JP2019108372A - Nanoparticles stabilized with nitrophenylboronic acid compositions - Google Patents

Abstract

To provide nanoparticles comprising a polyol-containing polymer and a nitrophenylboronic acid-containing polymer, for effective and specific delivery of a compound of interest.SOLUTION: A nanoparticle comprises a polyol-containing polymer coupled through a reversible covalent linkage to a nitrophenylboronic acid-containing polymer. The nitrophenylboronic acid-containing polymer is configured to be present in an environment external to the nanoparticle.SELECTED DRAWING: None

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 the Invention
The present disclosure relates to carrier nanoparticles, in particular, nanoparticles suitable for delivering a compound of interest, and related compositions, methods and systems.

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

  Some for delivery of different classes of biomaterials and biomolecules, typically with a desired biological activity and / or chemical activity, either for pathological examination, therapeutic treatment or basic biological research. Methods are known and used.

  Used with compounds of varying complexity, size and chemistry as the number of molecules suitable for use as chemical agents or biopharmaceuticals (eg, drugs, biologics, therapeutic agents or imaging agents) increases Development of a suitable delivery system has proven to be particularly difficult.

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

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

  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 to a polyol containing polymer, and the nanoparticles are configured such that the boronic acid containing polymer is present in the external environment of the nanoparticles. One or more compounds of interest can be carried by the nanoparticles as part of or attached to the polyol containing polymer and / or the boronic acid containing polymer.

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

  According to another aspect, a method of delivering a compound to a target is described. The method comprises contacting a target with a nanoparticle described herein, wherein the compound is a polyol containing polymer or a boronic acid containing polymer of the nanoparticles described herein. Included in

  According to another aspect, a system for delivering a compound to a target is described. This system comprises at least a polyol containing polymer and a boronic acid containing polymer, which can be linked together via reversible covalent bonds and collected in the nanoparticles described herein containing the compounds.

  According to another aspect, a method of administering a compound to an individual is described. The method comprises 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.

  According to another aspect, a system for administering a compound to an individual is described. This system can be linked to each other via a reversible covalent bond and collected in the nanoparticles described herein coupled to a compound to be administered to an individual according to the methods described herein , At least a polyol containing polymer and a boronic acid containing polymer.

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

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

  According to 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 a nitrophenylboronic acid group are described herein, which lowers the nanoparticle's pKa to increase its stability.

  Another aspect of the present disclosure provides a description of targeted nanoparticles. It may have only one single target ligand in some embodiments. This can facilitate delivery to specific targets of the nanoparticle, eg, cells presenting a binding partner for the target ligand of the particle. Targeted nanoparticles of this kind have advantages over nanoparticles with multiple target ligands, such as smaller overall size by having less surface ligands, as well as There are fewer ligands that mediate non-specific binding than affinity based interactions via the tei based interactions. Thus, nanoparticles that contain or carry a therapeutic agent can successfully target the target configuration (eg, cells or tissues) with only one targeting ligand, and the therapeutic agent can be It can be delivered to the target at high target ligand to therapeutic agent ratio. This aspect of the described nanoparticles significantly enhances the efficiency of producing such therapeutic agents, while at the same time 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 some implementations as flexible molecular structures suitable for carrying compounds of various sizes, dimensions and chemical properties. It can be used in form.

  The nanoparticles and related compositions, methods and systems described herein, in some embodiments, deliver 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 compounds.

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

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

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

  The nanoparticles and related compositions, methods and systems described herein, in some embodiments, have applications where controlled delivery of a compound of interest is desirable (such as therapeutic applications, diagnostic applications and clinical applications). It can be used in combination with (including but not limited to) medical applications. Further applications include biological analysis, veterinary applications, and delivery of compounds of interest to non-animal organs, in particular plant organs.

  The details of one or more embodiments of the present disclosure are set forth in the accompanying drawings and the detailed description and examples that follow. 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, explain the principles and practices of the present disclosure. Contribute to
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic of the relevant method for the formation of nanoparticles and associated in the absence of boronic acid containing compounds. Panel A shows a schematic of a polyol containing polymer (MAP, 4) and a compound of interest (nucleic acid) according to the embodiments described herein. Panel B shows the nanoparticles formed by the assembly of polymers comprising the polyols and compounds shown in panel A. FIG. 1 shows a schematic view of a nanoparticle and associated manufacturing method according to an embodiment 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 with a 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. 8 shows the formation of a complex comprising a polyol containing polymer according to the embodiments described herein and a compound of interest. In particular, FIG. 3 shows the results of a MAP gel shift assay using plasmid DNA in accordance with an embodiment of the present disclosure. The DNA ladder is loaded in lane 1. Lanes 2-8 show plasmid DNA combined with MAP with incrementally increased 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. 8 shows the formation of a complex comprising a polyol containing polymer according to the embodiments described herein and a compound of interest. In particular, 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 combined with MAP with incrementally increased charge ratio. 8 depicts the properties of nanoparticles according to some embodiments described herein. In particular, FIG. 5 shows particle size (dynamic light scattering (DLS) measurement) to charge ratio as well as zeta potential (properties related to surface charge of nanoparticles) to MAP-plasmid nanoparticles according to an embodiment of the present disclosure. The figure which illustrates the plot of a charge ratio is shown. 8 depicts the properties of nanoparticles according to some embodiments described herein. In particular, FIG. 6 shows a diagram illustrating particle size (DLS) to charge ratio as well as a plot of zeta potential to charge ratio for BA-pegylated MAP-plasmid nanoparticles according to an embodiment of the present disclosure. 16 shows salt stability of BA-pegylated MAP-plasmid nanoparticles in accordance with an embodiment of the present disclosure. Plot A: 5: 1 BA-PEG + np + 1X PBS 5 minutes later; Plot B: 5: 1 BA-PEG + np, dialysis 3 × w / 100 kDa + 1 × PBS 5 minutes; plot C: 5: 1 pre-pegylated w / BA-PEG + 1 × PBS 5 After minutes; plot D: 5: 1 pre-pegylated w / BA-PEG, dialysis 3 × w / 100 kDa + PBS 5 minutes later. Figure 15 shows delivery of an agent to human cells in vitro using nanoparticles according to the embodiments described herein. In particular, FIG. 8 is a relative light unit (RLU) pair that is 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 which illustrates the plot of a charge ratio is shown. FIG. 16 shows delivery of an agent to a target using nanoparticles according to the embodiments described herein. In particular, FIG. 9 shows a diagram illustrating a plot of cell survival to charge ratio after MAP / pGL3 transfection, in accordance with an embodiment of the present disclosure. Survival data are for the experiment shown in FIG. FIG. 16 shows delivery of multiple compounds to a target using nanoparticles according to the embodiments described herein. In particular, 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. FIG. 6 shows a plot of units (RLU) versus particle species. The term siCON refers to siRNA having a control sequence. FIG. 16 shows delivery of a compound to a target using nanoparticles according to the embodiments described herein. In particular, FIG. 11 shows relative light unit (RLU) vs. RLU for delivery of MAP / siGL3 into HeLa-Luc cells at a charge ratio of 5 +/− according to embodiments described herein. The figure which illustrates the plot of siGL3 density is shown. FIG. 1 shows a schematic diagram of the synthesis of a boronic acid containing polymer presenting a target ligand in accordance with some embodiments of the present disclosure. In particular, FIG. 12 shows a schematic for the synthesis of boronic acid-peg disulfide-transferrin in accordance with an embodiment of the present disclosure. FIG. 1 shows a schematic of the synthesis of nanoparticles according to some embodiments described herein. In particular, FIG. 13 shows a schematic for the formation of nanoparticles using Campothecin mucic acid polymer (CPT-mucic acid polymer) in water according to an embodiment of the present disclosure. FIG. 6 shows a table summarizing the particle size and zeta potential of nanoparticles formed from a CPT-munic acid polymer conjugated in water, prepared according to an embodiment of the present disclosure. FIG. 1 shows a schematic of the synthesis of nanoparticles according to some embodiments described herein. In particular, FIG. 15 shows the formation of boronic acid-pegylated nanoparticles with CPT-munic acid polymer and boronic acid-disulfide-PEG ₅₀₀₀ in water according to an embodiment of the present disclosure. Fig. 6 shows the salt stability of MAP-4 / siRNA stabilized by no PEG, nitropeg-PEG and 0.25 mol% Herceptin-PEG-nitro PBA. 10 × PBS was added at 5 minutes so that the solution prepared and obtained at a charge ratio of 3 (+/−) is in 1 × PBS. A and B are medium-sized MAP-CPT nanoparticles or targeted MAP- of targeted MAP-CPT nanoparticles at an increased ratio of Herceptin®-PEG-nitro PBA to nanoparticles in (A) BT-474. FIG. 7 shows intracellular uptake of CPT nanoparticles. (B) Medium-sized MAP-CPT nanoparticles or targets with or without free Herceptin® (10 mg / ml) in BT-474 cell line (filled bar) and MCF-7 cell line (white bar) Fig. 7 shows intracellular uptake of functionalized 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. The 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 concentrations of unconjugated CPT as a function of time. Biodistribution of MAP-CPT (5 mg CPT / kg) and targeted MAP-CPT (5 mg CPT / kg, 29 mg Herceptin® / kg) nanoparticles after 4 and 24 hours of treatment is shown. The injected dose (ID) percentage of total CPT per gram of tumor, heart, liver, spleen, kidney or lung. Biodistribution of MAP-CPT (5 mg CPT / kg) and targeted MAP-CPT (5 mg CPT / kg, 29 mg Herceptin® / kg) nanoparticles after 4 and 24 hours of treatment is shown. Total CPT percentage not conjugated in each organ in tumor, heart, liver, spleen, kidney and lung. 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. 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, BT-474 tumors taken from NCr nude mice treated with targeted MAP-CPT (5 mg CPT / kg, 29 mg Herceptin® / kg) at 29 mg Herceptin® / kg) (D) for 24 hours Confocal immunofluorescence micrographs of the sections are depicted. The left panel emits light at 440 nm (CPT, pink), the center left panel emits light at 519 nm (MAP, green), the center right panel emits light at 647 nm (Herceptin®, blue), and the right panel overlays the image. Figure 7 shows anti-tumor efficacy in NCr nude mice bearing BT-474 xenograft tumors. Mean tumor volume as a function of time, the group containing Herceptin dosing received once every two weeks, all other groups received dosing once every three 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 antitumor efficacy.

  Nanoparticles, as well as compositions, methods and systems that can be used in connection, methods for producing the described nanoparticles, delivery methods for the compounds contained in the nanoparticles, treatment methods with the described nanoparticles, and the described Kits for collecting nanoparticles are provided herein.

  The term "nanoparticle" as used herein denotes a composite structure of nanoscale dimensions. In particular, the nanoparticles are typically particles in the size range of about 1 to about 1000 nm, usually spherical, but different morphologies are possible depending on the nanoparticle composition. The portion of the nanoparticle that contacts the nanoparticle's external environment is generally defined as the surface of the nanoparticle. In the nanoparticles described in the present description, the size limitations are limited in two dimensions, thus the nanoparticles described herein comprise 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 nanoparticles according to the experimental design. For example, nanoparticles used for 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 , With a diameter of about 1 to about 100 nm. The term "targeting nanoparticle" refers to a nanoparticle conjugated to a targeting agent or targeting ligand.

Additional 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 are understood by one of ordinary skill in the art upon reading the present disclosure. The dimensions 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 a fluorescent label) and by those skilled in the art It is suitable for detecting other chemical properties that may be included by the additional techniques being performed.

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

  The terms "deliver" and "delivery" as described herein refer to the activity of controlling the spatial arrangement of the compounds, in particular controlling such arrangement. Thus, delivery of a compound in the sense of the present disclosure affects the positioning and movement of the compound under a specific time and a specific set of conditions, such that the positioning and movement of this compound under these conditions is Otherwise indicate the ability of this compound to be altered for positioning and movement.

  In particular, with respect to the reference endpoint, delivery of the compound indicates the ability of the compound to be ultimately placed on the selected reference endpoint by controlling the positioning and movement of the compound. In in vitro systems, delivery of a compound usually involves corresponding alteration of the chemical and / or biological detectable properties and activity of the compound. In in vitro systems, delivery of a compound also typically involves changes in the pharmacokinetics of the compound, and may also involve changes in pharmacodynamics.

  The pharmacokinetics of a compound show the absorption, distribution, metabolism and excretion of this compound from the system, typically produced by the body of an individual. In particular, the term "absorption" refers to the process by which the substance enters the body, the term "distribution" refers to the dispersion or dissemination of the substance through body fluids and tissues of the body, the term "metabolism" from the parent compound Indicating 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 processes the release of the compound, typically a drug, from the formulation. Show. The term "pharmacodynamics" refers to the physical effect of the compound on the body or microbe or parasite in the body or on the body, the mechanism of drug action and the relationship between drug concentration and effect. One skilled in the art can identify techniques and procedures that are suitable for the pharmacokinetic and pharmacodynamic characteristics and properties of the compound of interest and in particular the agent of interest, such as a drug.

  The term "agent" as used herein denotes a compound capable of exhibiting chemical or biological activity associated with a target. The term "chemical activity" as used herein denotes the ability of a molecule to carry out a chemical reaction. The term "biological activity" as used herein refers to the ability of a molecule to affect an organism. Examples of chemical activities of agents include the formation of covalent bonds or electrostatic interactions. Examples of biological activities of the agent include production and secretion of endogenous molecules, absorption and metabolism of endogenous or exogenous molecules, and activation or inactivation of gene expression including transcription and translation of the gene of interest .

  The term "target," as used herein, includes unicellular or multicellular organisms or any portion thereof, as well as any in vitro or in vivo biological system or any portion thereof Includes biological systems.

  The nanoparticles described herein, the boronic acid-containing polymer bound to the polyol-containing polymer, are arranged in the nanoparticles to be presented to the environmental exterior of the nanoparticles.

  The term "polymer" as used herein refers to large molecules typically composed of repeating structural units linked by covalent chemical bonds. Suitable polymers may be linear and / or branched and may be in the form of homopolymers or in the form of copolymers. 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 use, the polymer should 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. In particular, suitable polymers may have a molecular weight of about 100,000 or less.

  The terms "polyol containing polymer" or "polyol polymer" as used herein denote a polymer presenting a large number of hydroxyl functional groups. In particular, a polyol-containing polymer suitable for forming the nanoparticles described herein comprises at least one hydroxyl functional group for coupling interaction with at least one boronic acid of a boronic acid-containing polymer Includes polymers that present a part.

  The term "present" as used herein with respect to a compound or functional group indicates a bond that is performed to maintain the chemical reactivity of the bound compound or functional group. Thus, the functional groups present on the surface can, under appropriate conditions, perform one or more chemical reactions that chemically characterize the functional group.

The structural units forming the polymer include polyol monomers such as pentaerythritol, ethylene glycol and glycerin. Examples of polymers comprising polyols include polyesters, polyethers and polysaccharides. Examples of suitable polyester diols, and in particular, the general formula _{HO- (CH 2 CH 2 O)} p -H diol having p ≧ 1, such as polyethylene glycol, polypropylene glycol, and poly (tetramethylene ether ) Glycols, but not limited thereto. 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 of ordinary skill in the art. Examples of procedures for the synthesis of examples of polyol polymers have been previously described in the scientific literature and others are described in Examples 1-4. Additional procedures for producing polyol-containing polymers can be identified by one of ordinary skill in the art in light of the present disclosure.

  The terms "boronic acid containing polymer" or "BA polymer" as used herein denote a polymer comprising at least one boronic acid group presented for attachment to a hydroxyl group of a polyol containing polymer. In particular, the boronic acid containing polymers of the nanoparticles described herein include polymers comprising at least one structural unit of an alkyl or aryl substituted boronic acid comprising a chemical bond of carbon and boron. Suitable BA polymers include polymers in which the boronic acid is present in the terminal structural unit or any other suitable moiety to impart hydrophilic character to the resulting polymer. Examples of polyol-containing polymers include polymers having a molecular weight of about 40,000 or less, and 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 examples of polyol polymers have been previously described in the scientific literature and others are described in Examples 5-8. Additional procedures for making BA polymers can be identified by one of ordinary skill in the art in light of the present disclosure.

  In the nanoparticles described herein, the polyol polymer is coupled with the BA polymer. The terms "coupled" or "coupling" as used herein with respect to the bond between two molecules indicate an interaction that forms a reversible covalent bond. In particular, in the presence of a suitable culture medium, the boronic acid presented on the BA polymer interacts with the hydroxyl group of the polyol via a rapid and reversible covalent coupling interaction, and a suitable culture medium In the form of boronic acid ester. Suitable media include water and some aqueous solutions as well as further organic media which can be identified by the person skilled in the art. In particular, when contacted in aqueous medium, the BA polymer and the polyol react to form water as a by-product. It is known that this boronic acid-polyol interaction generally proceeds in an organic culture medium, although it is generally more preferable in an aqueous solution. Furthermore, the cyclic esters formed by 1,2 diols and 1,3 diols are generally more stable than their non-cyclic ester counterparts.

Thus, in the nanoparticles described herein, at least the boronic acid of the boronic acid containing polymer is attached to the hydroxyl groups of the polyol containing polymer by reversible covalent bonding. Boronic acid esters between BA polymers and polyol polymers are based, for example, on boron-11 nuclear magnetic resonance ( ¹¹ B NMR), depending on the specific chemistry and properties of the boronic acid and the polyols that make up the nanoparticles. It can be detected by methods and techniques identified by those skilled in the art, such as potentiometric titration, selected UV / visible light and fluorescence detection techniques.

  The nanoparticles resulting from the coupling interaction of the BA polymers described herein with the polyol polymers described herein present the BA polymers on the surface of the particles. In some embodiments, the nanoparticles can have a diameter of about 1 to about 1000 nm and spherical morphology, but the particle size and morphology are the BA and polyol polymers used to form the nanoparticles And by the compounds delivered in the nanoparticles according to the present disclosure.

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

  In some embodiments, the compound of interest to be delivered to the nanoparticles is attached to the polymer, typically a polyol polymer, via covalent or non-covalent bonds. Examples of such embodiments are provided by nanoparticles in which one or more moieties in at least one of the compound polyol polymer and the BA polymer are bound for one or more purposes.

  The terms "bind", "coupled" or "binding". As used herein, refers to connection or integration by bonding, linking, force or bonding to keep two or more components together, as this includes direct or indirect bonding. For example, where the first compound is directly attached to the second compound, and embodiments where one or more intermediate compounds, in particular an intermediate molecule, is placed between the first compound and the second compound. including.

In particular, in some embodiments, the compound may be attached to the polyol polymer or BA polymer via covalent attachment of the compound to a suitable portion of the polymer. An example of covalent attachment is the binding of the drug camptothecin to mucinic acid polymer as in Example 19 where the attachment to the mucic acid polymer is via a biodegradable ester bond linkage, as well as the attachment of transferrin to BA-PEG ₅₀₀₀ via PEGylation of transferrin. This is described in Example 9, which takes place.

  In some embodiments, the polymer (eg, of one or more functional groups capable of specifically binding to a corresponding functional group on the compound of interest, to allow attachment of the particular compound of interest) By addition) can be designed or modified. For example, in some embodiments, the nanoparticles can be PEGylated with BA-PEG-X, where X specifically reacts with thiols or nonspecifically with amines It may be a maleimide group or an iodoacetyl group or a free radical. The compound to be conjugated can then react with the maleimide or iodoacetyl group after modification to develop a thiol functional group. The compounds to be linked may also be modified by aldehyde or ketone groups, and these may be reacted via condensation reactions with diols on polyols to give acetals or ketals.

  In some embodiments, the compound of interest is attached to the polyol polymer or BA polymer through 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 noncovalent binding is described in Example 10.

  The compound of interest may be attached to the nanoparticle prior to, during or after formation of the nanoparticle, eg, via modification of the polymer and / or modification of any attached compound in the particle mixture. . Examples of procedures to carry out conjugation of compounds in nanoparticles are described in the Examples section. Further procedures for attaching a compound to a BA polymer, a polyol polymer or other components of the nanoparticles described herein (e.g., a previously introduced compound of interest) can be made by reading this disclosure by those skilled in the art. 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 nanoparticle binds with one or more agents used as a targeting ligand on the BA polymer and with one or more agents delivered to a selected target Bond on polyol polymer and / or BA polymer.

  The terms "targeting ligand" or "targeting agent" as used in the present disclosure, for the purpose of engaging a particular target, and in particular, for example by enabling nanoparticle cell receptor binding Fig. 6 shows any molecule that can be presented on the surface of nanoparticles 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. In particular, the targeting ligand may be an antibody to a specific surface cell receptor such as anti-VEGF, small molecules such as folate, as well as other proteins such as holo-transferrin.

  The ligand chosen is understood by those skilled in the art and may 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 TAT protein from HIV-1. TAT protein is viral transcriptional activation and actively enters the cell nucleus. Torchilin V. P. et al, PNAS. 98, 8786 8791, (2001). Suitable target ligands attached to the BA polymer typically include a flexible spacer such as poly (ethylene oxide) with boronic acid attached at its end (see Example 9).

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

  The section of polyol polymers and BA polymers for forming nanoparticles described herein can be performed in terms of compounds of interest and targets. In particular, the section of Polyol-containing polymers and BA-polymers suitable for forming nanoparticles described herein provides candidate Polyol polymers and BA-polymers and couplings in view of the present disclosure It can be practiced by selecting a polyol polymer and a BA polymer capable of forming an interaction, wherein the selected BA polymer and the polyol polymer are included in or bound to the polyol polymer and / or the BA polymer In view of the compound of and the targeting ligand, the polyol polymer has a chemical composition such that it is more hydrophilic than the BA polymer. The detection of relevant presentation in the environment external to BA polymer and nanoparticles on surface nanoparticles is carried out by detection of zeta potential which can carry out modification of the surface of the nanoparticles, as described in Example 12 It can be done. (See FIG. 6.) Further procedures for detecting particle surface charge and particle stability in salt solution are as exemplified in Example 12 (see FIG. 7 for details). And the like, as well as detection of charge of particle size such as, as well as further procedures which can be identified by the person skilled in the art.

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

During the ceremony
A has the following formula:

here,
R ₁ and R ₂ are independently selected from any carbon-based group or organic group having a molecular weight of about 10 kDa or less;
X is selected from aliphatic groups comprising one or more of -H, -F, -C, -N or -O; and Y is -OH or an organic having a hydroxyl (-OH) group moiety _{(-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) to but OH can be mentioned those Without limitation, in which 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 denotes a series of atoms that constitute part of a larger molecule or molecular species. In particular, moieties refer to constituents of repeating polymer structural units. Exemplary moieties include acid or base species, sugars, carbohydrates, alkyl groups, aryl groups and any other molecular constituents useful to form polymer structural units.

  The term "organic moiety" as used herein denotes a moiety comprising a carbon atom. In particular, organic moieties include natural and synthetic compounds, as well as 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. Synthetic organic groups refer to compounds prepared by reaction with other compounds.

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

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

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

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

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

In some embodiments, R 1 and / or R 2 have 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
During the ceremony
The spacer is independently selected from any organic moiety (specifically, optionally an heteroatom, such as an alkyl group comprising sulfur, nitrogen, oxygen or fluorine, a phenyl group or an alkoxy group may be mentioned),
The amino acid is selected from any organic group having a free amine group and a free carboxylic acid group;
n is 1 to 20; and Z ₁ is independently selected from -NH ₂ , -OH, -SH and -COOH.

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

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

It can be selected independently of.

  In some embodiments, (B) can be formed by any linear, branched, symmetrical or unsymmetrical compound linking 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 depend on the chemical nature of the compound to be covalently or non-covalently linked. Dependent.

  Examples of the cationic moiety of (B) for use with anionic cargoes are: amidine group, quaternary ammonium, primary amine group, tertiary amine group (protonated to less than its 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 sulfonates of the formula, nitrates of the formula, carboxylates of the formula, and organic groups having a phosphonate It is not limited.

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

In the formula, R ₅ is an electrophilic group that can be covalently bonded to A when A contains a nucleophile. R ₅ in this case includes, but is not limited to:
Among other things, -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 a secondary ein.

In particular, when the moiety (B) is a cationic moiety (B) used together with an anionic cargo, the "organic group" consists generally of C _m H _{2 m} where m ≧ 1 and other heteroatoms It may have a backbone having the formula and contain at least one of the functional groups comprising: Amidines of the formula (XIII), quaternary ammoniums of the formula (XIV), primary of the formula (XV) An amine group, a secondary amine group of formula (XVI), a tertiary amine group of formula (XVII), which is protonated below its pKa, and an imidazolium of formula (XVIII).

In embodiments where moiety (B) is an anionic moiety (B) used in conjunction with a cationic cargo, the "organic group" has a general formula consisting of C _m H _{2 m} and other heteroatoms where m ≧ 1 And have at least one functional group comprising: a sulfonate of formula (XIX), a nitrate of formula (XX), a carboxylate of formula (XXI), and a formula (XXII) Phosphonates.

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

In embodiments where (B) is comprised of a primary amine group of formula (XV) and / or a secondary amine group of formula (XVI), the compound incorporating a carboxylic acid group may form a peptide bond Can be combined via

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

here,
q is from 1 to 20; and in particular, may be 5;
p is 20-200; and L is a leaving group.

The term "leaving group" as used herein denotes a molecular fragment that separates a pair of electrons in an anisotropic bond linkage. In particular, the leaving group is a molecule of anionic or neutral, ability leaving group is separated, correlated to _the pK _a of the conjugate acid, low pK _a is associated with a better leaving group ability . Examples of anionic compounds, Cl ^-, Br ^- and I ^- halides and sulfonate esters, such as, for example, para - toluenesulfonate or "tosylate" (TsO ^-) and the like. Examples of neutral molecular leaving groups are water (H ₂ O), ammonia (NH ₃ ) and alcohols (ROH).

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

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

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

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

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

  In some embodiments, the polyol containing polymer may 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:

During the ceremony
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 alkynyl group, or an alkyl group having the formula -C _m H _{2 m-} where m 1 1 including aromatic groups (eg phenyl, biphenyl, naphthyl or anthracenyl) ,
r is 1 to 1000;
a is 0-3; and b is 0-3;
And wherein functional group 1 and functional group 2 are the same or different and are capable of binding to a target ligand, in particular a protein, an antibody or a peptide, or -OH, -OCH ₃ or-( Terminal groups such as X ₁ )-(Y ₁ ) -B (OH) _2- .

In some embodiments, R ₃ and R ₄ are (CH ₂ CH ₂ O) _t , where t is 2-2000, and in particular 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 phenyl 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, the functional groups 1 and / or 2 of the formula (XXXI) may be functional groups capable of binding to cargo, in particular they may be targeting ligands such as proteins, antibodies or peptides Or a terminal group such as —OH, —OCH ₃ or — (X)-(Y) -B (OH) ₂ .

  The term "functional group" as used herein denotes a specific group of atoms within or part of a molecular structure that is 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 one skilled in the art. In particular, functional groups in the sense of the present disclosure include carboxylic acids, amines, triarylphosphines, azides, acetylenes, sulfonyl azides, thioazides and aldehydes. In detail, for example, functional groups capable of binding to the corresponding functional groups in the target ligand can be selected to include the following binding partners: carboxylic acid group and amine group, azide and acetylene group, azide and triarylphosphine Groups, sulfonyl azides and thioazides, and aldehydes and primary amines. Additional functional groups can be understood by one skilled in the art by reading the present disclosure. As used herein, the term "corresponding functional group" refers to a functional group capable of reacting with another functional group. Thus, functional groups that can react with each other can be referred to as corresponding functional groups.

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

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

In formula, s is 20-300.

  Examples of agents and target ligands capable of binding to the nanoparticles of the present disclosure include organic molecules or inorganic molecules, and these include polynucleotides, nucleotides, aptamers, polypeptides, proteins, polysaccharides, macromolecular complexes (proteins And mixtures of polynucleotides and saccharides and / or polysaccharides, viruses, molecules with radioactive isotopes, antibodies or antibody fragments, but are not limited thereto).

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

  The term "aptamer" as used herein refers to an oligonucleic acid or peptide molecule that binds to a specific target. In particular, mucinic acid aptamers can be used for various molecular targets (eg small molecules, proteins, nucleic acids, and also cells, tissues and organisms), for example through repeated in vitro selection, ie through systematic evolution of ligands by exponential enrichment (SELEX). Even nucleic acid species that have been engineered to bind). Aptamers are useful in biotechnological and therapeutic applications because they provide molecular recognition properties that compete with that of the antibody. Peptide aptamers are peptides that are designed to specifically bind to and interfere with protein-protein interactions inside cells. In particular, peptide aptamers can be derived, for example, according to a selection strategy derived from 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 that bind to the transcription factor activation domain. In vivo binding of the peptide aptamer to its target via this selection strategy is detected as expression of a downstream yeast marker gene.

  The term "polynucleotide" as used herein denotes an organic linear, cyclic or branched polymer composed of two or more amino acid monomers and / or their analogues. 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" usually denote a polypeptide having less than 50 amino acid monomers. As used herein, the terms "amino acid", "amino acid monomer" or "amino acid residue" refer to any of the twenty naturally occurring amino acids, non-naturally occurring amino acids, and artificial amino acids, Includes both D and L enantiomers. In particular, non-naturally occurring amino acids include the D stereoisomer of naturally occurring amino acids. (These contain useful ligand building blocks because they are not susceptible to enzymatic degradation.) The term "artificial amino acids" can be easily coupled using standard amino acid coupling science but is not natural And a molecule having a molecular structure that does not resemble the amino acids present in The term "amino acid analog" is an amino acid in which one or more of the individual atoms are replaced with either different atoms, isotopes or different functional groups, but otherwise identical to the original amino acid from which this 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 building blocks other than one or more monomers or amino acid monomers. The terms monomer, subunit, or building block refers to a chemical compound that can be chemically coupled with another monomer of the same or different chemical nature to form a polymer under appropriate conditions. 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 as being specific to other proteins, DNA, RNA, lipids, metabolites, hormones, chemokines and other biological molecules, including small molecules, can be involved in the interaction 1 shows a polypeptide having secondary and tertiary structure of, but is not limited to. An example of a protein described herein is an antibody.

  The term "antibody" as used herein is a type of protein produced by active B cells after stimulation with an antigen that can specifically bind the antigen to promote an immune response in a biological system. Say Intact antibodies typically consist 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 specifically binding to one particular spatial and polar machinery of another biological molecule, called an "epitope", and thus is defined as 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, wherein at least two monoclonal antibodies bind to different antigenic epitopes. The different antigenic epitopes may be to the same target, different targets or a combination. Antibodies may be prepared by techniques well known in the art such as host immunization and serum collection (polyclonal), or continuous hybridoma cell line preparation and secreted protein collection (monoclonal).

  In some embodiments, the polyol polymer forms a non-covalent complex or forms a 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, wherein the drug is covalently linked to the polyol polymer. Examples of polyol polymers conjugated with drugs are described in detail in Examples 16-21. In these embodiments, drug-conjugated polyol polymers (referred to herein as "polyol polymer-drug conjugates") form nanoparticles, the structure of which on the surface is a molecule of BA. Present sites for interaction with

  In some embodiments, the nanoparticles further comprise a BA polymer formed to provide steric stability and / or targeted functionality of the nanoparticles. In particular, in these embodiments, the addition of the BA polymer enables the minimization of self aggregation and unwanted interactions with other nanoparticles, thus providing improved salt stability and serum stability. . For example, steric stability is provided by the BA polymer with PEG, as described herein by the example of nanoparticles described in Example 12.

  In such embodiments, the structure of the nanoparticles provides several advantages over prior art drug delivery methods, such as the ability to provide controlled release of one or more drugs. This feature may be provided, for example, by the use of biodegradable ester linkages between the drug and the polyol polymer. The person skilled in the art understands other connection possibilities which are suitable for this purpose. In these embodiments, another advantage is to provide specific targeting of the drug via the BA polymer moiety.

  In some embodiments, the BA polymer can include a boronic acid fluoride (Example 7) or a fluorocleavable boronic acid (Example 8) that can be used as an imaging agent in MRI or other similar techniques. . Such imaging agents may be routine 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 nanoparticles are modified liposomes. In these embodiments, the modified liposome comprises a lipid conjugated to 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 liposomal nanoparticles.

  The term "liposome" as used herein denotes 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, which is an aqueous environment suitable for containing the drug to be delivered internally. Such liposomes present an outer surface which may include suitable targeting ligands or cell surface receptors or molecules for specific recognition by other targets of interest.

  The term "conjugated" as used herein, means that 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 to show that it causes electron delocalization.

  In yet another embodiment 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 micelles comprise polyol polymers that have been modified to include hydrophobic polymer blocks.

  The term "hydrophobic polymer block" as used in the present disclosure denotes a segment of the polymer which 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 region in contact with the surrounding solvent, separating 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 arranged to form a nanoparticle which, when mixed with the agent to be delivered, is a modified micelle having the agent to be delivered within the nanoparticle. Such nanoparticle embodiments present a polyol polymer suitable on its surface to interact with the BA polymer. This 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 a hydrophilic A and a hydrophobic B in formula (I) or (II). This interaction provides similar or similar advantages to those 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 generally refers to various media acting as solvent, carrier, binder, excipient or diluent for the nanoparticles to be included in the composition as the active ingredient. Indicates one of the

  In some embodiments where 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, 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. It is disclosed to include in combination with

  The term "excipient" as used herein denotes 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 ability of the individual's body to absorb the nanoparticles. Suitable excipients also include any material that can be used to bulk the formulation containing the nanoparticles to allow for convenient and accurate dosing. In addition to their use in 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 the form of medication, different excipients may be used. Examples of excipients include anti-tack 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 denotes a diluting agent which serves to dilute or convey the active ingredient of the composition. Suitable diluents include any substance capable of reducing the viscosity of the pharmaceutical preparation.

  In a particular embodiment, the composition, in particular the pharmaceutical composition, may be formulated for systemic administration, including parenteral administration, more particularly intravenous, intradermal and intramuscular administration.

  Examples of compositions for parenteral administration include, but are not limited to, sterile aqueous solutions, injectable solutions or suspensions comprising nanoparticles. In some embodiments, the composition for parenteral administration comprises, in use, a powdered composition previously prepared in freeze-dried lyophilised form, a biologically compatible aqueous liquid (distilled water , Physiological solution or other aqueous solution).

  The term "lyophilization" (freeze-drying or low-temperature drying) refers to a dehydration process, which is typically used to preserve perishable substances or to make them easier to transport. Freeze-drying is accomplished by freezing the material, then depressurizing the ambient pressure, and applying sufficient heat sufficient to cause the frozen water in the material to digest directly from the solid phase into a gas.

  In pharmaceutical applications, freeze-drying is often used to extend the shelf life of products such as vaccines and other injectables. By removing the water from the substance and sealing the substance in a vial, the substance 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 comprises contacting the target with a nanoparticle described herein.

  In some embodiments, the method is provided for delivery of an agent to an individual, wherein the method comprises formulating suitable nanoparticles in accordance with various disclosed embodiments. The nanoparticles may also be formulated into a pharmaceutically acceptable composition in accordance with some embodiments of the present disclosure. The method comprises delivering the nanoparticles to the subject. The nanoparticles or nanoparticle formulations may be given orally, parenterally, topically or rectally to deliver the nanoparticles to the individual . These are delivered in a form suitable for each administration route. For example, the nanoparticle composition may be administered in the form of a pill or capsule, may be administered by injection, inhalation, eye drops, ointment, suppository, infusion, or may be administered rectally by suppository.

  The term "individual" as used herein includes one organism, which includes plants or animals, in particular higher animals, in particular the spine Animals, such as mammals, may be mentioned, in particular, but not limited to, humans.

  The terms "parenteral administration" and "administered parenterally" as used herein mean a mode of administration, usually by injection other than enteral and topical administration, without limitation Intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, intraperitoneal, transtracheal, subcutaneous, subepithelial, intraarticular, subcapsular, subarachnoid, Intrathecal and intrastemal injections and infusions.

  The phrases "systemic administration", "systemically administered", "peripheral administration" and "peripherally administered" as used herein refer to the nanoparticles or compositions thereof within the central nervous system. Aside from direct administration to, it means that it enters an individual's system and is therefore subject to processes such as metabolism, eg subcutaneous administration.

  Actual dosage levels of the active ingredient or agent in the pharmaceutical compositions described herein achieve the desired therapeutic response without poisoning the individual for the particular individual, composition and mode of administration. It may be varied to obtain an amount of active agent effective to

  These therapeutic polymer conjugates are administered orally and nasally such as by spray, rectally, vaginally, parenterally, intracisternally, and by powder, ointment or drops to human and other animals. It may be administered for therapy by any suitable route of administration, including topical administration, including buccal and sublingual.

  Regardless of the route of administration chosen, the therapeutic polymer conjugates which can be used in the suitable hydrated form and / or in the pharmaceutical form of the invention are pharmaceutically acceptable dosages by conventional methods known to the potter. It can be prescribed in form.

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

  The "drug" or "therapeutic agent" may be used in the treatment, prevention or diagnosis of a condition in an individual, or may otherwise be used to enhance the physical or mental well-being of the individual. Indicates active agent.

  The term "symptoms" as used herein, as a whole, or one or more parts thereof, usually associated with a complete physical, mental and possibly socially good condition. Indicates the physical condition of the individual's body that is incompatible with the physical condition, as a whole or in one or more parts thereof. The conditions described herein include, but are not limited to, disorders and diseases. Here, the term "disorder" refers to a symptom of a living individual with dysfunction of the body or some part thereof, and the term "disease" is a loss of normal function of the body or some part thereof Indicate the symptoms of a living individual, typically manifested by discriminatory signs or symptoms. Examples of symptoms include injuries, dysfunctions, disorders (including mental and physical disorders), syndromes, infections, abnormal behavior of an individual, and abnormal changes in the structure and function of the body or part of the individual. Not limited to these.

  "Treatment" as used herein refers to any activity that is part of a pharmaceutical or surgical, medical treatment 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 condition. This occurs at the following primary, secondary and tertiary levels of protection: (a) primary prevention avoids the onset of the disease; (b) secondary prevention aims at the treatment of the initial disease, thereby promoting disease progression. And (c) tertiary prevention reduces the adverse effects of already developed disease by reducing function restoration and disease related complications.

Examples of compounds delivered by the nanoparticles described herein that are suitable as a drug emit electromagnetic radiation (e.g. ¹⁰ B isotopes) and can be used in radiation treatment (e.g. boron neutrons) Supplements included. Additional therapeutic agents include any lipophilic or hydrophilic, synthetic or naturally occurring, biologically active therapeutic agents, 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.S. J. Examples of such therapeutic agents include small molecule drugs, antibiotics, steroids, polynucleotides (eg, genomic DNA, cDNA, mRNA, siRNA, shRNA, miRNA, antisense oligonucleotides, virus 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 relevant Active macromolecules such as, but not limited to, 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 may be covalently attached to the polymer in the hybrid. In some embodiments, the covalent bond is reversible (eg, through a biodegradable form such as a prodrug form or a disulfide bond) to provide 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 Included are 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 reading this disclosure.

In some embodiments, the compounds to be delivered are suitable for imaging cells or tissues of an individual. Examples of compounds that can be delivered by the nanoparticles described herein and suitable for imaging are, 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 positions where < ¹⁹ > F atoms may be present are on the BA polymer component, on the polyol polymer component or on the drug to be delivered. These and other modifications will be apparent to those skilled in the art.

  In some embodiments, the nanoparticles described herein can be used to deliver chemicals used in the agricultural industry. In another embodiment of the invention, the agent delivered by the nanoparticles described herein is a biologically active compound with microbiology 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 fungicides. Microbicides can also be used to treat municipal and industrial water (eg, cooling water, white water systems in papermaking) systems. Aqueous systems susceptible to microbial attack or degradation are also found in the leather industry, the textile industry, and the coatings or paint industry. Examples of such microbicides and their use individually and in combination are described in US Pat. Nos. 5,693,631, 6,034,081, and 6,060,466. And they are incorporated herein by reference. Compositions comprising active agents such as those described above may be used in the same manner as the active ingredient situation is known. Among other things, such a use is not a pharmaceutical use, so the hybrid polymer does not have to conform to the required toxicity profile 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 agents using the nanoparticles. . In some embodiments of this system, the nanoparticles provide a suitable component for preparing nanoparticles for administration to an individual.

  The systems described herein may be provided in the form of a kit of parts. For example, the polyol polymer and / or the BA polymer may be contained as molecules alone or in the presence of suitable excipients, vehicles or diluents.

  In the kit of parts, the polyol polymer, the BA polymer and / or the agent to be delivered are independently included in the composition, possibly in a composition together with a suitable vehicle carrier or auxiliary agent, and included in the kit. For example, the polyol polymer and / or the BA polymer may be included only in one or more compositions and / or may be included in a suitable vector. Also, the agent to be delivered may be included in the composition, along with a suitable vehicle carrier or adjuvant. Alternatively, the agent may be given by the end user and not included in the kit of parts. In addition, polyol polymers, BA polymers and / or agents may be included in various forms suitable for proper incorporation into nanoparticles.

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

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

  Further details regarding suitable carrier agents or auxiliary agents of the composition, and generally the manufacture and packaging of the kit, can be identified by one of ordinary skill in the art reading this disclosure.

  In some embodiments, the nanoparticles are prepared by preparing the individual components of the nanoparticles and then mixing the components in various orders to reach the desired mixed nanoparticle structure It can be done. The preparation and mixing of the components is carried out in suitable solutions known to the person skilled in the art.

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

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

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

  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 Be done. In other embodiments, nanoparticles comprised of polyol polymer-drug conjugates can be prepared by dissolving the nanoparticles in a suitable aqueous solution. In still further embodiments, nanoparticles comprised of polyol polymer-drug conjugates can be prepared by mixing polyol polymer-drug conjugates with BA polymers that provide or do not provide a targeting ligand.

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

  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 modified liposomes. In various embodiments, nanoparticles can be prepared by mixing a polyol polymer and a conjugated lipid with a BA polymer and then mixing the agent to be delivered. In other embodiments, nanoparticles can be prepared by mixing agents that deliver lipids conjugated with polyol polymers. In another embodiment, the nanoparticles are prepared by mixing the lipid conjugated with the polyol polymer with the agent to be delivered and then mixing with the BA polymer to prepare nanoparticles, which are modified liposomes. It can be done.

  The formation of nanoparticles in accordance with 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, gel shift is used to monitor and measure the uptake of nucleic acid agents into nanoparticles (Example 10). In some embodiments, suitable nanoparticle sizes and / or zeta potentials can be selected using known methods (Example 11).

  Further details regarding suitable carrier agents or auxiliary agents of the composition, and generally the manufacture and packaging of the kit, can be identified by one of ordinary skill in the art reading this disclosure.

Nanoparticles Having a Polymer Having Nitrophenylboronic Acid Nanoparticles having a polyol containing polymer conjugated to a nitrophenylboronic acid containing polymer are described herein. The polymer comprising the targeted nanoparticle polyol nanoparticle portion described may have one or more of any one of the following structural units:

A has the following formula:

Here, R ₁ and R ₂ are independently selected from any carbon-based group or organic group having a molecular weight of about 10 kDa or less; and X is -H, -F, -C, -N Or Y is selected independently from an organic moiety presenting -OH or -OH, and B is selected from aliphatic groups comprising one or more of -O; and B is a group of 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 acids are selected from free amines and organic groups with free carboxylic acid groups; n is any single number from 1 to 20 And Z ₁ is independently selected from —NH ₂ , —OH, —SH and —COOH; R ₁ and R ₂ may independently have the following formula:

In which 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 , A covalent bond linking one organic moiety to another, and Z ₁ is independently selected from -NH ₂ , -OH, -SH and -COOH; B is any one of the following: May be one:

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 as described above Is paired with any one of the A subunits of In more detailed embodiments, the polymers containing polyol nanoparticle segments of targeted nanoparticles as shown in structural units of formula (I) are:

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

The polymer containing the polyol nanoparticle segment of the targeting nanoparticle represented by the structural unit of formula (III) is

And
Here, n is an arbitrary single number of 1 to 20. In some embodiments of the described targeting nanoparticles, the polyol containing polymer is:

  In summary, any of the formulas (Formula VI, VII or VIII) for the lower part A is combined with any of the formulas (Formula XXIII or XIV) for the lower part B to obtain the described polyol containing polymer of nanoparticles It can be formed. In certain aspects described herein, the nanoparticle is any one of Formula (IX, X or XI) for Subportion A and any of Formulas (Formula XXIII or XIV) for Subportion B It may have a polyol containing polymer formed from one combination.

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

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

Here, s is any single number of 20-300. The polymers of Formulas 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 (Formula VI, VII or VIII) for the lower part A, in combination with any one of the formulas (Formula XXIII or XIV) for the lower part 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 nanoparticle is any one of Formula (IX, X or XI) for Subportion A and any of Formulas (Formula XXIII or XIV) for Subportion B It 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 nanoparticle is any one of Formula (IX, X or XI) for Subportion A and Formula (XXIII or XIV) for Subportion B It may have a polyol containing polymer formed from a combination with any one, which may then be coupled with a boronic acid containing polymer corresponding to any one of Formulas XXXIII, XXXIV, or XXXV.

  The nanoparticles described herein may 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 the lower part A (formula VI, VII or VIII) is combined with any one of the polymers of the lower part B (formula XXIII or XIV) and the polyol of the described nanoparticles 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 lower part A, the polymer of lower part B or the polymer with nitrophenylboronic acid may be formed with one or more therapeutic agents, such as small molecule chemotherapeutic agents or polynucleotides. In some embodiments, the polymer of Subpart A (Formula VI, VII or VIII) is combined with any one of the polymers of Subpart B (Formula X XIII or XIV) and the polyol-containing polymer of the described nanoparticles Nanoparticle segments can be formed, and the resulting polyol containing polymer can then be coupled with a nitrophenylboronic acid containing polymer. The polymer of lower part A, the polymer of lower part B or the polymer with nitrophenylboronic acid can be formed with one or more DNA, RNA or interfering RNA (eg shRNA, siRNA or miRNA). In some embodiments, the polymer of Subpart A (Formula VI, VII or VIII) is combined with any one of the polymers of Subpart B (Formula X XIII or XIV) and the polyol-containing polymer of the described nanoparticles Nanoparticle segments can be formed, and the resulting polyol containing polymer can then be coupled with a nitrophenylboronic acid containing polymer. The polymer of lower part A, the polymer of lower part B or the polymer with nitrophenylboronic acid may be formed with one or more chemotherapeutic agents. In some embodiments, the polymer of Subpart A (Formula VI, VII or VIII) is combined with any one of the polymers of Subpart B (Formula X XIII or XIV) and the polyol-containing polymer of the described nanoparticles Nanoparticle segments can be formed, and the resulting polyol containing polymer can then be coupled with a nitrophenylboronic acid containing polymer. The polymer of lower part A, the polymer of lower part B or the polymer with 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 targeting nanoparticle is any one of Formula (IX, X or XI) for Subportion A and Formula (Formula XXIII or XIV for Subportion 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 formulas XXXIII, XXXIV, or XXXV obtain.

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

A has the following formula:

Here, R ₁ and R ₂ are independently selected from any carbon-based group or organic group having a molecular weight of about 10 kDa or less; and X is -H, -F, -C, -N Or Y is selected independently from an organic moiety presenting -OH or -OH, and B is selected from aliphatic groups comprising one or more of -O; and B is a group of 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 acids are selected from free amines and organic groups with free carboxylic acid groups; n is any single number from 1 to 20 And Z ₁ is independently selected from —NH ₂ , —OH, —SH and —COOH; R ₁ and R ₂ may independently have the following formula:

In which 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 , A covalent bond linking one organic moiety to another, and Z ₁ is independently selected from -NH ₂ , -OH, -SH and -COOH; B is any one of the following: May be one:

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 as described above Is paired with any one of the A subunits of In more detailed embodiments, the polymers containing polyol nanoparticle segments of targeted nanoparticles as shown in structural units of formula (I) are:

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

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

And
Here, n is an arbitrary single number of 1 to 20. In some embodiments of the described targeting nanoparticles, the polyol containing polymer is:

  In summary, any of the Formulas (Formula VI, VII or VIII) for Subportion A, in combination with any of the Formulas for Formula B (Formulas XXIII or XIV), comprises the polyol-containing targeted nanoparticle described It can form a polymer. In certain aspects described herein, the targeting nanoparticle is any one of Formula (IX, X or XI) for Subportion A and Formula (XXIII or XIV) for Subportion B It may have a polyol containing polymer formed from any one combination.

  Desired targeting nanoparticles can also have boronic acid containing polymers that are reversibly covalently coupled to polyol containing polymers. 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 to the nanoparticle. In some embodiments, the boronic acid-containing polymer comprises at least one terminal boronic acid group and has the following general formula:

Here, 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, and Y ₁ is m ≧ 1 formula _-C m _{H 2m} - an alkyl group or an aromatic group, r is is any single number of 1 to 1000, a is any single number from 0 to 3, And b is any single number from 0 to 3, and functional group 1 and functional group 2 are the same or different, and any of -B (OH) ₂ , -OCH ₃ or -OH It can be selected independently or one. In some embodiments, these variable subportions of the described boronic acid containing polymers may be selected from: 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) is any one of -O-, and _{Y 1} is a phenyl group. In some embodiments, these variable subportions of the described boronic acid containing polymers may be selected from: 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 phenyl group, and r may have a value of 1, and a has a value of 0 And b may have a value of one. In detailed embodiments, the targeted nanoparticles described herein may comprise a boronic acid containing polymer having the following formula:

Here, s is any single number of 20-300.

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

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

Here, s is any single number of 20-300. The polymers of Formulas 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 (Formula VI, VII or VIII) for the lower part A, in combination with any one of the formulas (Formula XXIII or XIV) for the lower part B, describes the described targeting nano The polymer containing the polyol nanoparticle segment of the 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 acids. 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 targeting nanoparticle is any one of Formula (IX, X or XI) for Subportion A and Formula (XXIII or XIV) for Subportion 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 targeting nanoparticle is any one of Formula (IX, X or XI) for Subportion A and Formula (Formula XXIII or XIV for Subportion 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 formulas XXXI, XXXIII, XXXIV, or XXXV obtain.

  In some aspects described herein, formed from any of the polyol-containing polymeric nanoparticle segments described herein or a combination of polyol-containing polymeric nanoparticle segments and a boronic acid-containing polymer The nanoparticles are conjugated to the target ligand to form a targeted nanoparticle wherein the ratio of target ligand to nanoparticle is 3: 1. In some aspects described herein, formed from any of the polyol-containing polymeric nanoparticle segments described herein or a combination of polyol-containing polymeric nanoparticle segments and a boronic acid-containing polymer The nanoparticles are conjugated to the target ligand to form a targeted nanoparticle wherein the ratio of target ligand to nanoparticle is 1: 1. In some aspects described herein, formed from any of the polyol-containing polymeric nanoparticle segments described herein or a combination of polyol-containing polymeric nanoparticle segments and a boronic acid-containing polymer The nanoparticle is conjugated to one single target ligand to form a targeted nanoparticle. In some aspects, the described target ligand is conjugated to the boronic acid containing polymer at the end opposite to the boronic acid. The targeting ligand conjugated to the targeting nanoparticle described is a protein, a protein fragment, an amino acid peptide, or an aptamer derived from either an amino acid or a polynucleotide, or other known to bind to a target of interest It may be any one of high affinity molecules. In some embodiments, the targeting ligand which 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 chimeric protein having a portion thereof It may be If the targeting 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. In addition, the antibody may be humanized such that only a small portion of the variable regions comprising CDR segments or CDR segments are non-human antibodies, and the remainder of the antibodies are human antibodies. The antibodies described herein are of any isotype, such as IgG, IgM, IgA, IgD, IgE, IgY or another type of isoform known to be produced by a mammal. May be In some embodiments, the targeting 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 Subportion A is any one of Formulas (Formulas XXIII or XIV) for Subportion B: Combined to form the polyol-containing polymer segment of the described targeting nanoparticle, and further comprising any of 5, 4, 3, 2 or 1 boronic acid conjugated to a target ligand, such as phenylboronic acid or It was coupled to a nitrophenylboronic acid containing polymer.

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

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

  In certain aspects described herein, the targeting nanoparticle is any one of Formula (IX, X or XI) for Subportion A and Formula (XXIII or XIV) for Subportion 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 a 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 Subportion A is any one of Formulas (Formulas XXIII or XIV) for Subportion B: Combined to form the polyol-containing polymer segment of the described targeting nanoparticles and further 5, 4, 3, 2 or 1 boronic acid-containing polymers, such as phenylboronic acid or nitrophenylboronic acid conjugated with a target ligand Coupled to the Here, the targeted nanoparticles obtained are conjugated only to a single target ligand.

  In some aspects, a nanoparticle formed from any one of Formulas (VI, VII or VIII) for Subportion A is any one of Formulas (Formulas XXIII or XIV) for Subportion B: Together, they form the polyol-containing polymer segment of the described targeting nanoparticles and are further coupled to a single boronic acid-containing polymer conjugated to the target ligand, such as phenylboronic acid or nitrophenylboronic acid. Here, the targeted nanoparticles obtained are conjugated only to a single target ligand.

  In certain aspects described herein, the targeting nanoparticle is any one of Formula (IX, X or XI) for Subportion A and Formula (XXIII or XIV) for Subportion B It may have a polyol containing polymer formed from a combination with any one, which has the formula of any 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 targeted nanoparticles obtained are conjugated only to a single target ligand.

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

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

  In some embodiments, the targeting nanoparticles described herein are conjugated to a single target ligand. In some embodiments described herein, the targeting nanoparticle is any one of Formula (IX, X or XI) for Subportion A and Formula (Formula XXIII or XIV for Subportion 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 formulas XXXI, XXXIII, XXXIV, or XXXV obtain. It is further conjugated to a single target ligand selected from proteins, protein fragments, amino acid peptides or aptamers.

  In some embodiments, the targeting nanoparticles described herein are conjugated to a single target ligand. In some embodiments described herein, the targeting nanoparticle is any one of Formula (IX, X or XI) for Subportion A and Formula (Formula XXIII or XIV for Subportion B) ) May have a polyol-containing polymer formed from a combination with any one of the following, which is then any 5, 4, 3 corresponding to any one of the formulas XXXI, XXXIII, XXXIV, or XXXV , 2 or 1 may be coupled with a boronic acid containing polymer. It 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.

  In some embodiments described herein, the targeting nanoparticle is any one of Formula (IX, X or XI) for Subportion A and Formula (Formula XXIII or XIV for Subportion 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 formulas XXXI, XXXIII, XXXIV, or XXXV obtain. It 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.

  In some embodiments, the targeting nanoparticles described herein are conjugated to a single target ligand. In some embodiments described herein, the targeting nanoparticle is any one of Formula (IX, X or XI) for Subportion A and Formula (Formula XXIII or XIV for Subportion 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 formulas XXXI, XXXIII, XXXIV, or XXXV obtain. It is further conjugated to a single target ligand selected from a protein, a protein fragment, an amino acid peptide or an aptamer, wherein the resulting targeted nanoparticle only binds to a single target ligand Can not be

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

  In some embodiments described herein, the targeting nanoparticle is any one of Formula (IX, X or XI) for Subportion A and Formula (Formula XXIII or XIV for Subportion 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 formulas XXXI, XXXIII, XXXIV, or XXXV obtain. It is further conjugated to a single antibody, a cellular receptor, a ligand of a cellular receptor, or a chimeric protein having a protein or portion thereof, wherein the resulting targeted nanoparticle is a single Conjugated only to the target ligand.

  The targeted nanoparticles described herein may further comprise a compound. In some embodiments, the compound may be one or more therapeutic agents, such as 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.

  While various types of nanoparticles and targeting nanoparticles have been discussed that can be produced using the components described herein, the following detailed embodiments can be produced. In one embodiment, the targeted nanoparticle described is a therapeutic agent selected from a mucic acid containing polymer, camptothecin, epothilone, taxane or interfering RNA sequence, a formula covalently coupled with mucic acid polymer in a reversible covalent linkage It has a phenylboronic acid containing polymer having XXXI, XXXIII, XXXIV or XXXV. The targeting 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 the targeting ligand at the opposite end as the nanoparticle Here, this targeted nanoparticle comprises one single target ligand.

  In one embodiment, the targeted nanoparticle described is a therapeutic agent selected from a mucic acid containing polymer, camptothecin, epothilone, taxane or interfering RNA sequence, a formula covalently coupled with mucic acid polymer in a reversible covalent linkage It has a phenylboronic acid containing polymer having XXXI, XXXIII, XXXIV or XXXV. The targeting 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 the targeting ligand at the opposite end as the nanoparticle Here, this targeted nanoparticle comprises one single antibody.

  In one embodiment, the targeted nanoparticle described is a therapeutic agent selected from a mucic acid containing polymer, camptothecin, epothilone, taxane or interfering RNA sequence, a formula covalently coupled with mucic acid polymer in a reversible covalent linkage It has a phenylboronic acid containing polymer having XXXI, XXXIII, XXXIV or XXXV. The targeting 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, a mouse antibody, at the end opposite to the nanoparticle. A rabbit antibody, a non-human primate antibody, a canine antibody, or a rodent antibody, or a chimeric antibody consisting of any two of these antibodies (wherein the antibodies are IgG, IgD, IgM, IgE, IgA or IgY) And any one of the isoforms, wherein the targeting nanoparticle comprises one single antibody.

  In one embodiment, the targeted nanoparticle described is a therapeutic agent selected from a mucic acid containing polymer, camptothecin, epothilone, taxane or interfering RNA sequence, a formula covalently coupled with mucic acid polymer in a reversible covalent linkage It has a phenylboronic acid containing polymer having 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 as the nanoparticle Here, the targeted nanoparticle comprises one single cellular receptor.

  In one embodiment, the targeted nanoparticle described is a therapeutic agent selected from a mucic acid containing polymer, camptothecin, epothilone, taxane or interfering RNA sequence, a formula covalently coupled with mucic acid polymer in a reversible covalent linkage It has a phenylboronic acid containing polymer having XXXI, XXXIII, XXXIV or XXXV. The targeting 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 end opposite to the nanoparticle Here, the targeting nanoparticle comprises one single receptor ligand.

  The method system described herein is further described in the following examples. This is provided for illustrative purposes but is not intended to be limiting. Those skilled in the art understand the applicability of the features described in detail for nucleic acid detection and methods of 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. The 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, a plasmid containing the firefly luciferase gene, was extracted from bacteria expressing pGL3 and purified. siGL3 was purchased from Integrated DNA Technologies (sequences 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 mumate diamine-DMS polymers.

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

Example 2: N-BOC-protected munic acid diamine, (2)
A mixture of 8 g (33.6 mmol) of mucoic acid dimethyl ester (1; Example 1), 12.4 mL (88.6 mmol) of triethylamine and 160 mL of methanol under reflux in a continuously stirred 500 mL round bottom flask The mixture was heated at 85 ° C. for 0.5 hours, after which 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 refluxing overnight, the mixture is filtered, washed with methanol, recrystallized from methanol and dried under vacuum to give 9.4 g (19 mmol, 57%) of N-BOC-protected munic acid diamine Obtained. ¹ H NMR ((CD ₃ ) ₂ SO) δ 7.66 (m, 2 H), 6.79 (m, 2 H), 5.13 to 5.15 (d, 2 H), 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 munic acid diamine, (3)
Transfer 8 g (16.2 mmol) of N-BOC-protected mucinic acid diamine (2; Example 2) to a 500 mL round bottom flask containing 3 M HCl in methanol (160 mL) and continuously at 85 ° C. overnight While stirring, it 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 a diamine mucic acid. ¹ H NMR ((CD ₃ ) ₂ SO) δ 7.97 (m, 8 H), 5.35 to 5.38 (m, 2 H), 4.18 to 4.20 (m, 2 H), 3.82 (M, 2H), 3.35 to 3.42 (m, 8H), 2.82 to 2.90 (m, 4H). ESI / MS (m / z): 294.3 [M] ⁺ , 317.1 [M + Na] ⁺ , 333.0 [M + K] ⁺

Example 4: Mumic acid diamine-DMS copolymer (MAP), (4)
A 1.5 mL eppendorf tube was charged with 85.5 mg (0.233 mmol) of the bis (hydrochloride) salt of Example ₃ in 0.8 mL of 0.1 M NaHCO ₃ . Dimethylsuberimidate · 2HCl (DMS, Pierce Chemical Co., 63.6 mg, 0.233 mmol) was added and the solution was vortexed and centrifuged to dissolve the components. The resulting mixture was stirred at room temperature for 15 hours. The mixture was then diluted with 8 mL of water and brought to pH 4 by the addition of 1N HCl. The solution was then dialyzed in ddH ₂ O for 24 hours using a 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing). The dialysis solution was lyophilized to dryness to give 49 mg of a 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, 1 H), 168. 38 (s, 1 H), 71. 45 (s, 4 H), 71. 22 (s, 3 H), 42. 34 (s , 2H), 36.96 (s, 3 H), 32. 74 (s, 3 H), 28. 09 (s, 4 H), 26. 90 (s, 4 H). Mw [GPC] = 2520, Mw / Mn = 1.15.

  The 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 nucleic acids, eg, siRNA, to form nanoparticles. These nanoparticles need to have steric stability to be used in mammals, and if necessary, they could have the targeted drug involved. In order to perform these two functions, the nanoparticles may be decorated with PEG for steric rest and PEG-targeting ligands. To do so, a boronic acid containing PEG compound is prepared. For example, boronic acid containing PEG can be synthesized according to the following example.

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

Example 6: Boronic acid-disulfide-PEG ₅₀₀₀ , (6)
The 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, Lay San Bio Inc.) was added to a glass vial equipped with a stirrer. To this was added 110 mg of aldorithiol-2 (0.5 mmol, Aldrich) dissolved in 4 mL of methanol. The solution was stirred at room temperature for 2 hours after addition of 77 mg of mercaptophenylboronic acid (0.5 mmol, Aldrich) in 1 mL of methanol. The resulting solution was stirred at room temperature for a further 2 hours. The 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 into ddH ₂ O for 15 hours with a 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing). The dialyzed solution was lyophilized to dryness. ¹ H NMR (300 MHz, dDMSO) δ 8.12-8.00 (m), 7.83-7.72 (m), 7.72-7.61 (m), 7.61-7. 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)
The 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 described below.

The (2,3,5,6) -fluorocarboxyphenylboronic acid was dissolved in excess SOCl ₂ (about 100 equivalents) to which several drops of DMF were added. The mixture was refluxed for 2 hours under argon. The excess SOCl ₂ was removed under reduced pressure and the resulting residue was taken up in anhydrous dichloromethane. To this solution was added PEG ₅₀₀₀ -NH ₂ (1 equivalent) and triethylamine (30 equivalents) 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 into ddH ₂ O for 24 hours with a 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing). The dialyzed solution was lyophilized to dryness.

The fluorine containing compounds are useful to provide 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 (even for simple imaging or may allow for imaging and treatment by the addition of a therapeutic agent).

Example 8: Synthesis of (2,3,5,6) -tetrafluorophenylboronic acid-disulfide-PEG ₅₀₀₀ , (8)
The 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 described below.

Add 250 mg of PEG ₅₀₀₀ -SH (0.05 mmol, Lay San Bio Inc.) to a glass vial equipped with a stirrer. To this is added 110 mg of aldorithiol-2 (0.5 mmol, Aldrich) dissolved in 4 mL of methanol. This solution is stirred at room temperature for 2 hours after addition of 77 mg of (2,3,5,6) -fluoro-4-mercaptophenylboronic acid (0.5 mmol) in 1 mL of methanol. The resulting solution is stirred for a further 2 hours at room temperature. The 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 left to stand for 1 hour. The resulting precipitate is collected via centrifugation, washed several times with diethyl ether and dried. The dried solid is redissolved in water, filtered through a 0.45 μm filter and dialyzed into ddH ₂ O for 15 hours with a 3500 MWCO dialysis membrane. The dialyzed solution is lyophilized to dryness.

Example 9: Synthesis of boronic acid-PEG ₅₀₀₀ -transferrin, (9)
A targeting agent may be placed at the other end of the boronic acid derived PEG in the compounds of Examples 5-8, for example, according to the approach illustrated in FIG. 12 with reference to transferrin addition.

  Thus, the components of the system that contain nucleic acid as a therapeutic agent (a target ligand is a protein such as transferrin (FIG. 12), an antibody or antibody fragment, a peptide such as RGD or LHRH, a low concentration such as folate or galactose Or the like). Boronated PEGylated targeting agents can be synthesized as follows.

Specifically, the following procedure was performed to synthesize boronate PEG ₅₀₀₀ -transferrin according to the approach illustrated in FIG. A solution of 10 mg (0.13 μmol) nohuman holo-transferrin (iron rich) (Sigma Aldrich) in 1 mL of 0.1 M PBS buffer (p.H. 7.2) to 3.2 mg OPSS-PEG ₅₀₀₀ -SVA (5 equivalents, 0.64 μmol, Laysan Bio Inc.) was added. The resulting solution was stirred at room temperature for 2 hours. Pegylated transferrin 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 of OPSS-PEG ₅₀₀₀ pegylated transferrin in 100 μL was incubated at room temperature with 20 μL of 4-mercaptophenylboronic acid (1 μg / μL, 20 μg, 100 equivalents) for 1 hour at room temperature. After incubation, this solution was dialyzed twice on a YM-30,000 NMWI device (Millipore) to remove excess 4-mercaptophenylboronic acid and pyridyl-2-thione by-product.

Example 10: Formulation of MAP-nucleic acid particles-gel shift assay As illustrated in FIG. Mix 10 μl of MAP with different concentrations in DNAse and RNase free water and mix 0.5, 1, 1.5, 2, 2.5 and 5 charge ratios (“+” on polymer Charge vs. "-" charge on the nucleic acid was obtained. The resulting mixture was incubated at room temperature for 30 minutes. As shown in FIGS. 3 and 4, 10 μL of 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. The nucleic acids not contained in the nanoparticles migrate on the gel. These results provide guidance on the required charge ratio for nucleic acid content 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 water containing no DNase and RNAse, 10 μL of MAP, DNase and RNA degradation Mixed at various concentrations in enzyme free water to obtain charge ratios of 0.5, 1, 1.5, 2, 2.5 and 5. The resulting mixture was incubated at room temperature for 30 minutes. Then, 20 μL of the mixture was diluted to 70 μL with DNAse and RNAse free water for particle size measurement. The 70 μL solution was then diluted to 1400 μL with 1 mM KCl for zeta potential measurement. 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 Using Boronic Acid PEG _{5k As} illustrated in FIG. 2, 2 μg of plasmid DNA (0.45 μg / μL, 4 in DNAse-free and RNase-free water) .4 μl) was diluted to 80 μl in DNAse and RNAse free water. This plasmid solution is mixed with 4.89 μg of MAP (0.5 μg / μL, 9.8 μL) and diluted in 80 μL of DNAse and RNAse-free water to a charge ratio of 3 +/- and A final plasmid concentration of 0.0125 μg / μL was obtained. The resulting mixture was incubated at room temperature for 30 minutes. To this solution was added 480 μg of boronic acid PEG _5K (compound 6; Example 6), (20 μg / μL, 24 μL). The mixture is then incubated for an additional 30 minutes, dialyzed twice with DNAase and RNAse free water in 0.5 mL 100,000 MWCO membranes (BIOMAX, Millipore Corporation), DNAse and RNA Reconstitution was carried out in 160 μL of decomposing enzyme-free water. One half of this solution was diluted with 1.4 mL of 1 mM KCl for zeta potential measurement (FIG. 6). It should be noted that the zeta potential of the BA-containing nanoparticles showed a lower zeta potential than the nanoparticles not containing it. These results support the conclusion that BA-containing nanoparticles localize BA on the outside of the nanoparticles. The other half was used to measure particle size. Particle size was measured every minute for 5 minutes after the addition of 10.2 μL of 10 × PBS to obtain a final solution of 90.2 μL in 1 × PBS. The particle size was then measured every minute for another 10 minutes, as shown in FIG. BA-containing nanoparticles separated (by filtration) from non-particulate constituents are stable in PBS but particles without BA are not stable. These results support the conclusion that BA-containing nanoparticles are stable to aggregation in PBS and so have BA localized to their outside.

Example 13 Transfection of MAP / p DNA Particles into HeLa Cells HeLa cells are seeded in 24-well plates at 20,000 cells per well 48 hours prior to transfection and supplemented with 10% FBS Was grown in the culture medium. MAP particles were formulated to contain 1 μg of pGL3 in 200 μL of 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 added. Cells were then incubated for 5 hours at 37 ° C., 5% CO ₂ before adding 800 μL of growth medium supplemented with 10% FBS. After 48 hours of incubation, cell fractions were analyzed for cell viability using the MTS assay. The remaining cells were lysed in 100 μL of 1 × luciferase culture cell lysis reagent. Luciferase activity was determined by adding 100 μL of luciferase assay reagent to 10 ul of cell lysate, and biofluorescence was quantified using a Monolight 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: Cotransfection of MAP / pDNA particles and / or siRNA particles into HeLa cells HeLa cells are seeded in a 24-well plate at 20,000 cells per well 48 hours prior to transfection. , Grown in medium supplemented with 10% FBS. MAP particles were formulated to contain 1 μg pGL3 and 50 nM siGL3 in 200 μL Opti-MEM I at a charge ratio of 5 +/−. Particles containing only pGL3 or pGL3 and siCON were used as controls. Growth medium was removed, cells were washed with PBS and particle formulation added. Cells were then incubated for 5 hours at 37 ° C., 5% CO ₂ before adding 800 μL of growth medium supplemented with 10% FBS. After 48 hours of incubation, cells were assayed for luciferase activity and cell viability as described in Example 12. The results are shown in FIG. RLU is lower on transfection with siGL3 (the correct sequence), and both siGL3 and pGL3 have to 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 and supplemented with 10% FBS 48 hours prior to transfection It was grown in culture medium. MAP particles were formulated to contain 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 added. Cells were then incubated for 5 hours at 37 ° C., 5% CO ₂ before adding 800 μL of growth medium supplemented with 10% FBS. After 48 hours of incubation, cells were assayed for luciferase activity and cell viability as described in Example 12. The results are shown in FIG. These data suggest that alienation of endogenous genes may occur, as RLU decreases with increasing concentrations of siGL3.

Example 16 Synthesis of Mucic Acid Diozide, (10)
1 g (2.7 mmol) of munic acid diamine (Example 3) is 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 drop was made in a 250 mL round bottom flask. The mixture was allowed to react overnight at room temperature, with constant stirring. The solvent was then removed by vacuum pump and the product was filtered, washed with methanol and dried under vacuum to give 0.8 g (1.3 mmol, 46%) of mucic acid iodide. ¹ H NMR ((CD ₃ ) ₂ SO) δ 8.20 (s 2 H), 2 H), 7.77 (s, 2 H), 4.1 1 (m, 2 H), 4.0 3 (m, 2 H), 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 Mutate, (11)
7 mL of 0.1 M degassed sodium carbonate was added to 17 mg of L-cysteine and 0.4 g of iodide. The resulting suspension was refluxed at 150 ° C. for 5 hours until the solution became clear. The mixture was then cooled to room temperature and adjusted to pH 3 with 1N HCl. Then a slow addition of acetone was performed for product precipitation. After filtration, washing with acetone and vacuum drying, 60 mg of crude product was obtained.

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

Example 18: Polymer synthesis, (poly (mucic acid-DiCys-PEG)) (12)
Dry 12 mg (21.7 μmol) of dicysteine mucinate and 74 mg (21.7 μmol) of PEG-DiSPA 3400 under vacuum, then 0.6 mL of anhydrous DMSO under argon, 2 necks 10 mL round bottom Added in flask. After stirring for 10 minutes, 9 μL (65.1 μmol) of anhydrous DIEA was transferred to the reaction vessel under argon. The mixture was stirred overnight under argon. The polymer containing solution was then dialyzed using a 10 kDa membrane centrifuge and lyophilized to give 47 mg (58%) of poly (mucic acid-DiCys-PEG).

  The polyol containing polymer is an anionic AB polymer.

Example 19 Drug (Camptothecin, CPT) Covalent Attachment to a Mucic Acid Polymer, (13)
10 mg (2.7 μmol repeating units) of poly (mucic acid-DiCys-PEG) were dissolved in 1.5 mL of anhydrous DMSO in a glass bottle. After stirring for 10 minutes, 1.1 μL of DIEA (6.3 μmol), 3.3 mg (6.3 μmol) of TFA-Gly-CPT, 1.6 mg (8.1 μmol) of EDC and 0.7 mg (5. 9 μmol) of NHS 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 poly (mucic acid-DiCys-PEG) -CPT conjugate.

Example 20: Formulation of nanoparticles with CPT-munic acid polymer (13) in water (20)
The effective diameter of the poly (muc acid-DiCys-PEG) and poly (muc acid-DiCys-PEG) -CPT conjugates is determined by formulating this polymer in redistilled water (0.1 to 10 mg / mL), ZetaPALS ( Evaluation was made via dynamic light diffusion (DLS) using a Brookhaven Instrument Co) instrument. Three consecutive runs of 1 minute each were then recorded and averaged. The zeta potentials of both compounds were measured in a 1.1 mM KCl solution using a ZetaPALS (Brookhaven Instrument Co) instrument. Then, 10 consecutive automated runs at a target residue of 0.012 were performed and the results averaged (Figure 4). In particular, these two distributions were measured for the poly (mucic acid-DiCys-PEG) -CPT conjugate, the main distribution being 57 nm (60% of the total particle population). The second smallest distribution is also measured at 233 nm.

Example 21: Formulation of Boronic Acid-Pegylated Nanoparticles with CPT-Mutonic Acid Polymer (13) Boronic Acid-Disulfide-PEG ₅₀₀₀ (6) in Water: Boronic Acid Pegylated Poly (Muthate-DiCys-PEG) -CPT The nanoparticles are dissolved in redistilled water at a concentration of 0.1 mg / mL, and then Polymer 6 in water (BA-PEG) is also added to give poly (mucic acid-DiCys-PEG) -CPT. The ratio of PBA-PEG to diol on the mumate sugar in the conjugate is 1: 1. The mixture is incubated for 30 minutes, after which the effective diameter and zeta potential are measured using a ZetaPALS (Brookhaven Instrument Co) instrument.

Example 22 Targeting Nanoparticles for pDNA Delivery in Mice The plasmid pApoE-HCRLuc contains a gene expressing luciferase and is under the control of a liver specific promoter. Polymer (MAP) 4 (0.73 mg), polymer 6 (73 mg) and polymer 9 (0.073 mg) were combined in 5 mL water, then 1.2 mL pApoE-HCRLuc plasmid containing water was added (+3 The charge ratio of polymer 4 to plasmid is obtained). 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 implanted with Hepa-1-6 hepatoma cells and tumors were allowed to grow to approximately 200 mm ³ in size. The injection of targeted nanoparticles was administered i.v. v. I went there. The mice were imaged 24 hours after injection. The mice showed no signs of toxicity and luciferase expression was detected in the area of the tumor and not in the area of the liver.

Example 23 Synthesis and pKa Determination of PBA-PEG and Nitro-PBA-PEG In order to prepare stabilized targeted nanoparticles, the reversible covalent binding properties between boronic acid and MAP were used. 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. These acyl chloride species were then reacted with NH ₂ -PEG to form PBA-PEG and nitro PBA-PEG, respectively. Addition of bases of DMF and DIPEA was necessary to drive the synthesis reaction. However, the presence of these bases resulted in tetrahedral adduct formation by acidic PBA. Working up in 0.5 N HCl and subsequent equilibration to neutral pH by dialysis against water were performed to remove these adducts.

  For the synthesis of 200 mg (1.21 mM) of PBA-PEG, 3-carboxyphenylboronic acid dissolved in 5 mL of anhydrous tetrahydrofuran was added under argon to 18.7 μl (0.24 mM) of anhydrous dimethylformamide . 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 with constant stirring under venting. The solvent and excess reagents 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) of NH 2 -PEG 5 kDa-CO 2 H and 52 μl (0.3 mM) of dry DIPEA were added under argon. After overnight reaction under constant stirring, the solvent was removed under vacuum and the dried product was reconstituted in 0.5 N HCl. The solution was dialyzed against water through a 0.2 μm filter (Acrodisc®) and using a 3 kDa MWCO membrane filter device (Amicon®) until a constant pH was obtained. The supernatant was then filtered using a 0.2 μm filter (Acrodisc®) and lyophilized to give 377 mg (73%) of PBA-PEG-CO 2 H. 1 H NMR ((CD 3) 2 SO) δ 12.52 (s, 1 H), 8.39 (t, 1 H), 8.22 (s, 1 H), 8. 14 (s, 2 H), 7.88 (d , 1 H), 7.82 (d, 1 H), 7.39 (t, 1 H), 3.99 (s, 2 H), 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 carried out analogously for PBA-PEG-CO2 H except that the starting material was 3-carboxy 5-nitrophenylboronic acid. This reaction yielded 393 mg (76%) of nitro PBA-PEG-CO2H. 1 H NMR ((CD 3) 2 SO) δ 12.52 (s, 1 H), 8.89 (t, 1 H), 8.72 (s, 1 H), 8.68 (s, 1 H), 8.64 (s , 1 H), 8.61 (s, 2 H), 3.99 (s, 2 H), 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 measurement of the change in absorbance of PBA converted from the trigonal conformation (low pH) to the tetrahedral conformation (high pH). PH 7.4 was titrated with 1 N NaOH against a solution of 10-3 M PBA in 0.1 M PBS. 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 formation of nitro-PBA-PEG, the resulting compound was conjugated to an antibody. For conjugation, 36 mg (6.4 μM) of nitro PBA-PEG-CO 2 H, 12.3 mg (64 μM) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 11.1 mg (EDC) 96 μM) of NHS was dissolved in 2.4 ml of 0.1 M MES buffer, pH 6.0. The mixture was allowed to react on a rotary shaker at room temperature for 15 minutes. Excess reaction was dialyzed out with a 3 kDa molecular weight cut-off membrane filter device (AmiconTM). This activated carboxylic acid PEG compound was added to 20 mg (0.14 mM) of human IgG1 antibody (Herceptin®) in 0.1 M PBS, pH 7.2. The reaction was carried out on a rotary shaker for 2 hours at room temperature and then dialyzed 4 times against 1 × PBS at pH 7.4 using a 50 kDa molecular weight cut-off membrane filter device (AmiconTM). Mass spectrometry (MALDI-TOF) showed an average of 1-2 conjugates of the nitro PBA-PEG compound per antibody.

Example 26 Formulation and Characterization of Targeted MAP Nanoparticles The effects of PBA-PEG and nitro PBA-PEG on nanoparticle formulation and stability were examined. A solution of PBA-PEG was added to the solution of MAP at a charge ratio of 3 +/- (positive charge from MAP per negative charge from siRNA) in phosphate buffer pH 7.4. This mixture was allowed to stand at room temperature for 10 minutes for complex formation of PBA-PEG with diol-containing MAP. It was then added to an equal volume of siRNA in water and mixed by pipetting. After 10 minutes, testing and characterization were performed. The formulation of particles was stabilized with nitro PBA-PEG and different charge ratios were made in the same manner. Complex formation of PBA-PEG formed MAP / siRNA particles of 150-300 nm in size. MAP-4 (showing four methylene units between the charge center (amidine) and the binding site (polyol) resulted in smaller nanoparticles than when MAP-2 was used. However, upon addition of salt, all the aggregated particles showed that PBA-PEG can not give sufficient stability to the nanoparticles. When nitro PBA-PEG was used, the particles demonstrated stabilized nanoparticles at all charge ratios tested, 1 and 3 (+/−). The difference in particle stability imparted by PBA-PEG and nitro PBA-PEG indicates the importance of modifying the PEGylated boronic acid compound to have a stronger bond to the polyol.

  A particle size of 640 nm with a high zeta potential of +32 mV was observed for siRNA condensed with MAP-4 in the absence of the stabilizing agent PEG. 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 the result of the bond between the boronic acid and the diol. Interestingly, the particle size is further reduced to 82 nm when introducing 0.25 mol% IgG (Herceptin®) conjugated with nitro PBA-PEG, when the zeta potential remains the same there were.

  Salt stability was assessed for MAP-4 / siRNA nanoparticles conjugated with various PEG groups. Particle size was recorded for 5 runs at 1 minute using a zeta potential analyzer (DLS ZetaPALS instrument, Brookhaven Instruments, Holtsville, NY). The measurements were stopped and the particles were formulated by adding 10x PBS solution so that the resulting particles contained 1x PBS. The measurement was then resumed and ten consecutive runs of 1 minute each 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 stabilizing agent 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 To prepare targeted MAP-CPT nanoparticles, the reversible covalent binding properties between boronic acid and MAP (containing diol) were used. The coupling constant between PBA and MAP is as low as about 20 M ⁻¹ . In order to increase the coupling constant, PBA with an electron withdrawing group in the phenyl ring was used to increase the acidity of the boron atom and to evaluate the coupling constant of MAP. Commercially available 3-carboxy PBA and 3-carboxy 5-nitro PBA were converted to acyl chloride using oxalyl chloride. These acyl chloride species were then reacted with NH ₂ -PEG-CO ₂ H to form PBA-PEG-CO ₂ H and nitro PBA-PEG-CO ₂ H, respectively. Addition of bases of DMF and DIPEA was necessary to drive the synthesis reaction. However, the presence of these bases resulted in tetrahedral adduct formation by acidic PBA. Working up in 0.5 N HCl and subsequent equilibration to neutral pH by dialysis against water were performed to remove these adducts.

The pKa value of the modified PBA was determined by the change in absorbance due to the conformational change from PBA 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 PBA is present in reactive anionic tetrahedral form. The binding constant between PBA and MAP was found by competitive binding with Alizarin Red S in 0.1 M PBS, pH 7.4. This was chosen as a linker between IgG (Herceptin®) and MAP-CPT nanoparticles due to the low pKa value and high binding constant of MAP observed for nitro PBA-PEG-CO ₂ H.

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

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

  An average size of about 40 nm was observed by DLS and cryo-EM (Table 3) for targeted MAP-CPT nanoparticles. An increase of about 10 nm from non-targeted nanoparticles indicates binding of IgG (Herceptin®) to the nanoparticles (the amount was about 1 Herceptin® per nanoparticle) . In fact, cryo-EM images showed that the targeting nanoparticles were not spherical, but with 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: Cellular Uptake Study To Test HER2 Overexpressing Human Breast Cancer Cell Line (BT-474) for Cellular Uptake of Herceptin® Targeted MAP-CPT Nanoparticles Versus Non-Targeted Nano-Image Financing used. The 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) (in 24-well plates, respectively) Cellgro, Manassas, Va.) Was seeded at 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 was 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 Or Targeted MAP-CPT containing 10 mg / ml of free Herceptin®. Transfection was performed at 37 ° C. for 30 minutes. The formulation was then removed and cells were washed twice with cold PBS. For cell desorption and lysis, 200 μl RIPA buffer was added per well. The 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 in the BT-474 cell line that one Herceptin®-PEG-nitro PBA is sufficient per nanoparticle to achieve approximately 70% higher uptake compared to non-targeted nanoparticles (FIG. 17A). Uptake of targeted MAP-CPT in BT-474 cells showed alienation in the presence of free Herceptin® (FIG. 17B). In contrast, uptake of MAP-CPT nanoparticles in MCF-7 showed no dependence on targeting in the presence of free Herceptin® (FIG. 2B). These data indicate that there is receptor mediated uptake in BT-474 cells via binding of the HER2 receptor by targeted MAP-CPT nanoparticles. Cellular uptake of both targeted and non-targeted 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, long MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles. These studies were performed using 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 It was performed using 1 × PBS (pH 7.4), or 100 units / ml of butyrylcholinesterase (BCHE) or a combination of LDL and BCHE.

For equilibration, medium pipetted into 96-well plates was incubated at 37 ° C. in a humidification oven for 2 hours. The formulations were mixed into the corresponding medium and placed back in the oven. Samples were removed at predetermined times and immediately frozen at -80 ° C until analysis. For release in BALB / c mouse plasma and human plasma, incubate the sample in a humidified oven at 37 ° C. with 5% CO ₂ (a major pH buffered system in plasma at physiological pH levels carbonate (To maintain the 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 for 30 minutes at room temperature. Then, 80 μl of methanol was added and the mixture was incubated for 3 hours at room temperature for protein precipitation. The mixture is centrifuged at 14,000 g for 10 minutes at 4 ° C., the supernatant is 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 eluted CPT (at 7.8 min) 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. The solution was incubated at room temperature for 1 hour to release CPT from the parent polymer. Then 10 μl of 0.1 N HCl was added to convert the carboxylate CPT form to the lactate state. Thereafter, 73.5 μl of methanol were added and the mixture was incubated at room temperature for 3 hours. The samples were then centrifuged and processed as above. The polymer bound CPT concentration was determined from the difference between the total CPT concentration and the unconjugated CPT concentration.

  The release from CPT short, medium and long MAP-CPT nanoparticles all showed first order kinetics. The emission half-lives from CPT short, medium and long MAP-CPT nanoparticles were similar in all conditions tested. On the other hand, longer half-lives were 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 the short, medium and long MAP-CPT nanoparticles. These data indicate that hydrolysis plays an important role in the release of CPT. For the short, medium and long MAP-CPT nanoparticles, a 59 hour emission half-life was observed in BALB / c mouse plasma, while an extremely short 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 "high fat" than mouse plasma. Thus, to know the difference in release rates between human and mouse plasma, the contributions to esterase and fat CPT release rates were examined separately. 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 chosen to page out 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 extended the release rate. This effect is probably due to nanoparticle destruction by competitive hydrophobic interactions. Thus, nanoparticle destruction due to the presence of "fat" and subsequent CPT cleavage due to hydrolysis is believed to be different from the main mechanism of CPT release from MAP-CPT nanoparticles. The release from Herceptin® targeted MAP-CPT nanoparticles shows a longer half life than the non-targeted version. Since the presence of Herceptin® with a pI of 9.2 protects the nanoparticles by electrostatic interactions and thereby from some of the competing hydrophobic interactions, the stability of negatively charged nanoparticles Sex can be increased.

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

Example 30: Cytotoxicity Assay In vitro cytotoxicity of medium-sized MAP, nitro PBA-PEG, CPT, medium-sized MAP-CPT nanoparticles, targeted MAP-CPT nanoparticles and Herceptin®, two HER2 + breast cancer cell lines ( It was evaluated in 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 respectively incubated in RPMI-1640 medium, Dulbecco's modified Eagle's medium, McCoy's 5A modified medium, and Dulbecco's modified Eagle's medium (all: all , Supplemented with 10% fetal bovine serum). 3,000 cells per well were plated in 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 5% CO ₂ humidification oven at 37 ° C. for 2 hours, after which an absorbance measurement at 490 nm was made. Wells containing untreated cells were used as a control.

Medium MAP and nitro PBA-PEG give IC ₅₀ values of 500 μM and 1000 μM (highest concentration tested) respectively, which shows minimal cytotoxicity. The IC ₅₀ concentrations of CPT, medium-sized MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles are based on the content of CPT. Note that CPT was released gradually from medium sized MAP-CPT nanoparticles 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 over 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 sized MAP-CPT nanoparticles. The addition of Herceptin to BT-474 cells or SKBR-3 cells at a concentration of 0.001 to 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 previously (Phillips, et al., Cancer Res. 68, 9280-9290 (2008)). At Herceptin concentrations up to 0.5 μM, the HER2- cell line did not show any effect.

^A no value, no IC ₅₀ values were obtained over the concentration range of 0.001 to 0.5 μM.

Example 31: Pharmacokinetics study Plasma pharmacokinetics study at 10 mg / kg (CPT based) injection of short, medium and long MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles, female BALB / C mice were performed (FIG. 18). For these studies, nanoparticles formulated in 0.9 wt% NaCl (non-targeted nanoparticles) or PBS (targeted MAP-CPT nanoparticles) were injected 12-16 via bolus tail vein injection. Administered to age-old female BALB / c mice. At predetermined times, blood was collected via a saphenous vein with a blood collection tube (Microvette CB 300 EDTA, Sarstedt). The samples were immediately centrifuged at 10,000 g, 4 ° C. for 15 minutes, the supernatant removed and stored at -80 ° C. until analysis. The 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 for 30 minutes at room temperature. Then, 80 μl of methanol was added and the mixture was incubated for 3 hours at room temperature for protein precipitation. The mixture is centrifuged at 14,000 g for 10 minutes at 4 ° C., the supernatant is 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 eluted CPT (at 7.8 min) 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. The solution was incubated at room temperature for 1 hour to release CPT from the parent polymer. Then 10 μl of 0.1 N HCl was added to convert the carboxylate CPT form to the lactate state. Thereafter, 73.5 μl of methanol were added and the mixture was incubated at room temperature for 3 hours. The samples were then centrifuged and processed as above. The polymer bound CPT concentration was determined from the difference between the total CPT concentration and the unconjugated CPT concentration. 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 and approved by the California Institute of Technology Institutional Animal Care and Use Committee.

  Polymer-bound CPT for all nanoparticles showed a biphasic profile with rapid redistribution phase (α) and long extinction phase (β) (FIG. 18A). The quenching phase of medium and long MAP-CPT nanoparticles, in particular, extend the half life to 16.6 hours and 17.6 hours, respectively, and high curved area (AUC) of 2298 and 3636 μg × hour / ml, respectively I gave a value. Furthermore, they showed low volume distribution rates and clearance rates. Twenty-four hours after injection, 11.3% of the injected dose of medium-sized MAP-CPT nanoparticles and 20.5% of long-shaped MAP-CPT nanoparticles remained in circulation in plasma as polymer-bound CPT . In contrast, mice injected with CPT alone at 10 mg / kg show rapid clearance with an AUC of only 1.6 μg × hour / ml and a 0.01% dose remaining in circulation after 8 hours The Targeting of medium sized MAP-CPT nanoparticles enhanced the redistribution phase, extended the extinction phase to 21.2 hours and affected the pharmacokinetics with high AUC value of 2766 μg × hr / ml . The amount of unbound CPT in plasma for all nanoparticles was low at all time points (FIG. 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 without 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 (based on CPT) CPT-based), 5, 8 or 10 mg / kg (based on CPT) long-shaped MAP-CPT nanoparticles and 8 or 10 mg / kg (based on CPT) targeted MAP-CPT nanoparticles, days 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. Mouse weight and health were recorded and monitored for two weeks from the start of treatment. The MTD was defined as the highest dose with less than 15% weight loss and no treatment related death. The animals were euthanized by CO ₂ asphyxiation if the criteria for MTD were exceeded 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 are weighed twice weekly on days 0 and 7 and healthy Was monitored (Table 6). Mouse weight and health are not affected by treatment with high doses of medium sized MAP or nitro PBA-PEG, which indicates minimal toxicity for these polymer components. For the groups containing CPT, maximal weight loss was seen on days 3-5 of each treatment. Most groups recovered their lost weight. The study was discontinued if weight loss continued and exceeded an average of 15%. Some mice treated with 15 mg / kg medium MAP-CPT and 10 mg / kg long form MAP-CPT showed diarrhea and appeared weak. All other mice appeared healthy. MTD values were found to be 20, 10, 8 and 8 mg / kg (based on CPT) for short, medium, long and targeted MAP-CPT, respectively (Table 6).

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

Example 33: Biodistribution in Nude Mice To assess the biodistribution of targeted nanoparticles in mice, 7 week old NCr nude mice were subcutaneously implanted with 17β-estradiol pellets. Two days later, BT-474 carcinoma cells suspended in RPMI-1640 medium were injected subcutaneously in the right front flank at 10 million cells / animal. Treatment, tumors were initiated after reaching an average size of 260 mm ^3. The animals are randomized into 2 groups of 6 animals each and targeted to 5 mg CPT / kg of MAP-CPT nanoparticles (pH 7.4 in PBS) or 5 mg CPT / kg by intravenous injection in the tail vein Treated with immobilized MAP-CPT nanoparticles and 29 mg Herceptin® / kg (in PBS, pH 7.4). Blood was collected from these animals in each group via saphenous vein hemorrhage after 4 and 24 hours. The animals were then euthanized by CO ₂ nitrogen and soaked 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 <(R)> OCT (Sakura) and the other was collected in an eppendorf tube, both of which were immediately frozen at -80 <0> C to the point of treatment.

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

  The mean percentage injected dose (ID) of total CPT per gram of tumor, heart, liver, spleen, kidney and lung is shown in FIG. 19A. Four hours after dosing, 5.3 and 5.2% IDs of total CPT were 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 It was present per gram of tumor in mice that received MAP-CPT nanoparticles. These data show that targeting nanoparticles of substantially the same size and surface charge as the non-targeted version do not increase tumor localization over the non-targeted version of tumor localization and other reported observations Indicates that it was consistent with Mice treated with MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles produced similar CPT distributions in heart, liver, kidney and lung. However, in the spleen, relatively high amounts of total CPT accumulation for targeted MAP ^ CPT versus non-targeting were at 24 hours. This effect has been previously observed for humanized antibodies in mice.

  FIG. 19B shows the average percent unconjugated CPT in the organs of tumor, heart, liver, spleen, kidney and lung. The percentage of unconjugated CPT in heart, liver, spleen, kidney and lung was low at both 4 and 24 hours. This indicates 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 in tumors suggests cellular accumulation of CPT. Targeted nanoparticles show a slightly higher percentage of unconjugated CPT in mouse tumors than non-targeted nanoparticles at both 4 and 24 hours.

  The total concentration of CPT in plasma and the percentage of total unconjugated CPT in plasma was similar for both MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles (FIG. 19C). After 4 hours, 17 and 20 μg / ml of 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, which was 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 and rapid clearance of unconjugated CPT from plasma (FIG. 19D).

Example 33 Treatment of Tumors with Targeted Nanoparticles BT-474 Tumor-Bearing Ncr Nude Mice 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, mice were euthanized and tumors were excised and cut into 20-30 μm thick sections using a cryostat. Tumor sections were placed on Superfrost Plus slides (Fisher Scientific, Hampton, NH) and stored at -80 ° C until time of treatment. Slides were thawed and tissue sections were fixed directly on slide glass 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 fluoresces naturally with an emission at 440 nm. To identify the location of the MAP, PEG within the MAP was stained at 14 μg / ml with a rat anti-PEG primary antibody that recognizes an internal PEG unit and incubated overnight at 4 ° C. This was followed by a 1 hour incubation with 3 PBS washes and 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 on a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Germany) using a 63 × Plan-Neofluar 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 lasers and acquisition settings were set at the beginning of imaging and were not changed. Image analysis was performed on a Zeiss lsm image browser.

  FIG. 20A shows tumor sections of mice 4 hours after treatment with MAP-CPT nanoparticles. CPT signals were scattered. The signals for CPT and MAP appeared to co-localize in the superimposed images. Twenty-four hours after injection, accumulation of CPT signal was present in a punctate manner (FIG. 20B). The superimposed images suggested co-localization of CPT and MAP. For mice treated with targeted MAP-CPT nanoparticles, points showing CPT accumulation were observed both after 4 and 24 hours of treatment (FIGS. 20C and 20D). In the superimposed images, there was co-localization of CPT and MAP signals. The presence of Herceptin in targeted MAP-CPT nanoparticles is indicated by a strong blue signal in the Herceptin channel as compared to the weak background signal in the non-targeted version.

Example 33 Antitumor Efficacy Study in Nude Mice BT-474 is one of the most resistant breast cancer to anti-cancer drugs including camptothecin. To promote tumor growth in nude mice, 17β-estradiol pellets were implanted into 7 week old Ncr nude mice and 2 days later BT-474 tumor cell transplantation was performed. On day 0 (5 days after transplantation), the tumor size of each group was an average of 250 mm. The treatment was started on the first day. The animals were randomized into 9 groups of 6-8 animals each and treated with either of the following: MAP-CPT nanoparticles, 1 mg or 8 mg / kg (CPT based, in PBS, pH 7.4) CPT, 8 mg / kg (20% DMSO, 20% PEG 400, dissolved in 30% ethanol and 30% 10 mM pH 3.5 phosphoric acid); irinotecan, 80 mg / kg (5 w / v% dextrose solution, in D5 W) Herceptin®, 2.9 or 5.9 mg / kg (in PBS, pH 7.4); targeted MAP-CPT nanoparticles, CPT / kg and 2.9 mg Herceptin® / kg, or 1 mg CPT / kg and 5.9 mg Herceptin® / kg (in PBS, pH 7.4); or saline. All treatments were freshly prepared and given via tail vein intravenous injection. Injections were normalized at 150 μl per 20 g of mouse weight. 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 tumor volume exceeded 1000 mm ^3. Six weeks after the start of the treatment, the animals were euthanized by CO ₂ asphyxiation. The results of this study are shown in FIG. 21 and Table 7.

Tumors in the control group animals receiving saline grew rapidly (FIG. 21). After 28 days, five mice out of 8 animals has had tumors that exceeds 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 produce any tumor inhibition (P> 0.05) compared to the control group. Treatment-related deaths of 1 and 3 were recorded on day 9 and day 16 respectively, and on day 28 4 cases of euthanasia due to exceeding the size limit of the tumor were recorded. No animals survived until the last day of the study.

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

Animals that received MAP-CPT nanoparticles at 8 mg CPT / kg showed very significant tumor inhibition as compared to tumor inhibition of 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 had their tumor size reduced to zero. No deaths occurred in MTD studies using non-tumor bearing NCr nude mice treated with MAP-CPT, 8 mg CPT / kg, but on day 21 of the study, death of one case occurred due to weight loss The This may be due to the tumor burden added in this study and / or the mice used for this study being 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 control group (P <0.01). At day 11 of treatment, 7 out of 8 treated animals had tumors that shrunk to zero. However, on the final day of the study, two of the reduced tumors relapsed, giving rise to a total of 5 animals with 0 volumes of tumor, with a group mean tumor volume of 60 mm ³ (FIG. 22). Mice treated with 1 mg CPT / kg of MAP-CPT nanoparticles were quickly discarded as no antitumor effect was observed. Nevertheless, when Herceptin®, 5.9 mg / kg, is added to the MAP-CPT nanoparticles via the nitro PBA-BA linker in a low volume of 1 mg / kg as a targeting agent ( 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 until the last day of the study ( Figure 22). One non-tumor associated 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 indicates that, in addition to the natural activity of Herceptin®, there was an improvement in potency by the addition of a Herceptin® targeting agent.

Animals treated with Herceptin® 2.9 mg / kg gave rise to 2 animals with tumors that had shrunk to zero by the last day of the study. However, the average tumor size increased to 278 mm ³ from the start of the study. This result is significant (0.01 ≦ P ≦ 0.05) as compared to the control group. When the animals were treated with targeted MAP-CPT nanoparticles containing 0.5 mg CPT / kg and 2.9 mg Herceptin® / kg, the two tumors shrunk to zero. On the last day of the study, tumor size was reduced to 141 mm ³ on average. This result is very significant compared to the control group (P <0.01). These results further suggest the benefit of targeting tumor inhibition.

  Five weeks after 17β-estradiol pellet implantation, it was observed that several mice, regardless of treatment group, had swollen bladder and abdominal distension. This is likely due to the use of a 17β-estradiol pellet which results in hydronephrosis and urination in athymic nude mice. After six weeks of treatment, the symptoms worsened and many mice were observed to develop swollen bladder and abdominal distension, thereby being euthanized and ending the study.

^{The a} N _start is the number of animals at the _start of the study, and the N _end is the number of animals surviving 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 _Neuthan is the number of animals euthanized by having exceeded 1000 mm ^{3 of} tumor size limitation. ^The _{reduction to} ^c N ₀ is the number of animals whose tumors have shrunk to 0 on the last day of the study.

  The examples given above are provided to give those skilled in the art a method for making and using embodiments of the complete disclosure and the particles, compositions, systems and methods of the present disclosure, and we will Modifications of the above-described modes of practicing the present disclosure, which are obvious to those skilled in the art who do not intend to limit the scope considered, are intended 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 pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference was individually incorporated in its entirety.

  The entire disclosure of the documents (including patents, patent applications, magazines, articles, laboratory manuals, books or other disclosures) cited in the background art, abstract, detailed description and examples are hereby incorporated by reference. Be incorporated.

  It is understood that the present disclosure is not limited to particular compositions or biological systems, which 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. Do. The term "plural" includes two or more objects, unless the context clearly indicates 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 methods and materials similar or equivalent to those issued herein are in fact used to test specific examples of suitable materials and methods described herein. It is also good.

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

Claims (51)

A nanoparticle 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 in the environment outside the nanoparticles. Said nanoparticles. The polyol-containing polymer comprises at least one or more of the following structural units:
During the ceremony
A is the organic moiety of the following formula:
here,
R ₁ and R ₂ are independently selected from any carbon-based group or organic group having a molecular weight of about 10 kDa or less;
X is selected from aliphatic groups comprising one or more of -H, -F, -C, -N or -O; and Y is independently selected from organic moieties 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 described in. R ₁ and R ₂ independently have the following formula:
During the ceremony
d is 0 to 100;
e is 0 to 100;
f is 0 to 100;
Z is a covalent bond linking one organic moiety to another organic moiety; and Z ₁ is independently selected from -NH ₂ , -OH, -SH and -COOH. Nanoparticles. During the ceremony
X is C _n H _{2n + 1} , where n is 0 to 5; and wherein
The nanoparticle according to claim 2 or 3, wherein Y is -OH. A is the following:
Independently selected from the group consisting of
During the ceremony
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. The above A is as follows:
The nanoparticle according to any one of claims 2 to 5, which is independently selected from The above B is as follows:
here,
q is 1 to 20;
The nanoparticle according to any one of claims 2 to 6, wherein p is 20 to 200; and L is a leaving group. Nanoparticles according to claim 2, wherein the structural unit of formula (I) is
Nanoparticles according to claim 2, wherein the structural unit of formula (II) is
The structural unit of formula (III) is
And here
The nanoparticle according to claim 2, wherein n is 1 to 20. The nanoparticles 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:
During the ceremony
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 to 1000,
12. A nanoparticle according to any one of the preceding claims, wherein a is 0-3 and b is a nitrophenylboronic acid which is 0-3. 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 nanoparticle according to claim 12 or 13, wherein Y ₁ is a phenyl group having one or more nitro groups.   The nanoparticle according to any one of claims 12 to 14, wherein r = 1, a = 0 and b = 1. 16. The functional group 1 and the functional group 2 are the same or different and are independently selected from -B (OH) ₂ , -OCH ₃ , -OH. Nanoparticles described in. The nitrophenylboronic acid-containing polymer is as follows:
And
17. A nanoparticle according to any one of claims 12-16, wherein s = 20-300.   18. A nanoparticle according to any one of the preceding claims, further comprising a compound, wherein the compound forms part of the polyol containing polymer and / or the nitrophenylboronic acid containing polymer.   18. A nanoparticle according to any of the preceding claims, further comprising a compound, wherein the compound is attached to the polyol containing polymer and / or the nitrophenylboronic acid containing polymer.   20. The nanoparticle of any one of claims 1-19, 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 polynucleotide.   22. The nanoparticle of claim 21, wherein the polynucleotide is an interfering RNA.   23. The nanoparticle of any one of claims 1-22, further comprising one or more compounds, wherein at least one compound is a target ligand.   24. The nanoparticle according to claim 23, wherein the target ligand is a protein, an aptamer, a small molecule, an antibody or an antibody fragment.   25. The nanoparticle according to claim 23 or 24, wherein the target ligand is one single antibody.   26. The nanoparticle of claim 25, wherein the one single antibody is conjugated to the terminal portion of the nitrophenylboronic acid containing polymer.   The targeting ligand is selected from transferrin, a protein, an aptamer, a small molecule, an antibody, or an antibody fragment, and the nanoparticle is selected from a camptothecin, an epothilone, a taxane or a polynucleotide, or any combination thereof. 25. The nanoparticle of claim 23, further comprising.   A composition comprising the 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. 30. The composition of claim 29, wherein the nanoparticles further comprise ¹⁰ B as part of at least one nitrophenylboronic acid containing polymer, and wherein the composition is formulated for in vivo boron neutron activation therapy. object.   28. A method of delivering a compound to a target comprising contacting the target with the nanoparticle of 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 used to deliver the compound to the target.   28. A method of administering a compound to an individual, said method further comprising administering to said individual the nanoparticles of any one of claims 1-27, further comprising said 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 that can be interconnected via a reversible covalent bond. 28. The system wherein one polyol containing polymer and at least one nitrophenylboronic acid containing polymer are collected together with the compound in the nanoparticles of any one of claims 1-27 and administered to the individual. The following formula:
During the ceremony
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 to 1000,
Polymer containing nitrophenylboronic acid wherein a is 0-3 and b is 0-3. 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— The polymer according to claim 36 or 37, wherein Y ₁ is a nitrophenyphenyl group.   39. The polymer of 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.   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 one 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. The method of claim 1, comprising contacting the polyol-containing polymer with the nitrophenylboronic acid-containing polymer under time and conditions that allow the polyol-containing polymer to be coupled with the nitrophenylboronic acid-containing polymer. A method of preparing the nanoparticle according to any one of the above.   46. The method of claim 45, wherein the nanoparticles are configured such that the nitrophenylboronic acid containing polymer is in the external environment of the nanoparticles.   47. The method of claim 45 or 46, wherein the polyol containing polymer is a mucinous acid polymer. The nitrophenylboronic acid-containing polymer is as follows:
Is one of
48. The method of any one of claims 45-47, wherein s = 20-300.   28. A method comprising: nanoparticles comprising a polyol-containing polymer, a nitrophenylboronic acid-containing polymer, and instructions on how to conjugate the nanoparticles comprising the polyol-containing polymer with the nitrophenylboronic acid-containing polymer. A kit for producing the nanoparticle according to any one of the above.   50. The kit of claim 49, wherein the polyol containing polymer is a mucinous acid polymer. The nitrophenylboronic acid-containing polymer comprises:
Is one of
51. The kit of claim 49 or 50, wherein s = 20-300.