, where R ₁ G ₁ , R ₁ G ₂ , and R ₁ G ₃ are independently organic-based functional groups;
and B is an organic moiety that links one of _R1 and _R2 of the first A moiety to one of _R1 and _R2 of the second A moiety.

The term "moiety" as used herein refers to a series of atoms that make up part of a larger molecule or molecular species. In particular, moieties refer to repeating components of polymeric structural units. Exemplary moieties include acid or base species, sugars, carbohydrates, alkyl groups, aryl groups, and any other molecular components useful for forming polymeric structural units.

The term "organic moiety," as used herein, refers to a moiety that contains carbon atoms. In particular, organic moieties include natural and synthetic compounds, as well as compounds that contain heteroatoms. Exemplary natural organic moieties include, but are not limited to, most sugars, some alkaloids and terpenoids, carbohydrates, lipids and fatty acids, nucleic acids, proteins, peptides and amino acids, vitamins, and fats and oils. Synthetic organic groups refer to compounds that are prepared by reaction with other compounds.

In some embodiments, one or more compounds of interest may be bound to (A), (B), or (A) and (B).

In some embodiments, R ₁ and R ₂ independently have the formula:

式中、
ｄは、０～１００であり；
ｅは、０～１００であり；
ｆは、０～１００であり；
Ｚは、１つの有機部分を別の有機部分、詳細には、本明細書中で規定される部分Ａ又は部分Ｂに連結する共有結合であり、そして
Ｚ_１は、－ＮＨ_２、－ＯＨ、－ＳＨおよび－ＣＯＯＨから独立して選択される。

In the formula,
d is 0 to 100;
e is 0 to 100;
f is 0 to 100;
Z is a covalent bond linking one organic moiety to another organic moiety, specifically moiety A or moiety B as defined herein, and _Z1 is independently selected from _-NH2 , -OH, -SH and -COOH.

In some embodiments, Z is independently selected from -NH-, -C(=O)NH-, -NH-C(=O), -SS-, -C(=O)O-, -NH(=NH ₂ ⁺ )-, or -O-C(=O)-.

In some embodiments where structural unit A of the polyol-containing polymer has formula (IV), X can be C _v H _2v+1 , where v=0-5, and Y is --OH.

In some embodiments, R1 and/or R2 have the formula (V) where Z is -NH(=NH ₂ ⁺ )- and/or Z ₁ is NH ₂ .

In some embodiments, the polyol-containing polymer (A) of the particles described herein has the following formula:

から独立して選択され、
式中、
スペーサーは、任意の有機部分（詳細には、必要に応じてヘテロ原子、例えば硫黄、窒素、酸素又はフッ素を含むアルキル基、フェニル基又はアルコキシ基が挙げられ得る）から独立して選択され、
アミノ酸は、遊離のアミン基および遊離のカルボン酸基を有する任意の有機基から選択され；
ｎは、１～２０であり；且つ
Ｚ_１は、－ＮＨ_２、－ＯＨ、－ＳＨおよび－ＣＯＯＨから独立して選択される。

are independently selected from
In the formula,
spacers are independently selected from any organic moiety, which may in particular include alkyl, phenyl or alkoxy groups, optionally containing heteroatoms such as sulfur, nitrogen, oxygen or fluorine;
The amino acid is selected from any organic group having a free amine group and a free carboxylic acid group;
n is 1 to 20; and Z ₁ is independently selected from --NH ₂ , --OH, --SH, and --COOH.

In some embodiments, Z1 and NH2 and/or the sugar can be any monosaccharide, such as glucose, fructose, mannitol, sucrose, galactose, sorbitol, xylose, or galactose.

In some embodiments, one or more structural units (A) of the polyol-containing polymer of the particles described herein have the following formula:

から独立して選択され得る。

may be independently selected from

In some embodiments, (B) can be formed by any linear, branched, symmetrical or asymmetrical compound that links two (A) moieties via a functional group.

In some embodiments, (B) may be formed by a compound in which at least two crosslinkable groups link two (A) moieties.

In some embodiments, (B) comprises a neutral, cationic or anionic organic group, the nature and composition of which depends on the chemical nature of the compound to which it is covalently or non-covalently tethered.

Examples of cationic moieties in (B) for use with anionic cargoes include, but are not limited to, organic groups having amidine groups, quaternary ammonium, primary amine groups, tertiary amine groups (protonated below their pKa), tertiary amine groups, and imidazolium.

Examples of anionic moieties included in (B) for use with cationic cargoes include, but are not limited to, organic groups having the formula sulfonate, nitrate, carboxylate, and phosphonate.

In particular, one or more cationic or anionic moieties of (B) for use with anionic and cationic cargoes, respectively, may independently have the general formula:

式中、Ｒ_５は、Ａが求核生基を含む場合Ａに共有結合し得る求電子性基である。この場合におけるＲ_５としては、以下が挙げられるが、これらに限定されない：
他の幾つかの中でもとりわけ、－Ｃ（＝Ｏ）ＯＨ、－Ｃ（＝Ｏ）Ｃｌ、－Ｃ（＝Ｏ）ＮＨＳ、－Ｃ（＝ＮＨ_２ ^＋）ＯＭｅ、－Ｓ（＝Ｏ）ＯＣｌ－、－ＣＨ_２Ｂｒ、アルキルおよび芳香族エステル、末端アルキン、トシレート、およびメシレート。部分Ａが求電子末端基を含む場合、Ｒ_５は、－ＮＨ_２（第１級アミン）、－ＯＨ、－ＳＨ、Ｎ_３および第２級アインなどの求核性基を有する。

wherein _R5 is an electrophilic group that can be covalently bonded to A when A contains a nucleophilic group. _R5 in this case includes, but is not limited to, the following:
Among others, -C(=O)OH, -C(=O)Cl, -C(=O)NHS, -C(=NH ₂ ⁺ )OMe, -S(=O)OCl-, -CH ₂ Br, alkyl and aromatic esters, terminal alkynes, tosylates, and mesylates. When moiety A contains an electrophilic end group, R ₅ bears a nucleophilic group such as -NH ₂ (primary amines), -OH, -SH, N ₃ and secondary amines.

In particular, when moiety (B) is a cationic moiety (B) used with an anionic cargo, the "organic group" may have a backbone with the general formula consisting of _CmH2m , where m>1, and other _heteroatoms , and must contain at least one of the functional groups including: amidine of formula (XIII), quaternary ammonium of formula (XIV), primary amine group of formula (XV), secondary amine group of formula (XVI), tertiary amine group of formula (XVII) (protonated below its pKa), and imidazolium of formula (XVIII).

In embodiments where moiety (B) is an anionic moiety (B) used with a cationic cargo, the "organic group" may have a backbone with the general formula consisting of _{CmH2m ,} where m>1, and other heteroatoms, and must contain at least one of the functional groups including: sulfonates of formula (XIX), nitrates of formula (XX), carboxylates of formula (XXI), and phosphonates of formula (XXII).

In embodiments where (B) is constituted by a carboxylate (XXI), compounds containing primary amines or hydroxyl groups may also be attached via the formation of peptide or ester bonds.

In embodiments where (B) is comprised of a primary amine group of formula (XV) and/or a secondary amine group of formula (XVI), compounds containing a carboxylic acid group may be attached via formation of a peptide bond.

In some embodiments, portion (B) is independently selected from:

ここで、
ｑは、１～２０であり；且つ詳細には、５であってもよく、
ｐは、２０～２００であり；且つ
Ｌは、脱離基である。

here,
q is 1 to 20; and may particularly be 5;
p is 20 to 200; and L is a leaving group.

The term "leaving group" as used herein refers to a molecular fragment that separates a pair of electrons in anisotropic bond dissociation. In particular, leaving groups are anionic or neutral molecules, and the ability of the leaving group to dissociate is correlated to the pK _a of the conjugate acid, with a lower pK _a being associated with better leaving group ability. Examples of anionic compounds include halides such as Cl ^- , Br ^- and I ^- , and sulfonate esters, e.g., para-toluenesulfonate or "tosylate" (TsO ^- ). Examples of neutral molecular leaving groups are water (H ₂ O), ammonia (NH ₃ ), and alcohols (ROH).

Specifically, in some embodiments, L may be chlorine (Cl), methoxy (OMe), t-butoxy (OtBU), or N-succinimide (NHS).

In some embodiments, the structural unit of formula (I) may have the following formula:

In some embodiments, the structural unit of formula (II) may have the following formula:

In some embodiments, the structural unit of formula (III) may have the following formula:

の有機部分であり、ここで、
ｎは、１～２０であり；且つ詳細には、１～４である。

where:
n is 1-20; and particularly, 1-4.

In some embodiments, the polyol-containing polymer may have the following formula:

In some embodiments, the boronic acid-containing polymer contains at least one terminal boronic acid group and has the following structure:

式中、
Ｒ_３およびＲ_４は、任意の親水性有機ポリマーから独立して選択され、詳細には、独立して任意のポリ（エチレンオキシド）および双性イオン性ポリマーであってもよく、
Ｘ_１は、－ＣＨ、－Ｎ又は－Ｂの１つ以上を含有する有機部分であり、
Ｙ_１は、場合によりオレフィン基又はアルキニル基、又は芳香族基（例えば、フェニル、ビフェニル、ナフチル又はアントラセニル）を含むｍ≧１である式－Ｃ_ｍＨ_２ｍ－を有するアルキル基であってもよく、
ｒは、１～１０００であり；
ａは、０～３であり；且つ
ｂは、０～３であり；
且つ式中、官能基１および官能基２は、同一であるか又は異なっており、標的リガンドに結合可能であり、詳細には、タンパク質、抗体又はペプチド、又は－ＯＨ、－ＯＣＨ_３又は－（Ｘ_１）－（Ｙ_１）－Ｂ（ＯＨ）_２－などの末端基である。

In the formula,
_R3 and _R4 are independently selected from any hydrophilic organic polymer, and in particular may be independently any poly(ethylene oxide) and zwitterionic polymer;
_X1 is an organic moiety containing one or more of -CH, -N, or -B;
Y ₁ may be an alkyl group having the formula -C _m H _2m -, where m≧1, optionally including an olefinic or alkynyl group, or an aromatic group (e.g., phenyl, biphenyl, naphthyl, or anthracenyl);
r is 1 to 1000;
a is 0 to 3; and b is 0 to 3;
and wherein functional group 1 and functional group 2 are the same or different and are capable of binding to a target ligand, in particular a protein, an antibody or a peptide, or a terminal group such as -OH, _-OCH3 or -( _X1 )-( _Y1 )-B(OH) _2- .

In some embodiments, R ₃ and R ₄ are (CH ₂ CH ₂ O) _t , where t is 2-2000, particularly 100-300.

In some embodiments, X ₁ is -NH-C(=O)-, -S-S-, -C(=O)-NH-, -OC(=O)- or -C(=O)-O-, and/or Y ₁ is a phenylphenyl group.

In some embodiments, r may be 1, a may be 0, and b may be 1.

In some embodiments, Functional Group 1 and Functional Group 2 are the same or different and are independently selected from the following: -B(OH) ₂ , -OCH ₃ , -OH.

In particular, functional groups 1 and/or 2 of formula (XXXI) may be functional groups capable of binding to a cargo, in particular a targeting ligand, such as a protein, antibody or peptide, or may be a terminal group such as -OH, _-OCH3 or -(X)-(Y)-B(OH) ₂ .

The term "functional group" as used herein refers to a particular group of atoms in or part of a molecular structure that is responsible for a characteristic chemical reaction of the molecular structure or part thereof. Examples of functional groups include hydrocarbons, halogen-containing groups, oxygen-containing groups, nitrogen-containing groups, and phosphorus- and sulfur-containing groups, all of which can be identified by one of ordinary skill in the art. In particular, functional groups in the sense of the present disclosure include carboxylic acids, amines, triarylphosphines, azides, acetylenes, sulfonyl azides, thioazides, and aldehydes. In particular, for example, functional groups capable of binding to corresponding functional groups in a targeting ligand can be selected to include the following binding partners: carboxylic acid and amine groups, azides and acetylene groups, azides and triarylphosphine groups, sulfonyl azides and thioazides, and aldehydes and primary amines. Additional functional groups can be understood by one of ordinary skill in the art upon reading this disclosure. As used herein, the term "corresponding functional group" refers to a functional group that can react with another functional group. Therefore, functional groups that can react with each other can be said to be corresponding functional groups.

End groups refer to structural units that are the ends of a micro- or oligomeric molecule. For example, the end groups of a PET polyester can be alcohol or carboxylic acid groups. End groups can be used to determine the molar mass. Examples of end groups include: -OH, -COOH, _NH2 , and _OCH3 .

In some embodiments, the boronic acid-containing polymer may have the formula:

式中、ｓは、２０～３００である。

In the formula, s is 20 to 300.

Examples of drugs and targeting ligands that can be attached to the nanoparticles of the present disclosure include organic or inorganic molecules, including, but not limited to, polynucleotides, nucleotides, aptamers, polypeptides, proteins, polysaccharides, macromolecular complexes (including mixtures of proteins with polynucleotides, saccharides and/or polysaccharides, viruses, molecules bearing radioisotopes, antibodies or antibody fragments).

The term "polynucleotide" as used herein refers to an organic polymer of two or more monomers, including nucleotides, nucleosides, or analogs thereof. The term "nucleotide" refers to any of several compounds consisting of a purine or pyrimidine base and a ribose or deoxyribose sugar linked to a phosphate group, and is the basic structural unit of nucleic acids. The term "nucleoside" refers to a compound consisting of a purine or pyrimidine base (e.g., guanosine or adenosine) combined with deoxyribose or ribose, and is found especially in nucleic acids. The terms "nucleotide analog" or "nucleoside analog" refer to a nucleotide or nucleoside, respectively, in which one or more individual atoms are replaced with different atoms or different functional groups. Thus, the term "polynucleotide" includes nucleic acids of any length, particularly DNA, RNA, analogs and fragments thereof. Polynucleotides of three or more nucleotides are also referred to as "nucleotide oligomers" or "oligonucleotides."

The term "aptamer" as used herein refers to an oligonucleic acid or peptide molecule that binds to a specific target. In particular, mucic acid aptamers can include nucleic acid species that have been engineered to bind to various molecular targets (e.g., small molecules, proteins, nucleic acids, and even cells, tissues, and organisms) through, for example, repeated in vitro selection, i.e., SELEX (systematic evolution of ligands by exponential enrichment). Aptamers are useful in biotechnological and therapeutic applications because they offer molecular recognition properties that compete with those of antibodies. Peptide aptamers are peptides that are designed to specifically bind and interfere with protein-protein interactions inside cells. In particular, peptide aptamers can be derived, for example, following selection strategies derived from the yeast two-hybrid (Y2H) system. Specifically, according to this strategy, variable peptide aptamer loops that bind to a transcription factor binding domain are screened against a target protein bound to a transcription factor activation domain. In vivo binding of the peptide aptamer to its target via this selection strategy is detected as expression of a downstream yeast marker gene.

The term "polynucleotide" as used herein refers to an organic linear, cyclic or branched polymer composed of two or more amino acid monomers and/or analogs thereof. A "polypeptide" includes amino acid polymers of any length, including full-length proteins and peptides, as well as analogs and fragments thereof. Polypeptides of three or more amino acids are also referred to as protein oligomers, peptides or oligopeptides. In particular, the terms "peptide" and "oligopeptide" generally refer to polypeptides having fewer than 50 amino acid monomers. As used herein, the terms "amino acid", "amino acid monomer" or "amino acid residue" refer to any of the 20 naturally occurring amino acids, non-naturally occurring amino acids, and artificial amino acids, including both D and L optical isomers. In particular, non-naturally occurring amino acids include the D stereoisomers of naturally occurring amino acids. (These include useful ligand building blocks because they are less susceptible to enzymatic degradation.) The term "artificial amino acid" refers to molecules that can be readily coupled using standard amino acid coupling science, but have molecular structures that do not resemble naturally occurring amino acids. The term "amino acid analog" refers to an amino acid in which one or more individual atoms have been replaced with either a different atom, an isotope, or a different functional group, but which is otherwise identical to the original amino acid from which the analog is derived. All of these amino acids can be synthetically incorporated into peptides or polypeptides using standard amino acid coupling chemistry. The term "polypeptide" as used herein includes polymers that include one or more monomers or building blocks other than amino acid monomers. The terms monomer, subunit, or building block refer to chemical compounds that can be chemically bonded under appropriate conditions with another monomer of the same or different chemical nature to form a polymer. The term "polypeptide" is further intended to include polymers in which one or more building blocks are covalently bonded to each other by chemical bonds other than amide or peptide bonds.

The term "protein" as used herein refers to a polypeptide having a particular secondary and tertiary structure that can participate in interactions with other biological molecules, including, but not limited to, other proteins, DNA, RNA, lipids, metabolites, hormones, chemokines, and small molecules. An example of a protein described herein is an antibody.

The term "antibody" as used herein refers to a type of protein produced by activated B cells after stimulation with an antigen, capable of specifically binding to an antigen and promoting an immune response in a biological system. A complete antibody typically consists of four subunits, including two heavy chains and two light chains. The term antibody includes natural and synthetic antibodies, including, but not limited to, monoclonal antibodies, polyclonal antibodies, or fragments thereof. Examples of antibodies include IgA, IgD, IgG1, IgG2, IgG3, IgM, etc. Examples of fragments include Fab Fv, Fab'F(ab')2, etc. A monoclonal antibody is an antibody that is capable of specifically binding to, and is therefore defined as complementary to, one particular spatial and polar feature of another biological molecule, called an "epitope." In some forms, monoclonal antibodies may also have the same structure. A polyclonal antibody refers to a mixture of different monoclonal antibodies. In some forms, a polyclonal antibody may be a mixture of monoclonal antibodies, where at least two monoclonal antibodies bind to different antigenic epitopes. The different antigenic epitopes may be against the same target, different targets, or a combination. Antibodies may be prepared by techniques well known in the art, such as immunizing a host and collecting serum (polyclonal), or preparing continuous hybridoma cell lines and collecting secreted proteins (monoclonal).

In some embodiments, the polyol polymer forms a non-covalent complex or linkage with one or more compounds of interest to be delivered, according to the diagrams in Figures 1 and 2.

In some embodiments, the nanoparticle structure includes a drug and a polyol-containing polymer, where the drug is linked to the polyol polymer by a covalent bond. Examples of polyol polymers conjugated with drugs are detailed in Examples 16-21. In these embodiments, the polyol polymer conjugated to the drug (referred to herein as a "polyol polymer-drug conjugate") forms a nanoparticle, the structure presenting sites on its surface for interaction with BA molecules.

In some embodiments, the nanoparticles further comprise a BA polymer configured to provide steric stabilization and/or targeting functionality to the nanoparticles. Specifically, in these embodiments, the addition of the BA polymer allows for minimization of self-aggregation and unwanted interactions with other nanoparticles, thus providing improved salt and serum stability. For example, steric stabilization is provided by the BA polymer with PEG, as illustrated by the example of the nanoparticles described herein in Example 12.

In such embodiments, the nanoparticle structure provides several advantages over prior art drug delivery methods, such as the ability to provide controlled release of one or more drugs. This feature can be provided, for example, by the use of a biodegradable ester linkage between the drug and the polyol polymer. Those skilled in the art will appreciate other linkage possibilities suitable for this purpose. In these embodiments, another advantage is that it provides specific targeting of the drug via the BA polymer moiety.

In some embodiments, the BA polymer may include a fluorinated boronic acid (Example 7) or a fluorinated cleavable boronic acid (Example 8) that may be used as an imaging agent in MRI or other similar techniques. Such imaging agents may be useful for tracking the pharmacokinetics or pharmacodynamics of drugs delivered by the nanoparticles.

In some embodiments, the nanoparticle structure comprises a drug and a polyol polymer, where the nanoparticle is a modified liposome. In these embodiments, the modified liposome comprises a lipid conjugated via a covalent bond to a polyol polymer, whereby the surface of the liposome displays the polyol polymer. In these embodiments, the modified liposome formed to deliver the drug is contained within a liposomal nanoparticle.

The term "liposome" as used herein refers to a vesicular structure composed of lipids. The lipids typically have a long hydrocarbon chain and a tail group that includes a hydrophilic head group. The lipids are arranged to form a lipid bilayer, the interior of which is an aqueous environment suitable for containing the agent to be delivered. Such liposomes present an outer surface that may contain suitable targeting ligands or molecules for specific recognition by cell surface receptors or other targets of interest.

The term "conjugated," as used herein, refers to one molecule forming a covalent bond with a second molecule, including single and multiple (e.g., double) bonds (e.g., C=C-C=C-C) that affect each other and result in electron delocalization.

In still other embodiments of the present disclosure, the nanoparticle structure comprises a drug and a polyol, where the nanoparticle is a modified micelle. In these embodiments, the modified micelle comprises a polyol polymer that has been modified to include a hydrophobic polymer block.

The term "hydrophobic polymer block," as used in this disclosure, refers to a segment of a polymer that is itself hydrophobic.

The term "micelle," as used herein, refers to an aggregate of molecules dispersed in lipids. A typical micelle in aqueous solution forms an aggregate with a hydrophilic "head" region in the region in contact with the surrounding solvent, and sequestering a hydrophobic single tail region in the center of the micelle. In this disclosure, the head region may be, for example, a surface region of a polyol polymer, while the tail region may be, for example, a hydrophobic polymer block region of a polyol polymer.

In these embodiments, the polyol polymer having a hydrophobic polymer block is arranged to form a nanoparticle that, when mixed with the agent to be delivered, is a modified micelle with the agent to be delivered within the nanoparticle. Such nanoparticle embodiments present a polyol polymer on their surface suitable for interaction with a BA polymer, with or without targeting functionality according to the previous embodiment. In these embodiments, BA polymers that can be used for this purpose include those having a hydrophilic A and a hydrophobic B in formula (I) or (II). This interaction provides advantages similar or similar to those provided for the other nanoparticle embodiments described above.

In some embodiments, the nanoparticles or related components may be included in the composition together with an acceptable vehicle. The term "vehicle" as used herein generally refers to any of a variety of media that act as a solvent, carrier, binder, excipient, or diluent for the nanoparticles that are included in the composition as an active ingredient.

In some embodiments in which the composition is administered to an individual, the composition may be a pharmaceutical composition and the acceptable vehicle may be a pharma- ceutical acceptable vehicle.

In some embodiments, the nanoparticles may be included in a pharmaceutical composition together with an excipient or diluent. In particular, it is disclosed that in some embodiments, the pharmaceutical composition includes the nanoparticles in combination with one or more compatible and pharma- ceutically acceptable vehicles, in particular pharma-ceutically acceptable diluents or excipients.

The term "excipient" as used herein refers to an inert substance used as a carrier for the active ingredient of a pharmaceutical. Suitable excipients for the pharmaceutical compositions disclosed herein include any substance that enhances the ability of an individual's body to absorb the nanoparticles. Suitable excipients also include any substance that can be used to bulk a formulation containing nanoparticles to allow for convenient and accurate dosing. In addition to their use in single doses, excipients may be used in manufacturing procedures to aid in the manipulation of nanoparticles. Depending on the route of administration and the pharmaceutical form, different excipients may be used. Examples of excipients include, but are not limited to, anti-adherents, binders, coating disintegrants, fillers, flavorings (e.g., sweeteners) and colorings, glidants, lubricants, preservatives, and adsorbents.

The term "diluent" as used herein refers to a diluent agent that serves to dilute or carry the active ingredient of the composition. Suitable diluents include any material capable of reducing the viscosity of a pharmaceutical preparation.

In certain embodiments, the compositions, particularly pharmaceutical compositions, may be formulated for parenteral administration, more particularly systemic administration, including intravenous, intradermal and intramuscular administration.

Examples of compositions for parenteral administration include, but are not limited to, sterile aqueous solutions, injectable solutions, or suspensions containing nanoparticles. In some embodiments, compositions for parenteral administration may be prepared at the time of use by dissolving a powdered composition previously prepared in a freeze-dried lyophilized form in a biologically compatible aqueous liquid (distilled water, physiological solution, or other aqueous solution).

The term "lyophilization" (freeze-drying or cryo-drying) refers to a dehydration process typically used to preserve perishable materials or to make them easier to transport. Freeze-drying is accomplished by freezing the material and then reducing the ambient pressure and applying enough heat that the frozen water within the material decays directly from a solid phase to a gas.

In pharmaceutical applications, freeze-drying is often used to extend the shelf life of products such as vaccines and other injectable medications. By removing the water from the substance and sealing it in a vial, the substance can be easily reconstituted into its original form for storage, transportation and injection.

In some embodiments, the nanoparticles described herein are delivered to a predetermined target. In some embodiments, the target is an in vitro biological system, and the method includes contacting the target with the nanoparticles described herein.

In some embodiments, a method is provided for delivery of a drug to an individual, where the method includes formulating suitable nanoparticles according to various embodiments of the present disclosure. The nanoparticles may also be formulated into a pharma- ceutically acceptable composition according to some embodiments of the present disclosure. The method includes delivering the nanoparticles to the subject. To deliver the nanoparticles to the individual, the nanoparticles or nanoparticle formulations may be given orally, parenterally, topically, or rectally. They are delivered in a form suitable for each route of administration. For example, the nanoparticle composition may be administered in the form of a pill or capsule, by injection, inhalation, eye lotion, ointment, suppository, infusion, or rectally by suppository.

The term "individual" as used herein includes a single organism, including a plant or an animal, particularly including higher animals, particularly including vertebrates such as mammals, particularly including, but not limited to, humans.

The phrases "parenteral administration" and "administered parenterally," as used herein, refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, and intrasternal injection and infusion.

The terms "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered," as used herein, refer to administration of a nanoparticle or composition thereof other than directly into the central nervous system, such that it enters an individual's system and is thus subjected to processes such as metabolism, e.g., subcutaneous administration.

The actual dosage levels of the active ingredients or agents in the pharmaceutical compositions described herein may vary for a particular individual, composition and mode of administration to obtain an amount of active agent effective to achieve the desired therapeutic response without being toxic to the individual.

These therapeutic polymer conjugates may be administered to humans and other animals for therapy by any suitable route of administration, including oral administration, nasal administration, such as by spray, rectal administration, vaginal administration, parenteral administration, intracisternal administration, and topical administration, including buccal and sublingual administration, such as by powders, ointments or drops.

Regardless of the route of administration selected, the therapeutic polymer conjugates that may be used in suitable hydrated form and/or in the pharmaceutical form of the present invention may be formulated into pharma- ceutical acceptable dosage forms by conventional methods known to those skilled in the art.

In particular, in some embodiments, the compound being delivered is a drug for treating or preventing a condition in an individual.

"Drug" or "therapeutic agent" refers to an active agent that can be used in the treatment, prevention, or diagnosis of a condition in an individual, or that can otherwise be used to enhance the physical or mental well-being of an individual.

The term "condition" as used herein refers to a physical condition of an individual's body, as a whole or one or more of its parts, that is incompatible with the physical condition of the individual, as a whole or one or more of its parts, that is normally associated with complete physical, mental and possibly social well-being. Conditions described herein include, but are not limited to, disorders and diseases, where the term "disorder" refers to a condition in a living individual involving abnormal functioning of the body or any part thereof, and the term "disease" refers to a condition in a living individual in which the normal functioning of the body or any part thereof is impaired and is typically manifested by a distinguishing sign or symptom. Examples of conditions include, but are not limited to, injury, dysfunction, disorder (including mental and physical disorders), syndrome, infection, abnormal behavior of an individual, and abnormal changes in the structure and function of an individual's body or any part thereof.

"Treatment" as used herein refers to any activity that is part of medical treatment, pharmaceutical or surgical, for a condition.

The term "prevention," as used herein, refers to any activity that reduces the burden of mortality or morbidity from a condition in an individual. This occurs at the following primary, secondary, and tertiary prevention levels: (a) primary prevention avoids the onset of disease; (b) secondary prevention aims at early disease treatment, thereby increasing the chances of interventions that prevent disease progression and symptom appearance; and (c) tertiary prevention reduces the adverse effects of an already established disease by restoring function and reducing disease-related complications.

Examples of compounds that may be delivered by the nanoparticles described herein and that are suitable as drugs include compounds that emit electromagnetic radiation (e.g., ¹⁰ B isotopes) and can be used in radiation treatments (e.g., boron neutron capture). Additional therapeutic agents include any lipophilic or hydrophilic, synthetic or naturally occurring, biologically active therapeutic agent, including those known in the art. The Merck Index, An Encyclopedia of Chemicals, Drugs, and Biologicals, 13th Edition, 2001, Merck and Co., Inc., Whitehouse Station, N.J. Examples of such therapeutic agents include, but are not limited to, small molecule pharmaceuticals, antibiotics, steroids, polynucleotides (e.g., genomic DNA, cDNA, mRNA, siRNA, shRNA, miRNA, antisense oligonucleotides, viruses, and chimeric polynucleotides), plasmids, peptides, peptide fragments, small molecules (e.g., doxorubicin), chelators (e.g., deferoxamine (DESFERAL)), ethylenediaminetetraacetic acid (EDTA)), natural products (e.g., taxol, amphotericin), and other biologically active macromolecules such as proteins and enzymes. Reference is also made to U.S. Pat. No. 6,048,736, which lists active agents (therapeutic agents) that can be used as therapeutic agents with the nanoparticles described herein. Small molecule therapeutic agents are not only therapeutic agents in the hybrid particles, but in further embodiments can be covalently attached to the polymer in the hybrid. In some embodiments, the covalent bond is reversible (e.g., through a prodrug form or a biodegradable linkage such as a disulfide bond), providing another method of delivering the therapeutic agent. In some embodiments, the therapeutic agents that may be delivered by the nanoparticles described herein include chemotherapeutic agents, such as epothilones, camptothecin-based drugs, taxol, or nucleic acids such as plasmids, siRNA, shRNA, miRNA, antisense oligonucleotide aptamers, or combinations thereof, as well as other drugs that may be identified by one of skill in the art upon reading this disclosure.

In some embodiments, the compound being delivered is suitable for imaging cells or tissues of an individual. Examples of compounds that can be delivered by the nanoparticles described herein and that are suitable for imaging include compounds that contain isotopes such as ^19F isotopes for MR imaging, ^18F or ^64Cu for PET imaging.

In particular, the nanoparticles described herein can be made to include ¹⁹ F-containing BA polymers. For example, ¹⁹ F atoms can be incorporated into non-cleavable or cleavable BA polymer compounds. Other locations where ¹⁹ F atoms can be present are on the BA polymer component, on the polyol polymer component, or on the drug to be delivered. These and other modifications will be apparent to those skilled in the art.

In some embodiments, the nanoparticles described herein can be used to deliver chemicals used in the agricultural industry. In another embodiment of the invention, the agents delivered by the nanoparticles described herein are biologically active compounds that have microbiological and agricultural utility. These biologically active compounds include those known in the art. For example, suitable agricultural biologically active compounds include, but are not limited to, fertilizers, fungicides, herbicides, insecticides, and mildewicides. Microbicides can also be used to treat municipal and industrial water systems (e.g., cooling water, white water systems in papermaking). Aqueous systems susceptible to microbial attack or degradation are also found in the leather, textile, and coating or paint industries. Examples of such microbicides and their uses, individually and in combination, are described in U.S. Pat. Nos. 5,693,631, 6,034,081, and 6,060,466, which are incorporated herein by reference. Compositions containing active agents such as those described above can be used in the same manner as the active ingredient itself is known. In particular, since such uses are not pharmaceutical uses, the polymer of the composite need not meet the toxicity profile required for pharmaceutical use.

In certain embodiments, nanoparticles comprising a polyol polymer and a BA polymer may also be included in a system suitable for delivering any of the indicated compounds, particularly drugs, using nanoparticles. In some embodiments of this system, the nanoparticles provide suitable components for preparing the nanoparticles for administration to an individual.

The systems described herein may be provided in the form of a kit of parts. For example, the polyol polymer and/or the BA polymer may be included as the sole molecule or in the presence of a suitable excipient, vehicle or diluent.

In the kit of parts, the polyol polymer, BA polymer and/or the drug to be delivered may be included in the kit independently, possibly in a composition with a suitable vehicle carrier or auxiliary agent. For example, the polyol polymer and/or BA polymer may be included in one or more compositions alone and/or in a suitable vector. Also, the drug to be delivered may be included in a composition with a suitable vehicle carrier or auxiliary agent. Alternatively, the drug may be provided by the end user and not included in the kit of parts. Additionally, the polyol polymer, BA polymer and/or drug may be included in various forms suitable for proper incorporation into the nanoparticles.

Additional components may also be included in and constitute the microfluidic chip, reference standards, buffers, and additional components that may be identified by one of skill in the art upon reading this disclosure.

In the kits of the parts disclosed herein, the components of the kit may be provided with suitable interactions and other reagents necessary to carry out the methods disclosed herein. In some embodiments, the kit may contain the compositions in separate containers. Instructions for carrying out the assay, e.g., written or audio, on paper or on an electronic support (e.g., tape or CD-ROM), may also be included in the kit. The kit may also contain other packaged reagents and materials (e.g., wash buffers, etc.), depending on the particular method used.

Further details regarding suitable carrier agents or adjuvant agents for the compositions, and generally regarding the manufacture and packaging of kits, can be identified by one of skill in the art upon reading this disclosure.

In some embodiments, nanoparticles can be prepared by preparing the individual components of the nanoparticles and then mixing the components in various orders to arrive at the desired hybrid nanoparticle structure. The preparation and mixing of the components is carried out in a suitable solution known to those skilled in the art.

The term "mixing," as used herein, refers to the addition of one solution containing a molecule of interest to another solution containing another molecule of interest. For example, in the context of the present disclosure, an aqueous solution of a polyol polymer may be mixed with an aqueous solution of a BA polymer.

The term "solution" as used herein refers to any liquid phase sample containing a molecule of interest. For example, an aqueous solution of a polyol polymer may include the polyol polymer diluted in water or any buffer solution, particularly an aqueous solution.

In some embodiments, nanoparticles may be prepared by mixing a polyol polymer with the drug to be delivered (FIGS. 1 and 2) to form a polyol polymer-drug nanoparticle. In other embodiments, nanoparticles may be prepared by further mixing a BA polymer with the polyol polymer-drug nanoparticle. In other embodiments, nanoparticles may be prepared by mixing a polyol polymer with a BA polymer and then mixing the drug to be delivered. In yet other embodiments, nanoparticles may be prepared by simultaneously mixing a polyol polymer with a BA polymer and the drug to be delivered.

In some embodiments, nanoparticles are prepared by preparing nanoparticles comprised of polyol polymer-drug conjugates by forming a polyol polymer-drug conjugate according to various embodiments of the present disclosure. In other embodiments, nanoparticles comprised of polyol polymer-drug conjugates may be prepared by dissolving the nanoparticles in a suitable aqueous solution. In still further embodiments, nanoparticles comprised of polyol polymer-drug conjugates may be prepared by mixing the polyol polymer-drug conjugates with a BA polymer that may or may not provide a targeting ligand.

In some embodiments, nanoparticles can be prepared by mixing a polyol polymer with a hydrophobic polymer block having an agent to be delivered to prepare a modified micelle according to an embodiment of the present disclosure. In other embodiments, nanoparticles can be prepared by further mixing the modified micelle with a BA polymer. In still other embodiments, nanoparticles can be prepared by mixing a polyol polymer with a BA polymer, and then mixing the agent to be delivered to prepare a nanoparticle that is a modified micelle.

In some embodiments of the present disclosure, nanoparticles can be prepared by mixing lipids conjugated to polyol polymers with BA polymers and/or with an agent to be delivered to prepare modified liposomes. In various embodiments, nanoparticles can be prepared by mixing lipids conjugated to polyol polymers with BA polymers and then with an agent to be delivered. In other embodiments, nanoparticles can be prepared by mixing lipids conjugated to polyol polymers with an agent to be delivered. In other embodiments, nanoparticles can be prepared by mixing lipids conjugated to polyol polymers with an agent to be delivered and then with BA polymers to prepare nanoparticles that are modified liposomes.

The formation of nanoparticles according to some embodiments of the present disclosure can be analyzed by techniques and procedures known to those of skill in the art. For example, in some embodiments, a gel shift method is used to monitor and measure the uptake of a nucleic acid drug into the nanoparticles (Example 10). In some embodiments, a suitable nanoparticle size and/or zeta potential can be selected using known methods (Example 11).

Further details regarding suitable carrier agents or adjuvant agents for the compositions, and generally regarding the manufacture and packaging of kits, can be identified by one of skill in the art upon reading this disclosure.

Nanoparticles having a polymer with nitrophenylboronic acid Described herein are nanoparticles having a polyol-containing polymer conjugated to a nitrophenylboronic acid-containing polymer. The polymer comprising the polyol nanoparticle portion of the described targeted nanoparticles can have one or more of any one of the following structural units:

Ａは、以下の式：

A has the following formula:

ここで、Ｒ_１およびＲ_２は、約１０ｋＤａ以下の分子量を有する任意の炭素ベースの基又は有機基のいずれかから独立して選択され；Ｘは、－Ｈ、－Ｆ、－Ｃ、－Ｎ又は－Ｏの１つ以上を含む脂肪族基から選択され；且つＹは、－ＯＨ又は－ＯＨを提示する有機部分から独立して選択され、且つＢは、第１部分ＡのＲ_１およびＲ_２の１つに連結する有機部分であり、第２部分ＡのＲ_１およびＲ_２をポリマー中に有する。幾つかの実施形態において、Ｘは、Ｃ_ｎＨ_２ｎ＋１であってもよく、ここで、ｎは、０～５の任意の１つの数であり、Ｙは、－ＯＨである。幾つかの実施形態において、Ａは、以下のいずれか１つであり得る：

where R ₁ and R ₂ are independently selected from any carbon-based or organic group having a molecular weight of about 10 kDa or less; X is selected from an aliphatic group containing one or more of -H, -F, -C, -N or -O; and Y is independently selected from -OH or an organic moiety presenting -OH, and B is an organic moiety that links to one of R ₁ and R ₂ of the first moiety A and has R ₁ and R ₂ of the second moiety A in the polymer. In some embodiments, X can be C _n H _2n+1 , where n is any one of the numbers 0 to 5, and Y is -OH. In some embodiments, A can be any one of the following:

ここで、スペーサーは、任意の有機基から独立して選択され；アミノ酸は、遊離のアミンおよび遊離のカルボン酸基を有する有機基から選択され；ｎは、１～２０の任意の単一番号であり；且つＺ_１は、－ＮＨ_２，－ＯＨ、－ＳＨおよび－ＣＯＯＨから独立して選択され；Ｒ_１およびＲ_２は、独立して以下の式を有し得：

where spacer is independently selected from any organic group; amino acid is selected from organic groups having a free amine and a free carboxylic acid group; n is any single number from 1 to 20; and Z ₁ is independently selected from -NH ₂ , -OH, -SH, and -COOH; R ₁ and R ₂ can independently have the formula:

式中、ｄは、０～１００の任意の単一番号であり、ｅは、０～１００の任意の単一番号であり、ｆは、０～１００の任意の単一番号であり、Ｚは、１つの有機部分を別の有機部分に連結する共有結合であり、且つＺ_１は、－ＮＨ_２、－ＯＨ、－ＳＨおよび－ＣＯＯＨから独立して選択され；Ｂは、以下のいずれか１つであってもよく：

wherein d is any single number from 0 to 100, e is any single number from 0 to 100, f is any single number from 0 to 100, Z is a covalent bond linking one organic moiety to another organic moiety, and Z ₁ is independently selected from -NH ₂ , -OH, -SH and -COOH; B can be any one of the following:

ここで、ｑは、１～２０の任意の単一番号であり；ｐは、２０～２００の任意の単一番号であり；且つＬは、遊離基であり、これらのＢサブユニットは、上述のＡサブユニットのいずれか１つと対合される。より詳細な実施形態において、式（Ｉ）の構造単位に示される標的化ナノ粒子のポリオールナノ粒子セグメントを含有するポリマーは、以下：

wherein q is any single number from 1 to 20; p is any single number from 20 to 200; and L is a radical, and these B subunits are paired with any one of the A subunits described above. In more particular embodiments, the polymer containing the polyol nanoparticle segment of the targeted nanoparticles shown in the structural unit of formula (I) is as follows:

より詳細な実施形態において、式（ＩＩ）の構造単位に示される標的化ナノ粒子のポリオールナノ粒子セグメントを含有するポリマーは、以下：

In a more particular embodiment, the polymer containing the polyol nanoparticle segment of the targeted nanoparticles shown in the structural unit of formula (II) is:

であり、そして
式（ＩＩＩ）の構造単位に示される標的化ナノ粒子のポリオールナノ粒子セグメントを含有するポリマーは、以下：

and the polymer containing the polyol nanoparticle segment of the targeted nanoparticles shown in the structural unit of formula (III) is:

であり、
ここで、ｎは、１～２０の任意の単一番号である。記載の標的化ナノ粒子の幾つかの実施形態において、ポリオール含有ポリマーは、以下である：

and
where n is any single number from 1 to 20. In some embodiments of the described targeted nanoparticles, the polyol-containing polymer is:

In summary, any of the formulas for sub-moiety A (Formula VI, VII or VIII) can be combined with any of the formulas for sub-moiety B (Formula XXIII or XIV) to form the polyol-containing polymer of the described nanoparticle. In certain aspects described herein, the nanoparticle can have a polyol-containing polymer formed from a combination of any one of the formulas for sub-moiety A (Formula IX, X or XI) and any one of the formulas for sub-moiety B (Formula XXIII or XIV).

In some embodiments, the nitrophenylboronic acid-containing polymer contains nitrophenylboronic acid groups and has the following general formula:

ここで、Ｒ_３およびＲ_４は、独立して親水性有機ポリマーであり、Ｘ_１は、－Ｃ、－Ｎ又は－Ｂの１つ以上を含む有機部分であり、Ｙ_１は、ｍが≧１である式－Ｃ_ｍＨ_２ｍ－のアルキル基又は芳香族基であり、ｒは、１～１０００の任意の単一番号であり、ａは、０～３の任意の単一番号であり、且つｂは、０～３の任意の単一番号であり、且つ官能基１および官能基２は、同一であるか又は異なっており、－Ｂ（ＯＨ）_２、－ＯＣＨ_３又は－ＯＨのいずれか１つから独立して選択され得る。幾つかの実施形態において、記載のニトロフェニルボロン酸含有ポリマーのこれらの可変の下位部分は、以下から選択され得る：Ｒ_３およびＲ_４は、（ＣＨ_２ＣＨ_２Ｏ）_ｔであり得、ここで、ｔは、２～２０００の任意の単一番号であり；Ｘ_１は、－ＮＨ－Ｃ（＝Ｏ）－、－Ｓ－Ｓ－、－Ｃ（＝Ｏ）－ＮＨ－、－Ｏ－Ｃ（＝Ｏ）－又は－Ｃ（＝Ｏ）－Ｏ－のいずれか１つであり、且つＹ_１は、ニトロフェニル基である。幾つかの実施形態において、記載のニトロフェニルボロン酸含有ポリマーのこれらの可変の下位部分は、以下から選択され得る：Ｒ_３およびＲ_４は、（ＣＨ_２ＣＨ_２Ｏ）_ｔであり、ここで、ｔは、２～２０００の任意の単一番号であり；Ｘ_１は、－ＮＨ－Ｃ（＝Ｏ）－、－Ｓ－Ｓ－、－Ｃ（＝Ｏ）－ＮＨ－、－Ｏ－Ｃ（＝Ｏ）－又は－Ｃ（＝Ｏ）－Ｏ－のいずれか１つであり、且つＹ_１は、ニトロフェニル基であり、且つｒは、１の値を有し得、ａは、０の値を有し得、且つｂは、１の値を有し得る。ニトロフェニルボロン酸含有ポリマーの実施形態のそれぞれにおいて、ニトロ基は、オルト位、メタ位、又はパラ位のいずれであってもよい。なおさらなる実施形態において、ニトロフェニルボロン酸含有ポリマーは、フェニル環上に存在する、メチル基などのさらなる基を有し得る。詳細な実施形態において、本明細書中で記載される標的化ナノ粒子は、以下の式のいずれか１つを有するニトロフェニルボロン酸含有ポリマーを含み得る：

wherein R ₃ and R ₄ are independently a hydrophilic organic polymer, X ₁ is an organic moiety containing one or more of -C, -N or -B, Y ₁ is an alkyl or aromatic group of the formula -C _m H _2m -, where m is ≧1, r is any single number from 1 to 1000, a is any single number from 0 to 3, and b is any single number from 0 to 3, and functional group 1 and functional group 2 are the same or different and may be independently selected from any one of -B(OH) ₂ , -OCH ₃ or -OH. In some embodiments, these variable submoieties of the described nitrophenyl boronic acid containing polymers can be selected from the following: R ₃ and R ₄ can be (CH ₂ CH ₂ O) _t , where t is any single number from 2 to 2000; X ₁ is any one of -NH-C(=O)-, -S-S-, -C(=O)-NH-, -O-C(=O)-, or -C(=O)-O-, and Y ₁ is a nitrophenyl group. In some embodiments, these variable submoieties of the described nitrophenylboronic acid containing polymers may be selected from the following: R ₃ and R ₄ are (CH ₂ CH ₂ O) _t , where t is any single number from 2 to 2000; X ₁ is any one of -NH-C(=O)-, -S-S-, -C(=O)-NH-, -O-C(=O)-, or -C(=O)-O-, and Y ₁ is a nitrophenyl group, and r may have a value of 1, a may have a value of 0, and b may have a value of 1. In each of the embodiments of the nitrophenylboronic acid containing polymer, the nitro group may be in either the ortho, meta, or para position. In still further embodiments, the nitrophenylboronic acid containing polymer may have additional groups, such as methyl groups, present on the phenyl ring. In particular embodiments, the targeted nanoparticles described herein may include a nitrophenylboronic acid containing polymer having any one of the following formulas:

ここで、ｓは、２０～３００の任意の単一番号である。式ＸＸＸＩＩＩ、ＸＸＸＩＶおよびＸＸＸＶのポリマーは、フェニル環上のＰＥＧの位置を、ボロン酸基に関連してオルト位、メタ位、又はパラ位に変えるようにさらに改変され得る。

where s is any single number from 20 to 300. The polymers of Formula XXXIII, XXXIV and XXXV can be further modified to change the position of the PEG on the phenyl ring to the ortho, meta, or para position relative to the boronic acid group.

In summary, any one of the formulas (Formula VI, VII or VIII) for sub-moiety A can be combined with any one of the formulas (Formula XXIII or XIV) for sub-moiety B to form a polyol-containing polymer nanoparticle segment of the described nanoparticles, and the resulting polyol-containing polymer can then be coupled to a nitrophenylboronic acid-containing polymer. In some embodiments, the conjugation between the described polyol-containing polymer and the described nitrophenylboronic acid-containing polymer is mediated by at least one hydroxyl group of the boronic acid group. In certain aspects described herein, the nanoparticles can have a polyol-containing polymer formed from the combination of any one of the formulas (IX, X or XI) for sub-moiety A and any one of the formulas (Formula XXIII or XIV) for sub-moiety B, which can then be coupled to a boronic acid-containing polymer having formula XXX. In some embodiments described herein, the nanoparticles can have a polyol-containing polymer formed from a combination of any one of the formulas (IX, X, or XI) for submoiety A and any one of the formulas (formula XXIII or XIV) for submoiety B, which can then be coupled to a boronic acid-containing polymer corresponding to any one of the formulas XXXIII, XXXIV, or XXXV.

The nanoparticles described herein may further include a compound. In some embodiments, the compound may be one or more therapeutic agents, such as a small molecule chemotherapeutic agent or a polynucleotide. In some embodiments, the polynucleotide may be any one or more of DNA, RNA, or interfering RNA (e.g., shRNA, siRNA, or miRNA). In some embodiments, the small molecule chemotherapeutic agent may be one or more of camptothecin, epothilone, or taxane. The nanoparticles described herein may include a combination of a polynucleotide and one or more small molecules. In this regard, any one of the polymers of sub-portion A (Formula VI, VII, or VIII) may be combined with any one of the polymers of sub-portion B (Formula XXIII or XIV) to form a polyol-containing polymeric nanoparticle segment of the described nanoparticles, and the resulting polyol-containing polymer may then be coupled with a nitrophenylboronic acid-containing polymer. The polymer of sub-portion A, the polymer of sub-portion B, or the polymer with nitrophenylboronic acid may be formed with one or more therapeutic agents, such as a small molecule chemotherapeutic agent or a polynucleotide. In some embodiments, the polymer of sub-portion A (Formula VI, VII or VIII) can be combined with any one of the polymers of sub-portion B (Formula XXIII or XIV) to form a polyol-containing polymeric nanoparticle segment of the described nanoparticles, and the resulting polyol-containing polymer can then be coupled with a nitrophenylboronic acid-containing polymer. The polymer of sub-portion A, the polymer of sub-portion B or the polymer with nitrophenylboronic acid can be formed with one or more DNA, RNA or interfering RNA (e.g., shRNA, siRNA or miRNA). In some embodiments, the polymer of sub-portion A (Formula VI, VII or VIII) can be combined with any one of the polymers of sub-portion B (Formula XXIII or XIV) to form a polyol-containing polymeric nanoparticle segment of the described nanoparticles, and the resulting polyol-containing polymer can then be coupled with a nitrophenylboronic acid-containing polymer. The polymer of sub-portion A, the polymer of sub-portion B or the polymer with nitrophenylboronic acid can be formed with one or more chemotherapeutic agents. In some embodiments, the polymer of sub-moiety A (Formula VI, VII or VIII) can be combined with any one of the polymers of sub-moiety B (Formula XXIII or XIV) to form a polyol-containing polymeric nanoparticle segment of the described nanoparticles, and the resulting polyol-containing polymer can then be coupled with a nitrophenylboronic acid-containing polymer. The polymer of sub-moiety A, the polymer of sub-moiety B or the polymer with nitrophenylboronic acid can be formed with one or more DNA, RNA or interfering RNA (e.g., shRNA, siRNA or miRNA). In some embodiments, the conjugation between the described polyol-containing polymer and the described nitrophenylboronic acid-containing polymer is mediated by at least one hydroxyl group of the nitrophenylboronic acid group. In some embodiments described herein, the targeted nanoparticles can have a polyol-containing polymer formed from a combination of any one of the formulas (IX, X, or XI) for submoiety A and any one of the formulas (formula XXIII or XIV) for submoiety B, which can then be coupled to a nitrophenylboronic acid-containing polymer corresponding to any one of the formulas XXXIII, XXXIV, or XXXV.

Targeted Nanoparticles Described herein are targeted nanoparticles having a polyol-containing polymer conjugated to any 5, 4, 3, 2, or 1 targeting ligand. The polymer comprising the polyol nanoparticle portion of the described targeted nanoparticles can have one or more of any one of the following structural units:

Ａは、以下の式：

A has the following formula:

ここで、Ｒ_１およびＲ_２は、約１０ｋＤａ以下の分子量を有する任意の炭素ベースの基又は有機基のいずれかから独立して選択され；Ｘは、－Ｈ、－Ｆ、－Ｃ、－Ｎ又は－Ｏの１つ以上を含む脂肪族基から選択され；且つＹは、－ＯＨ又は－ＯＨを提示する有機部分から独立して選択され、且つＢは、第１部分ＡのＲ_１およびＲ_２の１つに連結する有機部分であり、第２部分ＡのＲ_１およびＲ_２をポリマー中に有する。幾つかの実施形態において、Ｘは、Ｃ_ｎＨ_２ｎ＋１であってもよく、ここで、ｎは、０～５の任意の１つの数であり、Ｙは、－ＯＨである。幾つかの実施形態において、Ａは、以下のいずれか１つであり得る：

where R ₁ and R ₂ are independently selected from any carbon-based or organic group having a molecular weight of about 10 kDa or less; X is selected from an aliphatic group containing one or more of -H, -F, -C, -N or -O; and Y is independently selected from -OH or an organic moiety presenting -OH, and B is an organic moiety that links to one of R ₁ and R ₂ of the first moiety A and has R ₁ and R ₂ of the second moiety A in the polymer. In some embodiments, X can be C _n H _2n+1 , where n is any one of the numbers 0 to 5, and Y is -OH. In some embodiments, A can be any one of the following:

ここで、スペーサーは、任意の有機基から独立して選択され；アミノ酸は、遊離のアミンおよび遊離のカルボン酸基を有する有機基から選択され；ｎは、１～２０の任意の単一番号であり；且つＺ_１は、－ＮＨ_２，－ＯＨ、－ＳＨおよび－ＣＯＯＨから独立して選択され；Ｒ_１およびＲ_２は、独立して以下の式を有し得：

where spacer is independently selected from any organic group; amino acid is selected from organic groups having a free amine and a free carboxylic acid group; n is any single number from 1 to 20; and Z ₁ is independently selected from -NH ₂ , -OH, -SH, and -COOH; R ₁ and R ₂ can independently have the formula:

式中、ｄは、０～１００の任意の単一番号であり、ｅは、０～１００の任意の単一番号であり、ｆは、０～１００の任意の単一番号であり、Ｚは、１つの有機部分を別の有機部分に連結する共有結合であり、且つＺ_１は、－ＮＨ_２、－ＯＨ、－ＳＨおよび－ＣＯＯＨから独立して選択され；Ｂは、以下のいずれか１つであってもよく：

wherein d is any single number from 0 to 100, e is any single number from 0 to 100, f is any single number from 0 to 100, Z is a covalent bond linking one organic moiety to another organic moiety, and Z ₁ is independently selected from -NH ₂ , -OH, -SH and -COOH; B can be any one of the following:

ここで、ｑは、１～２０の任意の単一番号であり；ｐは、２０～２００の任意の単一番号であり；且つＬは、遊離基であり、これらのＢサブユニットは、上述のＡサブユニットのいずれか１つと対合される。より詳細な実施形態において、式（Ｉ）の構造単位に示される標的化ナノ粒子のポリオールナノ粒子セグメントを含有するポリマーは、以下：

wherein q is any single number from 1 to 20; p is any single number from 20 to 200; and L is a radical, and these B subunits are paired with any one of the A subunits described above. In more particular embodiments, the polymer containing the polyol nanoparticle segment of the targeted nanoparticles shown in the structural unit of formula (I) is as follows:

より詳細な実施形態において、式（ＩＩ）の構造単位に示される標的化ナノ粒子のポリオールナノ粒子セグメントを含有するポリマーは、以下：

In a more particular embodiment, the polymer containing the polyol nanoparticle segment of the targeted nanoparticles shown in the structural unit of formula (II) is:

式（ＩＩＩ）の構造単位に示される標的化ナノ粒子のポリオールナノ粒子セグメントを含有するポリマーは、以下：

The polymer containing the polyol nanoparticle segment of the targeted nanoparticles shown in the structural unit of formula (III) is:

であり、
ここで、ｎは、１～２０の任意の単一番号である。記載の標的化ナノ粒子の幾つかの実施形態において、ポリオール含有ポリマーは、以下である：

and
where n is any single number from 1 to 20. In some embodiments of the described targeted nanoparticles, the polyol-containing polymer is:

In summary, any of the formulas for sub-moiety A (Formula VI, VII or VIII) can be combined with any of the formulas for sub-moiety B (Formula XXIII or XIV) to form the polyol-containing polymer of the targeted nanoparticles described. In certain aspects described herein, the targeted nanoparticles can have a polyol-containing polymer formed from a combination of any one of the formulas for sub-moiety A (IX, X or XI) and any one of the formulas for sub-moiety B (Formula XXIII or XIV).

The desired targeted nanoparticles may also have a boronic acid-containing polymer reversibly covalently coupled to the polyol-containing polymer. In some embodiments, the nanoparticles are configured such that the boronic acid-containing polymer is in the environment external to the nanoparticle. In still further embodiments, the boronic acid-containing polymer is conjugated to a targeting ligand at its end opposite the nanoparticle. In some embodiments, the boronic acid-containing polymer includes at least one terminal boronic acid group and has the following general formula:

ここで、Ｒ_３およびＲ_４は、独立して親水性有機ポリマーであり、Ｘ_１は、－Ｃ、－Ｎ又は－Ｂの１つ以上を含む有機部分であり、Ｙ_１は、ｍが≧１である式－Ｃ_ｍＨ_２ｍ－のアルキル基又は芳香族基であり、ｒは、１～１０００の任意の単一番号であり、ａは、０～３の任意の単一番号であり、且つｂは、０～３の任意の単一番号であり、且つ官能基１および官能基２は、同一であるか又は異なっており、－Ｂ（ＯＨ）_２、－ＯＣＨ_３又は－ＯＨのいずれか１つから独立して選択され得る。幾つかの実施形態において、記載のボロン酸含有ポリマーのこれらの可変の下位部分は、以下から選択され得る：Ｒ_３およびＲ_４は、（ＣＨ_２ＣＨ_２Ｏ）_ｔであり、ここで、ｔは、２～２０００の任意の単一番号であり；Ｘ_１は、－ＮＨ－Ｃ（＝Ｏ）－、－Ｓ－Ｓ－、－Ｃ（＝Ｏ）－ＮＨ－、－Ｏ－Ｃ（＝Ｏ）－又は－Ｃ（＝Ｏ）－Ｏ－のいずれか１つであり、且つＹ_１は、フェニル基である。幾つかの実施形態において、記載のボロン酸含有ポリマーのこれらの可変の下位部分は、以下から選択され得る：Ｒ_３およびＲ_４は、（ＣＨ_２ＣＨ_２Ｏ）_ｔであり、ここで、ｔは、２～２０００の任意の単一番号であり；Ｘ_１は、－ＮＨ－Ｃ（＝Ｏ）－、－Ｓ－Ｓ－、－Ｃ（＝Ｏ）－ＮＨ－、－Ｏ－Ｃ（＝Ｏ）－又は－Ｃ（＝Ｏ）－Ｏ－のいずれか１つであり、且つＹ_１は、フェニル基であり、且つｒは、１の値を有し得、ａは、０の値を有し得、且つｂは、１の値を有し得る。詳細な実施形態において、本明細書中で記載される標的化ナノ粒子は、以下の式を有するボロン酸含有ポリマーを含み得る：

wherein R ₃ and R ₄ are independently a hydrophilic organic polymer, X ₁ is an organic moiety containing one or more of -C, -N or -B, Y ₁ is an alkyl or aromatic group of the formula -C _m H _2m -, where m is ≧1, r is any single number from 1 to 1000, a is any single number from 0 to 3, and b is any single number from 0 to 3, and functional group 1 and functional group 2 are the same or different and may be independently selected from any one of -B(OH) ₂ , -OCH ₃ or -OH. In some embodiments, these variable submoieties of the described boronic acid containing polymers can be selected from the following: R ₃ and R ₄ are (CH ₂ CH ₂ O) _t , where t is any single number from 2 to 2000; X ₁ is any one of -NH-C(=O)-, -S-S-, -C(=O)-NH-, -O-C(=O)-, or -C(=O)-O-, and Y ₁ is a phenyl group. In some embodiments, these variable submoieties of the described boronic acid containing polymers may be selected from the following: R ₃ and R ₄ are (CH ₂ CH ₂ O) _t , where t is any single number from 2 to 2000; X ₁ is any one of -NH-C(═O)-, -S-S-, -C(═O)-NH-, -O-C(═O)-, or -C(═O)-O-, and Y ₁ is a phenyl group, and r may have a value of 1, a may have a value of 0, and b may have a value of 1. In particular embodiments, the targeted nanoparticles described herein may include a boronic acid containing polymer having the following formula:

ここで、ｓは、２０～３００の任意の単一番号である。

Here, s is any single number between 20 and 300.

In some embodiments, the boronic acid-containing polymer comprises nitrophenylboronic acid. In some embodiments, the nitrophenylboronic acid-containing polymer comprises nitrophenylboronic acid groups and has the following general formula:

ここで、Ｒ_３およびＲ_４は、独立して親水性有機ポリマーであり、Ｘ_１は、－Ｃ、－Ｎ又は－Ｂの１つ以上を含む有機部分であり、Ｙ_１は、ｍが≧１である式－Ｃ_ｍＨ_２ｍ－のアルキル基又は芳香族基であり、ｒは、１～１０００の任意の単一番号であり、ａは、０～３の任意の単一番号であり、且つｂは、０～３の任意の単一番号であり、且つ官能基１および官能基２は、同一であるか又は異なっており、－Ｂ（ＯＨ）_２、－ＯＣＨ_３又は－ＯＨのいずれか１つから独立して選択され得る。幾つかの実施形態において、記載のボロン酸含有ポリマーのこれらの可変の下位部分は、以下から選択され得る：Ｒ_３およびＲ_４は、（ＣＨ_２ＣＨ_２Ｏ）_ｔであり得、ここで、ｔは、２～２０００の任意の単一番号であり；Ｘ_１は、－ＮＨ－Ｃ（＝Ｏ）－、－Ｓ－Ｓ－、－Ｃ（＝Ｏ）－ＮＨ－、－Ｏ－Ｃ（＝Ｏ）－又は－Ｃ（＝Ｏ）－Ｏ－のいずれか１つであり、且つＹ_１は、ニトロフェニル基である。幾つかの実施形態において、記載のニトロフェニルボロン酸含有ポリマーのこれらの可変の下位部分は、以下から選択され得る：Ｒ_３およびＲ_４は、（ＣＨ_２ＣＨ_２Ｏ）_ｔであり、ここで、ｔは、２～２０００の任意の単一番号であり；Ｘ_１は、－ＮＨ－Ｃ（＝Ｏ）－、－Ｓ－Ｓ－、－Ｃ（＝Ｏ）－ＮＨ－、－Ｏ－Ｃ（＝Ｏ）－又は－Ｃ（＝Ｏ）－Ｏ－のいずれか１つであり、且つＹ_１は、ニトロフェニル基であり、且つｒは、１の値を有し得、ａは、０の値を有し得、且つｂは、１の値を有し得る。ニトロフェニルボロン酸含有ポリマーの実施形態のそれぞれにおいて、ニトロ基は、オルト位、メタ位、又はパラ位のいずれであってもよい。なおさらなる実施形態において、ニトロフェニルボロン酸含有ポリマーは、フェニル環上に存在する、メチル基などのさらなる基を有し得る。詳細な実施形態において、本明細書中で記載される標的化ナノ粒子は、以下の式のいずれか１つを有するボロン酸含有ポリマーを含み得る：

wherein R ₃ and R ₄ are independently a hydrophilic organic polymer, X ₁ is an organic moiety containing one or more of -C, -N or -B, Y ₁ is an alkyl or aromatic group of the formula -C _m H _2m -, where m is ≧1, r is any single number from 1 to 1000, a is any single number from 0 to 3, and b is any single number from 0 to 3, and functional group 1 and functional group 2 are the same or different and may be independently selected from any one of -B(OH) ₂ , -OCH ₃ or -OH. In some embodiments, these variable submoieties of the described boronic acid containing polymers can be selected from the following: R ₃ and R ₄ can be (CH ₂ CH ₂ O) _t , where t is any single number from 2 to 2000; X ₁ is any one of -NH-C(=O)-, -S-S-, -C(=O)-NH-, -O-C(=O)- or -C(=O)-O-, and Y ₁ is a nitrophenyl group. In some embodiments, these variable submoieties of the described nitrophenylboronic acid containing polymers may be selected from the following: R ₃ and R ₄ are (CH ₂ CH ₂ O) _t , where t is any single number from 2 to 2000; X ₁ is any one of -NH-C(=O)-, -S-S-, -C(=O)-NH-, -O-C(=O)-, or -C(=O)-O-, and Y ₁ is a nitrophenyl group, and r may have a value of 1, a may have a value of 0, and b may have a value of 1. In each of the embodiments of the nitrophenylboronic acid containing polymer, the nitro group may be in either the ortho, meta, or para position. In still further embodiments, the nitrophenylboronic acid containing polymer may have additional groups, such as methyl groups, present on the phenyl ring. In particular embodiments, the targeted nanoparticles described herein may include boronic acid containing polymers having any one of the following formulas:

ここで、ｓは、２０～３００の任意の単一番号である。式ＸＸＸＩＩＩ、ＸＸＸＩＶおよびＸＸＸＶのポリマーは、フェニル環上のＰＥＧの位置を、ボロン酸基に関連してオルト位、メタ位、又はパラ位に変えるようにさらに改変され得る。

where s is any single number from 20 to 300. The polymers of Formula XXXIII, XXXIV and XXXV can be further modified to change the position of the PEG on the phenyl ring to the ortho, meta, or para position relative to the boronic acid group.

In summary, any one of the formulas (Formula VI, VII or VIII) for sub-moiety A can be combined with any one of the formulas (Formula XXIII or XIV) for sub-moiety B to form a polymer containing a polyol nanoparticle segment of the targeted nanoparticle described, and the resulting polyol-containing polymer can then be coupled with a boronic acid-containing polymer, where the boronic acid-containing polymer is either phenylboronic acid or nitrophenylboronic acid. In some embodiments, the conjugation between the described polyol-containing polymer and the described boronic acid-containing polymer is mediated by at least one hydroxyl group of the boronic acid group. In certain aspects described herein, the targeted nanoparticle can have a polyol-containing polymer formed from the combination of any one of the formulas (IX, X or XI) for sub-moiety A and any one of the formulas (Formula XXIII or XIV) for sub-moiety B, which can then be coupled with a boronic acid-containing polymer having formula XXX. In some embodiments described herein, the targeted nanoparticles can have a polyol-containing polymer formed from a combination of any one of the formulas (IX, X, or XI) for submoiety A and any one of the formulas (formula XXIII or XIV) for submoiety B, which can then be coupled to a boronic acid-containing polymer corresponding to any one of the formulas XXXI, XXXIII, XXXIV, or XXXV.

In some aspects described herein, nanoparticles formed from either the polyol-containing polymeric nanoparticle segments described herein or combinations of polyol-containing polymeric nanoparticle segments and boronic acid-containing polymers are conjugated with a targeting ligand to form targeted nanoparticles with a targeting ligand to nanoparticle ratio of 3:1. In some aspects described herein, nanoparticles formed from either the polyol-containing polymeric nanoparticle segments described herein or combinations of polyol-containing polymeric nanoparticle segments and boronic acid-containing polymers are conjugated with a targeting ligand to form targeted nanoparticles with a targeting ligand to nanoparticle ratio of 1:1. In some aspects described herein, nanoparticles formed from either the polyol-containing polymeric nanoparticle segments described herein or combinations of polyol-containing polymeric nanoparticle segments and boronic acid-containing polymers are conjugated with one single targeting ligand to form targeted nanoparticles. In some aspects, the targeting ligand described is conjugated to the boronic acid-containing polymer at the end opposite the boronic acid. The targeting ligand conjugated to the described targeted nanoparticles may be any one of a protein, a protein fragment, an amino acid peptide, or an aptamer derived from either amino acids or polynucleotides, or any other high affinity molecule known to bind to the target of interest. In some embodiments, the targeting ligand, which is a protein or protein fragment, may be any one of an antibody, a cellular receptor, a ligand for a cellular receptor (e.g., transferrin), or a protein or chimeric protein having a portion thereof. If the targeting ligand is an antibody, the antibody may be a human, mouse, rabbit, non-human primate, dog, or rodent antibody, or a chimera consisting of any two of these antibodies. Furthermore, the antibody may be humanized such that only the CDR segments or a small portion of the variable region containing the CDR segments are non-human antibodies, while the remainder of the antibody is a human antibody. The antibodies described herein may be of any isotype, such as IgG, IgM, IgA, IgD, IgE, IgY, or another type of isotype known to be produced by mammals. In some embodiments, the targeting ligand may comprise only an antibody, a cellular receptor, or an amino acid peptide derived from a ligand for a cellular receptor that is responsible for binding to its target.

In some aspects, a nanoparticle formed from any one of the formulas (VI, VII or VIII) for sub-moiety A is combined with any one of the formulas (Formula XXIII or XIV) for sub-moiety B to form the polyol-containing polymer segment of the targeted nanoparticle described, further coupled to any of 5, 4, 3, 2 or 1 boronic acid, e.g., phenylboronic acid or nitrophenylboronic acid-containing polymers conjugated with a targeting ligand.

In some aspects, a nanoparticle formed from any one of the formulas (VI, VII or VIII) for sub-moiety A is combined with any one of the formulas (Formula XXIII or XIV) for sub-moiety B to form the polyol-containing polymer segment of the targeted nanoparticle described, further coupled to a single boronic acid, e.g., phenylboronic acid or nitrophenylboronic acid-containing polymer, conjugated with a targeting ligand.

In certain aspects described herein, the targeted nanoparticles may have a polyol-containing polymer formed from a combination of any one of the formulas (IX, X, or XI) for submoiety A and any one of the formulas (formula XXIII or XIV) for submoiety B, which may be coupled to a boronic acid-containing polymer of formula XXX coupled to a targeting ligand at the opposite end of the boronic acid, either 5, 4, 3, 2, or 1.

In certain aspects described herein, the targeted nanoparticles have a polyol-containing polymer formed from a combination of any one of the formulas (IX, X, or XI) for submoiety A and any one of the formulas (formula XXIII or XIV) for submoiety B, which can be coupled to a boronic acid-containing polymer of formula XXX coupled to a targeting ligand at the opposite end of the boronic acid.

In some aspects, a nanoparticle formed from any one of the formulas (VI, VII or VIII) for sub-moiety A is combined with any one of the formulas (Formula XXIII or XIV) for sub-moiety B to form the polyol-containing polymer segment of the targeted nanoparticle described, further coupled to 5, 4, 3, 2 or 1 boronic acid-containing polymer, e.g., phenylboronic acid or nitrophenylboronic acid, conjugated to a targeting ligand. Here, the resulting targeted nanoparticle is conjugated to only a single targeting ligand.

In some aspects, a nanoparticle formed from any one of formulas (VI, VII or VIII) for submoiety A is combined with any one of formulas (Formula XXIII or XIV) for submoiety B to form the polyol-containing polymer segment of the targeted nanoparticle described, and further coupled to a single boronic acid-containing polymer, e.g., phenylboronic acid or nitrophenylboronic acid, conjugated to a targeting ligand. Here, the resulting targeted nanoparticle is conjugated to only a single targeting ligand.

In certain aspects described herein, the targeted nanoparticles may have a polyol-containing polymer formed from a combination of any one of the formulas (IX, X, or XI) for submoiety A and any one of the formulas (formula XXIII or XIV) for submoiety B, which may be coupled to a boronic acid-containing polymer having any of 5, 4, 3, 2, or 1 of formula XXX, coupled to a targeting ligand at the opposite end of the boronic acid. Here, the resulting targeted nanoparticles are conjugated with only a single targeting ligand.

In certain aspects described herein, the targeted nanoparticles may have a polyol-containing polymer formed from a combination of any one of the formulas (IX, X, or XI) for submoiety A and any one of the formulas (formula XXIII or XIV) for submoiety B, which may be coupled to a boronic acid-containing polymer having formula XXX coupled to a targeting ligand at the opposite end of the boronic acid. Here, the resulting targeted nanoparticles are conjugated to only a single targeting ligand.

In some embodiments, the targeted nanoparticles described herein are conjugated to any targeting ligand. In some embodiments, the targeted nanoparticles described herein are conjugated to a single targeting ligand. In some embodiments described herein, the targeted nanoparticles may have a polyol-containing polymer formed from a combination of any one of formulas (IX, X, or XI) for submoiety A and any one of formulas (formula XXIII or XIV) for submoiety B, which may then be coupled to any one of 5, 4, 3, 2, or 1 boronic acid-containing polymers corresponding to any one of formulas XXXI, XXXIII, XXXIV, or XXXV. This is further conjugated to a targeting ligand selected from one or more of a protein, a protein fragment, an amino acid peptide, or an aptamer.

In some embodiments, the targeted nanoparticles described herein are conjugated to a single targeting ligand. In some embodiments described herein, the targeted nanoparticles may have a polyol-containing polymer formed from a combination of any one of the formulas (IX, X, or XI) for submoiety A and any one of the formulas (formula XXIII or XIV) for submoiety B, which may then be coupled to a boronic acid-containing polymer corresponding to any one of formulas XXXI, XXXIII, XXXIV, or XXXV. This is further conjugated to a single targeting ligand selected from a protein, a protein fragment, an amino acid peptide, or an aptamer.

In some embodiments, the targeted nanoparticles described herein are conjugated to a single targeting ligand. In some embodiments described herein, the targeted nanoparticles may have a polyol-containing polymer formed from a combination of any one of the formulas (IX, X, or XI) for submoiety A and any one of the formulas (formula XXIII or XIV) for submoiety B, which may then be coupled to any 5, 4, 3, 2, or 1 boronic acid-containing polymers corresponding to any one of formulas XXXI, XXXIII, XXXIV, or XXXV. This may be further conjugated to an antibody, a cellular receptor, a ligand for a cellular receptor, or a chimeric protein having a protein or portion thereof.

In some embodiments described herein, the targeted nanoparticles may have a polyol-containing polymer formed from a combination of any one of the formulas (IX, X, or XI) for submoiety A and any one of the formulas (formula XXIII or XIV) for submoiety B, which may then be coupled to a boronic acid-containing polymer corresponding to any one of the formulas XXXI, XXXIII, XXXIV, or XXXV. This may be further conjugated to a single antibody, a cellular receptor, a ligand for a cellular receptor, or a chimeric protein having a protein or portion thereof.

In some embodiments, the targeted nanoparticles described herein are conjugated to a single targeting ligand. In some embodiments described herein, the targeted nanoparticles may have a polyol-containing polymer formed from a combination of any one of the formulas (IX, X, or XI) for submoiety A and any one of the formulas (formula XXIII or XIV) for submoiety B, which may then be coupled to a boronic acid-containing polymer corresponding to any one of formulas XXXI, XXXIII, XXXIV, or XXXV. This is further conjugated to a single targeting ligand selected from a protein, a protein fragment, an amino acid peptide, or an aptamer, where the resulting targeted nanoparticle is conjugated to only a single targeting ligand.

In some embodiments, the targeted nanoparticles described herein are conjugated to a single targeting ligand. In some embodiments described herein, the targeted nanoparticles may have a polyol-containing polymer formed from a combination of any one of the formulas (IX, X, or XI) for submoiety A and any one of the formulas (formula XXIII or XIV) for submoiety B, which may then be coupled to any 5, 4, 3, 2, or 1 boronic acid-containing polymers corresponding to any one of formulas XXXI, XXXIII, XXXIV, or XXXV. This may be further conjugated to an antibody, a cellular receptor, a ligand of a cellular receptor, or a chimeric protein having a protein or a portion thereof. Here, the resulting targeted nanoparticle is conjugated to only a single targeting ligand.

In some embodiments described herein, the targeted nanoparticles may have a polyol-containing polymer formed from a combination of any one of the formulas (IX, X, or XI) for submoiety A and any one of the formulas (formula XXIII or XIV) for submoiety B, which may then be coupled to a boronic acid-containing polymer corresponding to any one of the formulas XXXI, XXXIII, XXXIV, or XXXV. This may be further conjugated to a single antibody, cellular receptor, ligand of a cellular receptor, or chimeric protein having a protein or portion thereof, where the resulting targeted nanoparticle is conjugated to only a single targeting ligand.

The targeted nanoparticles described herein may further comprise a compound. In some embodiments, the compound may be one or more therapeutic agents, such as a small molecule chemotherapeutic agent or a polynucleotide. In some embodiments, the polynucleotide may be one or more of DNA, RNA, or an interfering RNA (e.g., shRNA, siRNA, or miRNA). In some embodiments, the small molecule chemotherapeutic agent may be one or more of a camptothecin, an epothilone, or a taxane. The targeted nanoparticles described herein may comprise a combination of a polynucleotide and one or more small molecule chemotherapeutic agents.

Having discussed various types of nanoparticles and targeted nanoparticles that can be produced using the components described herein, the following detailed embodiments can be produced. In one embodiment, the targeted nanoparticle described comprises a mucic acid-containing polymer, a therapeutic agent selected from a camptothecin, an epothilone, a taxane, or an interfering RNA sequence, and a phenylboronic acid-containing polymer having the formula XXXI, XXXIII, XXXIV, or XXXV coupled to the mucic acid polymer by a reversible covalent bond. The targeted nanoparticle is configured to present the phenylboronic acid-containing polymer to the external environment of the nanoparticle, where the phenylboronic acid-containing polymer is conjugated to a targeting ligand at the end opposite the nanoparticle, where the targeted nanoparticle comprises one single targeting ligand.

In one embodiment, the targeted nanoparticle described comprises a mucic acid-containing polymer, a therapeutic agent selected from a camptothecin, an epothilone, a taxane, or an interfering RNA sequence, and a phenylboronic acid-containing polymer having formula XXXI, XXXIII, XXXIV, or XXXV coupled to the mucic acid polymer by a reversible covalent bond. The targeted nanoparticle is configured to present the phenylboronic acid-containing polymer to the external environment of the nanoparticle, where the phenylboronic acid-containing polymer is conjugated to a targeting ligand at an end opposite the nanoparticle, where the targeted nanoparticle comprises one single antibody.

In one embodiment, the targeted nanoparticle described comprises a mucic acid-containing polymer, a therapeutic agent selected from a camptothecin, an epothilone, a taxane, or an interfering RNA sequence, and a phenylboronic acid-containing polymer having the formula XXXI, XXXIII, XXXIV, or XXXV coupled to the mucic acid polymer by a reversible covalent bond. The targeted nanoparticle is configured to present the phenylboronic acid-containing polymer to the external environment of the nanoparticle, where the phenylboronic acid-containing polymer is conjugated at the end opposite the nanoparticle to any one of a human antibody, a mouse antibody, a rabbit antibody, a non-human primate antibody, a dog antibody, or a rodent antibody, or a chimeric antibody consisting of any two of these antibodies, where the antibody is any one of the isotypes IgG, IgD, IgM, IgE, IgA, or IgY, where the targeted nanoparticle comprises one single antibody.

In one embodiment, the targeted nanoparticle described comprises a mucic acid-containing polymer, a therapeutic agent selected from a camptothecin, an epothilone, a taxane, or an interfering RNA sequence, and a phenylboronic acid-containing polymer having formula XXXI, XXXIII, XXXIV, or XXXV coupled to the mucic acid polymer by a reversible covalent bond. The targeted nanoparticle is configured to present the phenylboronic acid-containing polymer to the external environment of the nanoparticle, where the phenylboronic acid-containing polymer is conjugated to a cellular receptor at an end opposite the nanoparticle, where the targeted nanoparticle comprises one single cellular receptor.

In one embodiment, the targeted nanoparticle described comprises a mucic acid-containing polymer, a therapeutic agent selected from a camptothecin, an epothilone, a taxane, or an interfering RNA sequence, and a phenylboronic acid-containing polymer having formula XXXI, XXXIII, XXXIV, or XXXV coupled to the mucic acid polymer by a reversible covalent bond. The targeted nanoparticle is configured to present the phenylboronic acid-containing polymer to the external environment of the nanoparticle, where the phenylboronic acid-containing polymer is conjugated to a receptor ligand at the end opposite the nanoparticle, where the targeted nanoparticle comprises one single receptor ligand.

The method systems described herein are further described in the following examples, which are provided for purposes of illustration and are not intended to be limiting. Those skilled in the art will appreciate the applicability of the features described in detail to methods of nucleic acid detection and detection of other targets, such as proteins, antigens, eukaryotic or prokaryotic cells, etc.

All chemical reagents were obtained from commercial suppliers and used as received without further purification. Polymer samples were analyzed on a Viscotek GPC System equipped with a TDA 302 triple detector array consisting of a refractive index (RI) detector, a differential viscometer, and a low angle light scattering detector. A 7.5% acetic acid solution was used as the eluent at a flow rate of 1 mL/min.

pGL3, a plasmid containing the firefly luciferase gene, was extracted and purified from bacteria expressing pGL3. siGL3 was purchased from Integrated DNA Technologies (sequence provided below). siCON1 (sequence provided below) was purchased from Dharmacon. HeLa cells were used to determine the efficacy of pDNA or siRNA delivery by cationic mucic acid diamine-DMS polymer.

Example 1: Synthesis of mucic acid dimethyl ester (1)
5 g (22.8 mmol) of mucic acid (Aldrich) was added to a 500 mL round bottom flask containing 120 mL of methanol and 0.4 mL of concentrated sulfuric acid. The mixture was refluxed at 85° C. overnight under constant stirring. The mixture was then filtered, washed with methanol, and then recrystallized from a mixture of 80 mL of methanol and 0.5 mL of triethylamine. After drying overnight under vacuum, 8.0 g (33.6 mmol, 71%) of mucic acid dimethyl ester was obtained. ¹ H NMR ((CD ₃ ) ₂ SO) δ 4.88-4.91 (d, 2H), 4.78-4.81 (m, 2H), 4.28-4.31 (d, 2H), 3.77-3.78 (d, 2H), 3.63 (s, 6H). ESI/MS (m/z): 261.0 [M+Na] ⁺

Example 2: N-BOC-protected mucic acid diamine, (2)
A mixture of 8 g (33.6 mmol) of mucic acid dimethyl ester (1; Example 1), 12.4 mL (88.6 mmol) of triethylamine and 160 mL of methanol was heated under reflux at 85° C. in a constantly stirred 500 mL round bottom flask for 0.5 h, after which 14.2 g (88.6 mmol) of N-BOC diamine (Fluka) dissolved in methanol (32 mL) was added. The reaction suspension was then returned to reflux. After refluxing overnight, the mixture was filtered, washed with methanol, recrystallized from methanol and dried under vacuum to give 9.4 g (19 mmol, 57%) of N-BOC-protected mucic acid diamine. ¹ H NMR ((CD ₃ ) ₂ SO) δ 7.66 (m, 2H), 6.79 (m, 2H), 5.13-5.15 (d, 2H), 4.35-4.38 (d, 2H), 4.08-4.11 (m, 2H), 3.78-3.80 (d, 2H), 2.95-3.15 (m, 8 H), 1.38 (s, 18). ESI/MS (m/z): 517.1 [M+Na] ⁺

Example 3: Synthesis of mucic acid diamine (3)
8 g (16.2 mmol) of N-BOC-protected mucic acid diamine (2; Example 2) was transferred to a 500 mL round bottom flask containing 3 M HCl in methanol (160 mL) and refluxed overnight at 85° C. with constant stirring. The precipitate was then filtered, washed with methanol and dried under vacuum overnight to give 5.7 g (15.6 mmol, 96%) of mucic acid diamine. ¹ H NMR ((CD ₃ ) ₂ SO) δ 7.97 (m, 8H), 5.35-5.38 (m, 2H), 4.18-4.20 (m, 2H), 3.82 (m, 2H), 3.35-3.42 (m, 8H), 2.82-2.90 (m, 4H). ESI/MS (m/z): 294.3 [M] ⁺ , 317.1 [M+Na] ⁺ , 333.0 [M+K] ⁺

Example 4: Mucate diamine-DMS copolymer (MAP), (4)
A 1.5 mL Eppendorf tube was charged with 85.5 mg (0.233 mmol) of the bis(hydrochloride) salt of Example 3 in 0.8 mL of 0.1 M NaHCO _3. Dimethylsuberimidate.2HCl (DMS, Pierce Chemical Co., 63.6 mg, 0.233 mmol) was added, and the solution was vortexed and centrifuged to dissolve the components. The resulting mixture was stirred at room temperature for 15 hours. The mixture was then diluted with 8 mL of water, and the pH was brought to 4 by the addition of 1 N HCl. The solution was then dialyzed in ddH ₂ O for 24 hours using a 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing). The dialysis solution was lyophilized to dryness, yielding 49 mg of a white fluffy powder. ^1H NMR (500MHz, dDMSO) δ 9.15 (bs), 7.92 (bs), 5.43 (bs), 4.58 (bs), 4.17 (bs), 3.82 (bs), 3.37 (bs), 3.28 (bs), 2.82 (bs), 2.41 (bs), 1.61 (bs) ), 1.28 (bs). ^13C NMR (126MHz, dDMSO) δ 174.88 (s, 1H), 168.38 (s, 1H), 71.45 (s, 4H), 71.22 (s, 3H), 42.34 (s, 2H), 36.96 (s, 3H), 32.74 (s, 3H), 28.09 (s, 4H) ), 26.90 (s, 4H). Mw[GPC]=2520, Mw/Mn=1.15.

Polymer 4 is an example of a cationic A-B type (the repeating structure is ABABAB...) polyol-containing polymer.

Example 5: Boronic acid-amide-PEG ₅₀₀₀ , (5)
The polymer of Example 4 is assembled with a nucleic acid, e.g., siRNA, to form nanoparticles. These nanoparticles need to be sterically stable for use in mammals, and optionally they could have a targeting agent included. To perform these two functions, the nanoparticles may be decorated with PEG for steric stability and PEG-targeting ligands. To do this, a PEG compound containing a boronic acid is prepared. For example, a boronic acid-containing PEG may be synthesized according to the following example.

332 mg of 4-carboxyphenylboronic acid (2 mmol) was dissolved in 8 mL of SOCl _2. To this, a few drops of DMF were added and the mixture was refluxed under argon for 2 hours. Excess SOCl ₂ was removed under reduced pressure and the resulting solid was dissolved in 10 mL of anhydrous dichloromethane. To this solution, 500 mg of PEG ₅₀₀₀ -NH ₂ (2 mmol) and 418 μL of triethylamine (60 mmol) dissolved in 5 mL of dichloromethane were dissolved at 0° C. under argon. The mixture was allowed to warm to room temperature and stirring was continued overnight. The solvent in dichloromethane was removed under reduced pressure and the resulting liquid was precipitated with 20 mL of diethyl ether. The precipitate was filtered, dried and redissolved in ddH ₂ O. The aqueous solution was then filtered through a 0.45 μm filter and dialyzed with a 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing) into ddH ₂ O for 24 hours. The dialyzed solution was lyophilized to dryness. ¹ H NMR (300 MHz, dDMSO) δ 7.92-7.77 (m), 4.44 (d), 4.37 (t), 3.49 (m), 2.97 (s).

Example 6: Boronic acid-disulfide-PEG ₅₀₀₀ , (6)
Cleavable (under reducing conditions) versions of the PEG compound of Example 5 can also be synthesized as follows.

250 mg of PEG ₅₀₀₀ -SH (0.05 mmol, LaySanBio Inc.) was added to a glass vial equipped with a stirrer. To this was added 110 mg of aldrithiol-2 (0.5 mmol, Aldrich) dissolved in 4 mL of methanol. This solution was stirred at room temperature for 2 hours after which 77 mg of mercaptophenylboronic acid (0.5 mmol, Aldrich) in 1 mL of methanol was added. The resulting solution was stirred for an additional 2 hours at room temperature. Methanol was removed under vacuum and the residue was dissolved in 2 mL of dichloromethane. 18 mL of diethyl ether was added to the dichloromethane solution and the mixture was allowed to stand for 1 hour. The resulting precipitate was collected via centrifugation, washed several times with diethyl ether, and dried. The dried solid was redissolved in water, filtered through a 0.45 μm filter, and dialyzed with a 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing) in ddH ₂ O for 15 hours. The dialyzed solution was lyophilized to dryness. ¹ H NMR (300 MHz, dDMSO) δ 8.12-8.00 (m), 7.83-7.72 (m), 7.72-7.61 (m), 7.61-7.43 (m), 3.72 (d, J=5.4), 3.68-3.15 (m), 3.01-2.83 (m).

Example 7: Synthesis of (2,3,5,6)-tetrafluorophenylboronic acid-PEG ₅₀₀₀ , (7)
Fluorinated versions of the boronic acid-containing PEG compounds of Example 5 can be synthesized and used as imaging agents with therapeutic nanoparticles. Fluorine atoms for imaging can be incorporated as described and illustrated below.

(2,3,5,6)-Fluorocarboxyphenylboronic acid was dissolved in excess SOCl ₂ (approximately 100 eq.) to which a few drops of DMF were added. The mixture was refluxed under argon for 2 hours. Excess SOCl ₂ was removed under reduced pressure and the resulting residue was dissolved in anhydrous dichloromethane. To this solution was added PEG ₅₀₀₀ -NH ₂ (1 eq.) and triethylamine (30 eq.) dissolved in dichloromethane at 0° C. under argon. The resulting mixture was allowed to warm to room temperature and stirring was continued overnight. The dichloromethane solvent was removed under reduced pressure and the resulting liquid was precipitated with diethyl ether. The precipitate was filtered, dried and redissolved in ddH ₂ O. The aqueous solution was then filtered through a 0.45 μm filter and dialyzed with a 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing) for 24 hours into ddH ₂ O. The dialyzed solution was lyophilized to dryness.

Fluorine-containing compounds are useful for providing ^{19 F} in nanoparticles, which can be detected by magnetic resonance spectroscopy using standard patient MRI. The addition of ¹⁹ F allows for imaging of the nanoparticles (which may be done for imaging alone, or may allow imaging and therapy with the addition of a therapeutic agent).

Example 8: Synthesis of (2,3,5,6)-tetrafluorophenylboronic acid-disulfide-PEG ₅₀₀₀ , (8)
Fluorinated versions of the boronic acid-containing cleavable PEG compounds of Example 5 can be synthesized and used as imaging agents with therapeutic nanoparticles. Fluorine atoms for imaging can be incorporated as described and illustrated below.

250 mg of PEG ₅₀₀₀ -SH (0.05 mmol, LaySanBio Inc.) is added to a glass vial equipped with a stirrer. To this, 110 mg of aldrithiol-2 (0.5 mmol, Aldrich) dissolved in 4 mL of methanol is added. This solution is stirred at room temperature for 2 hours after adding 77 mg of (2,3,5,6)-fluoro-4-mercaptophenylboronic acid (0.5 mmol) in 1 mL of methanol. The resulting solution is stirred for another 2 hours at room temperature. Methanol is removed under vacuum and the residue is dissolved in 2 mL of dichloromethane. 18 mL of diethyl ether is added to the dichloromethane solution and the mixture is allowed to stand for 1 hour. The resulting precipitate is collected via centrifugation, washed several times with diethyl ether, and dried. The dried solid is redissolved in water, filtered through a 0.45 μm filter, and dialyzed with a 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing) for 15 hours in ddH ₂ O. The dialyzed solution is lyophilized to dryness.

Example 9: Synthesis of Boronic Acid-PEG ₅₀₀₀ -Transferrin (9)
A targeting agent may be placed at the other end of the boronic acid derived PEG in the compounds of Examples 5-8, for example, following the approach illustrated in FIG. 12, which references transferrin attachment.

Thus, a component of the system may include a nucleic acid as a therapeutic agent (the targeting ligand may be a protein such as transferrin (FIG. 12), an antibody or antibody fragment, a peptide such as RGD or LHRH, a small molecule such as folate or galactose, etc.). A boronic acid PEGylated targeting agent may be synthesized as follows:

Specifically, the following procedure was carried out to synthesize boronate PEG ₅₀₀₀ -transferrin according to the approach illustrated in Figure 12. A solution of 10 mg (0.13 μmol) of human holo-transferrin (iron-rich) (Sigma Aldrich) in 1 mL of 0.1 M PBS buffer (pH 7.2) was added to 3.2 mg of OPSS-PEG ₅₀₀₀ -SVA (5 equivalents, 0.64 μmol, LaysanBio Inc.). The resulting solution was stirred at room temperature for 2 hours. PEGylated transferrin was purified from unreacted OPSS-PEG ₅₀₀₀ -SVA using Ultracel 50,000 MWCO (Amicon Ultra-4, Millipore) and from unreacted transferrin using a gel filtration column G3000SWxl (Tosoh Biosep) (confirmed by HPLC and MALDI-TOF analysis). 100 μg of OPSS-PEG ₅₀₀₀ PEGylated transferrin in 100 μL was then incubated with 20 μL of 4-mercaptophenylboronic acid (1 μg/μL, 20 μg, 100 equivalents) at room temperature for 1 hour. After incubation, the solution was dialyzed twice with a YM-30,000NMWI device (Millipore) to remove excess 4-mercaptophenylboronic acid and the pyridyl-2-thione by-product.

Example 10: Formulation of MAP-Nucleic Acid Particles - Gel Shift Assay As illustrated in Figure 1, 1 μg of plasmid DNA or siRNA (0.1 μg/μL, 10 μL) in DNase- and RNase-free water was mixed with 10 μL of MAP at various concentrations in DNase- and RNase-free water to obtain charge ratios ("+" charge on the polymer vs. "-" charge on the nucleic acid) of 0.5, 1, 1.5, 2, 2.5 and 5. The resulting mixtures were incubated at room temperature for 30 minutes. As shown in Figures 3 and 4, 10 μL of the 20 μL solution was loaded onto a 1% agarose gel with 3.5 μL of loading buffer and the gel was electrophoresed at 80 V for 45 minutes. Nucleic acid not contained in the nanoparticles migrates on the gel. These results provide an indication of the essential charge ratio for nucleic acid inclusion within the nanoparticles.

Example 11: Particle size and zeta potential of MAP-nucleic acid particles 1 μg of plasmid DNA (0.1 μg/μL, 10 μL) in DNase- and RNase-free water was mixed with 10 μL of MAP at various concentrations in DNase- and RNase-free water to obtain charge ratios of 0.5, 1, 1.5, 2, 2.5, and 5. The resulting mixture was incubated at room temperature for 30 minutes. 20 μL of the mixture was then diluted to 70 μL with DNase- and RNase-free water for particle size measurements. The 70 μL solution was then diluted to 1400 μL with 1 mM KCl for zeta potential measurements. Particle size and zeta potential measurements were performed using a ZetaPals dynamic light scattering (DLS) instrument (Brookhaven Instruments). The results are shown in FIG. 5.

Example 12: Particle size stabilization by PEGylation with boronate PEG _5k As illustrated in Figure 2, 2 μg of plasmid DNA in DNase- and RNase-free water (0.45 μg/μL, 4.4 μL) was diluted to 80 μL in DNase- and RNase-free water. This plasmid solution was mixed with 4.89 μg of MAP (0.5 μg/μL, 9.8 μL) and also diluted to 80 μL in DNase- and RNase-free water to obtain a charge ratio of 3 +/- and a final plasmid concentration of 0.0125 μg/μL. The resulting mixture was incubated at room temperature for 30 minutes. To this solution, 480 μg of boronate PEG _5k (compound 6; example 6), (20 μg/μL, 24 μL) was added. The mixture was then incubated for an additional 30 min, dialyzed twice in DNase- and RNase-free water using 0.5 mL 100,000 MWCO membrane (BIOMAX, Millipore Corporation), and reconstituted in 160 μL of DNase- and RNase-free water. Half of this solution was diluted with 1.4 mL of 1 mM KCl for zeta potential measurement ( FIG. 6 ). Note that the zeta potential of the BA-containing nanoparticles was lower than that of the non-containing nanoparticles. These results support the conclusion that the BA-containing nanoparticles localize BA on the outside of the nanoparticles. The other half was used to measure particle size. Particle size was measured every minute for 5 min after adding 10.2 μL of 10× PBS to give a final solution of 90.2 μL in 1× PBS. Particle size was then measured every minute for another 10 minutes, as shown in Figure 7. BA-containing nanoparticles separated (by filtration) from non-particle components are stable in PBS, whereas particles without BA are not. These results support the conclusion that BA-containing nanoparticles have BA localized on their exterior, since they are stable to aggregation in PBS.

Example 13: Transfection of MAP/pDNA particles into HeLa cells HeLa cells were seeded in 24-well plates at 20,000 cells per well 48 hours prior to transfection and grown in medium supplemented with 10% FBS. MAP particles were formulated to contain 1 μg pGL3 in 200 μL Opti-MEM I at various polymer to pDNA charge ratios (see Example 9). Growth medium was removed, cells were washed with PBS, and particle formulations were added. Cells were then incubated for 5 hours at 37° C., 5% CO ₂ , before adding 800 μL of growth medium supplemented with 10% FBS. After 48 hours of incubation, a fraction of cells was analyzed for cell viability using the MTS assay. The remaining cells were lysed in 100 μL of 1× Luciferase Culture Cell Lysis Reagent. Luciferase activity was determined by adding 100 μL of luciferase assay reagent to 10 ul of cell lysate, and bioluminescence was quantified using a Monolight luminometer. Luciferase activity is then reported as relative light units (RLU) per 10,000 cells. Results are shown in Figures 8 and 9.

Example 14: Co-transfection of MAP/pDNA and/or siRNA particles into HeLa cells HeLa cells were seeded at 20,000 cells per well in 24-well plates 48 hours prior to transfection and grown in medium supplemented with 10% FBS. MAP particles were formulated to contain 1 μg pGL3 and 50 nM siGL3 in 200 μL Opti-MEM I at a charge ratio of 5+/-. Particles containing only pGL3 or pGL3 and siCON were used as controls. Growth medium was removed, cells were washed with PBS, and particle formulations were added. Cells were then incubated for 5 hours at 37° C., 5% CO ₂ , before adding 800 μL of growth medium supplemented with 10% FBS. After 48 hours of incubation, cells were assayed for luciferase activity and cell viability as described in Example 12. The results are shown in Figure 10. The RLU is lower in transfections with siGL3 (correct sequence), and both siGL3 and pGL3 must be co-delivered.

Example 15: Transfection of MAP/siGL3 into HeLa cells HeLa cells were seeded at 20,000 cells per well in 24-well plates 48 hours prior to transfection and grown in medium supplemented with 10% FBS. MAP particles were formulated to contain 50 and 100 nM siGL3 at a charge ratio of 5+/- in 200 μL of Opti-MEM I. Growth medium was removed, cells were washed with PBS, and particle formulation was added. Cells were then incubated for 5 hours at 37°C, 5% _CO2 before adding 800 μL of growth medium supplemented with 10% FBS. After 48 hours of incubation, cells were assayed for luciferase activity and cell viability as described in Example 12. Results are shown in FIG. 11. Since RLU decreased with increasing concentrations of siGL3, these data suggest that alienation of the endogenous gene may occur.

Example 16: Synthesis of mucic acid diiodide, (10)
1 g (2.7 mmol) of mucic acid diamine (Example 3) was mixed with 3.8 mL (27.4 mmol) of triethylamine and 50 mL of anhydrous DMF, followed by the dropwise addition of 1.2 mL (13.7 mmol) of iodoacetyl chloride in a 250 mL round bottom flask. The mixture was allowed to react overnight at room temperature with constant stirring. The solvent was then removed by vacuum pump and the product was filtered, washed with methanol and dried under vacuum to give 0.8 g (1.3 mmol, 46%) of mucic acid diiodide. ¹ H NMR ((CD ₃ ) ₂ SO) δ 8.20 (s 2H), 2H), 7.77 (s, 2H), 4.11 (m, 2H), 4.03 (m, 2H), 3.79 (m, 2H), 3.11-3.17 (m, 2H), 1.78 (d, 2H). ESI/MS (m/z): 652.8 [M+Na] ⁺

Example 17: Synthesis of dicysteine mucate (11)
7 mL of 0.1 M degassed sodium carbonate was added to 17 mg of L-cysteine and 0.4 g of mucic acid diiodide. The resulting suspension was refluxed at 150° C. for 5 hours until the solution became clear. The mixture was then cooled to room temperature and adjusted to pH 3 with 1N HCl. A slow addition of acetone was then performed to precipitate the product. After filtration, washing with acetone, and drying under vacuum, 60 mg of crude product was obtained.

Example 17: Synthesis of dicysteine mucate (11)
20 mL of 0.1 M degassed sodium phosphate buffer at pH 7.5 in a 50 mL round bottom flask was added to 0.38 g L-cysteine (3.2 mmol) and 0.40 g (0.6 mmol) mucic acid diiodide. The resulting suspension was refluxed at 75° C. overnight, cooled to room temperature, and lyophilized. 80 mL of DMF was then added to the lyophilized light brown powder, and separation of the insoluble excess reagent and phosphate from the soluble product was achieved by filtration. DMF was removed under reduced pressure, and the product was vacuum dried to give 12 mg (0.02 mmol, 3%) dicysteine mucate.

Example 18: Polymer synthesis, (Poly(mucinic acid-DiCys-PEG)) (12)
12 mg (21.7 μmol) of dicysteine mucate and 74 mg (21.7 μmol) of PEG-DiSPA 3400 were dried under vacuum, then 0.6 mL of anhydrous DMSO was added under argon in a two-neck 10 mL round bottom flask. After stirring for 10 minutes, 9 μL (65.1 μmol) of anhydrous DIEA was transferred to the reaction vessel under argon. The mixture was stirred overnight under argon. The polymer-containing solution was then dialyzed using a 10 kDa membrane centrifuge and lyophilized to give 47 mg (58%) of poly(mucate-DiCys-PEG).

This polyol-containing polymer is an anionic AB polymer.

Example 19: Covalent attachment of a drug (camptothecin, CPT) to a mucic acid polymer (13)
10 mg (2.7 μmol repeat units) of poly(mucin acid-DiCys-PEG) was dissolved in 1.5 mL of anhydrous DMSO in a glass bottle. After stirring for 10 min, 1.1 μL of DIEA (6.3 μmol), 3.3 mg (6.3 μmol) of TFA-Gly-CPT, 1.6 mg (8.1 μmol) of EDC and 0.7 mg (5.9 μmol) of NHS were added to the reaction mixture. After stirring for 8 h, 1.5 mL of ethanol was added and the solvent was removed under reduced pressure. The precipitate was dissolved in water and unwanted materials were removed by filtration through a 0.2 μm filter. The polymer solution was then dialyzed against water through a 10 kDa membrane and then lyophilized to obtain the poly(mucin acid-DiCys-PEG)-CPT conjugate.

Example 20: Formulation of nanoparticles with CPT-mucinic acid polymer (13) in water (20)
The effective diameter of poly(mucin acid-DiCys-PEG) and poly(mucin acid-DiCys-PEG)-CPT conjugates was assessed via dynamic light scattering (DLS) using a ZetaPALS (Brookhaven Instrument Co) instrument by formulating the polymers in double distilled water (0.1-10 mg/mL). Three consecutive runs of 1 min each were then recorded and averaged. The zeta potential of both compounds was measured in 1.1 mM KCl solution using a ZetaPALS (Brookhaven Instrument Co) instrument. Ten consecutive automated runs at a target residual of 0.012 were then performed and the results averaged (Figure 4). Specifically, these two distributions were measured for the poly(mucinic acid-DiCys-PEG)-CPT conjugate, with the major distribution being at 57 nm (60% of the total particle population). The second minor distribution is also measured at 233 nm.

Example 21: Formulation of Boronic Acid-PEGylated Nanoparticles with CPT-Mucic Acid Polymer (13) Boronic Acid-Disulfide-PEG ₅₀₀₀ (6) in Water Boronic acid PEGylated poly(mucin acid-DiCys-PEG)-CPT nanoparticles are prepared by dissolving the polymer in double distilled water to a concentration of 0.1 mg/mL, followed by addition of polymer 6 (BA-PEG), also in water, to give a 1:1 ratio of PBA-PEG to diol on the mucin sugar in the poly(mucin acid-DiCys-PEG)-CPT conjugate. The mixture is incubated for 30 minutes, after which the effective diameter and zeta potential are measured using a ZetaPALS (Brookhaven Instrument Co) instrument.

Example 22: Targeted nanoparticles for pDNA delivery in mice Plasmid pApoE-HCRLuc contains a gene expressing luciferase and is under the control of a liver-specific promoter. Polymer (MAP) 4 (0.73 mg), polymer 6 (73 mg) and polymer 9 (0.073 mg) were combined in 5 mL of water, then 1.2 mL of pApoE-HCRLuc plasmid-containing water was added (to obtain a polymer 4 to plasmid charge ratio of +3). The particles were placed in D5W (5% glucose in water) by successive spin filtration and subsequent addition of D5W (starting with the starting formulation in water). Nude mice were implanted with Hepa-1-6 hepatoma cells and tumors were allowed to grow until they reached a size of approximately 200 ^mm3 . Injections of targeted nanoparticles were performed i.v. in the tail vein in an amount equivalent to 5 mg/plasmid kg mouse. Mice were imaged 24 hours after injection. Mice showed no signs of toxicity and luciferase expression was detected in the area of the tumor but not in the area of the liver.

Example 23: Synthesis and pKa Determination of PBA-PEG and NitroPBA-PEG The reversible covalent bond property between boronic acid and MAP was used to prepare stabilized targeted nanoparticles. Phenylboronic acid (PBA) has a high pKa of 8.8. To lower the pKa, PBA with an electron-withdrawing group on the phenyl ring was used to increase the acidity of the boron atom. Commercially available 3-carboxyPBA and 3-carboxy5-nitroPBA were converted to acyl chlorides using oxalyl chloride. These acyl chloride species were then reacted with NH ₂ -PEG to form PBA-PEG and nitroPBA-PEG, respectively. The addition of bases DMF and DIPEA was necessary for the synthesis reaction to proceed. However, the presence of these bases caused tetrahedral adduct formation with the acidic PBA. To remove these adducts, a workup in 0.5 N HCl followed by equilibration to neutral pH by dialysis against water was performed.

For the synthesis of 200 mg (1.21 mM) of PBA-PEG, 3-carboxyphenylboronic acid dissolved in 5 mL of anhydrous tetrahydrofuran was added to 18.7 μl (0.24 mM) of anhydrous dimethylformamide under argon. The reaction vessel was transferred to an ice bath and 195 μl (2.89 mM) of oxalyl chloride was added slowly under argon. The reaction was allowed to proceed for 2 hours at room temperature under venting and with constant stirring. The solvent and excess reagents were removed under vacuum. 37 mg (0.2 mM) of the dried acyl chloride compound was dissolved in 15 ml of anhydrous dichloromethane under argon. Then, 500 mg (0.1 mM) of NH2-PEG5kDa-CO2H and 52 μl (0.3 mM) of dry DIPEA were added under argon. After overnight reaction under constant stirring, the solvent was removed under vacuum and the dried product was reconstituted in 0.5 N HCl. The solution was passed through a 0.2 μm filter (Acrodisc®) and dialyzed against water using a 3 kDa MWCO membrane filter device (Amicon™) until a constant pH was obtained. The supernatant was then filtered using a 0.2 μm filter (Acrodisc®) and lyophilized to obtain 377 mg (73%) of PBA-PEG-CO2H. 1H NMR ((CD3)2SO)δ 12.52 (s, 1H), 8.39 (t, 1H), 8.22 (s, 1H), 8.14 (s, 2H), 7.88 (d, 1H), 7.82 (d, 1H), 7.39 (t, 1H), 3.99 (s, 2H), 3.35-3.62 (PEG peak) ．． MALDI-TOF (m/z): 5600 g/mol (NH2-PEG5kDa-CO2H, 5400 g/mol).

The synthesis of nitroPBA-PEG-CO2H was carried out in the same manner as for PBA-PEG-CO2H, except that the starting material was 3-carboxy 5-nitrophenylboronic acid. The reaction yielded 393 mg (76%) of nitroPBA-PEG-CO2H. 1H NMR ((CD3)2SO) δ 12.52 (s,1H), 8.89 (t,1H), 8.72 (s,1H), 8.68 (s,1H), 8.64 (s,1H), 8.61 (s,2H), 3.99 (s,2H), 3.35-3.62 (PEG peak). MALDI-TOF (m/z): 5600 g/mol (NH2-PEG5kDa-CO2H, 5400 g/mol).

The pKa of the synthesized PBA compounds was found by measuring the change in absorbance of PBA converted from trigonal conformation (low pH) to tetrahedral conformation (high pH). Solutions of 10-3M PBA in 0.1M PBS, pH 7.4 were titrated with 1N NaOH. The pH was recorded and the corresponding samples were taken for absorbance measurement at 268 nm.

Example 24: Antibody conjugation to nitroPBA-PEG After the formation of nitroPBA-PEG, the resulting compound was conjugated to an antibody. For conjugation, 36 mg (6.4 μM) of nitroPBA-PEG-CO2H, 12.3 mg (64 μM) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 11.1 mg (96 μM) of NHS were dissolved in 2.4 ml of 0.1 M MES buffer, pH 6.0. The mixture was reacted for 15 minutes at room temperature on a rotary shaker. The excess reactant was dialyzed off through a 3 kDa molecular weight cutoff membrane filter device (Amicon™). This activated carboxylic acid PEG compound was added to 20 mg (0.14 mM) of human IgG1 antibody (Herceptin®) in 0.1 M PBS, pH 7.2. The reaction was carried out for 2 hours at room temperature on a rotating shaker and then dialyzed four times against 1× PBS at pH 7.4 using a 50 kDa molecular weight cutoff membrane filter device (Amicon™). Mass spectrometry (MALDI-TOF) showed an average of 1-2 conjugates of nitro-PBA-PEG compound per antibody.

Example 26: Formulation and characterization of targeted MAP nanoparticles The effect of PBA-PEG and nitro-PBA-PEG on nanoparticle formulation and stability was examined. A solution of PBA-PEG was added to a solution of MAP at a charge ratio of 3+/- (positive charge from MAP per negative charge from siRNA) in phosphate buffer at pH 7.4. The mixture was left at room temperature for 10 minutes for complexation of PBA-PEG with diol-containing MAP. It was then added to an equal volume of siRNA in water and mixed by pipetting. After 10 minutes, testing and characterization was performed. The particle formulation was stabilized with nitro-PBA-PEG and different charge ratios were performed in the same manner. Complexation of PBA-PEG formed MAP/siRNA particles with sizes of 150-300 nm. MAP-4, which exhibits four methylene units between the charge center (amidine) and the binding site (polyol), resulted in smaller nanoparticles than when MAP-2 was used. However, upon addition of salt, all particles aggregated, indicating that PBA-PEG does not provide sufficient stability to the nanoparticles. When nitro-PBA-PEG was used, particles at all charge ratios tested, 1 and 3 (+/-), demonstrated stabilized nanoparticles. The difference in particle stability imparted by PBA-PEG and nitro-PBA-PEG indicates the importance of modifying the PEGylated boronic acid compound to have stronger binding to the polyol.

For siRNA condensed with MAP-4 in the absence of the stabilizer PEG, a particle size of 640 nm was observed with a high zeta potential of +32 mV. When nitroPBA-PEG was added to stabilize the particles, the size was reduced to 130 nm with a negative surface charge of -4 mV. The negative charge is the result of the bond between the boronic acid and the diol. Interestingly, when 0.25 mol% IgG (Herceptin®) conjugated with nitroPBA-PEG was introduced, the particle size was further reduced to 82 nm, while the zeta potential remained similar.

Salt stability was evaluated for MAP-4/siRNA nanoparticles with various PEG groups attached. Particle size was recorded for 5 runs at 1 min using a zeta potential analyzer (DLS ZetaPALS instrument, Brookhaven Instruments, Holtsville, NY). Measurement was stopped and 10x PBS solution was added to formulate the particles so that the resulting particles contained 1x PBS. Measurement was then resumed and 10 consecutive runs of 1 min each were recorded to study the effect of salt addition on particle stability. As shown in Figure 16, in the absence of the stabilizer PEG, the particles aggregated. In the presence of nitroPBA-PEG, the particles remained stable even under salt conditions.

Example 27: Formulation and characterization of targeted MAP-CPT nanoparticles The reversible covalent binding property between boronic acid and MAP (containing diol) was used to prepare targeted MAP-CPT nanoparticles. The binding constant between PBA and MAP is as low as about 20 M ⁻¹ . To increase the binding constant, PBA with an electron-withdrawing group on the phenyl ring was used to increase the acidity of the boron atom and evaluate the binding constant of MAP. Commercially available 3-carboxy PBA and 3-carboxy 5-nitro PBA were converted to acyl chlorides using oxalyl chloride. These acyl chloride species were then reacted with NH2-PEG-CO ₂ H to form PBA-PEG-CO ₂ H and nitro PBA-PEG-CO ₂ H, respectively. The addition of bases of DMF and DIPEA was required to drive the synthesis reaction. However, the presence of these bases resulted in tetrahedral adduct formation with the acidic PBA. To remove these adducts, a work-up in 0.5 N HCl followed by equilibration to neutral pH by dialysis against water was performed.

The pKa values of modified PBA were determined by the absorbance change due to the conformational change of PBA from trigonal to tetrahedral form with increasing pH (Table 2). PBA with electrophilic groups resulted in low pKa, with nitroPBA-PEG-CO ₂ H having the lowest pKa of 6.8. Thus, at physiological pH, most of the PBA exists in the reactive anionic tetrahedral form. The binding constant between PBA and MAP was found by competitive binding with Alizarin Red S in 0.1 M PBS, pH 7.4. Due to the low pKa value and high binding constant of MAP observed for nitroPBA-PEG-CO ₂ H, it was selected as the linker between IgG (Herceptin®) and MAP-CPT nanoparticles.

The conjugation reaction between IgG(Herceptin®) and NitroPBA-PEG-CO ₂ H proceeded via EDC coupling (described above). On average, 1-2 PEGs were conjugated per IgG(Herceptin®) antibody.

^ａムチン酸ジ（Ａｓｐ－アミン）あたり等量のＮ，Ｎ－ジイソプロピルエチルアミン（ＤＩＰＥＡ）を加えた。^ｂＭＷ、（Ｍｗ＋Ｍｎ）／２として決定した分子量；Ｍｗ，重量平均分子量；Ｍｎ，数平均分子量。^ｃＭｗ／Ｍｎとして決定した多分散性。

a Equivalent amount of N,N-diisopropylethylamine (DIPEA) was added ^per mucic acid di(Asp-amine). ^b MW, molecular weight determined as (Mw+Mn)/2; Mw, weight average molecular weight; Mn, number average molecular weight. ^c Polydispersity determined as Mw/Mn.

An average size of about 40 nm was observed for the targeted MAP-CPT nanoparticles by DLS and cryo-EM (Table 3). An increase of about 10 nm from the non-targeted nanoparticles indicates the binding of IgG (Herceptin®) to the nanoparticles (the amount was about 1 Herceptin® per nanoparticle). Indeed, the cryo-EM images showed that the targeted nanoparticles were not spherical but had protrusions indicating antibody binding. The surface charge of the targeted MAP-CPT nanoparticles was slightly higher than that of the MAP-CPT nanoparticles due to the presence of positive charges of Herceptin at pH 7.4 (pI of Herceptin®, 9.2).

Example 28: Cellular uptake studies A human breast cancer cell line overexpressing HER2 (BT-474) was used to test the cellular uptake of Herceptin® targeted MAP-CPT nanoparticles versus non-targeted nanoparticles. A HER2 negative breast cancer cell line (MCF-7) was used as a negative control. For these studies, 24-well plates were seeded with BT-474 (in RPMI-1640 medium supplemented with 10% fetal bovine serum) or MCF-7 (in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum) (Cellgro, Manassas, VA) at 20,000 cells per well and kept at 37°C in a humidified oven with 5% _CO2 . After 40 hours, the medium was replaced with 0.3 ml of fresh medium. The media contained medium MAP-CPT (40 μg CPT/ml), medium MAP-CPT with free Herceptin® at 10 mg/ml, targeted MAP-CPT at various target densities (40 μg CPT/ml) or targeted MAP-CPT with free Herceptin® at 10 mg/ml. Transfection was carried out at 37° C. for 30 min. The formulation was then removed and the cells were washed twice with cold PBS. For cell detachment and lysis, 200 μl of RIPA buffer was added per well. The lysed cells were then incubated at 4° C. for 15 min and centrifuged at 14,000 g for 10 min at 4° C. A portion of the supernatant was used for protein quantification by BCA assay. Separate proteins were added with an equal volume of 0.1 N NaOH, incubated overnight at room temperature, and fluorescence was measured at 370/440 nm using known concentrations of MAP-CPT as a standard.

We observed that one Herceptin®-PEG-nitroPBA per nanoparticle was sufficient to achieve approximately 70% higher uptake compared to non-targeted nanoparticles in the BT-474 cell line (Figure 17A). Uptake of targeted MAP-CPT in BT-474 cells showed inhibition in the presence of free Herceptin® (Figure 17B). In contrast, uptake of MAP-CPT nanoparticles in MCF-7 showed no dependency on targeting in the presence of free Herceptin® (Figure 2B). These data indicate that there is receptor-mediated uptake by targeted MAP-CPT nanoparticles in BT-474 cells via HER2 receptor binding. Cellular uptake of both targeted and non-targeted MAP-CPT nanoparticles also occurred via non-specific fluid-phase endocytosis.

Example 29: In Vitro Release Studies Experiments were conducted to evaluate the release of CPT from short, medium, long MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles. These studies were performed using 0.32 mg CPT/ml in 1x PBS (pH 6.5, 7, or 7.4); BALB/c mouse plasma, human plasma, and 1x PBS (pH 7.4) containing 3 mg/ml low density lipoprotein (LDL), or 100 units/ml butyrylcholinesterase (BCHE) or a combination of LDL and BCHE.

For equilibration, media pipetted into 96-well plates were incubated for 2 hours at 37°C in a humidified oven. Formulations were mixed into the corresponding media and placed back in the oven. Samples were removed at pre-determined time points and immediately frozen at -80°C until the time of analysis. For release in BALB/c mouse plasma and human plasma, incubations were performed in a humidified oven at 37°C with 5% _CO2 (to maintain the carbonate/carbon dioxide buffer system, which is the major pH buffer system in plasma, at physiological pH levels).

The amount of unconjugated CPT was determined by first mixing 10 μl of sample with 10 μl of 0.1 N HCl and incubating at room temperature for 30 min. Then, 80 μl of methanol was added and the mixture was incubated at room temperature for 3 h for protein precipitation. The mixture was centrifuged at 14,000 g for 10 min at 4°C, the supernatant was filtered through a 0.45 μm filter (Millex-LH), diluted 20-fold with methanol, and 10 μl of the resulting solution was injected into the HPLC. The peak area of eluted CPT (at 7.8 min) was compared with that of the control. To measure the total amount of CPT, 10 μl of the sample was mixed with 6.5 μl of 0.1 N NaOH. The solution was incubated at room temperature for 1 h to release CPT from the parent polymer. Then, 10 μl of 0.1 N HCl was added to convert the carboxylate CPT form to the lactate state. After that, 73.5 μl of methanol was added and the mixture was incubated at room temperature for 3 hours. The samples were then centrifuged and processed as above. The polymer-bound CPT concentration was determined from the difference between the total CPT concentration and the unconjugated CPT concentration.

The release of CPT from short, medium and long MAP-CPT nanoparticles all showed first-order kinetics. The release half-life of CPT from short, medium and long MAP-CPT nanoparticles was similar in all conditions tested. On the other hand, a longer half-life was observed for targeted MAP-CPT (Table 4). A strong dependence of release rate and pH was observed. With an increase in pH from 6.5 to 7.4, the release half-life was shortened from 338 to 58 hours for short, medium and long MAP-CPT nanoparticles. These data indicate that hydrolysis plays an important role in the release of CPT. For short, medium and long MAP-CPT nanoparticles, a release half-life of 59 hours was observed in BALB/c mouse plasma, whereas a significantly shorter half-life of 38 hours was observed in human plasma. It is known that mouse plasma has higher esterase activity than human plasma, while human plasma is more "fatty" than mouse plasma. Therefore, to understand the difference in release rates between human and mouse plasma, the contributions of esterases and fat to the CPT release rate were examined separately. Butyrylcholinesterase (BCHE) is present in both mouse and human plasma and was used to examine the contribution of esterases to the release rate. Low density lipoprotein (LDL) was chosen to document the contribution of "fat" to the release rate. These components were included in PBS (pH 7.4). The presence of BCHE was found not to affect the release rate (half-life of 61 hours compared to 58 hours in PBS pH 7.4 for short, medium and long MAP-CPT nanoparticles). However, the addition of LDL dramatically extended the release rate. This effect is likely due to nanoparticle destruction by competitive hydrophobic interactions. Therefore, nanoparticle destruction due to the presence of "fat" and subsequent CPT cleavage by hydrolysis appears to be a different main mechanism of CPT release from MAP-CPT nanoparticles. Release from Herceptin® targeted MAP-CPT nanoparticles shows a longer half-life than the non-targeted version. The presence of Herceptin®, which has a pI of 9.2, can increase the stability of negatively charged nanoparticles by electrostatic interactions and thereby shielding the nanoparticles from some of the competing hydrophobic interactions.

Abbreviations: PBS, phosphate buffered saline; LDL, low density lipoprotein; BCHE, butyrylcholinesterase. ^a PBS, pH 7.4, containing 3 mg/ml LDL. ^b PBS, pH 7.4, containing 100 units/ml BCHE. ^c PBS, pH 7.4, containing 3 mg/ml LDL and 100 units/ml BCHE.

Example 30: Cytotoxicity Assays The in vitro cytotoxicity of medium MAP, nitro-PBA-PEG, CPT, medium MAP-CPT nanoparticles, targeted MAP-CPT nanoparticles and Herceptin® was evaluated in two HER2+ breast cancer cell lines (BT-474, SKBR-3) and two HER2- breast cancer cell lines (MCF-7, MDA-MB-231) (Table 5). For these studies, cells were kept at 37°C in a humidified oven with 5% _CO2 . BT-474, MCF-7, SKBR-3 and MDA-MB-231 cell lines were incubated in RPMI-1640 medium, Dulbecco's modified Eagle's medium, McCoy's 5A modified medium and Dulbecco's modified Eagle's medium, respectively (all supplemented with 10% fetal bovine serum). 3,000 of the cells were plated per well in a 96-well plate. After 24 hours, the medium was removed and replaced with various concentrations of medium-sized MAP, nitroPBA-PEG, CPT, medium-sized MAP-CPT nanoparticles, targeted MAP-CPT nanoparticles or Herceptin® fresh medium. After 72 hours of incubation, the formulations were replaced with fresh medium and 20 μl of CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega) was added per well. This was incubated for 2 hours at 37° C. in a humidified oven with 5% CO ₂ , after which absorbance measurements were taken at 490 nm. Wells containing untreated cells served as controls.

Medium MAP and nitroPBA-PEG gave IC ₅₀ values of 500 μM and 1000 μM (the highest concentrations tested), respectively, indicating minimal cytotoxicity. The IC ₅₀ concentrations of CPT, medium MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles are based on the content of CPT. It should be noted that CPT was released slowly from medium MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles (see in vitro release studies). Furthermore, nanoparticle uptake by cells resulted in a long release time due to the acidic nature of the endosomes. These factors contribute to the IC ₅₀ values observed for medium MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles relative to CPT (Table 5). In HER2+ cell lines, targeted MAP-CPT nanoparticles gave lower IC ₅₀ values compared to MAP-CPT alone. On the other hand, targeting of HER2- cell lines did not affect IC _50. BT-474 was the most resistant cell line to CPT and therefore to the medium MAP-CPT nanoparticles. Addition of Herceptin at concentrations of 0.001-0.5 μM to BT-474 or SKBR-3 cells resulted in a consistent cell viability of about 60% compared to untreated controls. The lack of true IC ₅₀ values over this concentration range for these cell lines has been observed previously (Phillips, et al., Cancer Res. 68, 9280-9290 (2008)). At Herceptin concentrations up to 0.5 μM, no effect was observed in HER2- cell lines.

^ａｎｏ ｖａｌｕｅ、０．００１～０．５μＭの濃度範囲にわたり、ＩＣ_５０値が得られなかった。

^a no value, no IC ₅₀ value was obtained over the concentration range of 0.001-0.5 μM.

Example 31: Pharmacokinetic studies Plasma pharmacokinetic studies of short, medium and long MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles at 10 mg/kg (based on CPT) injection were performed in female BALB/c mice (Figure 18). For these studies, nanoparticles formulated in 0.9 wt% NaCl (non-targeted nanoparticles) or PBS (targeted MAP-CPT nanoparticles) were administered to 12-16 week old female BALB/c mice via bolus tail vein injection. At the designated time points, blood was collected via the saphenous vein into blood collection tubes (Microvette CB 300 EDTA, Sarstedt). Samples were immediately centrifuged at 10,000g for 15 min at 4°C and the supernatant was removed and stored at -80°C until the time of analysis. Analysis of unconjugated and polymer-bound CPT was as follows: The amount of unconjugated CPT was first determined by mixing 10 μl of the sample with 10 μl of 0.1 N HCl and incubating at room temperature for 30 min. Then, 80 μl of methanol was added and the mixture was incubated at room temperature for 3 h for protein precipitation. The mixture was centrifuged at 14,000 g for 10 min at 4° C., the supernatant was filtered through a 0.45 μm filter (Millex-LH), diluted 20-fold with methanol, and 10 μl of the resulting solution was injected into HPLC. The peak area of eluted CPT (at 7.8 min) was compared with that of the control. To measure the total amount of CPT, 10 μl of the sample was mixed with 6.5 μl of 0.1 N NaOH. The solution was incubated at room temperature for 1 h to release CPT from the parent polymer. Then, 10 μl of 0.1 N HCl was added to convert the carboxylate CPT form to the lactate state. Afterwards, 73.5 μl of methanol was added and the mixture was incubated at room temperature for 3 hours. The samples were then centrifuged and processed as above. The polymer-bound CPT concentration was determined from the difference between the total and unconjugated CPT concentrations. Non-compartmental modeling software PK Solutions 2.0 by Summit Research Services (Montrose, CO) was used for pharmacokinetic data analysis. All animals were treated in accordance with the National Institute of Health Guidelines for Animal Care and approved by the California Institute of Technology Institutional Animal Care and Use Committee.

Polymer-bound CPT for all nanoparticles showed a biphasic profile with a rapid redistribution phase (α) and a long elimination phase (β) (Figure 18A). The elimination phase of the medium and long MAP-CPT nanoparticles notably extended the half-life to 16.6 and 17.6 hours, respectively, and gave high area under the curve (AUC) values of 2298 and 3636 μg×hr/ml, respectively. Furthermore, they showed low volume distribution and clearance rates. 24 hours after injection, 11.3% of the injected dose of medium MAP-CPT nanoparticles and 20.5% of the long MAP-CPT nanoparticles still remained in the plasma circulation as polymer-bound CPT. In contrast, mice injected with only CPT at 10 mg/kg showed rapid clearance with an AUC of only 1.6 μg×hr/ml and 0.01% of the dose remaining in the circulation after 8 hours. Targeting of medium-sized MAP-CPT nanoparticles affected pharmacokinetics by increasing the redistribution phase and prolonging the elimination phase to 21.2 hours, with a high AUC value of 2766 μg×hour/ml. The amount of unbound CPT in plasma for all nanoparticles was low at all time points (Figure 18B).

Example 32: Determination of the maximum tolerated dose (MTD) in nude mice The MTD value was determined in female NCr nude mice and defined as the highest dose associated with less than 15% weight loss and no treatment-related deaths. In these experiments, 12-week-old female NCr nude mice were randomly divided into 13 groups with 5 mice per group. The formulations medium MAP or nitroPBA-PEG at 200 mg/kg, short MAP-CPT nanoparticles at 10, 15 or 20 mg/kg (based on CPT), medium MAP-CPT nanoparticles at 8, 10 or 15 mg/kg (based on CPT), long MAP-CPT nanoparticles at 5, 8 or 10 mg/kg (based on CPT) and targeted MAP-CPT nanoparticles at 8 or 10 mg/kg (based on CPT) were administered via intravenous injection in the tail vein on days 0 and 7. All injections were formulated in 0.9% w/v saline, except for Targeted MAP-CPT, which was formulated in PBS, pH 7.4. Mice weight and health were recorded and monitored for 2 weeks from the start of treatment. The MTD was defined as the highest dose associated with less than 15% weight loss and no treatment-related deaths. Animals were euthanized by _CO2 asphyxiation when they exceeded the criteria for the MTD or at the end of the study.

Mice treated with medium MAP, nitro-PBA-PEG, short, medium and long MAP-CPT and targeted MAP-CPT were weighed and monitored for health twice weekly on days 0 and 7 (Table 6). Mouse weight and health were not affected by treatment with high doses of medium MAP or nitro-PBA-PEG, indicating minimal toxicity for these polymeric components. For groups containing CPT, the greatest weight loss was seen on days 3-5 of each treatment. Most groups regained lost weight. If weight loss continued and exceeded an average of 15%, the study was discontinued. Some mice treated with medium MAP-CPT at 15 mg/kg and long MAP-CPT at 10 mg/kg showed diarrhea and appeared weak. All other mice appeared healthy. The MTD values were found to be 20, 10, 8 and 8 mg/kg (based on CPT) for short, medium, long MAP-CPT and targeted MAP-CPT, respectively (Table 6).

^ａＭＡＰ－ＣＰＴを含む全ての群は、ｍｇ ＣＰＴ／ｋｇに基づく。^ｂ最大体重減少百分率。^ｃ１５％を超える体重減少のために処分した動物。

^a All groups including MAP-CPT are based on mg CPT/kg. ^b Maximum percentage weight loss. ^c Animals culled due to weight loss greater than 15%.

Example 33: Biodistribution in Nude Mice To evaluate the biodistribution of targeted nanoparticles in mice, 7-week-old NCr nude mice were implanted subcutaneously with 17β-estradiol pellets. Two days later, BT-474 carcinoma cells suspended in RPMI-1640 medium were injected subcutaneously at 10 million cells/animal in the right front flank. Treatment was initiated after tumors reached an average size of 260 ^mm3 . Animals were randomized into two groups of six animals each and treated with 5 mg CPT/kg MAP-CPT nanoparticles (in PBS, pH 7.4) or 5 mg CPT/kg targeted MAP-CPT nanoparticles and 29 mg Herceptin®/kg (in PBS, pH 7.4) by intravenous injection into the tail vein. After 4 and 24 hours, blood was collected from the animals in each group via saphenous vein bleeding. The animals were then euthanized by _CO2 nitrogen and immersed in PBS. The tumor, lungs, heart, spleen, kidneys and liver were harvested and cut into two equal sized pieces. One piece was embedded in Tissue-Tek® OCT (Sakura) and the other was collected in an Eppendorf tube, both of which were immediately frozen at -80°C until the time of processing.

Organs were weighed and 100 mg of each was placed into a Lysing Matrix A homogenizer tube containing 0.64 cm (¼ inch) ceramic balls (MP Biomedicals, Solon, Ohio). 1 ml of RIPA lysis buffer (Thermo Scientific) was added and the tissue was homogenized at 6 m/s for 30 s using a FastPrep®-24 homogenizer (MP Biomedicals, Solon, Ohio). This was repeated three times with 1 min of cooling on ice between each run. Samples were then centrifuged at 14,000 g for 15 min at 4°C. The amount of unconjugated CPT was measured by first mixing 10 μl of the supernatant with 10 μl of 0.1 N HCl and incubating at room temperature for 30 min, then adding 80 μl of methanol and incubating at room temperature for 3 h for protein precipitation. The mixture was centrifuged at 14,000 g for 10 min at 4° C., the supernatant was filtered through a 0.45 μm filter (Millex-LH), and 10 μl of the resulting mixture was injected into HPLC. The peak area of eluted CPT (at 7.8 min) was compared with that of the control. To measure the total amount of CPT, 10 μl of the sample was mixed with 6.5 μl of 0.1 N NaOH. The solution was incubated at room temperature for 1 h to release CPT from the parent polymer. Then, 10 μl of 0.1 N HCl was added to convert the carboxylate CPT form to the lactone form. Then, 73.5 μl of methanol was added and the mixture was incubated at room temperature for 3 hours. The samples were then centrifuged and processed as above.

The mean percentage injected dose (ID) of total CPT per gram for tumor, heart, liver, spleen, kidney and lung is shown in FIG. 19A. Four hours after dosing, 5.3 and 5.2% ID of total CPT were present per gram of tumor in mice treated with MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles, respectively. Twenty-four hours after treatment, 2.6% ID of total CPT were present per gram of tumor in mice treated with MAP-CPT nanoparticles, while 3.2% ID of total CPT were present per gram of tumor in mice receiving targeted MAP-CPT nanoparticles. These data indicate that targeted nanoparticles of substantially the same size and surface charge as the nontargeted version did not increase tumor localization over that of the nontargeted version, consistent with other reported observations. Mice treated with MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles resulted in similar CPT distribution in the heart, liver, kidneys, and lungs. However, in the spleen, there was a relatively higher amount of total CPT accumulation for targeted MAP^CPT vs. non-targeted at the 24 hour time point. This effect has been previously observed for humanized antibodies in mice.

Figure 19B shows the average percentage of unconjugated CPT in each of the organs: tumor, heart, liver, spleen, kidney and lung. The percentage of unconjugated CPT in the heart, liver, spleen, kidney and lung was low at both 4 and 24 hours, indicating rapid clearance of free CPT from these organs. There was a significantly higher percentage of unconjugated CPT in the tumor at 24 hours compared to the 4 hour time point for both MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles. The retention of free CPT in the tumor suggests cellular accumulation of CPT. Targeted nanoparticles show a slightly higher percentage of unconjugated CPT than non-targeted nanoparticles in the tumors of mice at both 4 and 24 hours.

The total concentration of CPT in plasma and the percentage of CPT that was unconjugated in plasma were similar for both MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles (Figure 19C). After 4 hours, 17 and 20 μg/ml of total CPT remained in plasma for MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles, respectively. After 24 hours, the amount of total CPT remaining in plasma was the same for both MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles, at 12 μg/ml. The percentage of total CPT that was unconjugated in plasma was less than 3% for all conditions, indicating little release and rapid clearance of unconjugated CPT from plasma (Figure 19D).

Example 33: Treatment of tumors with targeted nanoparticles BT-474 tumor-bearing Ncr nude mice were treated with either MAP-CPT (5 mg CPT/kg) or targeted MAP-CPT (5 mg CPT/kg, 29 mg Herceptin/kg). After 4 and 24 hours, mice were euthanized and tumors were excised and sectioned at 20-30 μm thickness using a cryostat. Tumor sections were placed on Superfrost Plus slides (Fisher Scientific, Hampton, NH) and stored at -80°C until the time of processing. Slides were thawed and tissue sections were fixed directly on the slides with 10% formalin solution for 15 minutes. The slides were then washed three times with PBS for 5 minutes each and blocked in 5% goat serum blocking buffer for 1 hour. CPT is naturally fluorescent with an emission at 440 nm. To identify the location of MAPs, PEG within MAPs was stained with a rat anti-PEG primary antibody that recognizes internal PEG units at 14 μg/ml and incubated overnight at 4° C. This was followed by three PBS washes and a 1-hour incubation with 2 μg/ml Alexa Fluor® 488 goat anti-rat IgM secondary antibody. The slides were then washed three times with PBS and incubated for 1 hour with 2 μg/ml Alexa Fluor® 633 goat anti-human IgG secondary antibody to visualize Herceptin®. The slides were washed three more times with PBS, mounted with Prolong Gold Antifade reagent, and stored at 4° C. until imaging. Images were taken with a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Germany) using a 63x Plan-Neofluar oil objective. Two-photon excitation at 720 nm (emission filter BP 390-465 nm) was used to detect CPT. Excitation at 488 nm (emission filter LP 530) and 633 nm (emission filter BP 645-700 nm) were used to detect PEG and Herceptin, respectively. All laser and acquisition settings were set at the beginning of imaging and did not change. Image analysis was performed in the Zeiss lsm image browser.

Figure 20A shows tumor sections from mice treated with MAP-CPT nanoparticles 4 hours later. CPT signals were scattered. Signals for CPT and MAP appeared to be co-localized in the overlaid images. 24 hours after injection, there was a punctate accumulation of CPT signal (Figure 20B). The overlaid images suggested co-localization of CPT and MAP. For mice treated with targeted MAP-CPT nanoparticles, puncta indicating CPT accumulation were observed both 4 and 24 hours after treatment (Figures 20C and 20D). In the overlaid images, there was co-localization of CPT and MAP signals. The presence of Herceptin in the targeted MAP-CPT nanoparticles is indicated by the strong blue signal in the Herceptin channel compared to the weak background signal in the non-targeted version.

Example 33: Antitumor efficacy study in nude mice BT-474 is one of the most resistant breast cancers to anticancer drugs including camptothecin. To promote tumor growth in nude mice, 17β-estradiol pellets were implanted into 7-week-old Ncr nude mice, followed by BT-474 tumor cell implantation two days later. On day 0 (5 days after implantation), the tumor size of each group averaged 250 mm. Treatment was initiated on day 1. Animals were randomized into 9 groups with 6-8 animals per group and treated with either: MAP-CPT nanoparticles, 1 mg or 8 mg/kg (based on CPT, in PBS, pH 7.4); CPT, 8 mg/kg (dissolved in 20% DMSO, 20% PEG 400, 30% ethanol and 30% 10 mM pH 3.5 phosphate); irinotecan, 80 mg/kg (in 5% w/v dextrose solution, D5W); Herceptin®, 2.9 or 5.9 mg/kg (in PBS, pH 7.4); targeted MAP-CPT nanoparticles, CPT/kg and 2.9 mg Herceptin®/kg, or 1 mg CPT/kg and 5.9 mg Herceptin®/kg (in PBS, pH 7.4); or saline. All treatments were freshly prepared and given via intravenous injection in the tail vein. Injections were normalized at 150 μl per 20 g of mouse body weight. Treatments containing Herceptin® were given once a week for 2 weeks, and all other groups were given once a week for 3 weeks. Tumor size was recorded using caliper measurements (length x width ^2/2 ) three times a week, and animal health was continuously monitored. Animals were euthanized when tumor volumes exceeded ¹⁰⁰⁰ mm3. Six weeks after the start of treatment, animals were euthanized by _CO2 asphyxiation. The results of this study are shown in Figure 21 and Table 7.

Tumors in the saline-treated control group of animals grew rapidly (Figure 21). After 28 days, 5 of 8 mice had tumors exceeding the ¹⁰⁰⁰ mm3 size limit. All animals in this group were euthanized at this point.

The group treated with CPT (8 mg/kg) did not result in any tumor inhibition compared to the control group (P>0.05). One and three treatment-related deaths were recorded on days 9 and 16, respectively, as well as four euthanasias due to tumors exceeding size limits on day 28. No animals survived until the final day of the study.

Mice treated with irinotecan 80 mg/kg showed non-significant tumor inhibition compared to the control group (P>0.05). One tumor-related death occurred on day 11. On the final day of the study, tumor size reached an average of 575 ^mm3 .

Animals receiving MAP-CPT nanoparticles at 8 mg CPT/kg showed highly significant tumor inhibition compared to that of the control group (P<0.01). On the last day of the study, the average tumor size reached ⁶³ mm3, with 3 of 8 animals showing a reduction in tumor size to zero. No deaths occurred in the MTD study with non-tumor-bearing NCr nude mice treated with MAP-CPT, 8 mg CPT/kg, although one death occurred on day 21 of the study due to weight loss. This may be due to the tumor burden in this study and/or the fact that the mice used in this study were 7 weeks old, whereas 12 weeks old mice were used in the MTD study.

Animals treated with Herceptin® 5.9 mg/kg showed highly significant tumor inhibition compared to the control group (P<0.01). On the 11th day of treatment, 7 of 8 treated animals had tumors that had shrunk to zero. However, on the last day of the study, 2 of the shrunk tumors recurred, resulting in a total of 5 animals with zero tumor volume, with a group mean tumor volume of ⁶⁰ mm3 (Figure 22). Mice treated with MAP-CPT nanoparticles at 1 mg CPT/kg were early terminated due to the lack of observed antitumor effects. Nevertheless, when Herceptin®, 5.9 mg/kg, was added to the MAP-CPT nanoparticles as a targeted agent via a nitroPBA-BA linker at a low dose of 1 mg/kg (targeted MAP-CPT nanoparticles, 5.9 mg Herceptin®/kg and 1 mg CPT/kg), all tumors shrank to 0 on the 9th day of treatment and remained so until the last day of the study (Figure 22). One non-tumor-related death occurred on day 37. This result is highly significant compared to the control group (P<0.01). The combination of results observed for these three groups indicates that there was an improvement in efficacy by adding a Herceptin® targeted agent in addition to the inherent activity of Herceptin®.

Animals treated with 2.9 mg/kg Herceptin® resulted in two animals with tumors that shrank to 0 by the last day of the study. However, the average tumor size increased from the start of the study to ²⁷⁸ mm3. This result is significant compared to the control group (0.01≦P≦0.05). When animals were treated with targeted MAP-CPT nanoparticles containing 0.5 mg CPT/kg and 2.9 mg Herceptin®/kg, two tumors shrank to 0. On the last day of the study, tumor size shrank to an average of 141 ^mm3 . This result is highly significant compared to the control group (P<0.01). These results further suggest a benefit of targeted tumor inhibition.

Five weeks after 17β-estradiol pellet implantation, several mice, regardless of treatment group, were observed to have distended bladders and abdominal distension. This was likely due to the use of 17β-estradiol pellets, which produce hydronephrosis and urinary retention in athymic nude mice. Six weeks after treatment, symptoms worsened and many mice were observed to develop distended bladders and abdominal distension, which led to euthanasia and termination of the study.

^ａＮ_開始は、研究開始時の動物の数であり、Ｎ_終了は、研究の最後に生存していた動物の数である。^ｂＮ_ＴＲＤは、処置関連死の数、Ｎ_ＮＴＲＤは、非処置関連死の数、Ｎ_{ｅｕｔｈａｎ}は、腫瘍サイズ制限の１０００ｍｍ^３を超えたことによって安楽死させた動物の数である。^ｃＮ_{０まで縮小}は、研究の最終日に腫瘍が０まで縮小した動物の数である。

^a _Nstart is the number of animals at the start of the study, _Nend is the number of animals surviving at the end of the study. ^b _NTRD is the number of treatment-related deaths, _NNTRD is the number of non-treatment-related deaths, _Neuthan is the number of animals euthanized due to exceeding the tumor size limit of 1000 ^mm3 . ^c _{Nregression to 0} is the number of animals whose tumors regressed to 0 on the last day of the study.

The above examples are provided to give one of ordinary skill in the art a complete disclosure and how to make and use the disclosed particle, composition, system and method embodiments, and modifications of the above described modes of carrying out the disclosure that are obvious to one of ordinary skill in the art and are not intended to limit the scope of what the inventors contemplate regarding the disclosure are intended to be within the scope of the following claims. All patents and publications mentioned in this specification are indicative of the level of skill in the art to which this disclosure pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference was individually incorporated by reference in its entirety.

The entire disclosures of the documents cited in the Background, Summary, Detailed Description, and Examples (including patents, patent applications, journals, articles, laboratory manuals, books, or other disclosures) are hereby incorporated by reference.

It is understood that the present disclosure is not limited to a particular composition or biological system, which can, of course, vary. It should also be understood that the terms used herein are used only for the purpose of describing particular embodiments, and are not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. The term "plurality" includes two or more referents unless the content clearly dictates otherwise. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Any methods and materials similar or equivalent to those described herein may be used in practice to test the specific examples of suitable materials and methods described herein.

Many embodiments of the present disclosure have been described. However, it is understood that various modifications can be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims (21)

A nanoparticle comprising the following components (a) to (c):
(a) a modified micelle comprising a mucic acid polymer (MAP) modified to include a hydrophobic polymer block;
(b) a compound having the following structure:

A nitrophenylboronic acid-containing polymer having a mucic acid polymer conjugated thereto,
where s is between 20 and 300, * is the target ligand,
(c) a chemically or biologically active drug, said drug being contained within said modified micelle ;
wherein the nitrophenylboronic acid-containing polymer is bound to the mucic acid polymer by a reversible covalent bond between the boronic acid and the MAP diol such that the nanoparticle is configured such that the nitrophenylboronic acid-containing polymer is present in the external environment of the nanoparticle.
The nanoparticles. The nanoparticle of claim 1 , wherein the modified mucic acid polymer further comprises polymeric units of pentaerythritol, ethylene glycol, or glycerin. The nanoparticle of claim 1 or 2 , wherein the modified mucic acid polymer further comprises polyester, polyether or polysaccharide units . 4. The nanoparticle of claim 3 , wherein the polyester is a polycarbonate, polybutyrate or polyethylene terephthalate terminated with at least one hydroxyl end group. 4. The nanoparticle of claim 3 , wherein the polyether is HO-(CH ₂ CH ₂ O) _p -H, where p≧1. The nanoparticle of claim 5 , wherein the polyether is polyethylene glycol, polypropylene glycol or poly(tetramethylene ether) glycol. The nanoparticle of claim 3 , wherein the polysaccharide is cyclodextrin, starch, glycogen, cellulose, chitin or β-glycan. The nanoparticle of any one of claims 1 to 7 , wherein the chemically or biologically active drug is a therapeutic agent. The nanoparticle of claim 8 , wherein the therapeutic agent is a chemotherapeutic agent. The nanoparticle of claim 8 , wherein the therapeutic agent is a small molecule chemotherapeutic agent or a polynucleotide. The nanoparticle of claim 8 , wherein the therapeutic agent is a camptothecin, an epothilone, or a taxane. The nanoparticle of claim 8 , wherein the therapeutic agent is DNA, RNA or interfering RNA. The nanoparticle of claim 1 , wherein the targeting ligand is a protein, a protein fragment, a peptide, an aptamer or an antibody. The nanoparticle of claim 1 , wherein the targeting ligand is an antibody and is conjugated to a terminal portion of the nitrophenylboronic acid-containing polymer. The nanoparticle of claim 1 , wherein the targeting ligand is transferrin. A composition comprising the nanoparticles according to any one of claims 1 to 15 and a suitable solvent, carrier, binder, excipient and/or diluent. 17. The composition of claim 16, wherein the composition is a pharmaceutical composition and the suitable solvent, carrier, binder, excipient and/or diluent is a pharma- ceutically acceptable solvent, carrier, binder, excipient and / or diluent. A nanoparticle according to any one of claims 1 to 15 or a composition according to claim 16 for use in a method of treating a subject having a condition requiring treatment with a chemically or biologically active drug, said method comprising administering said nanoparticle or said composition to said subject. 20. The nanoparticle or composition of claim 18 , wherein the condition is a disease and the chemically or biologically active drug is effective in treating the disease. 20. The nanoparticle or composition of claim 18 , wherein the condition is cancer and the chemically or biologically active drug is a chemotherapeutic compound. 21. The composition of claim 20 , wherein the chemotherapeutic compound is a camptothecin, an epothilone, or a taxane.

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