JP2018058868A - Nanoparticles stabilized with nitrophenylboronic acid compositions - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide nanoparticles for delivering compounds of interest.SOLUTION: Described herein are nanoparticles comprising a polymer containing a polyol coupled to a polymer containing a boronic acid. The nanoparticles are each configured to present the polymer containing a boronic acid to an environment external to the nanoparticle. Targeted versions of the nanoparticles are also described, as are related compositions, methods and systems.SELECTED DRAWING: Figure 1

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 the invention.

(Field of Invention)
The present disclosure relates to carrier nanoparticles, in particular nanoparticles suitable for delivering compounds of interest, and related compositions, methods and systems.

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

  To deliver various classes of biomaterials and biomolecules with the desired biological and / or chemical activity, typically for either pathological examination, therapeutic treatment, or basic biological research, several These methods are known and used.

  As the number of molecules suitable for use as chemical or biopharmaceuticals (eg, drugs, biologics, therapeutic or imaging agents) increases, they are used with compounds of varying complexity, size and chemical nature. Therefore, the development of a suitable delivery system has proven particularly difficult.

  Nanoparticles are structures useful as carriers for the delivery of drugs by various methods of delivery. There are several nanoparticle delivery systems 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 a multifunctional tool for effective and specific delivery of a compound of interest. Specifically, in some embodiments, the nanoparticles described herein are flexible with a variety of sizes, dimensions, and chemistries for a given target, for the delivery and delivery of a wide range of molecules. Can be used as a secure system.

  According to one aspect, nanoparticles comprising a polyol-containing polymer and a nitrophenylboronic acid-containing polymer are described. In the nanoparticles, the boronic acid-containing polymer is coupled with the polyol-containing polymer, and the nanoparticles are 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 nanoparticles as part of or coupled to a polyol-containing polymer and / or a boronic acid-containing polymer.

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

  In accordance with another aspect, a method for delivering a compound to a target is described. The method includes contacting a target with the nanoparticles described herein, wherein the compound is a polyol-containing polymer or boronic acid-containing polymer of the nanoparticles described herein. Included in.

  In accordance with 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 are intercombinable via a reversible covalent bond and that are collected in the nanoparticles described herein containing the compound.

  In accordance with another aspect, a method for administering a compound to an individual is described. The method includes administering to the individual an effective amount of the nanoparticles described herein, wherein the compound is included in a polyol-containing polymer and / or a boronic acid-containing polymer.

  In accordance with another aspect, a system for administering a compound to an individual is described. This system can be interconnected via a reversible covalent bond and is collected in the nanoparticles described herein conjugated with a compound that is administered to an individual according to the methods described herein. At least a polyol-containing polymer and a boronic acid-containing polymer.

  In accordance with another aspect, a method for 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.

  In accordance with another aspect, several boronic acid-containing polymers are described and are described in detail in the following sections of this disclosure.

  In accordance with another aspect, several polyol-containing polymers are described, which are described in detail in the following sections of the disclosure.

  Nanoparticles having a polyol-containing polymer conjugated to a polymer having nitrophenylboronic acid groups are described herein, which lowers the pKa of the nanoparticle and increases its stability.

  Another aspect of the present disclosure provides a description of targeted nanoparticles. This may have only one single target ligand in some embodiments. This can facilitate delivery of the nanoparticle to a specific target, eg, a cell that presents a binding partner for the particle's target ligand. This type of targeted nanoparticles has advantages over nanoparticles with multiple target ligands, such as having a smaller overall size by having fewer surface ligands, as well as avid Through tee-based interactions, fewer ligands mediate non-specific binding than affinity-based interactions. Thus, nanoparticles that contain or carry a therapeutic agent can be successfully targeted to a target configuration (eg, a cell or tissue) using only one target ligand, High target ligand to therapeutic agent ratios can be delivered to the target. This aspect of the described nanoparticles significantly increases the efficiency of producing such therapeutic agents, while also reducing the need to use a large number of expensive antibodies that mediate targeting.

  The nanoparticles and related compositions, methods and systems described herein have been implemented in several ways as flexible molecular structures suitable for carrying compounds of various sizes, dimensions and chemistries. Can be used in form.

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

  The nanoparticles and related compositions, methods and systems described herein may in some embodiments depend on steric stability as well as specific targets such as tissues, specific cell types within tissues, and Even the arrangement between specific cells within a specific cell type can be used as a delivery system characterized by the ability to deliver compounds.

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

  The nanoparticles and related compositions, methods and systems described herein may, in some embodiments, have increased specificity and / or selectivity during targeting and / or It can be used to deliver a compound with increased target recognition of the compound as compared to a particular system.

  The nanoparticles and related compositions, methods and systems described herein may be used in some embodiments for applications where controlled delivery of the compound of interest is desired (such as therapeutic, diagnostic and clinical applications). Including, but not limited to medical applications). Further uses include biological analysis, veterinary applications, and delivery of compounds of interest to non-animal organs, particularly plant organs.

  The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the detailed description and examples below. Other features, objects, and advantages will be 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 present disclosure, and together with the detailed description and examples, illustrate the principles and practices of the present disclosure. Contribute to.
FIG. 2 shows a schematic diagram of a related method for forming a relationship in the absence of boronic acid including nanoparticles and compounds. Panel A shows a schematic diagram of a polyol-containing polymer (MAP, 4) and a compound of interest (nucleic acid) according to the embodiments described herein. Panel B shows nanoparticles formed by a collection of polymers comprising the polyol and compounds shown in panel A. FIG. 2 shows a schematic diagram of nanoparticles and related 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 an embodiment of the present disclosure along with the molecule of interest (nucleic acid). Panel B shows BA-pegylated stabilized nanoparticles formed by the assembly of polymers and compounds shown in Panel A. FIG. 6 illustrates the formation of a complex comprising a polyol-containing polymer and a compound of interest according to embodiments described herein. Specifically, FIG. 3 shows the results of a MAP gel shift assay using plasmid DNA according to an embodiment of the present disclosure. The DNA ladder is loaded in lane 1. Lanes 2-8 show plasmid DNA combined with MAP with increasing charge ratio. 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. 6 illustrates the formation of a complex comprising a polyol-containing polymer and a compound of interest according to embodiments described herein. Specifically, FIG. 4 shows the results of a MAP gel shift assay using siRNA according to an embodiment of the present disclosure. The DNA ladder is loaded in lane 1. Lanes 2-8 show siRNA in combination with MAP with increasing charge ratio. 2 illustrates the properties of nanoparticles according to some embodiments described herein. Specifically, FIG. 5 illustrates particle size (dynamic light scattering (DLS) measurement) to charge ratio as well as zeta potential (property related to the surface charge of the nanoparticles) versus MAP-plasmid nanoparticles according to embodiments of the present disclosure. The figure explaining the plot of a charge ratio is shown. 2 illustrates the properties of nanoparticles according to some embodiments described herein. Specifically, FIG. 6 shows a diagram illustrating a plot of particle size (DLS) to charge ratio as well as charge ratio plots for zeta potential versus BA-pegylated MAP-plasmid nanoparticles according to embodiments of the present disclosure. FIG. 4 shows the salt stability of BA-pegylated MAP-plasmid nanoparticles according to embodiments of the present disclosure. Plot A: 5: 1 BA-PEG + np + 1X PBS after 5 min; Plot B: 5: 1 BA-PEG + np, dialyzed 3X w / 100 kDa + 1X PBS after 5 min; Plot C: 5: 1 pre-pegylated w / BA-PEG + 1X PBS 5 After minutes; Plot D: 5: 1 pre-pegylated w / BA-PEG, dialyzed 3X w / 100 kDa + PBS 5 minutes later. FIG. 6 illustrates in vitro drug delivery to human cells using nanoparticles according to embodiments described herein. Specifically, FIG. 8 shows relative light unit (RLU) pairs, which are a measure of the amount of luciferase protein expressed from pGL3, for MAP / pGL3 transfection into HeLa cells according to an embodiment of the present disclosure. The figure explaining the charge ratio plot is shown. FIG. 6 illustrates delivery of a drug to a target using nanoparticles according to embodiments described herein. Specifically, FIG. 9 shows a diagram illustrating a plot of cell survival versus charge ratio after MAP / pGL3 transfection, according to an embodiment of the present disclosure. Survival data are for the experiment shown in FIG. FIG. 6 illustrates delivery of multiple compounds to a target using nanoparticles according to embodiments described herein. FIG. Specifically, FIG. 10 shows relative light 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 figure explaining the plot of unit (RLU) versus particle species is shown. The term siCON refers to siRNA having a control sequence. FIG. 6 illustrates delivery of a compound to a target using nanoparticles according to embodiments described herein. Specifically, FIG. 11 shows the relative light unit (RLU) pair for the delivery of MAP / siGL3 into HeLa-Luc cells at a charge ratio of 5 +/− according to embodiments described herein. The figure explaining the plot of siGL3 density | concentration is shown. FIG. 4 shows a schematic diagram of the synthesis of boronic acid-containing polymers presenting target ligands according to some embodiments of the present disclosure. Specifically, FIG. 12 shows a schematic diagram for the synthesis of boronic acid-PEG disulfide-transferrin according to an embodiment of the present disclosure. FIG. 2 shows a schematic diagram of the synthesis of nanoparticles according to some embodiments described herein. Specifically, FIG. 13 shows a schematic diagram for the formation of nanoparticles using Campothecin mucin acid polymer (CPT-mucin acid polymer) in water, according to an embodiment of the present disclosure. 2 shows a table summarizing the particle size and zeta potential of nanoparticles formed from CPT-mutic acid polymers conjugated in water prepared according to embodiments of the present disclosure. FIG. 2 shows a schematic diagram of the synthesis of nanoparticles according to some embodiments described herein. Specifically, FIG. 15 shows the formation of boronic acid-pegylated nanoparticles using CPT-mutic acid polymer and boronic acid-disulfide-PEG ₅₀₀₀ in water according to an embodiment of the present disclosure. Figure 3 shows the salt stability of MAP-4 / siRNA stabilized by peg-free, nitropeg-PEG and 0.25 mol% Herceptin-PEG-nitro PBA. 10x PBS was added at the 5 minute time point so that the solution prepared at a charge ratio of 3 (+/-) was in 1x PBS. A and B are (A) targeted MAP-CPT nanoparticles, medium MAP-CPT nanoparticles or targeted MAP- at an increased ratio of Herceptin®-PEG-nitro PBA to nanoparticles in BT-474. Figure 3 shows intracellular uptake of CPT nanoparticles. (B) Medium MAP-CPT nanoparticles or target with or without free Herceptin® (10 mg / ml) in BT-474 cell line (filled bar) and MCF-7 cell line (white bar) 2 shows intracellular uptake of conjugated MAP-CPT nanoparticles. A and B show 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 as a comparison. (A) Plasma concentration of polymer-bound CPT as a function of time. (B) Plasma concentration of unconjugated CPT as a function of time. The biodistribution of MAP-CPT (5 mg CPT / kg) and targeted MAP-CPT (5 mg CPT / kg, 29 mg Herceptin® / kg) nanoparticles after 4 and 24 hours of treatment is shown. Percent injected dose (ID) of total CPT per gram of tumor, heart, liver, spleen, kidney or lung. The biodistribution of MAP-CPT (5 mg CPT / kg) and targeted MAP-CPT (5 mg CPT / kg, 29 mg Herceptin® / kg) nanoparticles after 4 and 24 hours of treatment is shown. Percentage of total CPT unconjugated in each organ in tumor, heart, liver, spleen, kidney and lung. The biodistribution of MAP-CPT (5 mg CPT / kg) and targeted MAP-CPT (5 mg CPT / kg, 29 mg Herceptin® / kg) nanoparticles after 4 and 24 hours of treatment is shown. Total concentration of CPT in plasma. The biodistribution of MAP-CPT (5 mg CPT / kg) and targeted MAP-CPT (5 mg CPT / kg, 29 mg Herceptin® / kg) nanoparticles after 4 and 24 hours of treatment is shown. Percentage of total CPT not conjugated in plasma. (A) MAP-CPT at 4 hours (5 mg CPT / kg) (B) MAP-CPT at 24 hours (5 mg CPT / kg) (C) Targeted MAP-CPT at 4 hours (5 mg CPT / kg, 29 mg Herceptin® / kg) (D) BT-474 tumor taken from NCr nude mice treated with targeted MAP-CPT (5 mg CPT / kg, 29 mg Herceptin® / kg) at 24 hours Figure 2 illustrates a confocal immunofluorescence micrograph of a section. The left panel emits 440 nm (CPT, pink), the center left panel emits 519 nm (MAP, green), the center right panel emits 647 nm (Herceptin (registered trademark), blue), and the right panel overlays the image. Figure 9 shows anti-tumor efficacy in NCr nude mice with BT-474 xenograft tumors. Average tumor volume as a function of time, group containing Herceptin dosing once every 2 weeks, all other groups received dosing once every 3 weeks. 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), Shows anti-tumor efficacy.

  Nanoparticles and compositions, methods and systems that can be used in connection therewith, methods of producing the described nanoparticles, methods of treatment using the described nanoparticles, and described, for delivering compounds contained in the nanoparticles Kits for collecting nanoparticles are provided herein.

  The term “nanoparticle” as used herein refers to a composite structure of nanoscale dimensions. Specifically, the nanoparticles are typically particles with a size in the range of about 1 to about 1000 nm and are 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 limitation is limited in two dimensions, and thus the nanoparticles described herein include composite structures having a diameter of about 1 to about 1000 nm. Here, this 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 are typically And having a diameter of about 1 to about 100 nm. The term “targeted nanoparticle” refers to a nanoparticle conjugated to a target agent or target ligand.

Further desirable properties of the 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. Additional features will be appreciated by persons of ordinary skill in the art upon reading this disclosure. The size and properties of the nanoparticles can be detected by techniques known in the art. Examples of techniques for detecting particle size include, but are not limited to, dynamic light diffusion (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, the zeta potential zeta potential method. Further techniques are understood by 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 those skilled in the art Suitable for detecting other chemical properties that are included by further techniques to be performed.

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

  The terms “delivering” and “delivery”, as described herein, indicate an activity that affects the spatial arrangement of a compound, in particular, such arrangement. Thus, the delivery of a compound in the sense of the present disclosure affects the positioning and movement of the compound under a particular time and a particular set of conditions, so that the positioning and movement of this compound under these conditions , Otherwise indicates the ability of this compound to be altered with respect to positioning and movement.

  Specifically, with respect to the reference endpoint, delivery of the compound indicates the ability of the compound to be placed at the final selected reference endpoint by controlling the positioning and movement of the compound. In in vitro systems, delivery of a compound usually involves 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 alterations in the pharmacokinetics of the compound, and may be accompanied by alterations in pharmacodynamics.

  The pharmacokinetics of a compound show the absorption, distribution, metabolism and excretion of this compound, typically from the body of the individual, from the system. In particular, the term “absorption” refers to the process by which a substance enters the body, the term “distribution” refers to the dispersion or dissemination of the substance through bodily fluids and tissues, and the term “metabolism” refers to the parent compound. Indicates irreversible conversion to daughter metabolites, as well as the term “excretion” indicates the elimination of substances from the body. If the compound is in a formulation, pharmacokinetics also includes the release of the compound from the formulation, which describes the process of release of the compound, typically a drug, from the formulation. Show. The term “pharmacodynamics” refers to the physical effect of a compound, the mechanism of drug action and the relationship between drug concentration and effect on the body or on microorganisms or in parasites within or on the body. One skilled in the art can identify techniques and procedures suitable for the pharmacokinetic and pharmacodynamic characteristics and properties of the compound of interest and, in particular, the drug of interest, eg, drug.

  The term “agent” as used herein refers to a compound capable of exhibiting a chemical or biological activity associated with a target. The term “chemical activity” as used herein refers to the ability of a molecule to perform 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 drugs include covalent bond formation or electrostatic interactions. Examples of biological activity of drugs include the production and secretion of endogenous molecules, the absorption and metabolism of endogenous or exogenous molecules, and the activation or inactivation of gene expression, including transcription and translation of the gene of interest. .

  The term “target” as used herein includes unicellular organisms or multicellular organisms or any portion thereof and includes in vitro or in vivo biological systems or any portion thereof. Includes biological systems.

  The nanoparticles described herein, boronic acid-containing polymers attached to polyol-containing polymers, are arranged in the nanoparticles so that they are presented external to the environment of the nanoparticles.

  The term “polymer” as used herein refers to a large molecule composed of repeating structural units, typically joined by covalent chemical bonds. Suitable polymers may be linear and / or branched and may take the form of a homopolymer or a copolymer. If a copolymer is used, the copolymer may be a random copolymer or a branched copolymer. 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 use and applications, the polymer must have a low toxicity profile, in particular low toxicity or cytotoxicity. Suitable polymers include polymers having a molecular weight of about 500,000 or less. Specifically, suitable polymers can 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 presents a number of hydroxyl functional groups. In particular, polyol-containing polymers suitable for forming the nanoparticles described herein include at least hydroxyl functional groups due to coupling interactions with at least one boronic acid of the boronic acid-containing polymer. Includes polymers that present some.

  The term “presenting” as used herein with respect to a compound or functional group indicates a bond that is made to maintain the chemical reactivity of the bound compound or functional group. Thus, the functional group presented on the surface can undergo one or more chemical reactions that chemically characterize the functional group under appropriate conditions.

The 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 are diols, and in particular diols of the general formula HO— (CH ₂ CH ₂ O) _p —H having p ≧ 1, such as polyethylene glycol, polypropylene glycol, and poly (tetramethylene ether) ) Glycols, but not limited to. 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 having a molecular weight of about 500,000 or less, particularly 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 skilled in the art. Examples of procedures for the synthesis of polyol polymer examples have been previously described in the scientific literature, others are described in Examples 1-4. Additional procedures for making polyol-containing polymers can be identified by those skilled in the art in view of the present 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 that is presented for bonding to a hydroxyl group of a polyol-containing polymer. Specifically, the nanoparticulate boronic acid-containing polymers described herein include polymers comprising at least one structural unit of an alkyl or aryl substituted boronic acid containing a chemical bond between carbon and boron. Suitable BA polymers include those in which the boronic acid is present in the terminal structural unit or any other suitable moiety and imparts hydrophilic properties to the resulting polymer. Examples of polyol-containing polymers include polymers having a molecular weight of about 40,000 or less, particularly 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 skilled in the art. Examples of procedures for the synthesis of polyol polymer examples have been previously described in the scientific literature, others are described in Examples 5-8. Additional procedures for making BA polymers can be identified by those skilled in the art in view of the present disclosure.

  In the nanoparticles described herein, the polyol polymer is coupled with the BA polymer. The term “coupled” or “coupled” as used herein with respect to a bond between two molecules indicates 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 via a covalent bond interaction that is rapidly and reversibly coupled to a suitable medium. Forms boronate ester in it. Suitable media include water and some aqueous solutions and additional organic media that can be identified by one skilled in the art. Specifically, when contacted in an aqueous medium, the BA polymer and polyol react to produce water as a byproduct. This boronic acid polyol interaction is generally more preferred in aqueous solution, but is known to proceed in organic media. Furthermore, cyclic esters formed by 1,2 diols and 1,3 diols are generally more stable than their acyclic 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 boron-11 nuclear magnetic resonance ( ¹¹ B NMR), depending on, for example, the specific chemical properties and properties of the boronic acid and polyol that make up the nanoparticles. It can be detected by methods and art identified by those skilled in the art such as potentiometric titration, selected UV / visible light and fluorescence detection techniques.

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

In some embodiments, the compound of interest carried by the nanoparticles forms part of a BA polymer and / or polyol polymer. An example of such an embodiment is that one or more atoms of the polymer have been replaced by specific isotopes, such as specifically ¹⁹ F and ¹⁰ B, and thus for target imaging and / or Provided by nanoparticles, which are suitable as agents for providing radiation treatment to a target.

  In some embodiments, the compound of interest delivered to the nanoparticles is attached to the polymer, typically a polyol polymer, either covalently or non-covalently. An example of such an embodiment is provided by nanoparticles in which one or more moieties in at least one of the compound polyol polymer and BA polymer are bound to one or more purposes.

  The terms “bond”, “bonded” or “bond”. As used herein, refers to a connection or integration by coupling, coupling, force or coupling to keep two or more components together, as this includes direct or indirect coupling Embodiments in which, for example, the first compound binds directly to the second compound, as well as one or more intermediate compounds, particularly intermediate molecules, are placed between the first compound and the second compound. including.

Specifically, in some embodiments, the compound can be attached to the polyol polymer or BA polymer via a covalent bond of the compound to a suitable portion of the polymer. Examples of covalent linkages include Example 19 where the attachment of the drug camptothecin to the mucin polymer is via a biodegradable ester linkage linkage, as well as the transferrin attachment to BA-PEG ₅₀₀₀ via transferrin pegylation. Example 9 will be described.

  In some embodiments, the polymer can be coupled to a specific compound of interest (eg, one or more functional groups capable of specifically binding to the corresponding functional group on the compound of interest). (By addition) can be designed or modified. For example, in some embodiments, nanoparticles can be PEGylated with BA-PEG-X, where X reacts specifically with thiols or non-specifically with amines. , A maleimide group or an iodoacetyl group or a free radical. The coupled compound can then react with a maleimide group or an iodoacetyl group after modification to express the thiol functionality. The compounds to be bound may also be modified by aldehyde or ketone groups, which can react via condensation reactions with diols on the polyol to give acetals or ketals.

  In some embodiments, the compound of interest is bound to the polyol polymer or BA polymer via non-covalent bonds, such as ionic bonds and intermolecular interactions, between the compound to be bound and a suitable portion of the polymer. obtain. An example of non-covalent bonding is described in Example 10.

  The compound of interest can be bound to the nanoparticles before, during, or after the formation of the nanoparticles, for example, via modification of the polymer and / or modification of any bound compound in the particle hybrid . An example of a procedure for binding compounds in nanoparticles is described in the Examples section. Additional procedures for compound attachment to BA polymers, polyol polymers, or other components of the nanoparticles described herein (eg, pre-introduced compounds of interest) will occur to those skilled in the art upon reading this disclosure. Can be identified by

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

  The term “targeting ligand” or “targeting agent” as used in this disclosure is intended for the purpose of engaging a particular target, as well as in particular a specific by, for example, allowing the cell receptor binding of nanoparticles. Any molecule that can be presented on the surface of a nanoparticle for the purpose of cell recognition. Examples of suitable ligands include, but are not limited to, vitamins (eg, folic acid), proteins (eg, transferrin and monoclonal antibodies), peptides and polysaccharides. Specifically, the target ligand may be an antibody against a particular surface cell receptor, eg, a small molecule such as anti-VEGF, folic acid, as well as other proteins such as holo-transferrin.

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

  In some embodiments, at least one compound of a polyol polymer and / or BA polymer (including a target ligand) is delivered to a target, eg, an individual, that exerts a chemical or biological activity, eg, therapeutic activity. It may be a drug, in particular a drug.

  The polyol polymer and BA polymer sections to form nanoparticles described herein can be implemented in terms of the compound of interest and target. Specifically, the sections of suitable polyol-containing polymers and suitable BA polymers to form nanoparticles described herein provide candidate polyol polymers and BA polymers and couplings in light of the present disclosure. Can be implemented by selecting a polyol polymer and a BA polymer capable of forming an interaction, wherein the selected BA polymer and polyol polymer are included in or bound to the polyol polymer and / or BA polymer In terms of the compound and the target ligand, the polyol polymer has a chemical composition that is more hydrophilic than the BA polymer. Detection of the BA polymer on the surface nanoparticles and the relevant presentation in the environment external to the nanoparticles is performed by detection of zeta potential, which can perform surface modification of the nanoparticles, as described in Example 12. Can be done. (See FIG. 6.) Additional procedures for detecting particle surface charge and particle stability in salt solution include those exemplified in Example 12 (see FIG. 7 for details). Detection of particle size charges such as）) as well as additional procedures that can be identified by those skilled in the art.

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

Where
A is the following formula:

here,
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 comprising one or more of -H, -F, -C, -N or -O; and Y is -OH or an organic having a hydroxyl (-OH) group These include the moieties (—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. Without limitation, wherein R ₁ G ₁ , R ₁ G ₂ and R ₁ G ₃ are independently selected from organic-based functional groups),
And B is an organic moiety that connects one of _{R 1} and _{R 2} of the first part A to one of _{R 1} and _{R 2} of the second A portion.

  The term “moiety” as used herein refers to a series of atoms that make up a part of a larger molecule or molecular species. Specifically, a part refers to a constituent of a repeating polymer structural unit. Exemplary moieties include acid or base species, sugars, carbohydrates, alkyl groups, aryl groups and any other molecular components useful for forming polymer structural units.

  The term “organic moiety” as used herein refers to a moiety that includes a carbon atom. Specifically, organic moieties include natural and synthetic compounds, and compounds containing 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. A synthetic organic group refers to a compound prepared by reaction with another compound.

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

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

Where
d is 0-100;
e is 0-100;
f is 0-100;
Z is a covalent bond linking one organic moiety to another organic moiety, specifically, moiety A or moiety B as defined herein, and Z ₁ is —NH ₂ , —OH, Independently selected from -SH and -COOH.

In some embodiments, Z is —NH—, —C (═O) NH—, —NH—C (═O), —SS—, —C (═O) O—, —NH (═NH ₂ ⁺ )-or -O-C (= O)-.

In some embodiments where the structural unit A of the polyol-containing polymer has the formula (IV), X may be C _v H _{2v + 1} , where v = 0-5 and Y is − OH.

In some embodiments, R 1 and / or R 2 has the formula (V), wherein Z is —NH (═NH ₂ ⁺ ) — and / or Z ₁ is NH ₂ . is there.

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

Selected independently from
Where
The spacer is independently selected from any organic moiety, in particular may include heteroatoms such as alkyl, phenyl, or alkoxy groups containing a heteroatom 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 1-20; and Z ₁ is independently selected from —NH ₂ , —OH, —SH and —COOH.

  In some embodiments, Z1 and NH2 and / or 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 can have the following formula:

Can be selected independently.

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

  In some embodiments, (B) can 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 is related to the chemical nature of the compound that is covalently or non-covalently linked. Dependent.

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

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

  Specifically, one or more cationic or anionic moieties of (B) for use with anionic and cationic cargo, respectively, can independently have the following general formula:

In the formula, R ₅ is an electrophilic group that can be covalently bonded to A when A contains a nucleophilic group. R ₅ in this case includes, but is not limited to:
Among other several, -C (= O) OH, -C (= O) Cl, -C (= O) NHS, -C (= NH 2 +) OMe, -S (= O) OCl- , _-CH 2 Br, alkyl and aromatic esters, terminal alkyne, tosylate, and mesylate. When moiety A contains an electrophilic end group, R ₅ has a nucleophilic group such as —NH ₂ (primary amine), —OH, —SH, N ₃ and secondary ain.

In particular, when the moiety (B) is a cationic moiety (B) used with an anionic cargo, the “organic group” is generally composed of C _m H _2m and other heteroatoms where m ≧ 1 It may have a backbone having the formula and must contain at least one of the following functional groups: amidine of formula (XIII), quaternary ammonium of formula (XIV), primary of formula (XV) An amine group, a secondary amine group of formula (XVI), a tertiary amine group of formula (XVII) (protonated below its pKa), and an imidazolium of formula (XVIII).

In embodiments where moiety (B) is an anionic moiety (B) used with a cationic cargo, the “organic group” has the general formula consisting of C _m H _2m and other heteroatoms where m ≧ 1. And must contain at least one of the following functional groups: sulfonate of formula (XIX), nitrate of formula (XX), carboxylate of formula (XXI), and formula (XXII) Phosphonate.

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

In embodiments where (B) is composed of a primary amine group of formula (XV) and / or a secondary amine group of formula (XVI), the compound comprising the carboxylic acid group is capable of forming a peptide bond. Can be coupled via.

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

here,
q is 1-20; and in particular may 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 an anisotropic bond sequence. Specifically, the leaving group is an anion or a neutral molecule, and the ability of the leaving group to separate correlates with the pK _a of the conjugate acid, with a lower pK _a associated with better leaving group ability. . Examples of anionic compounds include halides and sulfonate esters such as Cl ⁻ , Br ⁻ and I ⁻ , such as para-toluene sulfonate or “tosylate” (TsO ⁻ ). Examples of neutral molecular leaving groups are water (H ₂ O), ammonia (NH ₃ ) and alcohol (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:

Where is the organic part of
n is 1 to 20; and, in particular, 1 to 4.

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

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

Where
R ₃ and R ₄ are independently selected from any hydrophilic organic polymer, and in particular may independently be any poly (ethylene oxide) and zwitterionic polymer,
X ₁ is an organic moiety containing one or more of —CH, —N or —B;
Y ₁ may optionally be an olefin group or an alkynyl group, or an alkyl group having the formula —C _m H _2m — where m ≧ 1, including an aromatic group (eg, phenyl, biphenyl, naphthyl or anthracenyl). ,
r is 1-1000;
a is 0-3; and b is 0-3;
And where functional group 1 and functional group 2 are the same or different and can bind to a target ligand, in particular, a protein, antibody or peptide, or —OH, —OCH ₃ or — ( X ₁ ) — (Y ₁ ) —B (OH) ₂ — and other terminal groups.

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

In some embodiments, X ₁ is —NH—C (═O) —, —S—S—, —C (═O) —NH—, —O—C (═O) — or —C ( = O) -O- and / or Y ₁ is a phenyphenyl 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: —B (OH) ₂ , —OCH ₃ , —OH.

In particular, functional group 1 and / or 2 of formula (XXXI) may be a functional group capable of binding to cargo and in particular may be a target ligand, for example a protein, antibody or peptide Or an end group such as —OH, —OCH ₃ 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 responsible for characteristic chemical reactions 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 those skilled in the art. In particular, functional groups within the meaning of the present disclosure include carboxylic acids, amines, triarylphosphines, azides, acetylenes, sulfonyl azides, thioazides and aldehydes. Specifically, for example, functional groups capable of binding to corresponding functional groups in the target ligand can be selected to include the following binding partners: carboxylic acid groups and amine groups, azide and acetylene groups, azide 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. Accordingly, functional groups that can react with each other can be referred to as corresponding functional groups.

The terminal group indicates a structural unit that is a terminal of a micromolecule or an oligomer molecule. For example, the end groups of PET polyester can be alcohol groups or carboxylic acid groups. End groups can be used to determine molar mass. Examples of end groups include: —OH, —COOH, NH ₂ and OCH ₃ .

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

In the formula, s is 20 to 300.

  Examples of drugs and target ligands that can bind to the nanoparticles of the present disclosure include organic or inorganic molecules, including polynucleotides, nucleotides, aptamers, polypeptides, proteins, polysaccharides, macromolecular complexes (proteins And polynucleotides, sugars and / or polysaccharides, viruses, molecules with radioisotopes, antibodies or antibody fragments).

  The term “polynucleotide” as used herein refers to an organic polymer composed of two or more monomers comprising nucleotides, nucleosides or analogs thereof. The term “nucleotide” refers to any compound 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 a nucleic acid. The term “nucleoside” refers to a compound consisting of a purine or pyrimidine base combined with deoxyribose or ribose (eg, guanosine or adenosine), and is particularly found in nucleic acids. The term “nucleotide analog” or “nucleoside analog” refers to a nucleotide or nucleoside in which one or more individual atoms are replaced with different atoms or different functional groups, respectively. Thus, the term “polynucleotide” includes nucleic acids of any length, in particular DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also referred to as a “nucleotide oligomer” or “oligonucleotide”.

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

  The term “polynucleotide” as used herein refers to an organic linear, circular or branched polymer composed of two or more amino acid monomers and / or analogs thereof. Hyogo “polypeptides” include amino acid polymers of any length, including full-length proteins and peptides, and 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 less than 50 amino acid monomers. As used herein, the term “amino acid”, “amino acid monomer” or “amino acid residue” refers to any of the 20 naturally occurring amino acids, non-naturally occurring amino acids, and artificial amino acids, Includes both D and L optical isomers. Specifically, non-naturally occurring amino acids include 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 acids” can be easily coupled using standard amino acid coupling science, The molecule | numerator which has the molecular structure which does not resemble the amino acid which exists in is shown. The term “amino acid analog” is an amino acid in which one or more individual atoms are replaced with either different atoms, isotopes or different functional groups, but otherwise identical to the original amino acid from which the analog is derived. Say. All these amino acids can be synthetically incorporated into a peptide or polypeptide using standard amino acid coupling chemistry. The term “polypeptide” as used herein includes polymers comprising one or more monomers or building blocks other than amino acid monomers. The term monomer, subunit, or building block refers to a chemical compound that can be chemically combined with another monomer of the same or different chemistry under appropriate conditions to form a polymer. The term “polypeptide” is further contemplated to include polymers in which one or more building blocks are covalently linked to each other by chemical bonds other than amide bonds or peptide bonds.

  The term “protein” as used herein is a specific that can participate in interactions with other biomolecules including other proteins, DNA, RNA, lipids, metabolites, hormones, chemokines and small molecules. Polypeptides having the secondary and tertiary structures of are shown below, but are not limited thereto. An example of a protein described herein is an antibody.

  The term “antibody” as used herein is a class of proteins that are produced by activated B cells following stimulation with an antigen and that can specifically bind to the antigen and promote an immune response in a biological system. Say. 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 and the like. Examples of fragments include Fab Fv, Fab'F (ab ') 2, and the like. A monoclonal antibody is an antibody that is capable of binding specifically to one particular spatial and polar mechanism of another biomolecule, termed an “epitope” and is therefore defined as being complementary. In some forms, the monoclonal antibodies may also have the same structure. Polyclonal antibody refers to a mixture of different monoclonal antibodies. In some forms, the polyclonal antibody may be a mixture of monoclonal antibodies in which at least two monoclonal antibodies bind to different antigenic epitopes. Different antigenic epitopes may be for the same target, different targets or combinations. Antibodies can be prepared by techniques well known in the art, such as host immunization and serum collection (polyclonal), or continuous hybridoma cell line preparation and secretion protein collection (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 illustrations of FIGS.

  In some embodiments, the nanoparticle structure comprises a drug and a polyol-containing polymer that is linked to the polyol polymer by a covalent bond. Examples of polyol polymers conjugated to drugs are described in detail in Examples 16-21. In these embodiments, a polyol polymer conjugated to a drug (referred to herein as a “polyol polymer-drug complex”) forms nanoparticles, and this structure is formed on the surface of BA molecules. Present the site for interaction with.

  In some embodiments, the nanoparticles further comprise a BA polymer formed to provide steric stability and / or target functionality of the nanoparticles. Specifically, in these embodiments, the addition of BA polymer allows for self-aggregation and minimization of unwanted interactions with other nanoparticles, thus providing improved salt and serum stability. . For example, steric stability is provided herein by BA polymers with PEG, as illustrated by the example of nanoparticles described 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 bond between the drug and the polyol polymer. Those skilled in the art will appreciate other connection possibilities suitable for this purpose. In these embodiments, another advantage is to provide a specific target for the drug via the BA polymer moiety.

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

  In some embodiments, the nanoparticle structure comprises a drug and a polyol polymer, wherein the nanoparticle is a modified liposome. In these embodiments, the modified liposome comprises a lipid conjugated with a polyol polymer via a covalent bond, whereby the surface of the liposome presents the polyol polymer. In these embodiments, the modified liposomes configured to deliver the drug are contained within the liposome nanoparticles.

  The term “liposome” as used herein refers to a vesicular structure composed of lipids. Lipids typically have a tail group that includes a long hydrocarbon chain and a hydrophilic head group. The lipids are arranged to form a lipid bilayer, an aqueous environment suitable for containing the drug to be delivered inside. Such liposomes present an outer surface that may contain molecules for specific recognition by a suitable target ligand or cell surface receptor or other target of interest.

  The term “conjugated” as used herein, one molecule forms a covalent bond with a second molecule, and single and multiple (eg, double) bonds (eg, C = C-C = C-C) and interact with each other, indicating electron delocalization.

  In still other embodiments of the present disclosure, the nanoparticle structure comprises a drug and a polyol, wherein 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 a lipid. Representative micelles in aqueous solution form aggregates with a hydrophilic “head” region in the area in contact with the surrounding solvent, isolating the hydrophobic single tail region at the micelle center. In the present 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 positioned to form nanoparticles that are modified micelles with the drug delivered within the nanoparticle when mixed with the drug to be delivered. Such nanoparticle embodiments present a suitable polyol polymer on the surface for interacting with the BA polymer. The BA polymer has or does not have the target functionality according to the previous embodiment. In these embodiments, BA polymers that can be used for that purpose include those having hydrophilic A and hydrophobic B in formula (I) or (II). This interaction provides similar or similar advantages as provided for the other nanoparticle embodiments described above.

  In some embodiments, nanoparticles or related components may be included in the composition together with an acceptable vehicle. The term “vehicle” as used herein refers to various media that act as solvents, carriers, binders, excipients or diluents for the nanoparticles that are usually included in the composition as the active ingredient. Indicates one of the following.

  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 pharmaceutically acceptable vehicle.

  In some embodiments, the nanoparticles may be included in the pharmaceutical composition together with an excipient or diluent. In particular, in some embodiments, the pharmaceutical composition comprises nanoparticles in one or more compatible and pharmaceutically acceptable vehicles, in particular pharmaceutically acceptable diluents or excipients. Including in combination.

  The term “excipient” as used herein refers to an inert substance used as a carrier for the active ingredient of a medicament. Suitable excipients for the pharmaceutical compositions disclosed herein include any substance that enhances the body's ability to absorb the nanoparticles. Suitable excipients also include any material that can be used to bulk up formulations containing nanoparticles to allow convenient and accurate dosing. In addition to its use in a single dose, excipients may be used in the manufacturing procedure to aid in the manipulation of the nanoparticles. Depending on the route of administration and pharmaceutical form, different excipients can be used. Examples of excipients include anti-blocking agents, binders, coating disintegrants, fillers, flavoring agents (eg, sweeteners) and colorants, lubricants, lubricants, preservatives, adsorbents, It is not limited to these.

  The term “diluent” as used herein refers to a diluting agent that is provided to dilute or carry the active ingredient of the composition. Suitable diluents include any substance that can reduce the viscosity of the pharmaceutical preparation.

  In certain embodiments, the composition, particularly the pharmaceutical composition, can 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, a composition for parenteral administration is prepared by using a powdered composition previously prepared in freeze-dried lyophilized form as a biologically compatible aqueous liquid (distilled water). , Physiological solutions or other aqueous solutions).

  The term “freeze drying” (freeze drying or low temperature drying) refers to a dehydration process that is typically used to store a perishable material or make it easier to transport. Freeze drying is accomplished by freezing the material, then reducing the ambient pressure and applying sufficient heat to allow the frozen water in the material to digest directly from the solid phase to the gas.

  In pharmaceutical applications, freeze drying is often used to extend the expiration date of products such as vaccines and other injectable drugs. By removing water from the material and sealing the material in a vial, the material can be easily reconstituted to its original form for storage, transport 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, the method is provided for delivery of a drug to an individual, wherein the method includes formulating suitable nanoparticles according to various embodiments of the present disclosure. The nanoparticles may also be formulated into a pharmaceutically acceptable composition according to some embodiments of the present disclosure. The method includes delivering nanoparticles to the subject. To deliver the nanoparticles to an individual, the nanoparticles or nanoparticle formulation may be given orally, parenterally, topically, or rectally. . These are delivered in a form suitable for each route of administration. For example, the nanoparticle composition may be administered in the form of pills or capsules, administered by injection, inhalation, eye drops, ointment, suppository, infusion, or rectally by suppository.

  The term “individual” as used herein includes one organism, which includes a plant or an animal, in particular a higher animal, in particular a spine. Examples include animals such as mammals, and details include, but are not limited to, humans.

  The phrases “parenteral administration” and “administered parenterally” as used herein refer to modes of administration, usually by injection, other than enteral and topical administration, and are not limited. Intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, epidermal, intraarticular, subcapsular, subarachnoid, Intramedullary and intrastemal injection and infusion are included.

  The phrases “systemic administration”, “systemically administered”, “peripheral administration” and “peripherally administered” as used herein refer to nanoparticles or compositions thereof within the central nervous system. By administration other than by direct administration, it is meant administration that enters the individual's system and thus undergoes a process such as metabolism, eg, subcutaneous administration.

  The actual dosage level of the active ingredient or drug in the pharmaceutical compositions described herein will achieve the desired therapeutic response for a particular individual, composition and mode of administration without being toxic to that individual. In order to obtain an amount of active agent effective to do, it can vary.

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

  Regardless of the route of administration chosen, suitable hydrated forms and / or therapeutic polymer conjugates that can be used in the pharmaceutical form of the present invention can be prepared by conventional methods known to the potters in a pharmaceutically acceptable dosage form. It can be formulated in form.

  Specifically, in some embodiments, the delivered compound is a drug for treating or preventing an individual's symptoms.

  A `` drug '' or `` therapeutic agent '' can be used in the treatment, prevention or diagnosis of symptoms in an individual, or else can be used to enhance a physical or mentally good condition of an individual. Indicates active agent.

  The term “symptom” as used herein usually refers to an individual as a whole or in one or more parts thereof, usually associated with a complete, physical, mental and possibly social condition. Indicates the physical state of an individual's body as a whole or in one or more parts thereof that is incompatible with the physical state. Symptoms described herein include, but are not limited to, disorders and diseases. Here, the term “disorder” refers to a symptom of a living individual with a dysfunction of the body or any part thereof, and the term “disease” refers to a loss of normal function of the body or any part thereof. , Typically indicates a symptom of a living individual that is manifested by an identifying sign or symptom. Examples of symptoms include injuries, dysfunctions, disorders (including mental and physical disorders), syndromes, infections, an individual's abnormal behavior, and abnormal changes in the structure and function of the individual's body or parts thereof. However, it is not limited to these.

  “Treatment” as used herein refers to any activity that is part of a medical treatment, either 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 an individual's symptoms. 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 causing disease progression. And increase the chances of interventions to prevent the appearance of symptoms; and (c) tertiary prevention reduces the adverse effects of disease that has already developed by restoring function and reducing disease-related complications.

Examples of compounds that are delivered by the nanoparticles described herein and that are suitable as drugs are compounds that emit electromagnetic radiation (eg, ¹⁰ B isotopes) and can be used in radiation treatments (eg, boron neutrons). Supplement). 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. et al. Examples of such therapeutic agents include small molecule pharmaceuticals, antibiotics, steroids, polynucleotides (eg, genomic DNA, cDNA, mRNA, siRNA, shRNA, miRNA, antisense oligonucleotides, viruses and chimeric polynucleotides), plasmids , Peptides, peptide fragments, small molecules (eg, doxorubicin), chelating agents (eg, deferoxamine (DESFERAL)), ethylenediaminetetraacetic acid (EDTA), natural products (eg, taxol, amphotericin), and other biologically Examples include, but are not limited to, active macromolecules such as proteins and enzymes. See also US Pat. No. 6,048,736, which lists active agents (therapeutic agents) that can be used as therapeutic agents with the nanoparticles described herein. The small molecule therapeutic agent is not only a therapeutic agent in the hybrid particle, but in a further embodiment it can be covalently bound to the polymer in the hybrid. In some embodiments, the covalent bond is reversible (eg, through a biodegradable linkage, such as a prodrug form or a disulfide bond), providing another method of delivering a therapeutic agent. In some embodiments, therapeutic agents that can be delivered by the nanoparticles described herein include chemotherapeutic agents such as epothilone, camptothecin-based drugs, taxol, or plasmids, siRNA, shRNA, miRNA, anti Nucleic acids such as sense oligonucleotide aptamers or combinations thereof, as well as other drugs that can be identified by one of ordinary skill in the art having read this disclosure.

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

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 may be present are on the BA polymer component, on the polyol polymer component, or on the drug being 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 agent delivered by the nanoparticles described herein is a biologically active compound with microbiological and agricultural utility. These biologically active compounds include those known in the art. For example, suitable agrobiologically active compounds include, but are not limited to, fertilizers, fungicides, herbicides, insecticides and powdery mildew fungicides. Microbicides can also be used to treat municipal and industrial water (eg cooling water, white water systems in papermaking) systems. Aqueous systems that are 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 are described individually and in combination in US 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 so that the active ingredient situation is known. In particular, because such use is not pharmaceutical use, the hybrid polymer need not conform to the toxicity profile required in 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 that use the nanoparticles. . In some embodiments of this system, the nanoparticles provide a suitable component for preparing nanoparticles for administration to an individual.

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

  In a partial kit, the polyol polymer, BA polymer and / or drug to be delivered is included in the kit, possibly in a composition, possibly together with a suitable vehicle carrier or adjuvant. For example, a polyol polymer and / or BA polymer may be included only in one or more compositions and / or included in a suitable vector. The agent to be delivered may also be included in the composition together with a suitable vehicle carrier or adjuvant. Alternatively, the drug may be given by the end user and not included in the partial kit. In addition, the polyol polymer, BA polymer and / or drug can be included in various forms suitable for proper incorporation into the nanoparticles.

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

  In the kit of parts disclosed herein, the components of the kit may be provided with suitable interactions and other reagents essential for performing the methods disclosed herein. In some embodiments, an individual kit may include the composition in a separate container. Instructions for performing the assay, eg written or audio, on paper or electronic support (eg tape or CD-ROM) may also be included in the kit. The kit may also include other packaging reagents and materials, such as wash buffers, depending on the particular method used.

  Further details regarding suitable carrier agents or adjuncts of the compositions, and generally the manufacture and packaging of kits, can be identified by those of ordinary skill in the art having read this disclosure.

  In some embodiments, the nanoparticles are prepared by preparing individual components of the nanoparticles and then mixing the components in various orders to arrive at the desired hybrid nanoparticle structure. Can be done. The preparation and mixing of the components is performed in suitable solutions 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 and another solution containing another molecule of interest. For example, in the context of the present disclosure, an aqueous solution of a polyol polymer can be mixed with an aqueous solution of a BA polymer.

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

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

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

  In some embodiments, nanoparticles can be prepared by preparing modified micelles according to embodiments of the present disclosure by mixing a polyol polymer with a hydrophobic polymer block having the drug to be delivered. In other embodiments, the nanoparticles can be prepared by further mixing the modified micelles with a BA polymer. In still other embodiments, the nanoparticles can be prepared by preparing nanoparticles that are modified micelles by mixing the polyol polymer and the BA polymer, followed by mixing the agent to be delivered.

  In some embodiments of the present disclosure, nanoparticles can be prepared by mixing a lipid conjugated to a polyol polymer with a BA polymer and / or an agent to be delivered to prepare a modified liposome. In various embodiments, nanoparticles can be prepared by mixing a lipid conjugated with a polyol polymer with a BA polymer and then mixing the agent to be delivered. In other embodiments, the nanoparticles can be prepared by mixing an agent that delivers a lipid conjugated with a polyol polymer. In other embodiments, the nanoparticles are prepared by mixing a polyol polymer and a lipid conjugated with a drug to be delivered, followed by mixing with a BA polymer to prepare nanoparticles that are modified liposomes. Can be done.

  Nanoparticle formation according to some embodiments of the present disclosure can be analyzed by techniques and procedures known to those skilled in the art. For example, in some embodiments, the gel shift method is used to monitor and measure the incorporation of nucleic acid drugs into 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 adjuncts of the compositions, and generally the manufacture and packaging of kits, can be identified by those of ordinary skill in the art having read this disclosure.

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

A is the following formula:

Wherein 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 —H, —F, —C, —N Or selected from aliphatic groups containing one or more of -O; and Y is independently selected from organic moieties presenting -OH or -OH, and B is R ₁ and R of the first moiety A an organic moiety that connects to one of the _2, with R ₁ and R ₂ of the second portion a in the polymer. In some embodiments, X may be C _n H _{2n + 1} , where n is any one number from 0 to 5 and Y is —OH. In some embodiments, A can be any one of the following:

Wherein the spacer is independently selected from any organic group; the amino acid is selected from an organic group having a free amine and a free carboxylic acid group; n is any single number from 1 to 20 Yes; and Z ₁ is independently selected from —NH ₂ , —OH, —SH and —COOH; R ₁ and R ₂ may independently have the following formula:

Where 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, and Z is 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 is any one of May be:

Where q is any single number from 1 to 20; p is any single number from 20 to 200; and L is a free radical, and these B subunits are defined above. Paired with any one of the A subunits. In a more detailed embodiment, the polymer containing the polyol nanoparticle segment of the targeted nanoparticle shown in the structural unit of formula (I) is:

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

And the polymer containing the polyol nanoparticle segment of the targeted nanoparticle represented by the structural unit of formula (III) is:

And
Here, n is an arbitrary 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 Formula A (Formula VI, VII or VIII) for subpart A can be combined with any of Formulas for Formula B (Formula XXIII or XIV) for Subpart B to produce a polyol-containing polymer of the described nanoparticles. Can be formed. In certain aspects described herein, the nanoparticles can be any one of formulas (IX, X or XI) for subpart A and any of formulas (Formula XXIII or XIV) for subpart B It may have a polyol-containing polymer formed from one combination.

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

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

Here, s is an arbitrary single number of 20 to 300. The polymers of formula XXXIII, XXXIV and XXXV can be further modified to change the position of 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 for formula A (formula VI, VII or VIII) for subpart A is combined with any one of formulas for formula (XXIII or XIV) for subpart B, Polyol-containing polymer nanoparticle segments can be formed and the resulting polyol-containing polymer can then be coupled with a nitrophenylboronic acid-containing polymer. In some embodiments, 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 be any one of formulas (IX, X or XI) for subpart A and any of formulas (Formula XXIII or XIV) for subpart B One can have a polyol-containing polymer formed from a combination with one, which can then be coupled with a boronic acid-containing polymer having the formula XXX. In some embodiments described herein, the nanoparticles are of any one of formula (IX, X or XI) for subpart A and formula (Formula XXIII or XIV) for subpart B It can have a polyol-containing polymer formed from a combination with any one, which can then be coupled with a boronic acid-containing polymer corresponding to any one of formulas XXXIII, XXXIV, or XXXV.

  The nanoparticles described herein can further comprise a compound. In some embodiments, the compound may be one or more therapeutic agents, such as small molecule chemotherapeutic agents or polynucleotides. In some embodiments, the polynucleotide may be any one or more of DNA, RNA or interfering RNA (eg, 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 comprise a combination of a polynucleotide and one or more small molecules. In this regard, any one of the polymers of subpart A (formula VI, VII or VIII) is combined with any one of the polymers of subpart B (formula XXIII or XIV) to form the nanoparticulate polyol described The containing polymer nanoparticle segments can be formed and the resulting polyol-containing polymer can then be coupled with a nitrophenylboronic acid-containing polymer. The polymer of the lower portion A, the polymer of the lower portion B or the polymer having nitrophenylboronic acid can be formed with one or more therapeutic agents, such as small molecule chemotherapeutic agents or polynucleotides. In some embodiments, the sub-part A polymer (formula VI, VII or VIII) is combined with any one of the sub-part B polymers (formula XXIII or XIV) to form the nanoparticulate polyol-containing polymer of The nanoparticle segments can be formed and the resulting polyol-containing polymer can then be coupled with a nitrophenylboronic acid-containing polymer. The polymer of the lower part A, the polymer of the lower part B or the polymer having nitrophenylboronic acid can be formed with one or more DNA, RNA or interfering RNA (eg, shRNA, siRNA or miRNA). In some embodiments, the sub-part A polymer (formula VI, VII or VIII) is combined with any one of the sub-part B polymers (formula XXIII or XIV) to form the nanoparticulate polyol-containing polymer of The nanoparticle segments can be formed and the resulting polyol-containing polymer can then be coupled with a nitrophenylboronic acid-containing polymer. The polymer of the lower part A, the polymer of the lower part B or the polymer having nitrophenylboronic acid can be formed with one or more chemotherapeutic agents. In some embodiments, the sub-part A polymer (formula VI, VII or VIII) is combined with any one of the sub-part B polymers (formula XXIII or XIV) to form the nanoparticulate polyol-containing polymer of The nanoparticle segments can be formed and the resulting polyol-containing polymer can then be coupled with a nitrophenylboronic acid-containing polymer. The polymer of the lower part A, the polymer of the lower part B or the polymer having nitrophenylboronic acid can be formed with one or more DNA, RNA or interfering RNA (eg, shRNA, siRNA or miRNA). In some embodiments, 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 have any one of formulas (IX, X or XI) for subpart A and formulas (Formula XXIII or XIV) for subpart B A polyol-containing polymer formed from a combination with any one of the following, which is then coupled with a nitrophenylboronic acid-containing polymer corresponding to any one of the formulas XXXIII, XXXIV, or XXXV obtain.

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

A is the following formula:

Wherein 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 —H, —F, —C, —N Or selected from aliphatic groups containing one or more of -O; and Y is independently selected from organic moieties presenting -OH or -OH, and B is R ₁ and R of the first moiety A an organic moiety that connects to one of the _2, with R ₁ and R ₂ of the second portion a in the polymer. In some embodiments, X may be C _n H _{2n + 1} , where n is any one number from 0 to 5 and Y is —OH. In some embodiments, A can be any one of the following:

Wherein the spacer is independently selected from any organic group; the amino acid is selected from an organic group having a free amine and a free carboxylic acid group; n is any single number from 1 to 20 Yes; and Z ₁ is independently selected from —NH ₂ , —OH, —SH and —COOH; R ₁ and R ₂ may independently have the following formula:

Where 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, and Z is 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 is any one of May be:

Where q is any single number from 1 to 20; p is any single number from 20 to 200; and L is a free radical, and these B subunits are defined above. Paired with any one of the A subunits. In a more detailed embodiment, the polymer containing the polyol nanoparticle segment of the targeted nanoparticle shown in the structural unit of formula (I) is:

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

Polymers containing polyol nanoparticle segments of targeted nanoparticles shown in the structural unit of formula (III) are:

And
Here, n is an arbitrary 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 subpart A (formula VI, VII or VIII) is combined with any of the formulas for subpart B (formula XXIII or XIV) to contain the polyol of the targeted nanoparticles described A polymer may be formed. In certain aspects described herein, the targeted nanoparticles are of any one of formula (IX, X or XI) for subpart A and formula (Formula XXIII or XIV) for subpart B It may have a polyol-containing polymer formed from any one combination.

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

Where R ₃ and R ₄ are independently hydrophilic organic polymers, X ₁ is an organic moiety comprising one or more of —C, —N or —B, and Y ₁ is m ≧≧ 1 is an alkyl group or an aromatic group of the formula —C _m H _2m —, 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 the functional group 1 and functional group 2 are the same or different and are, _-B (OH) 2, either -OCH ₃ or -OH Can be selected independently from one. In some embodiments, these variable sub-portions 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 —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 sub-portions 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 —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, and a has a value of 0. And b may have a value of 1. In a detailed embodiment, the targeted nanoparticles described herein can include a boronic acid-containing polymer having the formula:

Here, s is an arbitrary single number of 20 to 300.

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

Where R ₃ and R ₄ are independently hydrophilic organic polymers, X ₁ is an organic moiety comprising one or more of —C, —N or —B, and Y ₁ is m ≧≧ 1 is an alkyl group or an aromatic group of the formula —C _m H _2m —, 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 the functional group 1 and functional group 2 are the same or different and are, _-B (OH) 2, either -OCH ₃ or -OH Can be selected independently from one. In some embodiments, these variable sub-portions of the described boronic acid-containing polymers can be selected from: 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 —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 sub-portions of the described nitrophenylboronic 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 —NH—C (═O) —, —S—S—, —C (═O) —NH—, —O—C. Any one of (═O) — or —C (═O) —O—, and Y ₁ is a nitrophenyl group, and r may have a value of 1, and a is 0 And b may have a value of 1. In each of the embodiments of the nitrophenylboronic acid-containing polymer, the nitro group can be in the ortho, meta, or para position. In still further embodiments, the nitrophenylboronic acid-containing polymer can have additional groups, such as methyl groups, present on the phenyl ring. In detailed embodiments, the targeted nanoparticles described herein can include a boronic acid-containing polymer having any one of the following formulas:

Here, s is an arbitrary single number of 20 to 300. The polymers of formula XXXIII, XXXIV and XXXV can be further modified to change the position of 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 for Formula A (Formula VI, VII, or VIII) for subpart A is combined with any one of Formulas for Formula B (Formula XXIII or XIV) for Subpart B, and A polymer containing a polyol nanoparticle segment of particles can be formed, and the resulting polyol-containing polymer can then be coupled with a boronic acid-containing polymer, wherein the boronic acid-containing polymer is phenylboronic acid or nitro Any of phenylboronic acid. In some embodiments, 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 nanoparticles are of any one of formula (IX, X or XI) for subpart A and formula (Formula XXIII or XIV) for subpart B It can have a polyol-containing polymer formed from a combination with any one, which can then be coupled with a boronic acid-containing polymer having the formula XXX. In some embodiments described herein, the targeted nanoparticles have any one of formulas (IX, X or XI) for subpart A and formulas (Formula XXIII or XIV) for subpart B A polyol-containing polymer formed from a combination with any one of the following, which is then coupled with a boronic acid-containing polymer corresponding to any one of the formulas XXXI, XXXIII, XXXIV, or XXXV obtain.

  In some aspects described herein, formed from either a polyol-containing polymer nanoparticle segment described herein or a combination of a polyol-containing polymer nanoparticle segment and a boronic acid-containing polymer. The nanoparticles are conjugated with the target ligand to form targeted nanoparticles with a target ligand to nanoparticle ratio of 3: 1. In some aspects described herein, formed from either a polyol-containing polymer nanoparticle segment described herein or a combination of a polyol-containing polymer nanoparticle segment and a boronic acid-containing polymer. The nanoparticles are conjugated with the target ligand to form targeted nanoparticles with a 1: 1 ratio of target ligand to nanoparticle. In some aspects described herein, formed from either a polyol-containing polymer nanoparticle segment described herein or a combination of a polyol-containing polymer nanoparticle segment and a boronic acid-containing polymer. The nanoparticles are conjugated with one single targeting ligand to form targeted nanoparticles. In some aspects, the described target ligand is conjugated to the boronic acid-containing polymer at the opposite end to the boronic acid. Target ligands conjugated to the described targeted nanoparticles can be proteins, protein fragments, amino acid peptides, or aptamers derived from either amino acids or polynucleotides, or other known to bind to the target of interest. It may be any one of the high affinity molecules. In some embodiments, the target ligand that is a protein or protein fragment is any one of an antibody, a cellular receptor, a ligand for a cellular receptor (eg, transferrin), or a protein or portion thereof having a portion thereof. It may be. If the target ligand is an antibody, is this antibody a human, mouse, rabbit, non-human primate, dog, or rodent antibody? It may be a chimera consisting of any two of these antibodies. Furthermore, the antibody may be humanized such that only a small portion of the CDR segment or variable region comprising the CDR segment is a non-human antibody and the remainder of the antibody is a human antibody. The antibodies described herein may be of any isoform, such as IgG, IgM, IgA, IgD, IgE, IgY or another type of isoform known to be produced by mammals. May be. In some embodiments, the target ligand may comprise only an antibody responsible for binding to its target, a cellular receptor, an amino acid peptide derived from a ligand for a cellular receptor.

  In some aspects, a nanoparticle formed from any one of formulas (VI, VII, or VIII) for subpart A is any one of formulas (Formula XXIII or XIV) for subpart B Combined to form a polyol-containing polymer segment of the described targeted nanoparticles, and further a boronic acid, such as phenylboronic acid or any of 5, 4, 3, 2 or 1, conjugated with a target ligand Coupled to a nitrophenylboronic acid containing polymer.

  In some aspects, a nanoparticle formed from any one of formulas (VI, VII, or VIII) for subpart A is any one of formulas (Formula XXIII or XIV) for subpart B Combined to form the polyol-containing polymer segment of the described targeted nanoparticles and further coupled to a single boronic acid, eg, phenylboronic acid or nitrophenylboronic acid-containing polymer, conjugated with the target ligand.

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

  In certain aspects described herein, the targeted nanoparticles are of any one of formula (IX, X or XI) for subpart A and formula (Formula XXIII or XIV) for subpart B It can be coupled to a boronic acid-containing polymer of formula XXX having a polyol-containing polymer formed from a combination with any one and coupled to the target ligand at the opposite end of the boronic acid.

  In some aspects, a nanoparticle formed from any one of formulas (VI, VII, or VIII) for subpart A is any one of formulas (Formula XXIII or XIV) for subpart B Combined to form a polyol-containing polymer segment of the described targeted nanoparticles and further 5, 4, 3, 2 or 1 boronic acid-containing polymer, eg phenylboronic acid or nitrophenylboronic acid conjugated with a target ligand Coupled to. Here, the resulting targeted nanoparticles are only conjugated with a single target ligand.

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

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

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

  In some embodiments, the targeted nanoparticles described herein are conjugated to any target ligand. In some embodiments, the targeted nanoparticles described herein are conjugated to a single target ligand. In some embodiments described herein, the targeted nanoparticles have any one of formulas (IX, X or XI) for subpart A and formulas (Formula XXIII or XIV) for subpart B A polyol-containing polymer formed from a combination with any one of the following: 5, 4, 3, 2 corresponding to any one of formulas XXXI, XXXIII, XXXIV, or XXXV Alternatively, it can be coupled with any one of the boronic acid-containing polymers. This further conjugates to a target ligand selected from one or more of a protein, protein fragment, amino acid peptide or aptamer.

  In some embodiments, the targeted nanoparticles described herein are conjugated to a single target ligand. In some embodiments described herein, the targeted nanoparticles have any one of formulas (IX, X or XI) for subpart A and formulas (Formula XXIII or XIV) for subpart B A polyol-containing polymer formed from a combination with any one of the following, which is then coupled with a boronic acid-containing polymer corresponding to any one of the formulas XXXI, XXXIII, XXXIV, or XXXV obtain. This further conjugates to a single target ligand selected from proteins, protein fragments, amino acid peptides or aptamers.

  In some embodiments, the targeted nanoparticles described herein are conjugated to a single target ligand. In some embodiments described herein, the targeted nanoparticles have any one of formulas (IX, X or XI) for subpart A and formulas (Formula XXIII or XIV) for subpart B A polyol-containing polymer formed from a combination with any one of the following: any 5, 4, 3, corresponding to any one of formulas XXXI, XXXIII, XXXIV, or XXXV It can be coupled with two or one boronic acid containing polymers. This is further conjugated to a chimeric protein having an antibody, a cellular receptor, a ligand for the cellular receptor, or a protein or part thereof.

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

  In some embodiments, the targeted nanoparticles described herein are conjugated to a single target ligand. In some embodiments described herein, the targeted nanoparticles have any one of formulas (IX, X or XI) for subpart A and formulas (Formula XXIII or XIV) for subpart B A polyol-containing polymer formed from a combination with any one of the following, which is then coupled with a boronic acid-containing polymer corresponding to any one of the formulas XXXI, XXXIII, XXXIV, or XXXV obtain. This is further conjugated to a single target ligand selected from proteins, protein fragments, amino acid peptides or aptamers, where the resulting targeted nanoparticles only bind to a single target ligand It is not converted.

  In some embodiments, the targeted nanoparticles described herein are conjugated to a single target ligand. In some embodiments described herein, the targeted nanoparticles have any one of formulas (IX, X or XI) for subpart A and formulas (Formula XXIII or XIV) for subpart B A polyol-containing polymer formed from a combination with any one of the following: any 5, 4, 3, corresponding to any one of formulas XXXI, XXXIII, XXXIV, or XXXV It can be coupled with two or one boronic acid containing polymers. This is further conjugated to a chimeric protein having an antibody, a cellular receptor, a ligand for a cellular receptor, or a protein or part thereof. Here, the resulting targeted nanoparticles are only conjugated to a single target ligand.

  In some embodiments described herein, the targeted nanoparticles have any one of formulas (IX, X or XI) for subpart A and formulas (Formula XXIII or XIV) for subpart B A polyol-containing polymer formed from a combination with any one of the following, which is then coupled with a boronic acid-containing polymer corresponding to any one of the formulas XXXI, XXXIII, XXXIV, or XXXV obtain. This is 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, wherein the resulting targeted nanoparticles are a single It is conjugated only to the target ligand.

  The targeted nanoparticles described herein can further comprise a compound. In some embodiments, the compound may be one or more therapeutic agents, such as small molecule chemotherapeutic agents or polynucleotides. In some embodiments, the polynucleotide may be any one or more of DNA, RNA or interfering RNA (eg, shRNA, siRNA or miRNA). In some embodiments, the small molecule chemotherapeutic agent may be one or more of camptothecin, epothilone, or 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 described targeted nanoparticles are a therapeutic agent selected from a mucin-containing polymer, a camptothecin, an epothilone, a taxane or an interfering RNA sequence, a reversible covalent bond with a mucin acid polymer Having a phenylboronic acid-containing polymer with XXXI, XXXIII, XXXIV or XXXV. The targeted nanoparticle is configured to present the phenylboronic acid-containing polymer to the external environment of the nanoparticle, wherein the phenylboronic acid-containing polymer is conjugated to the target ligand at the opposite end of the nanoparticle. Here, the targeted nanoparticles comprise one single targeting ligand.

  In one embodiment, the described targeted nanoparticles are a therapeutic agent selected from a mucin-containing polymer, a camptothecin, an epothilone, a taxane or an interfering RNA sequence, a reversible covalent bond with a mucin acid polymer Having a phenylboronic acid-containing polymer with XXXI, XXXIII, XXXIV or XXXV. The targeted nanoparticle is configured to present the phenylboronic acid-containing polymer to the external environment of the nanoparticle, wherein the phenylboronic acid-containing polymer is conjugated to the target ligand at the opposite end of the nanoparticle. Where the targeted nanoparticle comprises one single antibody.

  In one embodiment, the described targeted nanoparticles are a therapeutic agent selected from a mucin-containing polymer, a camptothecin, an epothilone, a taxane or an interfering RNA sequence, a reversible covalent bond with a mucin acid polymer Having a phenylboronic acid-containing polymer with XXXI, XXXIII, XXXIV or XXXV. The targeted nanoparticle is configured to present a phenylboronic acid-containing polymer to the external environment of the nanoparticle, wherein the phenylboronic acid-containing polymer is a human antibody, mouse antibody at the opposite end of the nanoparticle. A rabbit antibody, a non-human primate antibody, a canine antibody, or a rodent antibody, or a chimeric antibody comprising any two of these antibodies (wherein the antibody is an IgG, IgD, IgM, IgE, IgA or IgY Which is any one of the isoforms), wherein the targeted nanoparticle comprises one single antibody.

  In one embodiment, the described targeted nanoparticles are a therapeutic agent selected from a mucin-containing polymer, a camptothecin, an epothilone, a taxane or an interfering RNA sequence, a reversible covalent bond with a mucin acid polymer Having a phenylboronic acid-containing polymer with XXXI, XXXIII, XXXIV or XXXV. The targeted nanoparticle is configured to present a phenylboronic acid-containing polymer to the external environment of the nanoparticle, wherein the phenylboronic acid-containing polymer is conjugated to a cellular receptor at the opposite end of the nanoparticle. Where the targeted nanoparticle comprises one single cellular receptor.

  In one embodiment, the described targeted nanoparticles are a therapeutic agent selected from a mucin-containing polymer, a camptothecin, an epothilone, a taxane or an interfering RNA sequence, a reversible covalent bond with a mucin acid polymer Having a phenylboronic acid-containing polymer with XXXI, XXXIII, XXXIV or XXXV. The targeted nanoparticle is configured to present a phenylboronic acid-containing polymer to the external environment of the nanoparticle, wherein the phenylboronic acid-containing polymer is conjugated to a receptor ligand at the opposite end of the nanoparticle. Here, the targeted nanoparticle comprises one single receptor ligand.

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

  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 differential 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, which is a plasmid containing the firefly luciferase gene, was extracted from bacteria expressing pGL3 and purified. 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 with cationic diamine mucinate-DMS polymer.

Example 1: Synthesis of mucin acid dimethyl ester, (1)
5 g (22.8 mmol) of mucin acid (Aldrich) was added to a 500 mL round bottom flask containing 120 mL methanol and 0.4 mL concentrated sulfuric acid. The mixture was refluxed at 85 ° C. overnight under constant stirring. This mixture was then filtered, washed with methanol and then recrystallized from a mixture of 80 mL methanol and 0.5 mL triethylamine. After drying under vacuum overnight, 8.0 g (33.6 mmol, 71%) of mucin acid dimethyl ester was obtained. ¹ H NMR ((CD ₃ ) ₂ SO) δ 4.88 to 4.91 (d, 2H), 4.78 to 4.81 (m, 2H), 4.28 to 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 mucinic acid diamine, (2)
A mixture of 8 g (33.6 mmol) mucin acid dimethyl ester (1; Example 1), 12.4 mL (88.6 mmol) triethylamine and 160 mL methanol was refluxed in a continuously stirred 500 mL round bottom flask. At 85 ° C. for 0.5 h, then 14.2 g (88.6 mmol) N-BOC diamine (Fluka) dissolved in methanol (32 mL) was added. The reaction suspension was then returned to reflux. After overnight reflux, the mixture was filtered, washed with methanol, recrystallized from methanol and dried under vacuum to obtain 9.4 g (19 mmol, 57%) of N-BOC-protected mucinic acid diamine. Obtained. ¹ H NMR ((CD ₃ ) ₂ SO) δ 7.66 (m, 2H), 6.79 (m, 2H), 5.13 to 5.15 (d, 2H), 4.35 to 4.38 (D, 2H), 4.08 to 4.11 (m, 2H), 3.78 to 3.80 (d, 2H), 2.95 to 3.15 (m, 8H), 1.38 (s , 18). ESI / MS (m / z): 517.1 [M + Na] ⁺

Example 3: Synthesis of mucin diamine (3)
8 g (16.2 mmol) of N-BOC-protected mutated diamine (2; Example 2) was transferred to a 500 mL round bottom flask containing 3M HCl in methanol (160 mL) and continuously at 85 ° C. overnight. While stirring, the mixture was refluxed. The precipitate was then filtered, washed with methanol and dried overnight under vacuum to give 5.7 g (15.6 mmol, 96%) of mucin diamine. ¹ H NMR ((CD ₃ ) ₂ SO) δ 7.97 (m, 8H), 5.35 to 5.38 (m, 2H), 4.18 to 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: Dimethyl mucin-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 addition of 1N HCl. The solution was then dialyzed for 24 hours in ddH ₂ O using a 3500 MWCO dialysis membrane (Pierce-plated dialyzation tubing). The dialyzed solution was lyophilized to dryness to give 49 mg of white fluffy powder. ¹ H NMR (500 MHz, 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). ¹³ C NMR (126 MHz, 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 AB type (repeating structure is ABABAB ...) polyol-containing polymer.

Example 5: Boronic acid-amide-PEG ₅₀₀₀ , (5)
The polymer of Example 4 is collected with a nucleic acid, such as siRNA, to form nanoparticles. These nanoparticles need to have the steric stability used in mammals, and if necessary, they could have the targeted drug included. In order to perform these two functions, the nanoparticles may be decorated with PEG for steric rest and PEG-targeting ligands. To do this, a PEG compound containing boronic acid is prepared. For example, boronic acid-containing PEG can be synthesized according to the following examples.

The 332mg of 4-carboxyphenyl boronic acid (2 mmol), was dissolved in SOCl ₂ in 8 mL. To this was added a few drops of DMF 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 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 warmed to room temperature and stirring was continued overnight. The solvent dissolved 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 against ddH ₂ O for 24 hours on a 3500 MWCO dialysis membrane (Pierce-plated dialyzation tubing). The dialyzed solution was lyophilized until dry. 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)
A cleavable version (under reducing conditions) of the PEG compound of Example 5 can also be synthesized as follows.

250 mg of PEG ₅₀₀₀ -SH (0.05 mmol, RaySanBio 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. The solution was stirred at room temperature for 2 hours after adding 77 mg mercaptophenylboronic acid (0.5 mmol, Aldrich) in 1 mL methanol. 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 against ddH ₂ O for 15 hours on a 3500 MWCO dialysis membrane (Pierce plated dialyzing tubing). The dialyzed solution was lyophilized until dry. 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)
A fluorinated version of the boronic acid-containing PEG compound of Example 5 can be synthesized and used as an imaging agent with therapeutic nanoparticles. Fluorine atoms for imaging can be incorporated as described and explained below.

(2,3,5,6) - fluoro-carboxyphenyl boronic acid, dissolved in an excess of SOCl ₂ (about 100 equivalents), To this was added a few drops of DMF. The mixture was refluxed for 2 hours under argon. 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 against ddH ₂ O for 24 hours on a 3500 MWCO dialysis membrane (Pierce-plated dialyzation tubing). The dialyzed solution was lyophilized until dry.

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

Example 8: Synthesis of (2,3,5,6) -tetrafluorophenylboronic acid-disulfide-PEG ₅₀₀₀ , (8)
A fluorinated version of the boronic acid-containing cleavable PEG compound of Example 5 can be synthesized and used as an imaging agent with therapeutic nanoparticles. Fluorine atoms for imaging can be incorporated as described and explained below.

Add 250 mg PEG ₅₀₀₀ -SH (0.05 mmol, RaySanBio Inc.) to a glass vial equipped with a stirrer. To this is added 110 mg of aldrithiol-2 (0.5 mmol, Aldrich) dissolved in 4 mL of methanol. The solution is stirred for 2 hours at room temperature after adding 77 mg (2,3,5,6) -fluoro-4-mercaptophenylboronic acid (0.5 mmol) in 1 mL methanol. The resulting solution is stirred for another 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 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. This dried solid is redissolved in water, filtered through a 0.45 μm filter, and dialyzed in ddH ₂ O for 15 hours on a 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing). The dialyzed solution is lyophilized until dry.

Example 9: Synthesis of boronic acid-PEG ₅₀₀₀ -transferrin, (9)
The target 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 refers to transferrin addition.

  Thus, the components of a system comprising a nucleic acid as a therapeutic agent are (target ligands are proteins such as transferrin (FIG. 12), antibodies or antibody fragments, peptides such as RGD or LHRH, low levels such as folic acid or galactose. Molecule, etc.). The boronic acid PEGylated target agent can be synthesized as follows.

Specifically, according to the approach illustrated in FIG. 12, the following procedure was performed to synthesize boronic acid PEG ₅₀₀₀ -transferrin. A solution of 10 mg (0.13 μmol) nohuman 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 obtained from unreacted OPSS-PEG _5000- SVA using Ultracel 50,000 MWCO (Amicon Ultra-4, Millipore) and from unreacted transferrin using gel filtration column G3000SWxl (Tosoh Biosep). Purified (confirmed by HPLC and MALDI-TOF analysis). Then 100 μg OPSS-PEG ₅₀₀₀ pegylated transferrin in 100 μL was incubated at room temperature with 20 μL 4-mercaptophenylboronic acid (1 μg / μL, 20 μg, 100 eq) for 1 hour at room temperature. After incubation, the solution was dialyzed twice with a YM-30,000 NMWI device (Millipore) to remove excess 4-mercaptophenylboronic acid and pyridyl-2-thione byproduct.

Example 10: MAP-Nucleic Acid Particle Formulation—Gel Shift Assay As illustrated in FIG. Mix with 10 μL of MAP at various concentrations in DNA- and RNase-free water and charge ratios of 0.5, 1, 1.5, 2, 2.5 and 5 (“+” on polymer) Charge vs. “−” charge on the nucleic acid). The resulting mixture was incubated for 30 minutes at room temperature. As shown in FIGS. 3 and 4, 10 μL of a 20 μL solution was loaded onto a 1% agarose gel with 3.5 μL loading buffer and the gel was electrophoresed at 80 V for 45 minutes. Nucleic acids that are not contained in the nanoparticles move on the gel. These results provide an derivation for the required charge ratio for nucleic acid inclusions 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 DNA-free and RNA-free enzyme-free water, 10 μL of MAP, and DNA-degrading enzyme and RNA degradation Mixing at various concentrations in enzyme-free water gave charge ratios of 0.5, 1, 1.5, 2, 2.5 and 5. The resulting mixture was incubated for 30 minutes at room temperature. The 20 μL mixture was then diluted to 70 μL with DNA- and RNase-free water for particle size measurement. This 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 diffusion (DLS) apparatus (Brookhaven Instruments). The results are shown in FIG.

Example 12: Particle size stabilization by pegylation with boronic acid PEG _{5k As} illustrated in FIG. 2, 2 μg of plasmid DNA (0.45 μg / μL, 4 in DNA-free and RNase-free water) .4 μL) was diluted to 80 μL of DNA- and RNase-free water. This plasmid solution was mixed with 4.89 μg MAP (0.5 μg / μL, 9.8 μL) and diluted to 80 μL in DNA- and RNase-free water to give a 3 +/− charge ratio and A final plasmid concentration of 0.0125 μg / μL was obtained. The resulting mixture was incubated for 30 minutes at room temperature. To this solution, 480 μg of boronic acid PEG _5K (Compound 6; Example 6), (20 μg / μL, 24 μL) was added. The mixture was then incubated for an additional 30 minutes and dialyzed twice with 0.5 mL 100,000 MWCO membrane (BIOMAX, Millipore Corporation) in DNA- and RNA-free enzyme-free water. Reconstitution was made in 160 μL of water containing no degrading enzyme. 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 showed a lower zeta potential than the nanoparticles without it. These results support the conclusion that BA-containing nanoparticles localize BA outside the nanoparticles. The other half was used to measure the particle size. The particle size was measured every minute for 5 minutes after 10.2 μL of 10 × PBS was added to finally obtain a 90.2 μL solution in 1 × PBS. The particle size was then measured every minute for another 10 minutes, as shown in FIG. BA-containing nanoparticles separated from non-particle components (by filtration) are stable in PBS, whereas particles without BA are not stable. These results support the conclusion that BA-containing nanoparticles have a BA localized outside of them as 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 supplemented with 10% FBS. Grown in prepared medium. 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 formulation was added. Cells were then incubated for 5 hours at 37 ° C., 5% CO ₂ before adding growth medium supplemented with 800 μL of 10% FBS. After 48 hours incubation, cell fractions were analyzed for cell viability using the MTS assay. The remaining cells were lysed in 100 μL of 1X luciferase cultured cell lysis reagent. Luciferase activity was determined by adding 100 μL of luciferase assay reagent to 10 ul of cell lysate, and biofluorescence was quantified using Monolitum luminometer. The luciferase activity is then reported as relative light units (RLU) per 10,000 cells. The results are shown in FIG. 8 and FIG.

Example 14: Co-transfection of MAP / pDNA particles and / or siRNA particles into HeLa cells HeLa cells were seeded in 24-well plates at 20,000 cells per well 48 hours prior to transfection. Grow 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 formulation was added. Cells were then incubated for 5 hours at 37 ° C., 5% CO ₂ before adding growth medium supplemented with 800 μL of 10% FBS. After 48 hours of incubation, cells were assayed for luciferase activity and cell viability as described in Example 12. The result is shown in FIG. RLU is lower in transfection 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 in 24-well plates at 20,000 cells per well 48 hours prior to transfection and supplemented with 10% FBS. Grown in medium. MAP particles were formulated to include 50 and 100 nM siGL3 in 200 μL Opti-MEM I at a charge ratio of 5 +/−. 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% CO ₂ before adding growth medium supplemented with 800 μL of 10% FBS. After 48 hours of incubation, cells were assayed for luciferase activity and cell viability as described in Example 12. The result is shown in FIG. Since RLU decreases with increasing concentrations of siGL3, these data suggest that alienation of the endogenous gene may occur.

Example 16: Synthesis of mucin diiodide, (10)
1 g (2.7 mmol) of diamine mucinate (Example 3) was mixed with 3.8 mL (27.4 mmol) of triethylamine and 50 mL of anhydrous DMF, followed by 1.2 mL (13.7 mmol) of iodoacetyl chloride. The addition was performed 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 a vacuum pump and the product was filtered, washed with methanol and dried under vacuum to give 0.8 g (1.3 mmol, 46%) of mucin 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 mucinate, (11)
7 mL of 0.1 M degassed sodium carbonate was added to 17 mg L-cysteine and 0.4 g mucinate diiodide. The resulting suspension was refluxed at 150 ° C. for 5 hours until the solution was clear. The mixture was then cooled to room temperature and adjusted to pH 3 with 1N HCl. A slow addition of acetone was then made for product precipitation. After filtration, washing with acetone and vacuum drying, 60 mg of crude product was obtained.

Example 17: Synthesis of dicysteine mucinate, (11)
20 mL of 0.1 M degassed sodium phosphate buffer in a 50 mL round bottom flask at pH 7.5 was added 0.38 g L-cysteine (3.2 mmol) and 0.40 g (0.6 mmol) mucinic acid. Added to 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 insoluble excess reagent and phosphate from 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 mucinate.

Example 18: Polymer synthesis, (Poly (mutic acid-DiCys-PEG)) (12)
12 mg (21.7 μmol) of dicysteine mucinate and 74 mg (21.7 μmol) of PEG-DiSPA 3400 were dried under vacuum, then 0.6 mL of anhydrous DMSO was added under 10 mL round bottom under argon. Added in a flask. After 10 minutes of stirring, 9 μL (65.1 μmol) 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 (mutic acid-DiCys-PEG).

  This polyol-containing polymer is an anionic AB polymer.

Example 19: Covalent attachment to drug (camptothecin, CPT) mucin polymer, (13)
10 mg (2.7 μmol repeat units) of poly (mutic acid-DiCys-PEG) was dissolved in 1.5 mL anhydrous DMSO in a glass bottle. After 10 minutes of stirring, 1.1 μL DIEA (6.3 μmol), 3.3 mg (6.3 μmol) TFA-Gly-CPT, 1.6 mg (8.1 μmol) EDC and 0.7 mg (5. 9 μmol) of NHS was added to the reaction mixture. After stirring for 8 hours, 1.5 mL of ethanol was added and the solvent was removed under reduced pressure. The precipitate was dissolved in water and unwanted material was 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 a poly (mutic acid-DiCys-PEG) -CPT conjugate.

Example 20: Formulation of nanoparticles with CPT-mutic acid polymer (13) in water (20)
Effective diameters of poly (mutic acid-DiCys-PEG) and poly (mutic acid-DiCys-PEG) -CPT conjugate were formulated in double distilled water of this polymer (0.1-10 mg / mL) and ZetaPALS ( Evaluation was via dynamic light diffusion (DLS) using a Brookhaven Instrument Co) device. Three consecutive runs of 1 minute each were then recorded and averaged. The zeta potential of both compounds was measured in a 1.1 mM KCl solution using a ZetaPALS (Brookhaven Instrument Co) apparatus. Ten consecutive automated runs were then performed with a target residue of 0.012 and the results averaged (FIG. 4). Specifically, these two distributions were measured for poly (mutic acid-DiCys-PEG) -CPT conjugate, the main distribution being 57 nm (60% of the total particle population). The second least distribution is also measured at 233 nm.

Example 21: Boronic acid-PEGylated nanoparticles with CPT-mutic acid polymer (13) Boronic acid-disulfide-PEG ₅₀₀₀ (6) Formulation in water Boronic acid PEGylated poly (mutic acid-DiCys-PEG) -CPT Nanoparticles were dissolved in double-distilled water at a concentration of 0.1 mg / mL, followed by addition of polymer 6 (BA-PEG) in water to obtain poly (mutic acid-DiCys-PEG) -CPT. The ratio of PBA-PEG to diol on the mucin sugar in the conjugate is 1: 1. This 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 that expresses 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 (+3 To obtain a charge ratio of polymer 4 to plasmid of The particles were placed in D5W (5% glucose in water) by continuous spin filtration followed by the addition of D5W (starting with the starting formulation in water). Nude mice were transplanted with Hepa-1-6 hepatoma cells and the tumors were allowed to grow to a size of approximately 200 mm ³ . Targeted nanoparticle injections i.v. in the tail vein in an amount equal to 5 mg / kg plasmid mouse. v. I went there. Mice were imaged 24 hours after injection. The mice showed no signs of toxicity and luciferase expression was detected in the tumor area and not in the liver area.

Example 23: Synthesis of PBA-PEG and nitro PBA-PEG and measurement of pKa The reversible covalent property between boronic acid and MAP was used to prepare stabilized targeted nanoparticles. The pKa of phenylboronic acid (PBA) is as high as 8.8. In order to lower the pKa, PBA with an electron withdrawing group in the phenyl ring was used to increase the acidity of the boron atom. Commercially available 3-carboxy PBA and 3-carboxy 5-nitro PBA were converted to acyl chloride using oxalyl chloride. Then these acyl chloride _species, is reacted with NH 2-PEG, formed PBA-PEG and nitro PBA-PEG, respectively. The addition of DMF and DIPEA bases was necessary to proceed with the synthesis reaction. However, the presence of these bases resulted in tetrahedral adduct formation by acidic PBA. In order to remove these adducts, work in 0.5N HCl and subsequent 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) oxalyl chloride was added slowly under argon. The reaction was allowed to proceed for 2 hours at room temperature under continuous venting with constant stirring. Solvent and excess reagent were removed under vacuum. 37 mg (0.2 mM) of the painted dry acyl chloride compound was dissolved in 15 ml of anhydrous dichloromethane under argon. Then 500 mg (0.1 mM) NH2-PEG5 kDa-CO2H and 52 [mu] l (0.3 mM) 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 0.5N HCl. This solution was dialyzed against water using a 3 kDa MWCO membrane filter device (Amicon ™) through a 0.2 μm filter (Acrodisc®) until a constant pH was obtained. The supernatant was then filtered using a 0.2 μm filter (Acrodisc®) and lyophilized to give 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-PEG5 kDa-CO2H, 5400 g / mol).

  The synthesis of nitro PBA-PEG-CO2H was performed in the same way for PBA-PEG-CO2H, except that the starting material was 3-carboxy 5-nitrophenylboronic acid. This reaction yielded 393 mg (76%) of nitro PBA-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-PEG5 kDa-CO2H, 5400 g / mol).

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

Example 24: Antibody Conjugation to Nitro PBA-PEG After generation of nitro PBA-PEG, the resulting compound was conjugated to an antibody. For conjugation, 36 mg (6.4 μM) nitro PBA-PEG-CO 2 H, 12.3 mg (64 μM) 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 11.1 mg (EDC) 96 μM) NHS was dissolved in 2.4 ml of 0.1 M MES buffer, pH 6.0. This mixture was allowed to react on a rotary shaker at room temperature for 15 minutes. Excess reaction was removed by dialysis through a 3 kDa molecular weight cut-off 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 performed on a rotary shaker at room temperature for 2 hours and then dialyzed 4 times against 1 × PBS at pH 7.4 using a 50 kDa molecular weight cut-off membrane filter device (Amicon ™). Mass spectrometry (MALDI-TOF) showed an average of 1-2 nitro PBA-PEG compound conjugates per antibody.

Example 26: Targeted MAP Nanoparticle Formulation and Characterization The effects of PBA-PEG and nitro PBA-PEG on nanoparticle formulation and stability were tested. The solution of PBA-PEG was added to the MAP solution in a phosphate buffer at pH 7.4 at a charge ratio of 3 +/− (positive charge derived from MAP per negative charge derived from siRNA). This mixture was left at room temperature for 10 minutes for complexation of PBA-PEG with diol-containing MAP. This was then added to an equal volume of siRNA in water and mixed by pipetting. After 10 minutes, testing and characterization were performed. The particle formulation was stabilized with nitro PBA-PEG and different charge ratios were performed in the same manner. PBA-PEG complex formation formed MAP / siRNA particles with a size of 150-300 nm. MAP-4 (representing 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 the salt, all aggregated particles showed that PBA-PEG did not give the nanoparticles sufficient stability. When nitro PBA-PEG was used, the particles demonstrated stabilized nanoparticles at all charge ratios tested, 1 and 3 (+/−). The difference in particle stability conferred by PBA-PEG and nitro PBA-PEG indicates the importance of modifying the PEGylated boronic acid compound to have a stronger bond to the polyol.

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

  Salt stability for MAP-4 / siRNA nanoparticles with various PEG groups attached was evaluated. The particle size was recorded for 5 runs at 1 minute using a zeta potential analyzer (DLS ZetaPALS instrument, Brookhaven Instruments, Holtsville, NY). Since the measurement was stopped and 10 × PBS solution was added to formulate the particles, the resulting particles contained 1 × PBS. Measurements were then resumed and 10 consecutive runs each minute were recorded to study the effect of salt addition on particle stability. As shown in FIG. 16, the particles aggregated in the absence of the stabilizer PEG. When nitro PBA-PEG was present, the particles remained stable even under salt conditions.

Example 27: Formulation and characterization of targeted MAP-CPT nanoparticles The reversible covalent properties between boronic acid and MAP (containing diol) were used to prepare targeted MAP-CPT nanoparticles. The binding constant between PBA and MAP is as low as about 20M- ¹ . In order to increase the binding constant, PBA with an electron withdrawing group in 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 chloride using oxalyl chloride. Then these acyl chloride species, is 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 DMF and DIPEA bases was necessary to proceed with the synthesis reaction. However, the presence of these bases resulted in tetrahedral adduct formation by acidic PBA. In order to remove these adducts, work in 0.5N HCl and subsequent equilibration to neutral pH by dialysis against water was performed.

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

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

^a Equivalent amount of N, N-diisopropylethylamine (DIPEA) was added per di (Asp-amine) mucinate. ^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 untargeted nanoparticles indicates binding of IgG (Herceptin®) to the nanoparticles (amount was about 1 Herceptin® per nanoparticle). . In fact, from the cryo-EM images, it was found that the targeted nanoparticles were not spherical but had protrusions indicating antibody binding. The surface charge of targeted MAP-CPT nanoparticles was slightly higher than that of MAP-CPT nanoparticles due to the presence of Herceptin's positive charge at pH 7.4 (Herceptin® pI, 9 .2).

Example 28: Cell uptake studies A human breast cancer cell line (BT-474) overexpressing HER2 was tested to test the cellular uptake of Herceptin® targeted MAP-CPT nanoparticles versus non-targeted nanopicture financing. used. A HER2 negative breast cancer cell line (MCF-7) was used as a negative control. For these studies, BT-474 (in RPMI-1640 medium supplemented with 10% fetal calf serum) or MCF-7 (Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum) were placed in 24-well plates, respectively ( Cellgro, Manassas, Va.) Was seeded with 20,000 cells per well and kept at 37 ° C. in a humidified oven with 5% CO ₂ . After 40 hours, the medium was replaced with 0.3 ml of fresh medium. Each medium is medium MAP-CPT (40 μg CPT / ml), medium MAP-CPT containing free Herceptin® at 10 mg / ml, targeted MAP-CPT (40 μg CPT / ml) at various target densities Alternatively, it included targeted MAP-CPT with free Herceptin® at 10 mg / ml. Transfections were performed at 37 ° C for 30 minutes. The formulation was then removed and the cells were washed twice with cold PBS. For cell detachment and lysis, 200 μl RIPA buffer was added per well. Lysed cells were then incubated at 4 ° C. for 15 minutes and centrifuged at 14,000 g for 10 minutes at 4 ° C. A portion of the supernatant was used for protein quantification by BCA assay. To another protein, an equal volume of 0.1 N NaOH was added, incubated overnight at room temperature, and fluorescence was measured at 370/440 nm using a known concentration of MAP-CPT as a standard.

  Observe that one Herceptin®-PEG-nitro PBA per nanoparticle is sufficient to achieve about 70% higher uptake compared to non-targeted nanoparticles in the BT-474 cell line (FIG. 17A). Incorporation of targeted MAP-CPT in BT-474 cells showed alienation in the presence of free Herceptin® (FIG. 17B). In contrast, the incorporation of MAP-CPT nanoparticles in MCF-7 did not show any dependence on targeting in the presence of free Herceptin® (FIG. 2B). These data indicate that there is receptor-mediated uptake through binding of HER2 receptor by targeted MAP-CPT nanoparticles in BT-474 cells. Cellular uptake of both targeted and untargeted MAP-CPT nanoparticles also occurred via non-specific liquid phase endocytosis.

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

For equilibration, the medium pipetted into 96 well plates was incubated at 37 ° C. in a humidified oven for 2 hours. The formulation was mixed into the corresponding medium and returned to the oven. Samples were removed at predetermined time points and immediately frozen at −80 ° C. until the time of analysis. For release in BALB / c mouse plasma and human plasma, incubation was carried out in a humidified oven at 37 ° C. with 5% CO ₂ (carbonic acid, which is the main pH buffer system in plasma at physiological pH levels). / To maintain a carbon dioxide buffer system).

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

  Release from CPT short, medium and long MAP-CPT nanoparticles all showed first order kinetics. Release half-lives from CPT short, medium and long MAP-CPT nanoparticles were 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. As the pH increased from 6.5 to 7.4, the release half-life decreased from 338 hours to 58 hours for short, medium and long MAP-CPT nanoparticles. These data indicate that hydrolysis plays an important role in CPT release. For short, medium and long MAP-CPT nanoparticles, a 59-hour release half-life was observed in BALB / c mouse plasma, while a significantly shorter 38-hour half-life was observed in human plasma. . Mouse plasma is known to have higher esterase activity than human plasma, while human plasma is known to be “higher fat” than mouse plasma. Therefore, the contribution of esterase and fat to the CPT release rate was tested individually to see the difference in release rate between human and mouse plasma. Butyrylcholinesterase (BCHE) is present in both mouse and human plasma and was used to test the contribution of esterase to the release rate. Low density lipoprotein (LDL) was selected to account for the contribution to the release rate of “fat”. These components were included in PBS (pH 7.4). The presence of BCHE was found not to affect the release rate (61 hours compared to 58 hours at pH 7.4 in PBS for short, medium and long MAP-CPT nanoparticle nanoparticles). Half life). However, the addition of LDL dramatically increased the release rate. This effect is probably due to nanoparticle destruction due to competitive hydrophobic interactions. Therefore, nanoparticle destruction due to the presence of “fat” and subsequent CPT cleavage by hydrolysis is considered to be different from the 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® with a pI of 9.2 protects the nanoparticles from electrostatic interactions and thereby some of the competing hydrophobic interactions, thus stabilizing the negatively charged nanoparticles Can be increased.

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

Example 30: Cytotoxicity Assay The in vitro cytotoxicity of medium MAP, nitro PBA-PEG, CPT, medium MAP-CPT nanoparticles, targeted MAP-CPT nanoparticles and Herceptin® was determined using 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% CO ₂ . BT-474, MCF-7, SKBR-3 and MDA-MB-231 cell lines were incubated in RPMI-1640 medium, Dulbecco's modified Eagle medium, McCoy's 5A modified medium and Dulbecco's modified Eagle medium, respectively (all Supplemented with 10% fetal bovine serum). 3,000 cells per well were plated into 96 well plates. After 24 hours, the medium was removed and replaced with various concentrations of medium MAP, nitro PBA-PEG, CPT, medium MAP-CPT nanoparticles, targeted MAP-CPT nanoparticles or Herceptin® fresh medium. After 72 hours of incubation, the formulation was replaced with fresh medium and 20 μl of CellTiter 96® AQueous One Solution cell proliferation assay (Promega) was added per well. This was incubated in a humidified oven with 5% CO ₂ at 37 ° C. for 2 hours, after which the absorbance was measured at 490 nm. Wells containing untreated cells were used as controls.

Medium MAP and nitro PBA-PEG give IC ₅₀ values of 500 μM and 1000 μM (highest concentration tested), respectively, indicating minimal cytotoxicity. The IC ₅₀ concentration of CPT, medium MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles is based on the content of CPT. Note that CPT was gradually released from medium and targeted MAP-CPT nanoparticles (see in vitro release studies). Furthermore, nanoparticle uptake by cells resulted in long release times due to the acidic nature of endosomes. These factors contribute to IC ₅₀ values from CPT observed for medium sized MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles (Table 5). In the HER2 + cell line, targeted MAP-CPT nanoparticles resulted in lower IC ₅₀ values compared to MAP-CPT alone. On the other hand, HER2- cell lines, targeting did not affect _{IC 50.} BT-474 was the most resistant cell line to CPT and thus to medium MAP-CPT nanoparticles. Herceptin addition to BT-474 cells or SKBR-3 cells at a concentration of 0.001-0.5 μM resulted in a constant 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 so far (Phillips, et al., Cancer Res. 68, 9280-9290 (2008)). At Herceptin concentrations up to 0.5 μM, the HER2-cell line showed no effect.

IC ₅₀ values were not obtained over ^a no value, 0.001-0.5 μM concentration range.

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

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

Example 32: Determination of Maximum Tolerated Dose (MTD) in Nude Mice MTD values were determined in female Ncr nude mice and defined as the highest dose with less than 15% weight loss and no treatment-related death. In these experiments, 12 week old female NCr nude mice were randomly divided into 13 groups, each containing 5 animals. Formulation medium MAP or nitro PBA-PEG 200 mg / kg, short MAP-CPT nanoparticles 10, 15 or 20 mg / kg (based on CPT), medium MAP-CPT nanoparticles 8, 10 or 15 mg / kg ( CPT based), 5, 8 or 10 mg / kg long MAP-CPT nanoparticles (based on CPT) and 8 or 10 mg / kg targeted MAP-CPT nanoparticles (based on CPT), day 0 and 7 On the day, it was administered via intravenous injection of the tail vein. All injections except targeted MAP-CPT were formulated in 0.9 w / v% saline and targeted MAP-CPT was formulated in PBS, pH 7.4. Over two weeks from the start of treatment, the mice's weight and health were recorded and monitored. MTD was defined as the highest dose with less than 15% weight loss and no treatment related death. Animals were euthanized by CO ₂ asphyxiation when they exceeded the criteria for 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 twice weekly on days 0 and 7 and healthy Were monitored (Table 6). Mouse body weight and health are not affected by treatment with high doses of medium MAP or nitro PBA-PEG, indicating minimal toxicity for these polymer components. For the group containing CPT, the greatest weight loss was seen on days 3-5 of each treatment. Most groups regained lost weight. The study ceased if weight loss continued and averaged over 15%. Some mice treated with medium MAP-CPT 15 mg / kg and long MAP-CPT 10 mg / kg showed diarrhea and appeared weak. All other mice looked healthy. 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).

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

Example 33: Biodistribution in nude mice To assess the biodistribution of targeted nanoparticles in mice, 17-week-old NCr nude mice were implanted subcutaneously with 17β-estradiol pellets. Two days later, BT-474 cancer type cells suspended in RPMI-1640 medium were subcutaneously injected into the right front flank at 10 million cells / animal. Treatment was initiated after the tumor reached an average size of 260 mm ³ . The animals were randomized into two groups of 6 animals each, and 5 mg CPT / kg MAP-CPT nanoparticles (in PBS, pH 7.4) or 5 mg CPT / kg target by intravenous injection into the tail vein Treated with immobilized MAP-CPT nanoparticles and 29 mg Herceptin® / kg (pH 7.4 in PBS). Blood was collected from these groups of animals via saphenous vein bleeding after 4 and 24 hours. The animals were then euthanized with CO ₂ nitrogen and immersed in PBS. Tumor, lung, heart, spleen, kidney and liver were collected 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 treatment.

  Organs were weighed and 100 mg each was placed in a Lysing Matrix A homogenizer tube with addition of 0.64 cm (1/4 inch) ceramic spheres (MP Biomedicals, Solon, Ohio). 1 ml of RIPA lysis buffer (Thermo Scientific) was added and the tissue was homogenized for 30 seconds at 6 m / sec using a FastPrep®-24 homogenizer (MP Biomedicals, Solon, Ohio). This was repeated 3 times with 1 minute cooling on ice between each time. The sample was then centrifuged at 14,000g for 15 minutes at 4 ° C. The amount of unconjugated CPT is first mixed with 10 μl of supernatant with 10 μl 0.1 N HCl and incubated for 30 minutes at room temperature, then 80 μl methanol is added at room temperature for protein precipitation. Measured by incubating for 3 hours. This mixture was centrifuged at 14,000 g for 10 minutes 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 the HPLC. The peak area of the eluted CPT (at 7.8 minutes) was compared to that of the control. To measure the total amount of CPT, 10 μl of sample was mixed with 6.5 μl of 0.1 N NaOH. This solution was incubated at room temperature for 1 hour to release CPT from the parent polymer. 10 μl of 0.1N HCl was then added to convert the carboxylate CPT form to the lactone form. 73.5 μl of methanol was then added and the mixture was incubated for 3 hours at room temperature. The sample was then centrifuged and processed as above.

  The average percentage injected dose (ID) of total CPT per gram of 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 was present per gram of mouse tumor treated with MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles, respectively. Twenty-four hours after treatment, 2.6% ID of total CPT is present per gram of tumor in mice treated with MAP-CPT nanoparticles, while 3.2% ID of total CPT is targeted. Was present per gram of tumor in mice receiving MAP-CPT nanoparticles. These data show that targeted nanoparticles of substantially the same size and surface charge as the non-targeted version did not increase tumor localization over that of the non-targeted version, and other reported observations Indicates a match. Mice treated with MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles resulted in similar CPT distribution in the heart, liver, kidney and lung. However, in the spleen, a relatively high amount of total CPT accumulation for targeted MAP ^ CPT versus untargeted was at the 24 hour time point. This effect has been observed previously for humanized antibodies in mice.

  FIG. 19B shows the average percentage of unconjugated CPT in each organ of 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. This indicates a rapid clearance of free CPT from these organs. In tumors at 24 hours, there was a significantly higher percentage of unconjugated CPT for both MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles compared to 4 hours. The prevalence of free CPT within the tumor suggests cellular accumulation of CPT. Targeted nanoparticles show a slightly higher percentage of unconjugated CPT than non-targeted nanoparticles in mouse tumors at both 4 and 24 hours.

  The total concentration of CPT in plasma and the percentage of CPT that was all unconjugated in plasma were similar for both MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles (FIG. 19C). After 4 hours, 17 and 20 μg / ml total CPT remained in the 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, 12 μg / ml. The percentage of total CPT that was unconjugated in plasma was less than 3% for all conditions. This indicates a slight release of unconjugated CPT from plasma and rapid clearance (FIG. 19D).

Example 33 Treatment of Tumors with Targeted Nanoparticles BT-474 tumor-bearing Ncr nude mice were either MAP-CPT (5 mg CPT / kg) or targeted MAP-CPT (5 mg CPT / kg, 29 mg Herceptin / kg). It was treated by. After 4 and 24 hours, the mice were euthanized and the tumors were excised and sectioned 20-30 μm thick using a cryostat. Tumor sections were placed on Superfrost Plus glass slides (Fisher Scientific, Hampton, NH) and stored at −80 ° C. until treatment. Glass slides were thawed and tissue sections were fixed directly on glass slides with 10% formalin solution for 15 minutes. The slides were then washed 3 times for 5 minutes each with PBS and blocked for 1 hour in 5% goat serum blocking buffer. CPT emits light at 440 nm and fluoresces naturally. To identify the location of MAP, PEG in MAP was stained at 14 μg / ml with rat anti-PEG primary antibody that recognizes internal PEG units and incubated overnight at 4 ° C. This was followed by 3 PBS washes and 1 hour incubation with 2 μg / ml Alexa Fluor® 488 goat anti-rat IgM secondary antibody. The slides were then washed 3 times with PBS and incubated with 2 μg / ml Alexa Fluor® 633 goat anti-human IgG secondary antibody for 1 hour 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 Plane-Neofluor oil objective. CPT was detected using 2-photon excitation at 720 nm (emission filter BP 390-465 nm). PEG and Herceptin were detected using excitation at 488 nm (emission filter LP 530) and 633 nm (emission filter BP 645-700 nm), respectively. All laser and acquisition settings were set at the beginning of the imaging and were not changed. Image analysis was performed in a Zeiss lsm image browser.

  FIG. 20A shows a tumor section of a mouse 4 hours after treatment with MAP-CPT nanoparticles. CPT signals were scattered. The signals for CPT and MAP appeared to be localized together in the superimposed images. 24 hours after injection, accumulation of CPT signal was punctate (FIG. 20B). Overlapped images suggested co-localization of CPT and MAP. For mice treated with targeted MAP-CPT nanoparticles, points indicating CPT accumulation were observed both at 4 hours and 24 hours after treatment (FIGS. 20C and 20D). There was co-localization of the CPT and MAP signals in the overlaid images. The presence of Herceptin in the targeted MAP-CPT nanoparticles is indicated by a strong blue signal in the Herceptin channel compared to the weak background signal in the non-targeted version.

Example 33: Anti-tumor efficacy study in nude mice BT-474 is one of the most resistant breast cancers to anti-cancer agents including camptothecin. In order to promote tumor growth in nude mice, 17β-estradiol pellets were transplanted into 7-week old Ncr nude mice and BT-474 tumor cell transplantation was performed 2 days later. On day 0 (5 days after transplantation), the average tumor size in each group was 250 mm. Treatment started on day 1. Animals were randomized into 9 groups of 6-8 animals in each 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 phosphoric acid); 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 prepared fresh and given via intravenous injection of the tail vein. Injections were normalized at 150 μl per 20 g body weight of mice. Treatment with Herceptin® was given once a week for 2 weeks and all other groups were given once a week for 3 weeks. Tumor size, using calipers measurement (length × width ^2/2) was recorded 3 times a week, animal health, and continuously monitored. Animals were euthanized when the tumor volume exceeded 1000 mm ³ . Six weeks after the start of treatment, the animals were euthanized by CO ₂ asphyxiation. The results of this study are shown in FIG.

Tumors in control animals that received saline grew rapidly (FIG. 21). After 28 days, 5 out of 8 mice had tumors exceeding the size limit of 1000 mm ³ . All animals in this group were euthanized at this point.

  The group treated with CPT (8 mg / kg) did not cause any tumor inhibition compared to the control group (P> 0.05). Treatment-related deaths of 1 and 3 were recorded on days 9 and 16, respectively, and on day 28, 4 euthanasias due to exceeding the tumor size limit were recorded. None of the animals survived until the last day of the study.

Mice treated with irinotecan 80 mg / kg showed insignificant tumor inhibition compared to the control group (P> 0.05). One tumor-related death occurred on day 11. On the last day of the study, the tumor size reached an average of 575 mm ³ .

Animals receiving MAP-CPT nanoparticles at 8 mg CPT / kg showed very 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 mm ³ and 3 out of 8 mice were reduced to 0 tumor size. In an MTD study using non-tumor bearing NCr nude mice treated with MAP-CPT, 8 mg CPT / kg, no deaths occurred, but on the 21st day of the study there was one death due to weight loss It was. This may be due to the addition of tumor burden in this study and / or the mice used in this study were 7 weeks old, while in the MTD study, 12 weeks old mice were used. There is.

Animals treated with Herceptin® 5.9 mg / kg showed very significant tumor inhibition compared to the control group (P <0.01). On day 11 of treatment, 7 out of 8 treated animals had tumors that had shrunk to zero. However, on the last day of the study, two of the reduced tumors recurred, yielding a total of 5 animals with 0 volume tumor, and the group mean tumor volume was 60 mm ³ (FIG. 22). Mice treated with 1 mg CPT / kg with MAP-CPT nanoparticles were quickly disposed of because no anti-tumor effect was observed. Nevertheless, when Herceptin®, 5.9 mg / kg is added to MAP-CPT nanoparticles as a target drug via a nitro PBA-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 were reduced to 0 on day 9 of treatment and remained intact until the end of the study ( FIG. 22). One non-tumor-related death occurred on day 37. This result is very significant compared to the control group (P <0.01). The combination of results observed for these three groups shows that in addition to the original activity of Herceptin®, there was an improvement in efficacy by adding Herceptin® targeted agent.

Animals treated with Herceptin® 2.9 mg / kg yielded 2 animals with tumors reduced to 0 by the end of the study. However, the average tumor size increased to 278 mm ³ from the start of the study. 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 zero. On the last day of the study, the tumor size was reduced to an average of 141 mm ³ . This result is very significant compared to the control group (P <0.01). These results further suggest a benefit of targeting tumor inhibition.

  Five weeks after transplantation of 17β-estradiol pellets, it was observed that several mice regardless of treatment group had swollen bladder and abdominal distension. This is likely due to the use of 17β-estradiol pellets that produce hydronephrosis and urinary retention in athymic nude mice. After 6 weeks of treatment, symptoms worsened and many mice were observed to develop swollen bladder and abdominal distension, thereby being euthanized and terminating the study.

^a N _start is the number of animals at the _start of the study and N _end is the number of animals that were alive at the _end of the study. ^b N _TRD is the number of treatment-related deaths, N _NTRD is the number of non-treatment-related deaths, and N _euthan is the number of animals euthanized by exceeding the tumor size limit of 1000 mm ³ . ^c N _o reduction to 0 is the number of animals whose tumor has reduced to 0 on the last day of the study.

  The examples set forth above are provided to provide those skilled in the art with a complete disclosure and method of making and using embodiments of the disclosed particles, compositions, systems and methods, with respect to this disclosure by the inventors. Modifications of the above-described manner of carrying out the present disclosure that are apparent to those of ordinary skill in the art without intending to limit the scope considered are contemplated to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which this disclosure belongs. All references cited in this disclosure are incorporated by reference to the same extent that each reference is incorporated individually by reference.

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

  It will be appreciated that the present disclosure is not limited to a particular composition or biological system, but can of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is 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. To do. The term “plurality” includes two or more objects, unless the content clearly dictates otherwise. Unless defined otherwise, 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 method and material similar or equivalent to those issued herein may be used to test specific examples of suitable materials and methods described herein. Also good.

  A number of embodiments of the disclosure have been described. However, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims (51)

Nanoparticles comprising a polyol-containing polymer and a nitrophenylboronic acid-containing polymer,
The nitrophenylboronic acid-containing polymer is linked to the polyol-containing polymer by a reversible covalent bond, and the nanoparticles are such that the nitrophenylboronic acid-containing polymer is present in the environment outside the nanoparticles. Said nanoparticles composed. The polyol-containing polymer comprises at least one or more of the following structural units:
Where
A is the organic moiety of the following formula:
here,
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 comprising one or more of -H, -F, -C, -N or -O; and Y is independently selected from an organic moiety presenting -OH or -OH ,
And B is an organic moiety that polymer by connecting one of R ₁ and R ₂ of the first said part A to one of R ₁ and R ₂ of the second said part A of claim 1 Nanoparticles according to 1. R ₁ and R ₂ independently have the following formula:
Where
d is 0-100;
e is 0-100;
f is 0-100;
Z is a covalent be a bond linking certain organic moiety to another organic moiety; and Z _{1 is} -NH 2, -OH, independently selected from -SH and -COOH, according to claim 2 Nanoparticles. Where
X is C _n H _{2n + 1} , where n is 0 to 5; and
The nanoparticle according to claim 2 or 3, wherein Y is -OH. A is the following:
Independently selected from the group consisting of
Where
The spacer is independently selected from any organic group,
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} -NH 2, -OH, independently selected from -SH and -COOH, nanoparticles according to any one of claims 2-4. Said A is the following:
Nanoparticles according to any one of claims 2 to 5, which are selected independently from each other. Said B is:
here,
q is 1-20;
The nanoparticle according to any one of claims 2 to 6, wherein p is 20 to 200; and L is a leaving group. The nanoparticle according to claim 2, wherein the structural unit of formula (I) is:
The nanoparticle according to claim 2, wherein the structural unit of formula (II) is:
The structural unit of formula (III) is:
And where
The nanoparticle according to claim 2, wherein n is 1-20. The nanoparticle according to claim 1, wherein the polyol-containing polymer is:
The polymer comprises at least one terminal nitrophenylboronic acid group and has the following general formula:
Where
R ₃ and R ₄ are independently hydrophilic organic polymers;
X ₁ is an organic moiety containing one or more of -C, -N or -B;
Y ₁ is a phenyl group having one or more nitro groups;
r is 1-1000;
The nanoparticle according to any one of claims 1 to 11, comprising a nitrophenylboronic acid wherein a is 0 to 3 and b is 0 to 3. R ₃ and _{R 4} are _{(CH 2 CH 2 O) t} , wherein, t is 2 to 2000, the nanoparticles of claim 12. X ₁ is —NH—C (═O) —, —S—S—, —C (═O) —NH—, —O—C (═O) — or —C (═O) —O—. The nanoparticles according to claim 12 or 13, wherein Y ₁ is a phenyl group having one or more nitro groups.   15. Nanoparticles according to any one of claims 12 to 14, wherein r = 1, a = 0 and b = 1. Functional group 1 and functional group 2 are the same or different and are and _{-B (OH) 2, -OCH 3} , is independently selected from -OH, any one of claims 12 to 15 Nanoparticles according to 1. The nitrophenylboronic acid-containing polymer is:
And
The nanoparticle according to any one of claims 12 to 16, wherein s = 20 to 300.   18. Nanoparticles according to any one of claims 1 to 17, further comprising a compound, wherein the compound forms part of the polyol-containing polymer and / or nitrophenylboronic acid-containing polymer.   The nanoparticle according to any one of claims 1 to 17, further comprising a compound, wherein the compound is bound to the polyol-containing polymer and / or nitrophenylboronic acid-containing polymer.   The nanoparticle according to any one of claims 1 to 19, further comprising a compound, wherein the compound is a therapeutic agent.   21. The nanoparticle of claim 20, wherein the therapeutic agent is a small molecule chemotherapeutic agent or a polynucleotide.   24. The nanoparticle of claim 21, wherein the polynucleotide is an interfering RNA.   23. A nanoparticle according to any one of claims 1 to 22, further comprising one or more compounds, wherein at least one compound is a target ligand.   24. The nanoparticle of claim 23, wherein the target ligand is a protein, aptamer, small molecule, antibody or antibody fragment.   25. A nanoparticle according to claim 23 or 24, wherein the target ligand is a single antibody.   26. The nanoparticle of claim 25, wherein the single antibody is conjugated to a terminal portion of the nitrophenylboronic acid-containing polymer.   The target ligand is selected from transferrin, protein, aptamer, small molecule, antibody, or antibody fragment, and the nanoparticle is a therapeutic agent selected from camptothecin, epothilone, taxane or polynucleotide, or any combination thereof. 24. The nanoparticle of claim 23, further comprising.   28. A composition comprising nanoparticles according to any one of claims 1 to 27 and suitable vehicles and / or excipients.   29. The composition of claim 28, wherein the composition is a pharmaceutical composition and the suitable vehicle and / or excipient is a pharmaceutically acceptable vehicle and / or excipient. Wherein the nanoparticles further comprises a ^{10 B} as part of at least one nitro-phenyl boronic acid-containing polymer, wherein the composition is formulated for Inbibohou containing neutron activation therapy composition of claim 29 object.   28. A method of delivering a compound to a target comprising contacting the target with a nanoparticle according to any one of claims 1-27.   32. The method of claim 31, wherein the target is a cancer cell in a mammalian body.   28. A system for delivering a compound to a target, comprising the nanoparticles of any one of claims 1 to 27, wherein the system is used to deliver the compound to the target.   28. A method of administering a compound to an individual, the method further comprising administering to the individual a nanoparticle according to any one of claims 1 to 27 further comprising the compound.   A system for administering a compound to an individual, the system comprising at least one polyol-containing polymer and at least one nitrophenylboronic acid-containing polymer capable of being interconnected via a reversible covalent bond, wherein the at least one 28. The system wherein one polyol-containing polymer and at least one nitrophenylboronic acid-containing polymer are collected with the compound in the nanoparticles of any one of claims 1-27 and administered to the individual. The following formula:
Where
R ₃ and R ₄ are independently hydrophilic organic polymers;
X ₁ is an organic moiety containing one or more of —CH, —N or —B;
Y ₁ is a phenyl group having one or more nitro groups;
r is 1-1000;
a is 0-3, and b is 0-3 A polymer containing nitrophenylboronic acid. R ₃ and _{R 4} are independently _{(CH 2 CH 2 O) t} , wherein, t is 2 to 2000, the polymer of claim 36. X ₁ is —NH—C (═O) —, —S—S—, —C (═O) —NH—, —O—C (═O) — or —C (═O) —O—. 38. The polymer according to claim 36 or 37, wherein Y ₁ is a nitrophenyphenyl group.   39. A polymer according to claim 36, 37 or 38, wherein r is 1, a is 0 and b is 1. Functional group 1 and functional group 2 are the same or different and are and _{-B (OH) 2, -OCH 3} , is independently selected from -OH, any one of claims 36 to 39 The polymer described in 1.   41. The polymer of any one of claims 36-40, further comprising a target ligand.   42. The polymer of claim 41, wherein the target ligand is an antibody.   42. The polymer of claim 41, wherein the target ligand is a single antibody.   43. The polymer of claim 41 or 42, wherein the antibody is conjugated to a terminal portion of the nitrophenylboronic acid-containing polymer.   28. Contacting the polyol-containing polymer with the nitrophenylboronic acid-containing polymer for a time and under conditions that allow the polyol-containing polymer to be coupled with the nitrophenylboronic acid-containing polymer. The method of preparing the nanoparticle of any one of these.   46. The method of claim 45, wherein the nanoparticles are configured such that the nitrophenylboronic acid-containing polymer is present in an environment external to the nanoparticles.   47. The method of claim 45 or 46, wherein the polyol-containing polymer is a mucin acid polymer. The nitrophenylboronic acid-containing polymer is:
Any one of
48. A method according to any one of claims 45 to 47, wherein s = 20-300.   28. Including instructions for nanoparticles comprising a polyol-containing polymer, nitrophenylboronic acid-containing polymers, and methods for conjugating nanoparticles comprising the polyol-containing polymer and nitrophenylboronic acid-containing polymers. A kit for producing the nanoparticles according to any one of the above.   50. The kit of claim 49, wherein the polyol-containing polymer is a mucin acid polymer. The nitrophenylboronic acid-containing polymer is:
Any one of
51. The kit according to claim 49 or 50, wherein s = 20-300.